irProyes^ - IRIS PAHO Home

Pouo tcb l4at, RI

irProyes^

A:MERICRAN HEALTH ORGANIZATION-~ n;of Research Development and Coordination

WORLD HEALTH ORGANIZATION

19 76

Researchin Progress

1976

-/Ref: RD 15/1.

.,AN AM'ECBCAN HE[aALTH O©.RGANIZATM©NDepartment of Research Development and Coordination_

WORLD HEALTH ORGANIZATION

·. - 1 ,.i.,

-) ü, A o )

1 ¿t -6

FOREWORD

The PAHO research program will complete 15 years in June1976. From a modest beginning, the program has progressed to aninvestment of about five million dollars a year on research andresearch training schemes in a wide variety of fields relevant tohealth problems in the Region. Although funds come mainly fromsources outside the Organization's regular budget, PAHO plays animportant catalytic function in securing these funds and in iden-tifying projects that can effectively use them.

During the last few years the biomedical community of theAmericas has gradually come to regard PAHO as a desirable sourceof research support, a reflection in part (we like to think) ofthe program's good performance. We are grateful to the PAHOAdvisory Committee on Medical Research for maintaining at a highscientific level a program that is specifically attuned to theneeds and opportunities for research in Latin America and theCaribbean.

This new edition summarizes 151 projects--ranging from basiclaboratory research to clinical, epidemiologic, and methodologicstudies--in which PAHO currently participates as grantor, grantee,or collaborator. We hope it will contribute to narrowing the gapbetween ongoing research and formal publication of its findings.

M. Martins da Silva, M.D., ChiefDepartment of Research Development

and Coordination

CONTENTS

Page

NUTRITION AND FOOD SCIENCE

Protein Requirements of Children of Preschool Age - Rice-Bean DietsGuellet mo Auroyave .............. ... ... 1..................

Amino Acid Requirements of Preschoolchildren Gu emno Atoyave ....... 3

Nutrient Sources Ríeaxdo Bt an ..................................... 5

Chemical and Nutritional Characterization of Basic Foods andTheir Utilization Rcaxdo BeLan .................................. 7

Corn Fortification: A Field Demonstration Model Juan Jos6 UJLLutía .... 10

Evaluation of Sugar Fortification with Vitamin A at the National LevelGuw.etmo AtLoyave and Ivan D. Beghin ................................ 12

Utilization of Agricultural and Industrial Byproducts in AnimalNutrition R¿catd Besan ......................................... 15

Graduate Courses in Food Science Technology and Animal NutritionRicatdo Bre4sani ..................................................... 18

Preservation and Processing of Foods for Human ConsumptionRicatdo Bte6sani ........................................ 20

Assessment of and Nutritional Losses Due to Malabsorption in LowIncome Rural Guatemalan Communities Robxtto E, Schneidet andDoSií Calelway ........................................ 22

Development and Evaluation of Measures to Reduce Food Waste Caused byIntestinal Diseases Robetto E. SchneideA and Morrl i Shi4man ....... 24

Effect of Food Supplementation During Pregnancy and Lactation onInfant Morbidity and Mortality Robextt E. Kleín and Aaton Lechtig 26

Protein-Calorie Malnutrition: Effects, Mechanisms of Adaptation,Pathophysiology, and Treatment Fetxnando E. V¿teti andNoel W. Solomon ........................................ 28

Have Modern Health Practices Removed the Safeguard of BacterialInterference, Shortened Breast-Feeding, and Increased InfantMalnutrition? John Kennele and MauthaUe Kauw ...................... 36

Mainutrition, Infection, and Human Leukocyte Metabolism OcaOJ Pneda 38

Hematologic Investigations in the Central American PopulationFernando E. V¿tei ................................................... 40

Antibody Response to Measles Vaccine in Malnourished ChildrenJuan Joseé Uwutía and Míicha~e Katz ................................... 43

Interaction Between Nutrition and Productivity of AgriculturalLaborers Fet'uando E. Viteti ....................................... 45

Page

Longitudinal Study of the Effects of Mainutrition on Physical Growthand Mental Development RobeRt E. Kleín ............................ 48

Cross Cultural Studies in Cognitive Development Robert E. Keiín andJetome Kagan ........................................................ 61

Evaluation of the Effects of Nutrition Education on Nutritional StatusCawtbbean Food and Nut'itÁion InYtí,tute .............................. 63

Program of Social Studies In46¿tute. of Nuwitkoon of CenvtraAmetica and Panama .................................................. 65

METABOLIC DISEASES

Epidemiological and Metabolic Studies of Cholelithiasis in ChileVtcente Valdivieío ................................................... 67

DENTAL HEALTH

Dental Caries Research in Two Colombian CommunitiesGeotge M. Gilezspie, Betutam Cohen, and Willíam H. Bowen ............ 70

ALCOHOL AND DRUG ABUSE

Epidemiology of Alcoholism in Latin America MwaLiyn E. Katatsky ...... 72

ENDOCRINOLOGY

Pathophysiology of Endemic Goiter and Cretinism in Ecuador andTheir Prevention with lodinated Oil Rodtigo FieJto-BeenCtez ........ 74

GENETICS

Multidisciplinary Studies on Primitive Populations in Latin AmericaJame, V. NeeQ ....................................................... 77

Clinical and Genetic Investigations on the Alport Syndrome(Uruguayan Kindred) Míxímo E. Dtet6, ............................... 81

PERINATOLOGY AND FERTILITY

Sociodemographic and Biological Determinants of Natural Fertilityin Guatemalan Villages Robert E. Klein, Hean4n Delgado,Chalesl TellRe, and John Boongaaxts ................................. 83

Sociological, Economic, and Psychological Determinants of BirthSpacing in Guatemala Robett E. KRe¿n .............................. 85

Page

Effects of Immunization with Human Chorionic Somatomammotropin(HCS) on Reproduction Míguel A. Ca4&telano .......................... 88

Isolation, Characterization, and Elucidation of the BiologicalFunction of a New Human Placental Hormone Jorge Btovetto-Ctuz ....... 90

Relationship of Oxytocin and Ovarian Contractility in the RatRiícaBdo Roca .......................................................... 92

Evaluation of Monitoring in Labor Induction J. C. Cuadto andRuben Belitzky ........................................................ 95

Effects of Membrane Rupture on Labor Progress and on CephalicMolding RicPtdo L. Schwatcz ..................................... 97

Intrauterine Growth Charts of a Normal Population at Sea LevelVíoleJta Cuwbelo ........................................ 102

Growth and Development of Low-Birthweight Newborns Miguel Mate ..... 104

Perinatal Factors Affecting Child Development Daniel Foneca andRa'R Rugga ....................................................... 106

Antibacterial Defenses in the Newborn Juan Jo4é UAuwtia andGerald T. Kech ...................................................... 108

Factors Affecting Perinatal Mortality and Morbidity in DiabeticsS. V. Pose and R. Betzky ............................................ 110

Implementation of Simple System for Collecting and ProcessingPerinatal Data Raúl BSejá' and R. Betzky ........................... 112

IMMUNOLOGY

Mechanisms of Anaphylactic Reactions Ivan Mo.ta ........................ 114

Regulatory Mechanism of Reaginic Antibodies Ivan Mota .................. 116

Adjuvant Effect of Synthetic Double-Stranded Ribonucleic AcidsIvan Mota ......................................... 118

Study and Quantitation of Body Fluids by Means of ImmunonephelometryJotge Lizana .......................................................... 120

CHRONIC DISEASES AND CANCER

Epidemiologic Study of Myocardial Ischemia, High-Density Lipoproteinand Other Coronary Risk-Factors in Trinidad G. J. Me ............ 123

Control Mechanisms of DNA Synthesis in Human Lymphocytes andLeukemic Celis Jos4é Modoh .......................................... 125

Latin American Cancer Research Information Project Jorge Ltvak ........ 127

iii

Page

MALARIA AND OTHER PARASITIC DISEASES

Sporozoite-lnduced Immunity in Mammalian MalariaRuth S. Nussenzweig ........... ....................................... 129

Interrelationship of Trypanosoma cruzi Infection and ChronicChagas' Cardiomyopathy in a Rural Population Kenneth E. Mott,Rodney Hoff, J. Stauf4er Lehman, Jt., Richard H. Mortow, Jr., andThomas H. WeIler ..................................................... 132

Natural History of Schistosomiasis Mansoni in a Rural Population ofNortheastern Brazil J. Stauferh Lehman, Jr., Kenneth E. Mott,Richavd H. Mortow, Jr., Rodney Hofd, and Thomau H. WeUlex ............ 135

Human Filarias and Their Vectors in Trinidad Patcick J. S. Ham¿lton 137

Kabowra Fly (Simuliidae) Studies in Guyana Patxick J. S. Haml¿2ton 139

INFECTIOUS DISEASES

Studies of Arthropod-Borne Viruses Wi¿Jiam F. Scherer andRobert W. Dickeman .................................................. 141

Early Biopsy Diagnosis of Rabies Donald C. Blenden .................... 144

Epidemiology of Rabies C. O. R. Everard ............................... 146

Epidemiology of Leptospirosis in Trinidad and GrenadaC. O. R. Evetahd ..................................................... 148

Diarrhea in a Closed Community Catibbean Epidem¿ology CentAe .......... 150

Infectious Diseases Epidemiology in South American IndianPopulations Francis L. Black ....................................... 152

Cellular &tudies on Rheumatic and Glomerulonephritis Patientsin Trinidad John B. Zabriskíe ...... ............................... 157

Hepatitis B in Trinidad LesQ61e Spence and Miles C. Wi¿iam6s ........... 159

MULTIDISCIPLINARY STUDIES

Organization and Development of a Multidisciplinary ResearchProgram in the Amazon Area MauWic¿o Ma~tnts da Sieva andKenne.th E. D xon ................................................... ........ 161

DISEASE SURVEILLANCE

Caribbean Epidemiology Centre PattIck J. S. Ham~ton ............... 164

iv

Page

MYCOLOGY

Spherulin and Coccidioidin: Cross-Reactions in Dermal Sensitivityto Histoplasmin and Paracoccidioidin Hit¿e B8. Levíne ............. 168

Studies on the Development of Serologic Antigen(s) forParacoccidioides brasiliensis Yeasts Grown in VitroCharlotte C. Campbell ............................................... 171

Production of a Latex-Paracoccidioidin Reagent for the SerologicDiagnosis of Paracoccidioidomycosis Angela Re;ttepo-Moteno ........ 173

Study of Cross-Reactivity Among Fungal Skin-Test AntigensJohn D. Schne¿dau, Jr. ............................................. 175

Usefulness of the Indirect Immunofluorescence Test in LungAspergillosis Ricahdo Negton¿ ..................................... 177

Determination of Serum Antibodies to Aspergillus in the MaracaiboPopulation GuRemo0 Caa,6 Ríncbn ................................. 180

Experimental Taxonomic Studies'of Certain Hyphomycetes Pathogenicto Man Gavty T. Cole .............................................. 182

Keratinophilic Fungi and Adiaspiromycosis in Ontario A. Baketu.pigel ... 184

Causal Agents of Granulomas and Mycetomas Which Form Grey ColoniesDante BorteL¿ ........................................ 186

Deep Mycoses in Patients Showing Abnormal Chest X-raysHumbetto Campins .................................................... 188

MYCOBACTERIUM INFECTIONS

Fernandez and Mitsuda Reactions in Leprosy Patients Using Humanand Armadillo Antigens Jack W. MileaX ............................. 190

Mycobacteria and Poor Housing in Leprogenous FociGurUetmo MuFioz Riva ............................................... 192

Experimental Infection of the Armadillo with Mycobacterium ulceransRíchatd E. KxLeg .................................................... 194

Contribution to the Study of the Armadillo Model Jacinto Convut ...... 196

FOOT-AND-MOUTH DISEASE AND VESICULAR STOMATITIS

Improvement of Techniques for Foot-and-Mouth Disease (FMD)Research EduÁardo R. Centeno, ALbino A. Fexnandez, Antonio Vieita,and Maria Elma V. Feuteita .......................................... 199

v

Page

Pathogenesis, Transmission, and Reservoir of Foot-and-MouthDisease Virus (FMDV) Paui Sutm£noet, Paulo A. de Mello, Ivo Gomes,and Antonio Veitua ................................................... 202

Investigation of FMD Virus in Milk and Milk Products Paul SutmVnlolel,FeiLx Rozenberg, Vicente Astudillo, and Hertán Málaga ............... . 206

Selection of Strains for Foot-and-Mouth Disease Vaccine ProductionAlbno A. Fetnandez, Daniel Abaracon, Magnuz S. Sondahl, andEduatrdo R. Centeno ....................................... ............ 208

Production, Purification, Concentration, and Treatment of Foot-and-Mouth Disease Virus (FMDV) Antigen for Vaccine ProductionDanvel Abatacon, Homexo V. Giacomett¿, Eduatdo R. Centeno, andJul¿o Mesquita ..................................................... .. 210

Foot-and-Mouth Disease Virus (FMDV) Inactivation Hanz Bahnemann,Dantie Abatuacon, and Hometo V. Giocometti ............................ 213

Adjuvants for Foot-and-Mouth Disease (FMD) Vaccines Daniel Abatacon,Homero V. Giacometti, Paulo A. de Mello, and JulRo Mezquta .......... 215

Field Application of Inactivated Foot-and-Mouth Disease Oil AdjuvantedVaccine Paulo A. de Mello, Vicente AstudRllo, and Ivo Gomes ........ 218

Modified Live Foot-and-Mouth Disease Virus Vaccines Cartos Betenal,Paul Sutmoblle, Paulo A. de Mello, and Kleise F. Costa ............... 221

Quality Control of Foot-and-Mouth Disease (FMD) Vaccine Albíno A.Fetnandez, vao Gomez, Antonio Vieira, and Maria V. FeuteiLta .......... 226

Diagnosis of Vesicular Diseases of Livestock Albnao A. Fetnandez,Magnus S. Sondahl, vao Gomez, and Eduardo R. Centeno ................. 229

The Management of Mouse and Guinea Pig Colonies Under Tropicaland Subtropical Conditions Paul W. Schkillng ....................... 233

ZOONOSES

Determination of Rabies Antibodies by CounterimmunoelectrophoresisAna Marta 0. de Díaz ................................................. 236

Antigenic Differences Among Rabies Virus Strains Ana Matia 0. de DVíaz .. 237

Biological Characteristics of Rabies Virus StrainsAna Matxa 0. de Dáz ................................................ 239

Rabies Vaccine of Tissue Culture Origin 0. P. Largh¿ ................. 241

Comparative Effect of Adjuvants in Suckling Mouse Brain RabiesVaccine for Bovine Use Ana Maxza 0. de Díaz ....................... 243

Evaluation of Serological Tests Used in the Diagnosis of HumanBrucellosis Rail Casas Olascoaga and Atno Twuouetzky .............. 245

vi

Page

Demonstration Project of Bovine Brucellosis Eradication in LargeDairy Farms of Argentina RaúU Ca&sU O1ascoaga .................... . 248

Pathogenesis of Ram Epididymitis Caused by Brucella ovisExperimental Studies in Laboratory Animals AlbeAto Cuba-Capat6 .... 250

Experimental Infection of Male Goats with Brucella ovis

Caíiimíto Gatcia Caítll¿ o ............................................ 252

Evaluation of the Card Test with Nondisposable Material in theDiagnosis of Brucellosis RaúQl Cacuac Ozcoaga ..................... 254

Typing of Brucella Strains Isolated from Man and Animals inLatin America Cacsmt0o Gatc.ía Ca¿o ............................ 256

Evaluation of Vaccines Against Swine BrucellosisCau-~ni'o Gatcía Caw~JÁ¿o and V. C. F. Cedto ......................... 258

Studies on the Growth and Dissociation of the Rev 1 Vaccine Strainof Brucella melitensis in Controlled Liquid Culture EnvironmentsSandord S. Elbeg ................................................... 261

Study of Inactivated Brucella Vaccines Ca6Zm-to Gatca Ca~utiXe o ...... 264

Factors Affecting the Storage of Lyophilized VaccinesCa¿nu'Lo Gatca CattLeo ............................................ 266

Immunity in Guinea Pigs Conferred by Strain 19 Vaccine AgainstDifferent Biotypes of B. abortus Prevalent in Latin AmericaCasimxto Gatcta CaAtAílo ............................................ 268

Proficiency Testing of Brucella abortus Strain 19 VaccineCac¿mi'to Gatcía CattU¿o ............................................ 270

Evaluation of Four Variants of the Indirect Hemagglutination Testfor Human Hydatidosis Diagnosis V. M. Vateta-DZaz ................. 272

Penetration of Macromolecules into Hydatid CystsV. M. Vatela-Díaz ................................................... 274

Modification of the Immunoelectrophoresis Test for the Diagnosisof Human Hydatidosis V. M. Vaiteza-D~iaz ............................ 276

Evaluation of Immunodiagnostic Tests for Human Hydatid DiseaseV. M. Vatea~-DaZz ................................................... 278

Evaluation of the Crossed-Over Electrophoresis Test for theImmunodiagnosis of Human Hydatidosis V. M. Vateta-DZaz ............ 280

Evaluation of a Serologic Screening Approach for DetectingHydatidosis Cases in Field Situations V. M. VaAtela-Díaz ........... 282

Evaluation of Double Diffusion Tests for the Immunodiagnosisof Human Hydatidosis V. M. Vatela-Díaz ............................ 284

vi i

Page

Epidemiologic Studies on Hydatid Disease in Bolivia, Ecuador,and Guatemala Amax S. Thakuh and Hcatort L6pez Ada0so ............... 285

Epidemiologic Studies on Hydatid Disease in ArgentinaAma,% S. Thak-uL and H-ctor L6pez Adatoz ............................... 287

Estimation of Economic Loss in Sheep Due to Hydatid Diseasein Uruguay Amat S. ThakuW .......................................... 289

Structure and Function of Pilot Hydatid Control Programs inArgentina, Uruguay, Peru, Chile, and Brazil Amah S. Thakuh andJo0s Gemaná Rodrlguez To0r4e ......................................... 291

Drugs for the Treatment of Canine Echinococcosis Amah S. Thakuht ........ 293

Fine Structure of the Germinal Membrane of Echinococcus granulosusCysts V. M. VaxeQa-Dlaz ................... ..................... 295

Practical Methods of Killing Scolices of Echinococcus granulosusto Make Viscera Safe for Dog Feeding Amat S. Thakur ................ 297

Induction of Immunity Against Larval E. granulosus UsingX-Irradiated Eggs Amah S. Thaktu- ................................... 299

Effect of Bunamidine Hydrochloride and Embay 8440 on Eggs (Free orWithin Proglottids) of Echinococcus granulosus Amac S. Thakuh ...... 301

Survival and Viability Criteria of Hydatid Cysts MaintainedIn Vitro V. M. Vatela-Díaz ......................................... 303

Surveys of Cysticercosis and Fascioliasis in Man and Animals inthe Americas AmotL S. Thakut ........................................ 305

Isolation of Serotype Fort-Bragg in Barbados and its Association toLeptospirosis of Cattle and Man Donald M. Myes .................... 306

Equine Leptospirosis: Serologic Studies and Isolations of SerotypeHardjo and Leptospira Biflexa Strains from Horses of ArgentinaDonald M. Myeu ...................................................... 308

Histologic and Hematologic Studies in Apparently Normal Laboratoryand Wild Animal Species ALbeto Cuba-Capcat6 ........................ 310

The Armadillo as a Possible Reservoir Host for Leptospiral SerotypeHardjo Infection in Cattle Donald M. MyQet ......................... 312

Influence of Leptospiral Growth Media on Microagglutination SerumTiters in the Detection of Leptospiral AgglutininsDonald M. Mye ...................................................... 314

Determination of the Efficacy of the Outer Envelope of Leptospiraas a Potential Vaccine and/or Diagnostic Reagent Ronald H. Jonesz ... 316

Effects of Tuberculin Test Sensitivity by Tuberculin Potency,Test Site, and Test Interpretation Scheme in Argentine CattleInfected with Mycobacterium bovis J. D. Roswwu .................... 318

viii

Page

Transport Media for Bovine Tuberculous Lymph NodesIsabel N. de Kantot .................................................. 320

Isolation of Mycobacteria from Two Species of Armadillos:Dasypus hybridus (Mulita) and Chaetophractus villosus (Peludo)lsabel N. de Kanto ........................................ .......... 322

Development of Microbiological Criteria for the Control ofCheese in Argentina Fetnaando Quevedo ............................... 324

New Method for Microbiological Sampling of Animal Carcasses andSurfaces FeAtnando Quevedo .......................................... 326

Salmonellae and Fecal Coliforms as Pollution Indices in SuperficialWaters of the Greater Buenos Aires Area FeAtnando Quevedo ........... 328

Pathology of Laboratory Animals Albetto Cuba-Copat6 ................... 330

HEALTH STATISTICS

Inter-American Investigation of Mortality in Childhood Ruth R. Puflet ,. 332

ENVIRONMENTAL SCIENCES AND ENGINEERING

Rural Sanitation Ftanc.¿co Campos ..................................... 337

Chemical and Bacteriologic Quality of the Water of Lake PeñuelasJortge Cac6tíe.o ......................................................... 338

Sewage Treatment with Tower-Like Filters Atu/to Pazoz ................. 340

Sedimentation Tanks Juan Pablo Sch4fní¿ ............................... 341

Stabilization Ponds Juan Pablo Sch n ............................... 343

Stream Pollution in the Watershed of the Lake Amatitlan andin the Villalobos River CatLoS E. Munoz PalacíoS ................... 345

Toxic Parameters in the Water Supply Systems of the MajorCities in Chile Ftanc¿6co Unda Opazo ............................... 347

Air Pollution in Guatemala City RodoLdo Gonzdlez Moutszo .............. 349

Mathematical Models for Self-Purification Constants inSurface Waters Josz M. Gundeach ................................... 350

OPERATIONS RESEARCH

National System for the Maintenance of Health Care FacilitiesJohn E. Donahue and John Z. Andexs ................................... 351

ix

Page

Information System for the Control of Supporting Servicesin Hospital Wards Johge OxUtz and Jorge Peña ....................... 353

BIOMEDICAL COMMUNICATIONS

PAHO Regional Library of Medicine and the Health SciencesM. Maxuin da Síeva and Amadot Negíhe .......................... 355

Strengthening the Brazilian Biomedical Information NetworkJohn D. Wilkes ....................................................... 358

x

PROTEIN REQUIREMENTS OF CHILDREN OF PRESCHOOL AGE - RICE-BEAN DIETS

PROBLEM

The amounts of protein from egg, milk, and corn-bean mixtures required bychildren between the ages of 2-3 years has been studied extensively in the last 3years. Available information indicates that egg and milk protein meet nitrogen andindividual amino acid requirements when given at a level of 1.2 g of protein/kg/day.Daily intake of 1.6 g of corn and bean protein also fulfills nitrogen and amino acidrequirements when about 50 percent of the protein is provided by beans and 50 percentby corn. Many underdeveloped populations in some Central American countries live ona rice-bean, instead of a corn-bean diet. The objective of the present study wastherefore to determine the amount of rice-bean protein necessary to maintain growth,normal biochemical characteristics, and nitrogen retention in children aged 2-3 years.

METHOD

Four boys 2-3 years of age, fully recovered from malnutrition, were studied.Full recovery was documented by anthropometric, clinical, and biochemical parameters.The children received a diet in which the only protein source was rice and beans.Mineral and vitamin supplements were given. The proportion of rice and beans givenwas 70 percent rice and 30 percent black beans, which provides the most balanced pro-tein diet according to the UN Food and Agriculture and World Health Organizations.The diets provided 100 cal/kilo/day, 30 percent of the calories coming from vegetablefat. Two of the children received initially 1.75 g protein/kg/day and, in 3-weekperiods, had their protein intake reduced sequentially to 1.50, 1.25, 1.00, and 0.75g/kg/day. The 2 other children followed a reverse sequence. The children were fol-lowed daily by clinical, anthropometric, and biochemical methods. They were free tomove about and play, except for the last 3 days of each 3-week period when they wereconfined to bed for nitrogen balance determination. The main values measured were:daily body weight, weekly height, hemoglobin and hematocrit, serum proteins andelectrophoresis, and essential-to-nonessential amino acid ratio.

RESULTS TO DATE

During the total study period the four subjects developed hepatitis. Despitethe interfering effect of this superimposed morbidity the following conclusions weremade:

(1) Plasma amino acid results strongly suggest that intakes of 0.75 and 1.00g/kg/day are less than desired, and that 1.50 and 1.75 g/kg/day are adequate. Theintermediate intake of 1.25 g could not be evaluated because of the unfortunate in-terfering effect of hepatitis on this biochemical parameter.

(2) On the bases of anthropometric measurements and nitrogen balance, theprotein of the rice-bean combination studied seems excellent. Although direct com-parison with previous, independently run trials is not in order, the protein of thiscereal-legume mixture does not appear inferior to that of egg or milk. An intake ofas low as 1 g protein/kg/day seemed sufficient. An intake of 1.25 g/kg/day maintainedample positive nitrogen balance even in children suffering from mild hepatitis.

1

SIGNIFICANCE

The results of this investigation and those of previous studies with corn andbeans, strongly support the feasibility of attacking or preventing the problem ofdietary protein deficiency with cereal-legume combinations. In recent years therehas been increasing evidence that a diet with adequate amount of leguminous seedscomplementing a cereal staple, contains enough protein, both in quality and in quan-tity, and that protein calorie deficiency is mostly due to insufficient availabilityand consumption. Although it is recognized that diets containing animal proteins aremore desirable and more appealing, it is not possible to predict at the moment whenthese animal products will be abundant enough for consumption in the underdevelopedworld. Consequently, the demonstrated feasibility of providing adequate proteins incereal-legume combinations is extremely important as a short-term approach to theproblem.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. Guillermo Arroyave, Division of Physiological Chemistry,Institute of Nutrition of Central America and Panama (INCAP),Guatemala City, Guatemala.

Funded by: World Health Organization.

Timetable: 1968 - continuing.

2

AMINO ACID REQUIREMENTS OF PRESCHOOLCHILDREN

PROBLEM

Data on amino acid requirements are slowly being gathered, but there are stillimportant gaps of information which have to be filled.

The specific aim of this project is to determine the minimum requirements foressential amino acids of children of preschool age. This information will permit theformulation of the ideal amino acid composition of a protein for their adequate proteinnutrition. This knowledge could then be used as the basic pattern for determining thenutritional value of food proteins intended for feeding children of this age.

METHOD

The study is being conducted in healthy male children 2-3 years old, fully re-covered from mainutrition according to anthropometric, clinical, and biochemical crite-ria. The children receive a diet that provides 1.2 g of milk protein/kg/day and 100 kgcal/kg/day, 20 percent of the total calories coming from vegetable fat, The childrenalso receive adequate mineral and vitamin supplements. The requirements for each essen-tial amino acid are being investigated in six children, three following a descending,and three an ascending design. In the descending design they receive a diet consistingof 0.3 to 0.5 g of milk protein/kg/day plus a mixture of amino acids equivalent to 0.7to 0.9 9 of milk protein/kg/day. In a sequence of 6- to 9-day periods, the level ofeach specific amino acid is progressively decreased or increased until the intake ofthat amino acid is that provided by the milk protein alone. The main parameters beingmeasured are: daily body weight, weekly height, serum proteins and electrophoresis,4-day nitrogen balances, serum and red cell free amino acid hydroxiproline excretion,creatiniñe-height index, and basal oxygen consumption.

RESULTS TO DATE

Methionine and lysine were studied first because they are the two most limitingamino acids in legume seeds and cereals respectively, foods that are the basic staplesof the diets of low socioeconomic groups. The effect of decreasing simultaneously thelevels of methionine and cystine was also studied. In each case, lowering the levelof the amino acid until its contents in the diet was that provided by 0.3 g of milkprotein/kg/day, still maintained positive nitrogen balances above 20 mg of nitrogenper kilo of body weight per day, estimated necessary retention for adequate growth rateat this age. These results suggest that within the relatively short periods of study,the children were able to compensate the low levels of the essential amino acid in thediet with some of the amino acid present in the body pool. The results obtained fromother parameters studied are being analyzed to determine if they can indicate, withmore sensitivity, whether the requirement of each particular amino acid is being met.

SIGNIFICANCE

The availability of data on amino acid requirements of preschoolchildren wouldpermit the application of a rational approach to the problem of predicting protein

3

nutritional quality for this age group. This prediction is essential for more efficientplanning of programs aimed at eliminating protein malnutrition. The availability ofexcellent facilities, well-tested techniques, and new approaches to the study of pro-tein metabolism may permit a more accurate estimation of requirements of amino acidsthan was previously accomplished.

PUBLICATIONS

None thus far.

OTHER DATA

Dr. Guillermo Arroyave, Division of Physiological Chemistry,Institute of Nutrition of Central America and Panama (INCAP),Guatemala City, Guatemala.

U.S. National Institutes of Health.

1974 - continuing.

4

Grantee:

Funded by:

Timetable:

NUTRIENT SOURCES

PROBLEM

The availability of protein, as well as calories and other nutrients, for eitherhuman or animal nutrition, is still a problem in Central America. The problem willbecome even more difficult to solve in the future because of increased demands forthose nutrients. There is therefore an urgent need to find alternate sources of foodand to develop new nutrients through chemical and nutritional studies of nonconventionalagricultural products, or through the utilization of agricultural and industrial by-products.

METHOD

The program is divided into three parts. The first one includes study ofmaterials not used in the past; the second evaluates the utilization of agriculturalor industrial byproducts; and the third focuses on combinations of various sources togive food products of a desirable composition and nutritional value. Research method-ology varies, therefore, according to the study being conducted: standard chemicaland biochemical methods are used, as well as any required toxicological assays. Variousspecies of experimental animais are used depending on the use to be made of the sourceof nutrients under study. The following projects are being carried out:

(1) Effect of milk and meat protein replacement by soybean protein on thenutritive quality of diets.

(2) Protein complementation between soybean flour and wheat gluten and betweensoybean flour and corn gluten.

(3) Protein complementation between soybean and sesame.

(4) Morro or Jicaro (Crescentia alata) as a source of nutrients in CentralAmerica.

(5) Chemical composition and nutritive value of malanga (Xanthosoma violaceum).

(6) Analytical techniques to determine amino acids and protein content of foods.

(7) Chemical and biological evaluation of diets of four communities from thelongitudinal study on nutrition and mental development.

RESULTS TO DATE

High-protein foods of good quality and of high-energy content may be preparedby appropriate combination of various protein sources. Howeveri caution should beexercised in producing excessive amounts of texturized vegetable protein to replacemeat protein, since there is a resulting significant decrease in quality. Finally,various tubers may be used as sources of carbohydrates to alleviate the demand oncereal grains. In addition, other sources of oil, such as the Jicara seed, are asgood in quality as conventional oilseed oils.

5

SIGNIFICANCE

These studies are important because (a) they uncovered greater sources ofnutrients; (b) they made better use of available and natural resources; (c) they pro-duced foods of improved nutritional quality; and (d) they decreased the pressure madeon existing nutrient sources and increased the availability of food products.

PUBLICATIONS

1. Braham, J. E. and R. Bressani. Effect of different levels of gossypol onthe transaminase activity, non-essential to essential amino acid ratio andiron and nitrogen retention in rats. J Nutrition 105:348, 1975.

2. Bressani, R. et al. Efecto del gosipol libre de diferentes harinas dealgodón sobre el crecimiento de ratas y niveles de lisina libre y gosipollibre en órganos, músculo y suero de animales. Arch Latinoam Nutr 25:47,1975.

3. ----- et al. Whole soybeans as a means of increasing protein and caloriesin maize-based diets. J Food Sci 39:577, 1974.

4. -----. Nutritional contribution of soy protein to food systems. J Am OilChem Soc 52:254A, 1975.

OTHER DATA

Grantee: Dr. Ricardo Bressani, Sources of Nutrients Program, Divisionof Agricultural and Food Sciences, Institute of Nutrition ofCentral America and Panama (INCAP), Guatemala City, Guatemala.

Funded by: Research Corporation.


6

CHEMICAL AND NUTRITIONAL CHARACTERIZATIONOF BASIC FOODS AND THEIR UTILIZATION

PROBLEM

Cereals are the most important food items and contribute significant amounts ofcalories and protein to the diet of a large segment of the population because of theirhigh consumption. Cereal protein is, however, of a poor nutritive value for both youngand adult persons and the content of protein in cereals is relatively low. The most im-portant sources of supplementary protein in the diet are food legumes, but beans are noteasily available because of their low yield. In addition, bean consumption is limitedbecause beans cannot be stored easily; seeds harden, leading to an increase in energyconsumption and deterioration of their nutritional quality. Furthermore, they have an-tiphysiological compounds and are low in sulfur-containing amino acids.

The general objective of this research is to improve the nutritional quality ofthese foods through a variety of interventions such as soil fertility, genetic control,supplementation, storage, and processing.

METHOD

No specific method can be described because the nature of the work depends on thespecific objectives of the various research and application projects carried out underthis program. In general, however, methodology involves chemical analysis and nutri-tional value determination of cereals and legumes. It also involves the determinationof the effects of processing on the nutritional and rheologic characteristics of thesefoods and their utilization either individually or in combination. The following in-vestigations are being carried out:

(1) Effect of storage on the physical and nutritive value stability of cerealsand food legumes.

(2) Chemical composition and protein quality of soybean varieties grown invarious localities in Guatemala.

(3) Studies on the possible relationship between seed coat pigments andprotein quality of beans.

(4) Processing of grain sorghum by lime-cooking for the preparation oftortillas.

(5) Biological evaluation of protein quality in human adults.

(6) The effect of maturation and germination of corn on its protein quality.

(7) The nutritional role of the germ toward the protein quality of corn.

(8) Evaluation of the protein quality of texturized vegetable protein.

7

RESULTS TO DATE

The information obtained emphasizes again the poor protein quality of common cornand other cereal grains and the favorable effect that results from correcting the defi-ciency with either protein or amino acids. The implications of the low protein qualityof cereal grains for human populations should be considered before proposing that aneven higher consumption of this cereal can fulfill the essential amino acid needs ofadults. Even though other foods are consumed with corn and other cereal grains, theirquality must be such that it can correct the amino acid deficiencies in cereals. Theamount of beans that are usually consumed with corn and other cereals are not totallycapable of correcting these deficiencies. Technological information on both corn andcorn supplemented with proteins is needed in order to make better use of corn in thepreparation of other foods, and to be able to give the physical and organoleptic char-acteristics to corn-based foods. The information obtained on tortilla supplemented withwhole soybeans is of paramount importance, since the tortilla thus obtained has a higherprotein and calorie content and a better protein quality than the unsupplemented tor-tilla. The data obtained indicate that among food legumes, cowpeas and pigeon peas aresuperior to common beans in protein nutritive value. This difference in quality isclosely related to the concentration of trypsin inhibitors in the raw material, whichis lower in cowpea than in anyiof the other species studied. Therefore, efforts shouldbe directed to promote the use of this legume by the general population or as a com-ponent of other foods. Aside from the trypsin inhibitor concentration, the manner ofcooking affects the protein nutritive value of beans. Excessive heat or heat appliedto ground beans decrease protein quality. In the first case, because of destruction orinactivation of essential amino acids, and in the second, because the ground materialdoes not allow good heat transfer. Extrusion cooking or drum cooking gives good qualityproducts. Other factors involved are probably tannins in the seed coat of the bean. Asindicated above, the eatability of beans deteriorates rapidly soon after harvest. Itappears that a properly controlled thermal treatment reduces significantly the hardeningof the seed and its biodeterioration. This finding has tremendous implications, sincethrough the use of this processing technique, large losses of such a valuable food canbe avoided. Poor storage of both cereal grains and food legumes result in high physicallosses as well as in nutritive value.

SIGNIFICANCE

As previously indicated, cereals and food legumes constitute a very importantpart of the diet of large groups of the Central American population. Obviously, anyimprovement on the quality of these foods will result in improvement in the nutritionalstatus of the population.

PUBLICATIONS

1. Bressani, R. Legumes in human diets and how they might be improved. In:Milner, M. (ed.), Nutritional Improvement of Food Legumes by Breeding.John Wiley and Sons, Inc., 1975.

2. -----. A new assessment of needed research. In: Milner, M. (ed.),Nutritional Improvement of Food Legumes by Breeding. John Wiley andSons, Inc., 1975.

3. Elías, L. G. et al. Evaluación de la calidad de la- proteína de alimentosde bajo contenido proteínico. Arch Latinoam Nutr 24:81, 1974.

8

4. ----- and R. Bressani. Nutritional factors affecting the consumption ofleguminous seeds. Arch Latinoam Nutr 24:365, 1974.

5. ----- et al. Effect of germination and maturation on the nutritive valueof common beans (Phaseolus vulgaris), p. 139, 1973. In: Jaffé, W. G.(ed.), Nutritional Aspects of Common Beans and Other Legume Seeds as Animaland Human Foods. Proceedings of a meeting held in Ribeirao Preto, 3razil,6-9 November 1973.

6. Gómez Brenes, R. A. et al. Mejoramento del valor nutritivo del maíz por mediode infusiones de lisina y triptofano. Arch Latinoam Nutr 24:243, 1974.

7. ----- et al. Chanqes in chemical composition and nutritive value of commonbeans and other legumes during house cooking, p 93, 1973. In: Jaffé, W. G.(ed.), Nutritional Aspects of Common Beans and Other Legume Seeds as Animaland Human Foods. Proceedings of a meeting held in Ribeirao Preto, Brazil,--9 November, 1972.

8. Murillo, B. et al. Influencia de la densidad calórica sobre la utilizacionde la proteina en dietas elaboradas a base de maíz y frijol. Arch LatinoamNutr 24:223, 1974.

OTHER DATA

Grantee: Dr. Ricardo Bressani, Basic Food Program, Division of Agriculturaland Food Sciences, Institute of Nutrition of Central America andPanama (INCAP), Guatemala City, Guatemala.



9

CORN FORTIFICATION: A FIELD DEMONSTRATION MODEL

PROBLEM

The shortage and poor quality of food consumed in many developing areas willnot be rapidly overcome by orthodox methods. Other measures must therefore be in-vestigated, particularly those with a theoretical potential significance. Metabolicward experiments in animals and humans have shown that the addition of small amountsof the limiting amino acids to foods such as corn results in a remarkable improvementin their quality. Such fortification has not been investigated extensively underfield conditions, however.

The purpose of the present research is to conduct a fortified corn trial ina whole rural community in which a significant amount of basic information on growth,nutrition, and infection was collected during the 8 years preceding the initiation ofthe project. Fortification makes the biologic value of corn comparable to that ofmeat. If this measure is effective, an improvement in fetal growth, infant mortality,and general health will be observed in the community.

METHOD

The study is being conducted in the Indian village of Santa María Cauqué in theGuatemalan highlands. The corn is fortified at 8 percent at the mill. At this con-centration it provides 7.82 percent soy bean flour, 0.12 percent lysine, and a mixtureof vitamins A, B1, and B2, niacin, and iron. The PER of corn fortified by this methodis increased to 2.76.

Following supplementation, the biological effect of fortification was monitoredby measuring fetal and postnatal growth in all village children. A morbidity surveil-lance was conducted in a cohortof children O to 3 years old, to determine the incidence,duration, and severity of infectious disease. At the same time, the dietary intake,morbidity, and health status of pregnant women were kept under observation, and themortality of infants and 2- to 5-year-old children determined.

RESULTS TO DATE

During 1975 an analysis was made of the effect of corn fortification fromJune 1972 to May 1975. Preliminary findings indicate that the intervention has beenbeneficial in families fortifying their corn at high concentration. Mortality de-creased by 50 percent in infants and very noticeably in 2- to 5-year-old children.Morbidity during weaning was reduced by 33 percent. These changes appeared to beindependent of socioeconomic class. Though corn fortification was not proved to havea significant effect on postnatal growth, a positive tendency for better growth in-crements was found in 3- to 5-year-old children. No change in fetal growth was found;pregnant women's dietary intake, was, however, improved, especially during the first6 months of pregnancy.

So far, corn fortification has been found to be very beneficial to children'shealth. It is proposed to'extend this study increasing the level of fortification to 10percent, to bring positive changes in other factors associated with nutrition.

10

SIGNIFICANCE

The available data suggest that corn fortification improves the health statusof individuals in a community without any sanitation intervention. It is expectedthat this research will continue for 2 or more years in order to replicate the findingsof the morbidity survey in a larger sample of children. If the results already observedin the *reduction of morbidity and mortality are definitely proved, then corn fortifica-tion in a whole region or even at national level, might change the health status of acommunity.

PUBLICATIONS

1. Caparelli, E. and L. J. Mata. Microflora of maize prepared as tortillas.Appl Microbiol 29:802-06, 1975.

2. Mata, L. J. et al. A model for maize fortification with soy bean flour,lysine, and other nutrients in a low socioeconomic rural community. In:Bressani, R., J. E. Braham, and M. Béhar (eds.). Nutritional Improvementof Maize, Institute of Nutrition of Central America and Panama (INCAP),Guatemala City, 1973.

3. Urrutia, J. J. et al. Infection and low birthweight in a developingcountry. Am J Dis Child 129:558-61, 1975.

4. ----- et al. Reporte preliminar del efecto biológico de la fortificacióndel maíz con harina de soya y lisina. In: Memorias de la Primera Conferencia Latinoamericana sobre la Proteína de Soya, México, 1975, in press.

5. ----- and L. J. Mata. Infección en la mujer embarazada y su influenciaen el producto de la concepción. In: Aspectos Perinatales del PartoPrematuro y del Retardo de Crecimiento Intrauterino, Montevideo, Uruguay,in press.

OTHER DATA

Grantee: Juan José Urrutia, Division of Environmental Biology, Instituteof Nutrition of Central America and Panama (INCAP), GuatemalaCity, Guatemala.

Funded by: U.S. Agency for International Development (USAID).

Timetable: 1971-1976.

11

EVALUATION OF SUGAR FORTIFICATION WITH VITAMIN AAT THE NATIONAL LEVEL

PROBLEM

Epidemiological evidence has revealed a serious and widespread problem ofvitamin A deficiency in the populations of Central America. To attack this problem,a process for the fortification of sugar with vitamin A at the national leve! hasbeen developed. Sugar fortification has been legally approved in Guatemala andCosta Rica, and it is expected that other Central American countries will take thesame step.

Although vitamin A fortification of sugar has demonstrated effectiveness in thelaboratory and in pilot studies with small population groups, its effectiveness andcost have to be assessed at the national or regional level. The objectives of thísproject are, therefore, to evaluate at the national level: (1) the biological effec-tiveness of fortification; (2) the delivery system of the fortified sugar; and(3) the cost of the intervention.

Baseline information has been secured about the vitamin A status of the popu-lation in which the fortification is to be applied, before the intervention, and willcontinue to be collected at 6-month intervals during a 2-year period.

METHOD

The major variables to be studied will be:

(1) Intake of sugar and of food sources of vitamin A;

(2) Blood serum retinol levels in children under 6 years of age;

(3) Retinol content of human milk;

(4) Ocular signs of vitamin A deficiency in preschoolchildren;

(5) Skin examination in all persons in the study sample;

(6) Retinol content of liver samples collected from coroner's autopsies;

(7) Hospital and clinic registration of cases of blindness and eye lesionsattributed to hypovitaminosis A;

(8) Collecting and reporting of suspected cases of hypervitaminosis A;

(9) Cost of the fortification and control.

The above studies are being conducted on population samples of each majorecological regions of the countries. Results will serve to determine the value ofthis public health nutritional intervention, not only for Central America, but alsofor other countries or regions where the application of the process may provenecessary.

12

RESULTS TO DATE

Baseline information about the vitamin A status in the population has been col-lected in 12 rural Guatemalan villages. The studies were carried out in a representa-tive sample of each community, which involved: (1) 360 family dietary surveys; (2) 750clinical examinations, including over 500 eye examinations in preschoolchildren; (3) 543blood serum retinol determinations in preschoolchildren; and (4) 283 determinations ofretinol in breast milk from lactating women.

Nearly 100 liver samples from coroner's autopsies have been collected with thecooperation of the department of legal medicine and pathology of the general hospital,San Juan de Dios, of Guatemala City, for the estimation of liver reserves. Hospital andclinic registration of blindness cases and eye lesions attributed to hypovitaminosis Aare being carried out with the cooperation of the ophthalmological hospital, Dr. RodolfoRobles, in Guatemala City, and the National Committee for the Blind of Guatemala, infour rural communities covered by its specialized medical services. The data are nowbeing processed in order to compute and interpret the results obtained, and will be re-ported in the near future.

SIGNIFICANCE

This research is based on the premise that the application of a public healthintervention without adequate built-in evaluation is wasteful, since the potential ofthe intervention for similar situations elsewhere would be undetermined. In the in-stance of sugar fortification with vitamin A, this is of utmost importance since thismeasure has never been applied in the world. Guatemala and Costa Rica are the firsttwo countries which have officially decided to implement the process at industrial andnationwide scale.

Most underdeveloped country populations are suffering from vitamin A deficiency.It is therefore expected that, if the program is proved effective, many countries willbe interested in considering its implementation. Specifically the study is of greatsignificance for the six member countries of INCAP. Only after proper assessment ofits effectiveness and cost-benefit ratio, will government authorities be in a positionto determine whether they should continue with this public health measure as it is,modify it, or discontinue it.

PUBLICATIONS

1. Aguilar, J. R. et al. Rapid method for adding vitamin A to sugar atan industrial level. X International Congress of Nutrition, Kyoto,Japan, August 3-9, 1975. Symposia and Free Communications, pp. 339,1975.

2. Arroyave, G. et al. Manual de operaciones para la fortificaci6nde azúcar con vitamin A. Instituto de Nutrición de Centro Américay Panamá (P-2301), 1975, Guatemala City, Guatemala.

13

Drs. Guillermo Arroyave, Division of Physiological Chemistry,and ¡van D. Beghin, Division of Applied Nutrition, Instituteof Nutrition of Central America and Panama (INCAP), GuatemalaCity, Guatemala.

U.S. Agency for International Development (USAID).

1975-1976.

14

OTHER DATA

Grantee:

Funded by:

Timetable:

UTILIZATION OF AGRICULTURAL AND INDUSTRIALBYPRODUCTS IN ANIMAL NUTRITION

PROBLEM

There is an increasing interest in Central America to spur the development oflivestock, particularly ruminants, because more land is allotted to cash and food cropsand because during the dry season significant losses in animal production take place.On the other hand, there are various potential sources of feed from agroindustries,which are not being properly utilized, and which in some cases contribute to the con-tamination of the environment. The purpose of this study is to learn of the chemicaland nutritional value of these agroindustrial byproducts, and to determine how theycan be used in animal production. The study also includes economic evaluation of thebyproducts in comparison with standard of reference feeds.

METHOD

The byproducts to be studied, coffee pulp, sugar cane bagasse, cottonseed hulls,and others, are analyzed for their chemical composition and screened biologically invitro. After this stage they are given to various animal species as the main feed oras ingredient to a complete feed. The evaluation is carried out by measuring animalperformance, feed consumption, and biological utilization. Byproducts are processedeither to eliminate undesirable compounds or to preserve them. Processing includesdehydration or silage production. The resulting products are also evaluated as in-dicated broadly above.

RESULTS TO DATE

Efficient and economic systems of animal production were developed using 3-dayold calves fed only limited amounts of milk. These animals were marketed 18 monthslater at 450 kg. At that point they were receiving complete rations that containedthe appropriate concentration of the agroindustrial byproducts (30 percent coffeepulp for ruminants and 20 percent for swines). Antiphysiological factors still beingidentified in coffee pulp were found to act, together with caffeine, in decreasinganimal performance. The study also showed that most of these byproducts can be pre-served safely by ensilage or dehydration. The costs of these processes are underinvestigation.

The following projects are being carried out in connection with this study:

(1) Effect of coffee pulp on the sulfur-containing fraction in the urine ofrats.

(2) Effect of extrusion on the chemical composition and on in-vitro digesti-bility of dehydrated coffee pulp.

(3) Growing chicks as experimental animals for the evaluation of low-proteinrations.

15

(4) Chemical and nutritional evaluation of coffee pulp treated with sodiummetabisulfite in broilers.

(5) Chemical changes and degree of nitrogen extraction of kikuyu (Pennisetumclandestinum) grass during vegetative growth.

(6) Nutritive value of dehydrated kikuyu grass for rabbits.

(7) Effect of alkaline hydrolysis and/or extrusion on the chemical compositionand in-vitro digestibility of sawdust and studies on the processing of coffee pulp byvarious techniques.

SIGNIFICANCE

The efficient utilization of agroindustrial byproducts have economic as well aspublic health implications. The economic aspects are related to an increased avail-ability of feeds and ingredients, and to better use of land. From the public healthpoint of view, these materials cause less contamination of the environment.

PUBLICATIONS

1. Braham, J. E. et al. Utilización de urea en terneros de dos días a cincoy doce semanas de edad. Arch Latinoam Nutr 24:201, 1974.

2. Bressani, R. et al. The use of coffee processing waste as animal feed.Proceedings of the Conference on Animal Feeds qf Tropical and SubtropicalOrigin. London, Tropical Products Institute, 1975.

3. Cabezas, M. T. et al. Pulpa y pergamino de café. V. Absorción y retenciónde nitrógeno en terneros alimentados con raciones elaboradas con pulpa decafé. Turrialba 24:90, 1974.

4. ----- et al. Pulpa y pergamino de café. VI. Adaptación del ganado bovinoa la pulpa de café. Turrialba 24:160, 1974.

5. Gómez Brenes, R. A. et al. Comparación del cerdo criollo y Duroc Jerseyen cuanto a crecimiento y utilización del alimento. Turrialba 24:29, 1974.

6. Jarquin, R. et al. Crecimiento de terneros utilizando cantidades limitadasde leche y formulaciones con niveles proteicos diferentes. Turrialba 24:250,1974.

7. ----- et al. Pulpa y pergamino de café. IX. Uso de la pulpa de café en laalimentación de cerdos en la fase de crecimiento y acabado. Turrialba24:353, 1974.

8. ----- et al. Pulpa y pergamino de café. VII. Utilización de pergamino decafé en la alimentación de ruminantes. Turrialba 24:168, 1974.

16

Dr. Ricardo Bressani, Animal Nutrition Program, Division ofAgricultural and Food Sciences, Institute of Nutrition ofCentral America and Panama (INCAP), Guatemala City, Guatemala.

Research Corporation (RC) and International DevelopmentResearch Centre (IDRC).

1971 - continuing (RC);1975 - continuing (IDRC).

17

OTHER DATA

Grantee:

Funded by:

Timetable:

GRADUATE COURSES IN FOOD SCIENCE TECHNOLOGY AND ANIMAL NUTRITION

PROBLEM

The purpose of these courses is to train Latin American personnel, preferablyCentral American, in the utilization, directly or indirectly through animal nutrition,of raw materials and industrial wastes found in Latin America. The courses are de-signed to train students for academic or research activities in the area of foodscience, with special emphasis on food technology or animal nutrition. After allrequirements have been fulfilled the students are awarded a Master of Science (MS)degree by the University of Guatemala.

METHOD

Research activities leading to a Master of Science Degree dissertation.

RESULTS TO DATE

During 1975 twelve students were enrolled in the courses: 7 in Animal Nutritionand 5 in Food Technology. Of these students, 8 have been promoted to the second year.The geographical distribution of the students is as follows: 3 from Nicaragua, 2 fromPeru, 1 from Bolivia, 1 from Guatemala, and 1 from El Salvador. The six students whograduated in 1975 were from Guatemala (1), Ecuador (1), Mexico (1), Brazil (1), Vene-zuela (1), and Nicaragua (1). In 1976, 10 students were selected. They were: 1 fromNicaragua, 1 from Argentina, 2 from El Salvador, 1 from Chile, 1 from Mexico, 1 fromBolivia, 1 from Guatemala, 1 from Haiti, and 1 from the Dominican Republic.

SIGNIFICANCE

There has been a noticeable lack of communication between the food industryand animal production in Central America. Students graduating from these courseswill fill the gap between these two activities and will promote interest in the de-velopment of the food industry and animal nutrition projects both from the academicand practical points of view. Central American universities lack a curriculum in foodscience or food technology and a significant contribution of the graduates of thesecourses would be the implementation at the university level of food science and foodtechnology programs in their respective universities.

PUBLICATIONS

From 1973 to 1975, 14 theses have been published and distributed to academicand governmental offices in Central America.

1. Argueta, C. Optimization of the chemical and chemical enzymaticprocesses for the extraction of proteins from legume and oil seeds.

18

2. Contreras, I. Utilization of the morro (Crescentia alata) fruit inanimal feeds.

3. Daqui, L. Chemical and nutritional characteristics of coffee pulp ensilagedwith Napier grass and corn fodder.

4. Del Busto, A. Methodology for the protein evaluation in foods.

5. Estrada, E. Caffein and tannins as limiting factors in coffee pulpfor the feeding of calves.

6. Gonzalez, D. Relationship between the pigments in the seed coat oflegumes (P. vulgaris) and the degree of toxicity, digestibility, andnutritive value of the latter.

7. Hernandez, M. Effect of the cooking conditions on the residual toxicactivity, amino acid availability, and protein value of some legumeseeds.

8. Mayorga, I. Partial substitution of wheat flour (semolina) and improve-ment of nutritive value in pastas and spaghetti by amino acid and proteinsupplementation.

9. Nuñez, E. 1. Effect of several solvents on the extraction and digestibilityof different protein fractions in beans.

10. Roque de Daqui, V. A short method for the evaluation of protein qualityin humans.

11. Rosales, F. Coffee pulp in feed formulations for swine.

12. Ruiloba, E. Physical, chemical, organoleptic and nutritional changes inbeans during storage.

13. Ruiloba, H. Supplementation of rice through amino acid infusions.

14. Vargas, E. Nutritive value of coffee pulp.

OTHER DATA

Grantee: Dr. Ricardo Bressani, Academic Program, Division of Agriculturaland Food Sciences, Institute of Nutrition of Central Americaand Panama (INCAP), Guatemala City, Guatemala.



19

PRESERVATION AND PROCESSING OF FOODS FOR HUMAN CONSUMPTION

PROBLEM

In recent years, emphasis has been given to the increased production of basicfoods. Some increases in production are already apparent but they may not continueunless ways are found to preserve them, so that they will retain their physical, culi-nary, and nutritional properties. It is also of interest to diversify their uses sothat there is always a demand for them. On the other hand, countries are trying todevelop foods that will not make them dependent on imports from other nations, and thereis an interest also in developing new nutrients and fortified foods, to meet nutritionalneeds. Interest also exists in developing vegetable proteins to complement meat pro-ducts, as well as other animal-derived foods.

The purpose of this program is to develop, from data obtained by other studies,new foods of high nutritional quality, preserve food products, and diversify their uses.

METHOD

Staple foods are subjected to a variety of storage conditions, processed by dif-ferent techniques using pilot plant equipment, or enriched with different nutrients,and the effects of these interventions evaluated by chemical, physical, and biochemicalmethods. In some instances, certain food components are substituted by others to pro-duce the type of food preferred by people. The substitutions are evaluated as above.The projects pursued during 1976 are:

(1) Extrusion cooking of mixtures of cowpea/corn and cowpea/cassava.

(2) Simple extruder cookers and their efficiency to inactivate antiphysiolog-ical compounds in oilseeds and food legumes.

(3) Effect of cooking on the nutritional quality of supplemented and unsupple-mented bakery products, pasta, bread, and cookies.

(4) Comparison of the protein quality of corn/whole soybean mixtures processedby different techniques.

RESULTS TO DATE

Important advances have been obtained so far. High protein and calorie contain-ing foods have been prepared with different processing techniques, oilseeds, cerealgrains, or legume foods. Bakery products, bread, cookies, and pasta products contain-ing up to 40 percent corn flour have been developed, retaining the characteristic ofthe bakery products made from wheat flour. Basic staple foods have been improved nu-tritionally by the addition of small amounts of protein concentrates, and new sourcesof protein have been obtained from nonedible legume foods. Simple and economic proce-dures have been elaborated for the preservation of cereal grains and food legumes.

20

SIGNIFICANCE

Various aspects of the above results are quite significant. First, foods ofhigh nutritional quality are available to replace animal-derived foods. There areimportant economic implications as well and a certain freedom from dependence on foodsimported from other nations. A reduction in the reported 40 percent post-harvest lossesof food crops in developing countries will increase their availability.

PUBLICATIONS

1. Molina, M. R. et al. Interrelaciones entre tiempo de remojo, tiempo decocción, valor nutritivo y otras características del frijol (Phaseolusvulgaris). Arch Latinoam Nutr 24:469, 1974.

2. ----- and R. Bressani. Protein-starch extraction and nutritive value ofthe jack-bean and jack-bean protein isolate, p. 153, 1973. In: Jaffé, W. G.(ed.), Nutritional Aspects of Common Beans and Other Legume Seeds as Animaland Human Foods. Proceedings of a meeting held in Ribeirao Preto, Brazil,6-9 November 1973.

3. ----- et al. Pulpa y pergamino de café. VIII. Estudios básicos sobre ladeshidratación de la pulpa de café. Turrialba 24:280, 1974.

4. ----- et al. Extraction of nitrogenous constituents from the jack-bean(Canavalia ensiformis). Agr Food Chem 22:309, 1974.

5. ----- et al. Production of high-protein quality pasta products using asemolina-corn-soyflour mixture. 1. Influence of thermal processing ofcorn flour on pasta quality. Cereal Chem 52:240, 1975.

6. ----- et al. Interrelationships between storage, soaking time, cookingtime, nutritive value and other characteristics of the black bean(Phaseolus vulgaris). J. Food Sci 40:587, 1975.

OTHER DATA

Grantee: Dr. Ricardo Bressani, Food Technology Program, Division ofAgricultural and Food Sciences, Institute of Nutrition ofCentral America and Panama (INCAP), Guatemala City, Guatemala.



21

ASSESSMENT OF AND NUTRITIONAL LOSSES DUE TO MALABSORPTIONIN LOW INCOME RURAL GUATEMALAN COMMUNITIES

PROBLEM

A significant number of asymptomatic and apparently healthy individuals from lowsocioeconomic groups of preindustrialized countries suffer mild-to-moderate degrees ofintestinal malfunction, characterized by GI bacterial overgrowth, abnormal low absorp-tion of d-xylose, and/or vitamin B12, as well as by the presence of nonspecific mor-phologic alterations in their duodenojejunal mucosa. Although the pathogenesis of thisentity, often called subclinical or tropical malabsorption, remains obscure, it iscommonly accepted that it is an acquired condition related to the unsanitary environ-ments in which individuals live, the high incidence of parasitic diseases, the kind ofdiet consumed, and the high incidence of chronic mild-to-moderate malnutrition.

Ideally, subclinical malabsorption should be studied without removing the in-dividuals from their own environments, but the extent to which this can be accomplishedis limited. Most tests of absorption and absorptive functions do not lend themselvesto broad screening programs or field studies, especially under the conditions prevailingin rural areas.

The purpose of this study is to develop a field test suitable for the screeningof subclinical malabsorption of food nutrients, and capable of detecting alterations inthe gastrointestinal ecology of individuals subjected to poor nutrition and low levelsof sanitation as occurs in rural Guatemala. Furthermore, it is intended to determinethe extent to which the diet of these individuals, characterized by its high content ofpoorly digestible carbohydrates, influences their gastrointestinal ecology and the uti-lization of nutrients.

METHOD

The tests performed consist of measuring different gases originating in theintestine as a result of bacterial action, in samples of exhaled air after the oraladministration of carbohydrate substrates. The extent to which the gastrointestinaltract of man can absorb these carbohydrates is variable, since several of them are non-absorbable and others absorbed only partially. The portion of compounds which is notabsorbed is usually metabolized and degraded by intestinal flora; the results of thisaction is the formation of intestinal gases, especially hydrogen (H2) and methane (CH4),which are exhaled through breath. Both gases can be measured by gas liquid chromato-graphy in samples of expired air taken hourly after the administration of the sugarsubstrate. The substrates that will be used are d-xylose, raffinose, lactose, lactulose,and different carbohydrates mixtures coming from black beans and corn.

The response of rural subjects to these substrates will help clarify the basisof observed low urinary excretion of xylose in Guatemalan rural populations. Speci-fically, the time sequence of H2 and CH4 formation may help to detect the presence ofsignificant upper gastrointestinal tract bacterial overgrowth, as well as the volume ofH2 produced. This finding will determine the amount of nonabsorbed carbohydrate avail-able for bacterial fermentation. Time of sampling, dosages, and conditions will be

adjusted to provide the simplest test scheme compatible with clear diagnosis of absorp-tive capacity and determination of intestinal bacterial overgrowth. Microbiologicstudies of the intestinal flora will be performed in a small group of volunteers andresults used as characteristics for adult individuals living in rural areas of Guatemala.

22

A metabolic balance study will also be carried out to quantify the extent of fecalloss of nutrients due to malabsorption and to the characteristics of the rural diet. Forthis purpose, a highly absorbable formula diet based on egg white protein will be pre-pared, as well as a typical rural diet. Both diets will be administered for 14 consecu-tive days in amounts required by the World Health Organization in protein allowance perkilogram of body weight and energy sufficient to maintain constant body weight. Othernutritional levels will be raised to meet standards by supplementation if needed. Car-bohydrate breath analysis will be performed periodically in these subjects using raffi-nose, d-xylose, and carbohydrates derived from black beans and corn.

Studies will be replicated in a small number of healthy North American male adultsin the Human Nutrition Laboratory of the University of California at Berkeley. Compari-son of the responses should clarify the basis of low urinary excretion of xylose inGuatemalan adults and how the rural Guatemalan diet is handled by individuals living inenvironments with different levels of sanitation.

RESULTS TO DATE

None thus far.

SIGNIFICANCE

Chronic subclinical malabsorption is widespread in tropical areas. Its impact interms of health, food waste, productivity, and general economics ts not known. Thisproject aims at shedding light into those areas in order to be able to design inter-vention programs that can be evaluated on their cost effectiveness. The development ofnoninvasive techniques for the study of malabsorptive problems will contribute to thewider application of evaluation procedures to intervention programs.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. Roberto E. Schneider, Institute of Nutrition of CentralAmerica and Panama (INCAP), Guatemala City, Guatemala, andDr. Doris Calloway, Department of Nutritional Sciences,University of California, Berkeley, California.

Funded by: U.S. Department of Health Education and Welfare, NationalInstitutes of Health.


23

DEVELOPMENT AND EVALUATION OF MEASURES TO REDUCEFOOD WASTE CAUSED BY INTESTINAL DISEASES

PROBLEM

The objectives of this project are:

(1) To measure the prevalence of gastrointestinal diseases (including parasiticdiseases) commonly transmitted under insanitary conditions, and their effect on intes-tinal malabsorption and nutritional status in rural communities of Guatemala.

(2) To evaluate the severity and prevalence of intestinal malabsorption andfood waste due to these conditions.

(3) To determine the effectiveness of a combination of environmental sanitationmeasures (introduction of a chlorinated house-to-house water supply system togetherwith a sanitary education program) to decrease the prevalence of gastrointestinal dis-ease and the waste of ingested food caused by malabsorption.

(4) To determine the health and economic benefits associated with such a pro-gram.

METHOD

This project started in April 1972 in two communities of 960 to 980 inhabitantseach, very similar in their geographic, social, ethnic, and economic characteristics,as well as in their gastrointestinal disease prevalence. One of these villages, witha poor quality water supply system became the control village. The other, with acentral water source suitable for development into a more sophisticated house-to-housesystem became the experimental community. Both communities were evaluated longitudi-nally in areas related to health, nutrition, sanitation, economics, and malabsorption.The latter is evaluated by performing at regular intervals d-xylose absorption testsin adult males from both communities. A subgroup was admitted to a rural metabolicward where detailed absorption studies were performed to evaluate the significance ofd-xylose malabsorption and also to determine the actual waste of ingested food.

Following the longitudinal studies, improvement of the water supply system becameeffective in the experimental community; this was complemented with the initiation ofa sanitary education program in April 1975. It is contemplated to follow both communi-ties until April 1978 to evaluate the effect of the interventions performed.

RESULTS TO DATE

Except for the presence of a very primitive communal area in the experimentalvillage, both communities have grown without any plan of urban development. Most oftheir houses are in very poor conditions (shacks); latrines or systems for disposal ofgarbage and excreta practically do not exist. A very significant degree of environ-mental fecalization demonstrated by high incidence of intestinal parasitism and positivestool cultures for shigella-salmonella is also present in both. The incidence of diar-rhea is very high,especially during the rainy season and in the poorest areas of bothcommunities. Children under the age of 6 are at high risk not only for diarrhea but

24

also for respiratory diseases. Variable degrees of malnutrition were seen in bothcommunities where regular daily intakes consist mainly of corn (tortillas) and beans,providing an average of 2,200 to 2,500 kcal/day/adult males. The average cost of theabove mentioned diet fluctuates between Q 0.40 and 0.45.

Despite individual variations, the prevalence of d-xylose malabsorption in adultmales has remained fairly stable since 1972 with minor fluctuations (29 percent-32 per-cent) contrasting with that of healthy controls (7 percent-10 percent). Detailed ab-sorption studies carried out in 1973 and 1974 at the rural metabolic ward, showed that56 percent-64 percent of the subjects studied presented calorie malabsorption, losing200 to 300 calories/day/individual above the average observed in normal absorbers. Theadditional fecal calorie loss represents 5 percent-6 percent of the daily cost of thediet regularly consumed by these individuals.

It is too early to be able to evaluate the effect of the sanitation improvementsintroduced in the experimental community.

SIGNIFICANCE

Chronic subclinical malabsorption is widespread in tropical areas. Its impactin terms of health, food waste, productivity, and general economics is not known. Thisproject aims at shedding light into these areas to be able to design intervention pro-grams that can be evaluated for their cost effectiveness.

PUBLICATIONS

1. Schneider, R. E, et al. Prevalence of d-xylose malabsorption in ruralcommunities of Guatemala. Summaries V International Congress of Gastro-enterology. Mexico, October 1974.

OTHER DATA

Grantee: Dr. Roberto E, Schneider, Institute of Nutrition of CentralAmerica and Panama (INCAP), Guatemala City, Guatemala, andDr. Morris Shiffman, University of North Carolina, School ofPublic Health, Chapel Hill, North Carolina, U.S.A.

Funded by: U.S. Agency for International Development.


25

EFFECT OF FOOD SUPPLEMENTATION DURING PREGNANCYAND LACTATION ON INFANT MORBIDITY AND MORTALITY

PROBLEM

Almost 1 out of 10 children born alive in developing countries dies during thefirst year of life. In contrast, the percentage in well-to-do societies is roughlyone out of fifty. Although it is widely believed that the high levels of morbidityand mortality reported in developing countries are due to environmental factors asso-ciated with poverty, the precise role of nutrition during pregnancy and the period oflactation has yet to be defined.

Intervention programs designed to reduce infant mortality have generally focusedcn the control of communicable infectious diseases and have paid relatively little atten-tion to maternal nutrition pe.r se. The proposed research will directly address itselfto the role of nutrition during pregnancy and lactation, as an important factor in thehigh levels of infant morbidity and mortality in developing nations. The results ofthis study will also be applicable to public health problems among minority groups indeveloped countries, since these groups generally show similar patterns of infant mor-bidity and mortality to those reported for developing nations.

METHOD

The experimental design for the present study grows out of the.basic researchdesign of the longitudinal study of malnutrition, physical growth and mental develop-ment. For the purpose of the present study, infants will be divided into two groupsreceiving, one high, and the other low food supplementation.

Food supplementation is the experimental treatment and the two groups of childrento be studied are defined by the number of supplemented calories ingested either by themother during pregnancy and lactation and/or by the child during the first year of life.Infant mortality and morbidity during the first year of life are the primary outcomesof dependent variables.

RESULTS TO DATE

Preliminary results indicate that child mortality decreases as the level ofmaternal supplementation increases.

SIGNIFICANCE

Research findings on the effect of nutrition on infant mortality and morbiditywill have important implications in national planning in underdeveloped countries. Inaddition, more detailed knowledge of the relationship between physical growth and mor-bidity and mortality during infancy will be obtained. Indicators of high risk of mor-bidity and mortality at birth will be developed and refined, and in turn will allowmore efficient utilization of the scarce health resources. Finally, general and

26

specific recommendations will be made for public health officials in underdevelopedcountries, about the role of nutrition in infant morbidity and mortality during thefirst year of life.

PUBLICATIONS

None thus far.

OTHER DATA

Drs. Robert E. Klein and Aaron Lechtig, Division of HumanDevelopment, Institute of Nutrition of Central America andPanama (INCAP), Guatemala City, Guatemala.

U.S. Agency for International Development.

1975-1978.

27

Grantee:

Funded by:

Timetable:

PROTEIN-CALORIE MALNUTRITION: EFFECTS, MECHANISMS OFADAPTATION, PATHOPHYSIOLOGY, AND TREATMENT

PROBLEM

Protein and calorie deficiencies are interrelated and highly prevalent in devel-oping countries. There is a definite need to understand better: (a) the effects ofvarious degrees of protein and calorie deficiencies; (b) the interaction between mal-nutrition, diarrhea, and malabsorption; (c) the interrelationships between protein andcalorie utilization, both during depletion and during recovery, including the effectsof calorie balance, expenditure, and activity pattern; (d) the interactions betweenfood intake, hormonal regulation, and body composition during recovery from malnutri-tion; and (e) muscle metabolism during caloric restriction.

In recent years efforts have concentrated on: (a) gastrointestinal physiologyin protein-calorie mainutriton (PCM) and during recovery, in the presence or absenceof diarrhea; (b) caloric intake, activity patterns, and body composition; (c) hor-monal interactions in PCM; and (d) muscle metabolism in calorie deficiency.

METHOD

Longitudinal studies on gastrointestinal alterations were conducted in childrenadmitted to INCAP's Clinical Center with severe PCM, with and without diarrhea. Thestudies included morphology, microbiology, and physiology of the small intestine atdifferent levels. Intubations of the gastrointestinal tract were performed under basalconditions and after stimulation with a fat meal or intravenous administration of cho-lecystokynine. Samples of intestinal contents were studied to determine intraluminalevents of fat absorption with emphasis on micelle formation, lipolytic activity, andbile composition in conjugated and free bile acids, as well as-bacterial flora. Mea-surements of bile acid pools and turnover rates were also accomplished.

Recently, noninvasive techniques have been used in the study of the gastrointes-tinal alterations in PCM and diarrhea. Careful validation of results with the usualtechniques is essential before these very promising tools can be applied to the charac-terization of gastrointestinal function in population groups. This work is being donein cooperation with the University of Chicago and Argonne Laboratories in the UnitedStates.

Effects of caloric intake, growth increase, and recovery from PCM were investi-gated in an animal model and a methodology was set up to study children recovering fromPCM. The methodology included various activity regimens, energy balance, and body com-position studies. In conjunction with this experimental design, growth hormone andinsulin production were measured in response to physiologic and maximal stimuli.

Muscle metabolism in caloric restriction was studied in rats through a hind-limb perfusion technique. Two groups of rats were studied: a control group and agroup of adult males fed one-third of the controls' caloric intake. Arterial venousdifferences in metabolites and muscle composition were measured in both groups, beforeand after electrical stimulation of the sciatic nerve.

28

RESULTS TO DATE

Children with protein-calorie malnutrition, with or without diarrhea, had adecreased concentration of conjugated bile acids and a moderate increase in free bileacids, both primarily and secondarily. This was associated with increased anaerobicand facultative flora in the upper gastrointestinal tract and with a decreased capac-ity to form lipid micelles. This general characteristic was enhanced or made moresevere by the presence of diarrhea. As the child recovered, intestinal flora decreased,conjugated bile acid concentration increased, and free bile concentration remained es-sentially constant. As a result, lipid-micelle formation was also increased. The pre-sence of diarrhea in fully recovered children was accompanied by an increase in uppergastrointestinal flora, a marked increase in free bile acids, and a decrease in con-jugated bile acids. Micelle formation from lipid meal was also reduced. The tauro-cholate pool in malnourished children was markedly reduced but attained normal valueswhen the child recovered. The presence of diarrhea reduced the half-life of taurocho-late to one-fifth of the normal value, suggesting a marked fecal loss of bile acids indiarrhea. All the above changes were also present, but to a lesser degree, in healthychildren from low socioeconomic settings with poor hygiene. In this population, alter-ations in the morphology of the intestinal mucosa and in bile acid composition appearedas early as 3 months after birth.

Deconjugation of 13C glycocholate and production of 13C0o from deconjugated gly-cine have been detected by means of breath analyses in malnourished children withdiarrhea. Increased breath H2 has been substantiated in children with carbohydrateintolerance.

Results of studies in the rat showed a marked decrease in efficiency of calorieand protein utilization when activity was decreased. A method to stimulate adequateactivity patterns through games and progressive exercise regimens was developed forchildren recovering from malnutrition. As in the animal model, children with a higherdegree of physical activity recovered faster than children treated under usual hospi-tal conditions. Recovery was evaluated by gains in weight, height, creatinine excre-tion and basal oxygen consumption. Growth hormone and insulin radioimmunoassays arenow standardized and samples of these substances, taken from children recovering fromkwashiorkor under different activity patterns, are being studied. Different stimuliare being tested for this purpose.

To conduct more exhaustive studies in this area, we have successfully reproducedkwashiorkor and marasmus-like pictures in the rat.

Studies of muscle metabolism in calorie-deprived rats have shown that these ani-mals break their muscles at a very fast rate when electrically stimulated, liberatinga large fraction of myofibrillar nitrogen. At the same time, lactic acid and ammoniaproduction are markedly enhanced. Amino acid analysis of the perfusates is under way.

SIGNIFICANCE

The significance of the research carried out in this project is multiple. Mostimportant is an understanding of the basic mechanisms and effects of protein and calo-rie deficiency with emphasis on diarrhea and malabsorption. This should result in fun-damental knowledge necessary for a rational treatment and the adoption of certain pre-ventive measures. The possible benefit of physical activity in terms of efficiencyof food utilization and growth and the reverse, the adverse effects of restrictedactivity, should be further investigated. The interactions between caloric and pro-tein deprivation are becoming more evident as a consequence of these studies.

29

PUBLICATIONS

1. Alleyne, G. A. 0. et al. Indices of body composition in infantile malnutri-tion: total body potassium and urinary creatinine. Am J Clin Nutr 23:875-78, 1970.

2. Alvarado, J. and F. E. Viteri. Erythropoietic changes during recovery ofprotein calorie malnutrition and its relation to lean body mass. In:Abstract Volume, Xlll International Congress of Hematology, Munich, August2-8, 1970. Munich, J. F. Lehmanns Verlag, 1970, p. 300.

3. ----- et al. Desnutrición proteínico-calórica: El uso de la biopsia mus-cular percutánea en la valoración de la recuperación nutricional. Rev ColMed (Guatemala) 21:100-10, 1970.

4. ----- et al. Tratamiento hospitalario de la desnutrición proteínico caló-rica severa. Rev Col Med (Guatemala) 21:231-45, 1971. Also in: El Pro-blema de la Desnutrición Proteínico Calórica en el Istmo Centraoamericano.Monograph No. 7, Institute of Nutrition of Central America and Panama(INCAP), 1971, p. 245 (Guatemala).

5. ----- et al. Vitamin B-12 absorption in protein-calorie malnourished chil-dren and during recovery. Influence of protein depletion and of diarrhea.Am J Clin Nutr 26:595-99, 1973.

6. Arroyave, G. et al. Influencia de la ingesta proteínica sobre las inmuno-globulinas séricas en pre-escolares. In: X Congreso Internacional deMicrobiología. Resúmenes. México, D.F., Editorial Muñoz, S.A., VIII,1970, p. 72.

7. Béhar, M. et al. Protein quality of fish protein concentrate. Fed Proc30:297, 1971. (Abstract No. 579).

8. ----- and F. Viteri. Protein-calorie malnutrition progress in food andnutrition science, vol. 1, No. 2, pp. 123-37. Pergamon Press (1975).

9. Beghin, 1. and F. E. Viteri. Nutritional rehabilitation centers: anevaluation of their performance. J Trop Pediat 19:759-68, 1973.

10. Bressani, R. et al. Effect of amino acid supplementation of white rice fedto children. Arch Latinoam Nutr 21:347-60, 1971.

11. ----- et al. The quality of various animal and vegetable proteins with anote on the endogenous and fecal nitrogen excretion of children. ArchLatinoam Nutr 22:227-41, 1972.

12. Contreras, C. et al. Effect of protein-calorie malnutrition on musclecreatine, phosphokinase activity. Fed Proc 30:231, 1971, (Abstract No. 214).

13. ----- and F. E. Viteri. Sialic acids content of red blood cells from pro-tein calorie malnourished children and during recovery and from normalchildren and adults. Can J Physiol Pharmacol 51:853-58, 1973.

14. Lechtig, A. et al. Inmunoglobulinas séricas de la desnutrición proteínicocalórica de niños pre-escolares. Arch Latinoam Nutr 20:321-32, 1970.

15. ----- et al. Influencia de la ingesta de proteínas sobre la concentraciónde inmunoglobulinas séricas en niños pre-escolares. Arch Latinoam Nutr20:333-43, 1970.

30

41. ----- and 0. Pineda. Effects on body composition and body function.Psychological effects. In: Blix, G., Y. Hofvander, and B. Vahlquist(eds.), Famine. A Symposium Dealing with Nutrition and Relief Operationsin Time of Disaster. Stockholm, Sweden, Almsqvist & Wiksell, 1971, p. 25-40. (Symposia of the Swedish Nutrition Foundation IX).

42. ----- et al. Reply to Drs. Méndez and Burkirk, (Letter to the Editor).Am J Clin Nutr 24:386-87, 1971.

43. - .---- Effect of activity restriction and exercise on growth of calorically-restricted rats. Fed Proc 30:645, 1971 (Abstract No. 2512).

44. -----. Composición corporal y requerimientos calóricos y proteínicos enrelación a la edad. Rev Col Med (Guatemala), 21:148-73, 1970. Also in:El Problema de la Desnutrición Proteínico Calórica en el Istmo Centro-americano, Monograph No. 7. Institute of Nutrition of Central America andPanama, Guatemala, 1971, p. 148.

45. ----- and J. Alvarado. Aspectos fisiológicos y respuestas adaptativas enla desnutrición calórica y en la desnutrición proteínica. Rev Col Med(Guatemala) 21:175-230, 1970. Also in: El Problema de la DesnutriciónProteínico Calórica en el Istmo Centroamericano. Monograph No. 7. Insti-tute of Nutrition of Central America and Panama, 1971, p. 175.

46. -----. Efecto de ejercicio e inactividad física sobre el crecimiento y laeficiencia de utilización de la dieta en ratas alimentadas ad libitum y conrestrición calórica. In: III Reun Soc Latinoam Nutrición and Seminariosobre Ambiente Biológico y Nutrición. Resúmenes de trabajos. Instituteof Nutrition of Central America and Panama, Guatemala 11-14 September 1972.

47. ----- et al. Evaluación de la calidad proteínica del maíz común, del maízOpaco-2 y del maíz común suplementado con amino ácidos y con otras fuentesde proteína. In: Bressani, R., J. E. Braham, and M. Béhar (eds.), Mejora-mento Nutricional del Maíz. Memorias de una conferencia de nivel inter-nacional. Institute of Nutrition of Central America and Panama, GuatemalaCity, 6-8 March 1972. Guatemala City, Talleres Gráficos del INCAP. Octo-ber 1972, pp. 195-208.

48. ----- and R. Bressani. The quality of new sources of protein and theirsuitability for weanlings and young children. Bull WHO 46:827-43, 1972.

49. ----- et al. The relative importance of protein nutriture in the malabsorp-tion of protein calorie malnutrition (PCM). Gastroenterology 60:729, 1971.

50. ----- and G. Arroyave. Protein-calorie mainutrition. In: Goodhart, R. S.and M. E. Shils (eds.), Modern Nutrition in Health and Disease. 5th ed.Philadelphia, Penn., Lea and Febiger, 1972.

51. -----. Creatinine-height index in malnourished children (Letter to theEditor). Nutr Rev 30:24-26, 1972.

52. -----. Nutritional evaluation of subclinically protein-calorie malnourishedpreschoolchildren. In: Memories of the IX International Congress of Nutri-tion, Mexico, September 3-9, 1972.

53. ----- et al. Intestinal malabsorption in malnourished children and duringrecovery. Duodenal content of lipase, nitrogen and micellar fat after fatstimulation. Arch Latinoam Nutr 22:613-27, 1972.

31

30. ----- and -----. Morphological aspects of the duodenojejunal mucosa inprotein-calorie malnourished children and during recovery. Am J Clin Nutr25:1092-1102, 1972.

31. ----- and -----. Capacidad para micelarizar grasa del contenido duodenalde niños con desnutrición proteínico-calórica (DPC), su relación con estadonutricional y diarrhea. Presented at the IX Cong Int Nutrición, Mexico,3-9 September 1972. Also in: III Reun Soc Latinoam Nutrición and Semina-rio sobre Ambiente Biológico y Nutrición. Resúmenes de trabajos. Insti-tute of Nutrition of Central America and Panama, Guatemala, 11-14 September1972, p. 47.

32. ----- and -----. Alteraciones de las sales biliares del contenido duodenalde niños con desnutrición proteínico-calórica (DPC). Su relación con esta-do nutricional y diarrhea. Presented at the IX Cong Int Nutrición, Mexico3-9 September 1972. Also in: III Reun Soc Latinoam Nutrición and Seminariosobre Ambiente Biológico y Nutrición.

33. ----- and -----. Relación entre la capacidad de micelarización de grasadel contenido duodenal de niños con DPC y niveles de actividad de lipasay concentración de sales biliares. Presented at the IX Cong Int Nutrición,Mexico, 3-9 September 1972. Also in: III Reun Soc Latinoam Nutrición andSeminario sobre Ambiente Biológico y Nutrición. Resúmenes de Trabajos.Institute of Nutrition of Central America and Panama, Guatemala City,11-14 September 1972, p. 49.

34. ----- and -----. Estudios sobre estado nutricional-diarrea. Mala absorciónintestinal. I. Micelarización de grasas. II. Lipasa Pancreática y III.Sales Biliares. In: XVI Cong Nac Pediatría, Guatemala City, 24 February-3 March 1973. Asociación Pediátrica de Guatemala (Sección de Resúmenes deTrabajos del Programa), 1973, Pp. 40-42.

35. ----- and -----. Some morphologic and functional alterations of the gastro-intestinal tract in protein-calorie malnourished children. Workshop onMalabsorption and Nutrition, National Academy of Sciences, Washington,April 1971. Am J Clin Nutr, in press.

36. ----- et al. Studies on the luminal events of lipid absorption in proteincalorie malnutrition (PCM) children: its relation with nutritional recoveryand diarrhea. I. Lipid micellarization capacity of the duodenal content.Am J Clin Nutr 27:777-87, 1974.

37. ----- et al. Studies on the luminal events of lipid absorption in proteincalorie malnutrition (PCM) children: its relation with nutritional recoveryand diarrhea. II. Alteration in the bile salts of the duodenal content.Am J Clin Nutr 27:788-96, 1974.

38. Smith, F. R. et al. Serum vitamin A, retinol-binding protein, and prealbu-min concentrations. II. Treatment including supplemental vitamin A. Am JClin Nutr 26:982-87, 1973.

39. Torún, B. et al. Effect of physical activity upon growth of children re-covering from protein-calorie malnutrition. 10th International Congress ofNutrition, Abstract 314, The Science Council of Japan, 1975.

40. Viteri, F. E. and J. Alvarado. The creatinine height index: its use in theestimation of the degree of protein depletion and repletion in protein calo-rie malnourished children. Pediatrics 46:696-706, 1970.

32

16. Martínez, C. and F. E. Viteri. Pérdidas sensibles e insensibles de nitro-geno en el pre-escolar. In: III Reunión de la Sociedad Latinoamericanade Nutrición y Seminario sobre Ambiente Biológico y Nutrición. Resúmes detrabajos. Institute of Nutrition of Central America and Panama, Guatemala

17. Mata, L. J. et al. Gastrointestinal flora of children with protein caloriemalnutrition. Am J Clin Nutr 25:1118-26, 1972.

18. Nichols, B. L. et al. Potassium and nitrogen requirements during recoveryfrom protein calorie malnutrition (PCM). In: Proceedings of the II WesternHemisphere Nutrition Congress. August 30-September 2, 1971. Miami Beach,Florida. Abstracts. Mount Kisko, New York. Futura Publishing Co., 1972,p. 387.

19. ----- et al. Therapeutic implications of electrolyte water and nitrogenlosses during recovery from protein calorie malnutrition. J Pediat 84:759-68, 1974.

20. ----- et al. Anomalies of the regulation of salt and water in protein calo-rie malnutrition. In: Gardner, L. I. and P. Amacher, (eds.), EndocrineAspects of Malnutrition Marasmus, Kwashiorkor,and Psychosocial Deprivation.Proceedings of a Symposium. Kroc Foundation, May 1973, Santa Ynez, Cali-fornia, pp. 363-98.

21. Nichols, B. and F. E. Viteri. On the pathogenesis of deranged nitrogenmetabolism in protein-calorie malnutrition. Reply to a letter to the editor,J Pediat 86:650-51, 1975.

22. Pineda, O. et al. Leukocyte enzyme adaptation to low protein-calorie diets.Fed Proc 30:231, 1971 (Abstract No. 215).

23. Schneider, R. E. Incidencia de la malabsorción intestinal en comunidadesrurales guatemaltecas. In: Memoria, XV Congreso Médico Centroamericano.San José, Costa Rica, December 1973.

24. -----. Cambios en la ecología gastrointestinal de niños guatemaltecos ob-servados durante sus primeros meses de vida. In: Memoria, XV CongresoMédico Centroamericano. San José, Costa Rica, December 1973.

25. -----. Alterations in the gastrointestinal ecology of Guatemalan childrenduring the first 18 months of age. Summaries of the V International Con-gress of Gastroenterology. Mexico, October 1974, in press.

26. -----. Descripción de una sonda que simplifica la intubación del intestinodelgado distal del niño. Rev Col Med (Guatemala) 24:196-98, 1974.

27. ----- and R. Chang. A pediatric tube and capsule for suction biopsy of thesmall 'intestinal mucosa designed for direct nasogastric intubation. Gut12:399-402, 1971.

28. ----- and F. Viteri. Duodenal micellar capacity in protein calorie mal-nourished (PCM) children and during recovery. Gastroenterology 60:714,1971 (Abstract).

29. ----- and -----. Descripción de una sonda que simplifica la intubación in-testinal por vía nasal en pacientes pediátricos. Rev Col Med (Guatemala)22:242-44, 1971.

33

54. -----. Infección y Nutrición. In: Meneghello, Julio (ed.), Pediatría,Vol. 1, Chapter 6, Nutrición Infantil. Buenos Aires, Argentina. EditorialInter-Médica, 1972, pp. 353-61.

55. ----- et al. Evaluation of the protein quality of common maize, opaque-2maize, and common maize supplemented with amino acids and other sources ofprotein. In: Bressani, R., J. E. Braham, and M. Béhar (eds.), Nutri-tional Improvement of Maize. Proceedings of an international conference.Institute of Nutrition of Central America and Paname, Guatemala City,March 6-8, 1972. Guatemala City, Talleres Gráficos del INCAP, 1973,pp. 191-204.

56. ----- et al. Intestinal malabsorption in malnourished children and duringrecovery. Relationships between severity of protein deficiency and the mal-absorption process. Am J Dig Dis 18:201-11, 1973.

57. ----- et al. Métodos de evaluación del estado nutricional proteínico caló-rico en pre-escolares de condiciones socio-económicos diferentes. Reper-cusión nutricional del sarampión en niños crónicamente subalimentados.Arch Latinoam Nutr 23:13-31, 1973.

58. -----. Efecto de la inactividad sobre el crecimiento de ratas alimentadascon una dieta adecuada, a niveles de ingestión calórica normal y restringi-dos. In: Nuevos Conceptos sobre Viejos Aspectos de la Desnutrición, Chap-ter IV. Desnutrici6n y Crecimiento Físico. Mexico Fondo Editorial Nestléde la Academia Mexicana de Pediatría, 1973, PP. 207-29.

59. ----- and B. Torún. Estudios sobre dieta-actividad física-crecimiento.I. Diseño experimental y resultado de medidas corporales. II. Composi-ción Corporal y III. Estudios metabólico-nutricionales. In: XVI CongNac Peditría, Guatemala City, 24 February-3 March 1973. Guatemala City,Asociación Pediátrica de Guatemala, 1973, pp. 36-38. (Sección de Resúmenesde Trabajos del Programa).

60. ----- et al. Digestibilidad y calidad biológica del frijol negro (Ph. vul-qaris) en niños pre-escolares. In: XVI Cong Nac Pediatría, Guatemala City,24 February-3 March 1973, PP. 38-39. (Sección de Resúmenes de Trabajos delPrograma).

61. ----- and J. Alvarado. Estudios sobre adaptación hematológica en la desnu-trición proteínico-patológica. Respuestas hematológicas a diversos nivelesde ingestión de proteínas. In: XVI Cong Nac Pediatría, Guatemala City,24 February-3 March 1973. Guatemala City, Asociación Pediátrica de Guate-mala, 1973, pp. 39-40 (Sección de Resúmenes de Trabajos del Programa).

62. ----- and R. E. Schneider. Gastrointestinal alterations in Protein-CalorieMalnutrition. Medical Clinics of North America 58:1487-1505, 1974.

63. ----- and -----. Alteraciones gastroingestinales en la desnutrición pordeficiencia de Calorías y Proteínas. Clínicas Médicas de Norte América58:1483-1502, 1974.

64. ----- and Moisés Béhar. Efectos de diversas infecciones sobre la nutricióndel pre-escolar especialmente el sarampión. Bol Of Sanit Panam 78:226-39,1975.

65. - .---- Nutritional evaluation of subclinically protein calorie malnourishedpreschoolchildren. In: Chávez, A., H. Bourges, and S. Basta (eds.). Nutri-tion. Proc 9th Int Congr Nutrition, Mexico 1972, Vol. 2, Basel, Switzerland,S. Karger, 1975, PP. 227-35.

34

Dr. Fernando E. Viteri, Institute of Nutrition of CentralAmerica and Panama (INCAP), Guatemala City, Guatemala, andDr. Noel W. Solomons, Department of Medicine, University ofChicago, Chicago, Illinois.

U.S. National Institutes of Health, Institute of Nutritionof Central America and Panama (INCAP), University of Chicago,and Nutrition Foundation.

1964-1976.

35

OTHER DATA

Grantee:

Funded by:

Timetable:

HAVE MODERN HEALTH PRACTICES REMOVED THE SAFEGUARD OF BACTERIAL INTERFERENCE,SHORTENED BREAST-FEEDING, AND INCREASED INFANT MALNUTRITION?

PROBLEM:

Rapid urbanization and other influences derived from industrial societiesaccount today for an increasingly greater rate of early weaning. This is commonlyobserved in the cities and it is becoming more frequent in rural areas. Furthermore,observations in Guatemala indicate that the incidence of breast-feeding is progres-sively decreasing in low income urban groups. This phenomenon has serious implica-tions not only because the child is deprived of human milk (which provides the safestfood and immune defense factors), but also because the population does not have theeconomic resources, education, and hygienic practices necessary for carrying out anappropriate supplementary feeding. It is planned to study whether the simple maneuverof increasing the time the mother has close contact with her infant immediately afterbirth, will result in extended breast-feeding with its nutritional and infection-resisting benefits, and will, at the same time, reduce the chance of contaminatingthe infant (and subsequently his family) with hospital organisms.

METHOD

To test whether present hospital routines result in reduced breast-feedingsecondary to a diminution of maternal responses, one group of mothers are given theirinfants nude in the first hour of life to hold skin to skin contact and to suckle forup to 45 minutes in a private room. A heat panel 4 feet above the infant maintainhis temperature. These mothers also have their babies for 5 additional hours eachday during their hospital stay. The control group of mothers have contact with theirbabies according to the usual hospital policies, a glimpse of the baby right afterbirth but no physical contact, identification at 8-12 hours, then a 20-30 minute breast-feeding every 4 hours. The babies are being prospectively followed up to 12 monthsof age, to determine the pattern of growth, breast-feeding, and morbidity.

RESULTS TO DATE

At the present, the analysis of data shows that children with early and prolongedcontact with their mothers have a tendency to develop less episodes of illness. Breast-feeding is more prolonged in the experimental than in the control group. The averageage at weaning is about 90 days more in the group of children with early contact.

SIGNIFICANCE

If this short period of early physical contact does increase the length ofbreast-feeding, the growth and development of the infant would then be expected toimprove greatly, and the mother would be provided with at least partial contraceptiveprotection. This study has the potential of showing that great benefits may be gainedfrom a simple intervention that can easily be provided with no cost.

36

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Drs. John Kennell and Marshall Klaus, Department of Pediatrics,Case Western Reserve University, Cleveland, Ohio, U.S.A.

Williams-Waterman Research Corporation.

1974-1977.

37

MALNUTRITION, INFECTION, AND HUMAN LEUKOCYTE METABOLISM

PROBLEM

The purpose of this investigation was to evaluate the extent to which malnutri-tion, alone or complicated with infection, may affect the bactericidal capacity of thepolymorphonuclear (PMN) leukocyte and if so, to investigate at which stage of phagocy-tosis is the biochemical damage produced. The project also aimed to relate clinicalrecovery of the malnourished child with functional recovery of the leukocyte.

METHOD

The rate of oxygen uptake was measured polarographically in resting and phago-cytizing leukocytes, with the aid of a Clark electrode. Phagocytosis was measured bycounting, at different time intervals, the number of bacteria present intra- and extra-cellularly. This procedure also permitted the evaluation of bactericidal capacity ofthe leukocyte. During the course of the study, phagocytosis and bactericidal capacitywere measured using Escherichia coli (K-12), Staphyloccus albus (mendita), and S. albus(DiGiorgio).

RESULTS TO DATE

The experimental part of this investigation was concluded in 1975. The dataobtained are being analyzed statistically but some conclusions may already be drawn.The rate of resting oxygen uptake by PMN leukocytes of malnourished children did notdiffer significantly from that of leukocytes from healthy, well-nourished children.Phagocytic stimulus of the cells induced an increase in respiration that was not ofthe same magnitude as that observed in healthy individuals. This response was roughly50 percent of the expected value. This observation was the basis for use of oxygenuptake in leukocytes for detection of malnutrition. Infection was also detected byincreased resting rate of uptake. The magnitude of the normal response was, hereagain, twice as high as that observed in malnourished children. In addition, leukocytesfrom malnourished children had lower phagocytic and bactericidal rates, with variousbacterial strains, especially those related to the myeloperoxidase system. Thus thebactericidal capacity of leukocytes from malnourished children was only about 40 per-cent of the normal response.

SIGNIFICANCE

The results obtained pointed up the important relationship between mainutritionand infection and permitted a better interpretation of previously observed phenomena.

Furthermore, this study drew attention to the usefulness of circulating PMNleukocytes in testing important metabolic alterations in the host, studies which couldbe applied to humans in the future.

38

PUBLICATIONS

1. Pineda, O. Mainutrition, infection, and human polymorphonuclear leukocytemetabolism. Fed Proc 31:710, 1972.

OTHER DATA

Dr. Oscar Pineda, Division of Physiological Chemistry, Instituteof Nutrition of Central America and Panama (INCAP), GuatemalaCity, Guatemala.

U.S. National Institute of Arthritis and Metabolic Diseases.

1973-1975.

39

Grantee:

Funded by:

Timetable:

HEMATOLOGIC INVESTIGATIONS IN THE CENTRAL AMERICAN POPULATION

PROBLEM

Epidemiologic studies and therapeutic trials in population groups in CentralAmerica have indicated that iron deficiency is the most important cause of nutritionalanemia. Folate deficiency, evident by biochemical analysis, apparently has no directinfluence on hemoglobin levels, although it may limit the response to iron administra-tion. In continuation of previously reported studies, we have concentrated on findingan adequate form of iron supplementation to the population. Various forms of iron havebeen tested, both at INCAP's Clinical Center and at the population level, with empha-sis on an iron salt that would behave more like the hem iron than like the inorganic(cereal-vegetable) iron.

METHOD

At INCAP's Clinical Center, patterns of iron absorption have been studied afterthe administration of rerrous sulfate, ferric orthophosphate, sodium iron pyrophos-phate, hemoglobin, and sodium iron EDTA administered alone and in combination withcorn and beans or with purified corn starch. Iron absorption from radiolabeled FeC1 3,Hb, NaFeEDTA and ferrous ascorbate has been measured in healthy adult volunteers andin iron deficient children to quantitate the value of fortifying sugar with iron, theinfluence of different foods on iron absorption and the interaction of various formsand dosages of iron in the process of absorption. 60CoC1 2 absorption is also beinginvestigated as a noninvasive technique that could be used in lieu of iron under cer-tain conditions and as an indicator of iron deficiency in population groups. At thepopulation level, a coastal group of anemic individuals were given therapeutic dosesof iron to bring to normal their iron nutrition. The population was then divided intotwo similar groups: one receiving sugar fortified with ferric orthophosphate andthe other receiving unfortified sugar. This test lasted 9 months. Similar studiesare proceeding in another population receiving sugar fortified with NaFeEDTA, to pro-vide 15 mg of ¡ron/100 g of sugar. The effect of sugar fortification with FeSO4and NaFeEDTA on the organoleptic properties of various dietary preparations is beingevaluated.

RESULTS TO DATE

Ferric orthosphosphate and sodium ¡ron pyrophosphate are essentially nonabsorb-able. The pattern of absorption of ferrous sulfate was different from that of sodiumiron EDTA which resembled more the pattern of the hem iron. Administration of ferroussulfate mixed with corn and beans changed the iron absorption and also apparently de-creased it, when compared to the effect of ferrous sulfate mixed with purified cornstarch. In contrast, the iron from hem and from sodium iron EDTA was better absorbedwith corn and beans or with corn starch; there appeared to be no difference betweenthe effects of these two foods.

Absorption of radioiron indicates that iron in NaFeEDTA forms a pool with inor-ganic iron (nonheme) which differs, however, from it in that the chelated iron seemsto be protected from food factors which modify, usually inhibiting, nonheme iron ab-sorption. The absorption of iron from NaFeEDTA added to sugar and consumed with mealsis often superior and so far never inferior to that from nonheme iron. Heme ironabsorption is higher than that from NaFeEDTA.

40

Tests of the effect of sugar fortified with ferric-orthophosphate at a popula-tion level corroborated the results obtained at the Clinical Center, that the iron inferric orthophosphate is essentially unabsorbable. The fortification of sugar withNaFeEDTA has now been accomplished at the technical level and its trial in a lowlandpopulation is indicating a successful reduction in the prevalence of iron deficiencyand in the incidence of ferropenic anemia.

SIGNIFICANCE

¡ron fortification and supplementation has not been accomplished in populationswho consume primarily cereals and legumes. The discovery of a form of iron that wouldbe absorbed adequately and that would interfere minimally with the organoleptic pro-perties of food, would greatly facilitate the implementation of iron-supplementationprograms.

PUBLICATIONS

1. Cifuentes, E. and F. E. Viteri. Physical fitness, iron deficiency andanemia in agricultural laborers of Central America. Fed Proc 31:719, 1972,(Abstract 2843).

2. Cook, J. D. et al. Nutritional deficiency and anemia in Latin America.A collaborative study. Blood 38:591-603, 1971.

3. ----- et al. Las carencias nutricionales y la anemia en Latinoamérica.Estudio en colaboración. Bol Of Sanit Panam 72:215, 1972.

4. Viteri, F. E. et al. Normal haematological values in the Central AmericanPopulation. Br J Haematol 23:189-204, 1972.

5. ----- and M. A. Guzmán. Haematological status of the Central Americanpopulation: prevalence of individuals with haemoglobin levels belownormal. Br J Haematol 23:725-35, 1972.

6. -----. Physical fitness and anemia. In: Shimazono, N. (ed.), Proceedingsof the International Symposium on Malnutrition and Functions of Blood Cells.Kyoto, Japan, November 27-30, 1972, pp. 559-83.

7. ----- and B. Torún. Anaemia and physical work capacity, Clinics in Haema-tology, 3:609-26, 1974.

8. ----- and E. Cifuentes. Capacidad física en trabajadores agrícolos de lacosta de Guatemala antes y después de terapia con hierro. Presented atthe Ill Reunión de la Sociedad Latinoamericana de Nutrición y Seminariosobre Ambiente Biológico y Nutrición. Resúmenes de trabajos. Instituteof Nutrition of Central America and Panama (INCAP), Guatemala 11-14September 1972, p. 63.

9. ----- and J. Alvarado. Estudios sobre adaptación hematológica en la des-nutrición proteTnico-patológica. Respuestas hematológicas a diversosniveles de ingestión de proteínas. In: XVI Congreso Nacional de Pedia-tría, Guatemala, 24 February-3 March 1973. Guatemala, Asociación Pediá-trica de Guatemala, 1973, PP. 39-40.

10. ----- et al. Anemias nutricionales en Centro América. Influencia de in-fección por uncinaria. Arch Latinoam Nutr 23:33-53, 1973.

41

11. ----- and B. Torún. ¡ron fortification of sugar: advantages of NaFeEDTAFeSO 4. lOth International Congress of Nutrition, Abstract 8211, The ScienceCouncil of Japan, 1975.

12. -----. ¡ron fortification of sugar to prevent iron deficiency in CentralAmerica. Submitted for publication, Lancet, 1976.

13. ----- et al. Fortificación del azúcar con la sal sódico-férrica de EDTApara aumentar la ingestión del hierro en Centro América. Enviado a publi-cación en Arch Latinoam Nutr, 1976.

OTHER DATA

Grantee: Dr. Fernando E. Viteri, Institute of Nutrition of CentralAmerica and Panama (INCAP), Guatemala City, Guatemala.

Funded by: World Health Organization.


42

ANTIBODY RESPONSE TO MEASLES VACCINE IN MALNOURISHED CHILDREN

PROBLEM

Although children are routinely vaccinated against measles in many countries ofthe world, there is little information regarding antibody responses of malnourishedchildren to this vaccine. Since measles virus is known to be able to produce a stateof latency that may be related to inadequate host response to the original infection andmay lead to subsequent disease processes, it is extremely important to determine whattype of antibody response malnourished children have to wild measles infection and tothe live attenuated viral vaccine and whether these two types of response lead to a stateof latent viral infection.

METHOD

Serum samples from 90 children who had natural measles were collected yearly andtested for measles antibodies. Children who had been immunized with the Moraten strainof measles in 1972 were prospectively studied for the persistence and type of measlesantibodies.

RESULTS TO DATE

Forty-nine out of fifty-eight children had a persistence of the measles antibodyreacting with the intranuclear antigen. This persistence was noted in the sera obtainedeven as late as 9 years after the original measles infection in all but 3 children whohad positive reactions to the NF antibody. The three had remained positive for 3 to 5years after measles, but then had lost the antibody. This type of persistence had notbeen previously observed for more than 5 months after the acute measles in the well-nourished American and German children. Clinical data on these children are being ana-lyzed for correlations between the type of serological reaction and the nutritional stateof each child. In addition, serial sera of children who had received live attenuatedmeasles vaccine (the Moraten strains) during May and June 1972 were collected in 1975.No nuclear antibody has been identified in these sera. However, in four children knownto have seroconverted after the vaccine, no antibodies were detected.

SIGNIFICANCE

Knowledge of the pattern of persistence of antibodies to measles virus will beuseful in determining the number of children showing evidence of latency of infectionafter natural measles. The ability of one- and second-degree malnourished children toseroconvert after immunization with the attenuated strain (Moraten) of measles virus,as well as the pattern of persistence of antibodies will be determined.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. Juan José Urrutia, Division of Environmental Biology,Institute of Nutrition of Central America and Panama (INCAP),Guatemala City, Guatemala, and Dr. Michael Katz, Faculty ofMedicine, Columbia University, New York, New York.

Funded by: U.S. National Institutes of Health (NIH).


44

INTERACTION BETWEEN NUTRITION AND PRODUCTIVITY OF AGRICULTURAL LABORERS

PROBLEM

Agricultural laborers' physical working capacity is limited by the availabilityof calories for activity derived from their total caloric intake, minus basal energyexpenditure. It has been previously shown that an increase in daily calorie intakefrom 2,700 to 3,500/cal produces a substantial increase in daily energy output. It hasalso been shown that even mild degrees of iron deficiency anemia decrease the capacityof agricultural laborers to perform near maximal exercise.

The purpose of this study is: (a) to test the hypothesis that calorie and ironsupplementation in the diet of agricultural laborers will result in increased workingcapacity and actual productivity, and (b) that better earnings and more energy off-the-job will result in an improvement in the nutrition and the general quality of life ofthe family of such laborers. Under these conditions, the availability of effective de-livery systems of energy-rich foodsis essential for the continuity of the beneficialeffects.

METHOD

Two communities in the lowland areas of Guatemala are studied through anthropo-metric and hematologic measurements, physiologic performance in standardized tests,energy expenditure in actual field situations, and socioeconomic, morbidity, and di-etary surveys of the family. Work output, income, and expenditures are constantlyevaluated throughout two agricultural cycles. Both communities receive vitamin supple-mentation, but in one community, workers' diet is supplemented by 800 extra caloriesper day. The measurements outlined before are continued through at least two fullagricultural cycles.

The effectiveness, feasibility, and costs of various systems for delivery ofenergy-rich foods to agricultural communities are also being explored.

RESULTS TO DATE

All basal data were collected during 1973 and supplementation started in themiddle of 1974. Findings are being analyzed but preliminary information indicates thatenergy supplementation produces an increment in physical activity at work, which resultsin an increase in productivity or in a threefold return on investment. Many factors,besides energy nutrition, affect productivity. The interactions between all possiblefactors are being investigated in a fairly closed environment (farm) and in an open com-munity with an active social organization (cooperative).

SIGNIFICANCE

This research is fundamental in guiding the distribution of scarce resources innutrition programs. Improving the work output of laborers and the quality of life oftheir families through supplementation of the main provider is a new and, most probably,very effective nutrition policy.

45

PUBLICATIONS

1. Cifuentes, E. and F. E. Viteri. Physical fitness, iron deficiency andanemia in agricultural. laborers of Central America. Fed Proc 31:719, 1972(Abstract 2843).

2. Immink, M. D. C. et al. Calorie supplementation, working capacity andproductivity in Guatemalan sugarcane cutters. lOth International Congressof Nutrition, Abstract 8203. The Science Council of Japan, 1975.

3. Torún, B. and F. E. Viteri. Response of Guatemalan rural men and soldiersto heavy exercise of short duration. Fed Proc 30:646, 1971 (AbstractNo. 2521).

4. Viteri, F. E. Considerations on the effect of nutrition on the body com-position and physical working capacity of young Guatemalan adults. In:Scrimshaw, N. S. and A. M. Altschul (eds.), Amino Acid Fortification ofProtein Foods. Report of an International Conference held at the Massa-chusetts Institute of Technology, September 16-18, 1969, Cambridge,Massachusetts, The MIT Press, 1971, pp. 350-75.

5. ----- et al. Determining energy cost of agricultural activities by res-pirometer and energy balance techniques. Am J Clin Nutr 24:1,418-30, 1971.

6. ----- et al. Caloric balance of agricultural labor populations in CentralAmerica. Fed Proc 31:719, 1972 (Abstract 2842).

7. -----. Physical fitness and anemia. In: Shimazono, N. (ed.), Proceedingsof the International Symposium on Mainutrition and Functions of Blood Cells,Kyoto, Japan, November 27-30, 1972, pp. 559-83.

8. ----- and E. Cifuentes. Physical fitness in anemic Guatemalan agriculturallaborers before and after iron therapy. Submitted for publication.

9. ----- and -----. Capacidad física en trabajadores agrícolas de la costade Guatemala antes y después de terapia con hierro. Presented at the IIIReun Soc Latinoam Nutrición and Seminario sobre Ambiente Biológico yNutrición . Resúmenes de trabajos. Institute of Nutrition of CentralAmerica and Panama, Guatemala City, 11-14 September 1972, p. 63.

10. ----- et al. Dietary intake, energy cost of work, total caloric expenditure,and caloric balance in two agricultural laborer populations of Guatemala.Submitted for publication.

11. ----- and B. Torún. Anemia and physical work capacity. Clinics in Haema-tology, 3:609-26, 1974.

12. ----- and -----. Ingestión calórica y trabajo físico de obreros agrícolasen Guatemala. Efecto de la suplementación alimentaria y su lugar en losprogramas de salud. Bol Of Sanit Panam 78:58-74, 1975.

13. ----- et al. Nutrición y Productividad. Efecto del consumo y suplementa-ción de energía (calorías) sobre la Productividad de trabajadores agrícolasen Centro América.En: Memorias XIV Reunión del Comité Asesor sobre Inves-tigaciones Médicas de la OPAS, Washington, Julio 1974.

46

14. ----- et al. Nutrition and Productivity. Effect of energy (calories)intake and implementation on the productivity of agricultural laborersin Central America. In: Proceedings of XIV Meeting of the AdvisoryCommittee on Medical Research. PAHO, Washington, July 1975.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Fernando E. Viteri, Institute of Nutrition of CentralAmerica and Panama (INCAP), Guatemala City, Guatemala.

Government of Guatemala, Pan American Health and EducationFoundation/World Health Organization, and W. K. KelloggFoundation.

1974-1977.

47

LONGITUDINAL STUDY OF THE EFFECTS OF MALNUTRITIONON PHYSICAL GROWTH AND MENTAL DEVELOPMENT

PROBLEM

Whereas the prevalence of severe protein-calorie mainutrition in young rural andurban poor children is often less than 3 percent, chronic moderate protein-calorie mal-nutrition affects nearly two-thirds of such children in most developing nations. It is,therefore, important to assess changes in growth and development which may result fromnutritional improvements in such a larger group.

The purposes of this study are to determine: (1) the modifications in physicalgrowth and mental development that occur in moderately malnourished preschoolchildren asa result of varying degrees of protein-calorie nutritional status; (2) at what age ade-quate nutritional supplements promote favorable changes in physical growth and mentaldevelopment; and (3) which cultural and socioeconomic factors affect physical growth andmental development.

METHOD

Four rural communities of eastern Guatemala are under study. Two of them receivea protein-calorie supplement and the other two, a caloric supplement. Dietary surveysshow that calories, not proteins, are limiting factors. All communities receive pre-ventive as well as curative medical services.

Pregnant and lactating mothers as well as all children from birth to 7 years ofage are studied. Subjects are assessed periodically at predetermined age levels by an-thropometric measurements, biochemical determinations, dietary and morbidity surveys,demographic and sociocultural studies, and neurological and psychological examinations.

RESULTS TO DATE

Caloric supplementation, whether or not accompanied by protein, decreases theprevalence of low birthweight by 50 percent. Caloric supplementation also improvesphysical growth in preschoolchildren, particularly in the first 3 years of life. Itis estimated that children with well-supplemented diets from conception to age 7, willnarrow the gap in height and weight between baseline values and the standards for well-nourished children by 40 percent to 50 percent. The high prevailing morbidity, par-ticularly gastrointestinal ailments, is growth-retarding. In addition, physical growthis related to a variety of socioeconomic indicators, such as housing quality and theextent of land owned and/or rented by the family.

Considerable progress has been made in testing the hypothesis that mild-to-moderate malnutrition affects mental development, particularly in 15- to 24-month oldinfants and in preschoolchildren aged 36 to 48 months. The socioeconomic status ofstudy children's families has also been found to influence mental test performances,and to interact with nutritional status. Thus children from low socioeconomic statusfamilies are much more likely to perform poorly in mental tests, as a consequence ofinadequate, unsupplemented diets than children from less impoverished families. Fur-thermore, findings indicated that the most damaging effect of malnutrition on subsequentmental development is felt at the crucial time of gestation.

48

SIGNIFICANCE

The majority of preschoolchildren in developing countries, which encompass over66 percent of the world population, are now malnourished to varying degrees. Growthand development rates, which are fastest in preschoolchildren, are seriously impairedby protein and calorie deficiencies as well as the infectious diseases prevalent insuch children.

This study is yielding valuable data on the effects of environmental conditionsof human growth and development potentials, including the influence of age of onset,duration, and severity of malnutrition.

The findings to date of a relationship between mainutrition during pregnancyand the first years of life and deficient intellectual development during the earlychildhood years are of crucial importance to developing countries. If data currentlybeing collected show that these detrimental effects persist beyond early childhood,then immediate measures to prevent early mainutrition should receive highest priorityin public health programs.

PUBLICATIONS

1. Adinolfi, A. A. and R. E. Klein. The value orientation of Guatemalansubsistence farmers: measurement and implications. J Soc Psychol 87:13-20,1972.

2. ----- et al. Aggressive reactions to frustration in urban Guatemalan chil-dren: the effects of sex and social class. J Pers Soc Psychol 25:227-33,1973.

3. Arroyave, G. et al. Relationship between serum vitamin A in pregnant womenand their newborns in two contrasting socioeconomic groups. In: Abstractsof Papers, Symposia and Free Communications of the Xth International Congressof Nutrition. Kyoto, Japan, August 3-9, 1975. Abstract No. 7314, pp. 333.

4. ----- et al. Vitamina A en sangre de embarazadas y sus recién nacidos endos grupos socioeconomicos. Arch Latinoam Nutr 25:283-90, 1975.

5. Ayala, G. et al. Utilización de la suplementación alimentaria como vehículopara la adición de fluor. (Abstract). Paper presented at Primer CongresoPeruano de Nutricion, Lima, Peru, March 1972.

6. Baker, G et al. Family planning factors in four Guatemalan Ladino villages.Paper presented at the Annual Latin American Conference of Arizona LatinAmerican Studies. Arizona State University, Tempe, May 9, 1973.

7. Beteta, C. E. et al. Estudio sobre incidencia de patología clínica, anoma-lías físicas menores y desarrollo neurológico en niños de poblaciones rura-les de Guatemala. Paper presented at Congreso Nacional de Pediatría,Guatemala, April 1972.

8; -----. Morbilidad intrauterina. Paper presented at IX Congreso Nacional dePediatría on "Factores que Afectan el Desarrollo durante el Primer Año deVida", Guatemala, March 13-15, 1975.

9. Blanco, R. et al. Retardation in appearance of ossification centers indeprived Guatemalan children. Hum Biol 44:525-36, 1972.

49

10. ----- et al. Sex differences in retardation of skeletal development in ruralGuatemala. Pediatrics 50:912-15, 1972.

11. ----- et al. Prevalence of brachmesophalangy V in Guatemalan rural children.Hum Biol 45:571-81, 1973.

12. ----- et al. Height, weight, and lines of arrested growth in young Guatemalanchildren. Am J Phys Anthrop 40:30-48, 1974.

13. Bradfield, R. B. et al. Predicting risk of low birth weight with maternalhair tissue. In: Abstracts of Papers, Symposia and Free Communications ofthe Xth International Congress of Nutrition. Kyoto, Japan, August 3-9, 1975.

14. Canosa, C. A. et al. The intervention approach: The Guatemala study. In:Moore, W. M., M. M. Siverberg, and M. S. Read (eds.), Nutrition, Growth, andDevelopment of North American Indian Children. Washington, D.C., U.S.Government Printing Office, DHEW Publication No. (NIH) 72-76, 1972, pp. 185-199.

15. Dayton, D. H. and R. A. Blanco. Timing of neurological development in ruralGuatemalan children. Pediatrics 53:726-36, 1974.

16. DDH/INCAP. Nutrición, crecimiento y desarrollo. Bol Of Sanit Panam 78:38-51, 1975.

17. Delgado, H. et al. Prenatal nutrition. Paper presented at Symposium onCurrent Concepts in Nutrition: Nutrition in the Life Cycle", University ofSouthern California, Los Angeles, September 29, 1973.

18. -----. Implicaciones para un servicio de atención médica, práctica yefectiva. Paper presented at IX Congreso Nacional de Pediatría on "Factoresque Afectan el Desarrollo Durante el Primer Año de Vida", Guatemala, March13-15, 1975.

19. -----. Algunas ideas con relación a la evaluación del técnico de saludrural. Paper presented at Taller Sobre Evaluación del Sistema de SaludRural, Cobán, Guatemala, April 11-13, 1975.

20. ----- et al. Nutritional status and the timing of deciduous tooth eruption.Am J Clin Nutr 28:216-24, 1975.

21. ----- et al. Efectos de la nutrición materna sobre la duración de la lactan-cia y de la amenorrea post-parto. Paper presented at 2o. Congreso Peruano deNutrición, Trujillo, Peru, July 11-17, 1975.

22. ----- et al. Programas de alimentación complementaria. Paper presented atSeminario Subregional para América Latina: Programas de Nutrición en losServicios Descentralizados de Salud en América Central, Guatemala, INCAP,November 1975.

23. ----- et al. Effect of improved nutrition on the duration of post-partumamenorrhea in moderate malnourished populations. In: Abstracts of Papers,Symposia and Free Communications of the Xth International Congress of Nutri-tion. Kyoto, Japan, August 3-9, 1975, Abstract No. 3325, p. 182.

24. ----- et al. Maternal nutrition: its effects on infant growth and develop-ment and birthspacing. Paper presented at the Harold C. Mack Symposium:Nutrition and Human Reproduction: Biochemical and Clinical Aspects, Detroit,Michigan, October 9-10, 1975. New York, Harper and Row, in press.

50

25. Engle, P. et al. The effects of malnutrition on mental development. Paperpresented at Pan American Health Organization's Seminar on Mental Retardation,Cartagena, Colombia, December 17-21, 1973.

26. ----- et al. Efecto de la desnutrición sobre el desarrollo mental. Paperpresented at the Seminario sobre Organización de Servicios para el RetrasadoMental, Publication Científica No. 293, Cartagena, Colombia, December 12-17,1973. Washington, D.C., Organización Panamericana de la Salud, 1974, pp. 73-80.

27. -----. The use of the vernacular languages in education. Language medium inearly school years for minority language groups. Paper in Applied Linquistics,Bilingual Education Series: 3. Arlington, Virginia: Center for AppliedLinguistics, 1975, 33 PP.

28. -----. Language medium in early school years for minority language groups.Rev Ed Res 45:283-325, 1975.

29. ----- et al. Effects of long-term nutritional supplementation on mentaldevelopment of rural village 2 and 3 year olds. Paper presented at theBiennial Meeting of the Society for Research in Child Development, Denver,Colorado, April 1975.

30. -----. The language debate: education in first or second language? In:P. R. Sanday (ed.), Anthropology and the Public Interest: Field Work andTheory. New York: Academic Press, 1976, in press.

31. Erdmenger, J. J. et al. Estudio en ratas del efecto de la suplementaciónproteínica de una dieta típica de una comunidad rural de Guatemala. ArchLatinoamer Nutr 22:179-90, 1972.

32. Guzmán, G. et al. Environmental factors affecting fetal and child growth.In: Pediatría XIV, Vol. 5. Buenos Aires: Editorial Medica Panamericana,1974, pp. 198-208.

33. Habicht, J. P. et al. Field measurement of breast milk consumed by sucklingchildren. In: Arroyo, P., S. S. Basta, H. Bourges, A. Chávez, M. Coronado,M. Muñoz, and S. E. Quiróz, (eds.), IX International Congress of NutritionAbstracts of Short Communications, September 1972, pp. 113.

34. ----- and R. E. Klein. The Latin American child's struggle to survive: anattempt to rank and eliminate problems in four villages of Guatemala. Paperpresented at the Institute of Latin American Affairs, Temple University,Philadelphia, April 1972.

35. ----- and -----. Possible factors affecting acceptability of fertility re-gulation methods in four Guatemalan Ladino villages. Paper presented at theWHO Task Force Symposium on Contraceptive Acceptability, Geneva, Switzerland,June 21-23, 1972.

36. ----- et al. Posibles factores que afectan la aceptación de métodos decontrol de la natalidad en cuatro comunidades ladinas de Guatemala. Paperpresented at the WHO Task Force on Contraceptive Acceptability, GenevaSwitzerland, June 21-23, 1972.

37. ----- et al. The effect on birthweight of timing of supplementation duringpregnancy. In: Arroyo, P., S. S. Basta, H. Bourges, A. Chávez, M. Coronado,M. Muñoz, and S. E. Quiróz (eds.), IX International Congress of Nutrition,Abstracts of Short Communications, September 1972, pp. 71.

51

38. - .----- The timing of the effect of supplementation feeding on growth ofrural preschoolchildren. In: Arroyo, P., S. S. Basta, H. Bourges, A. Chávez,M. Coronado, M. Muñoz, and S. E. Quir6óz(eds.), IX International Congress ofNutrition, Abstracts of Short Communications, September 1972, pp. 149-.

39. -----. The Guatemala study. In: Nutritional Supplementation and the Out-come of Pregnancy. Washington, D.C.: National Academy of Sciences, 1973,pp. 129-140.

40. ----- et al. Repercussions of lactation on nutritional status of the motherand the infant. In: Nutrition Congress, Vol. II, New York, S. Karger Basel,1973, PP. 101-08.

41. ----- et al. Protein-calorie nutrition and prenatal growth. Paper presentedat the Symposium of the United States and Japanese Mainutrition Panels ofthe U.S.-Japan Cooperative Medical Science Program, National Institute ofHealth, Bethesda, Maryland, July 24-25, 1973.

42. ----- et al. The effect of malnutrition during pregnancy on survival of thenewborn. Testimony presented at the Hearing of the Select Committee onNutrition and Human Needs, U.S. Senate, Washington, D.C., June 5, 1973.

43. ----- et al. Biochemical indices of nutrition reflecting ingestion of ahigh protein supplement in rural Guatemalan children. Am J Clin Nutr26:1046-52, 1973.

44. ----- et al. Relationships of birthweight, maternal nutrition and infantmortality. Nutr Rep Internat 7:533-46, 1973.

45. -----. Estandardización de métodos epidemiológicos cuantitativos sobre elterreno. Bol Of Sanit Panam 76:375-84, 1974.

46. -----. The effect of maternal nutrition during pregnancy on psychomotorperformance of child at six months of age. Talk presented at the Institutefor Research into Mental and Multiple Handicaps, London, England, March 15,1974.

47. -----. Human implications of animal studies in prenatal nutrition and neuro-logical development. In: Symposia of the Swedish Nutrition Foundation Xll.Uppsala: Almquist & Wiksell Informationindustri AB, 1974, pp. 111-14.

48. ----- and M. Béhar. Nutrición, planificación familiar y salud en la madrey en el niño. Talk presented for the "Seminario de Planificación Familiar",Academia Americana de Pediatría, San Salvador, El Salvador, February 24-27,1974.

49. ----- et al. Community control and quality control of medical primary carepersonnel. Paper presented at the XIV Congreso Internacional de Pediatría,Buenos Aires, Argentina, October 3-9, 1974.

50. ----- et al. Maternal nutrition, birthweight and infant mortality. In:Elliot, K. and J. Knight (eds.), Size at Birth (Ciba Foundation Symposium27). Amsterdam: Associated Scientific Publishers, Elsevier, 1974, pp. 353-77.

51. ----- et al. Height and weight standards for preschoolchildren: Are therereally ethnic differences in growth potential? Lancet 1:611-15, 1974.

52

52. ----- et al. Assessing nutritional status in a field study of mainutritionand mental development. Specificity, sensitivity and congruity of indicesof nutritional status. In: Symposia of the Swedish Nutrition Foundation XII,Uppsala: Almquist & Wiksells, 1974, pp. 35-42.

53. -----. Relation of maternal supplementary feeding during pregnancy to birth-weight and other sociobiological factors. In: Winick, M. (ed.), Nutritionand Fetal Development. New York, John Wiley & Sons, Chapter 7, 1974, pp. 127-45.

54. Himes, J. H. et al. The relationship between growth in stature and corticalbone in malnourished preschoolchildren in Guatemala. Paper presented at the43rd Annual Meeting of the American Association of Physical Anthropologists,Amherst, Mass., April 11-13, 1974.

55. ----- et al. Patrones de crecimiento del hueso compacto en niños ladinospreescolares del área rural de Guatemala. Paper presented at the XVIII Con-greso Nacional de Pediatría, Guatemala, February 11-16, 1974.

56. ----- et al. Patterns of cortical bone growth in moderately malnourishedpreschoolchildren. Hum Biol 47:337-50, 1975.

57. Hinshaw, R. et al. Environmental effects on child spacing and populationincrease in highland Guatemala. Curr Anthrop 13:216-30, 1972.

58. INCAP/DDH Publication/Working Group on Rural Medical Care: Delivery ofprimary care by medical auxiliaries: Techniques of use and analysis ofbenefits achieved in some rural villages of Guatemala. Washington, D.C.,PAHO/WHO Scientific Publication No. 278, Medical Auxiliaries, 1973.pp. 24-37.

59. Irwin, M. et al. The problem of establishing validity in cross-culturalmeasurements. Paper presented at the Conference on Issues in Cross-CulturalPsychology, New York Academy of Sciences, New York, October 1-3, 1975, inpress.

60. Kagan, J. and R. E. Klein. Cross-cultural perspectives on early development.Am Psychol 28:947-61, 1973.

61. ----- et al. Memory and meaning in two cultures. Child Develop 44:221-23,1973.

62. ----- et al. Studies in cognitive development. Paper presented at theConference on Issues in Cross-Cultural Psychology, New York Academy ofSciences, New York, October 1-3, 1975.

63. Klein, R. E. et al. Is big smart? The relation of growth to cognition.J Hlth Soc Behav 13:219-25, 1972.

64. ----- et al. Empirical findings with methodological implications in thestudy of malnutrition and mental development. In: Nutrition, the NervousSystem, and Behavior. Washington, D.C., PAHO, Scientific PublicationNo. 251, 1972, pp. 43-47.

65. ----- et al. Cross-cultural evaluation of human intelligence. In: Lipids,Malnutrition, and the Developing Brain. Amsterdam, Associated ScientificPublishers, 1972, pp. 249-65.

53

66. ----- and C. Yarbrough. Some considerations in the interpretation of psycho-logical data as they relate to the effects of malnutrition. Arch LatinoamerNutr 22:41-48, 1972.

67. ----- et al. Psychological test performance and indigenous conceptions ofintelligence. J Psychol84:219-22, 1973.

68. ----- et al. Some methodological problems in field studies of nutrition andintelligence. In: Kallen, D. J. (ed.), Nutrition, Development, and SocialBehavior. Washington, D.C., U.S. Government Printing Office, 1973. DHEWPublication No. (NIH) 73-242, pp. 61-75.

69. ------et al. Relationship of infant/caretaker interaction, social class andnutritional status to developmental test performance among Guatemalan infants.In: Burg Wartenstein Symposium No. 57 on Cultural and Social Influences inInfancy and Early Childhood, Austria, June 18-26, 1973. New York, Wenner-Gren Foundation for Anthropological Research, 1974, pp. 1-24.

70. ----- et al. Correlations of mild to moderate protein-calorie malnutritionamong rural Guatemalan infants and preschoolchildren. In: Symposia of theSwedish Nutrition Foundation XII. Uppsala: Almquist & Wiksell Information-industri AB, 1974, pp. 168-80.

71. ----- and A. A. Adinolfi. Measurement of the behavioral correlates of mal-nutrition. In: Prescott, J., M. S. Read, and D. F. Coursin (eds.), BrainFunction and Mainutrition: Neurophysiological Methods of Assessment. NewYork, John Wiley & Sons, Inc., 1975, pp. 73-82.

72. ----- et al. Effect of maternal nutrition on fetal growth and infant develop-ment. Paper presented at the Fourteenth Meeting of the PAHO Advisory Com-mittee on Medical Research, Washington, D.C., July 7-10, 1975.

73. ----- et al. Malnutrition and human behavior: A backward glance at anongoing longitudinal study. Paper presented at the Conference on Malnutri-tion and Behavior, Cornell University, Ithaca, New York, November 6-8, 1975.

74. ----- et al. Malnutrition, poverty, and the development of mental abilitiesin rural eastern Guatemala. Paper presented at the Conference on EcologicalFactors in Human Development, The University of Surrey, Guildford, England,July 13-17, 1975.

75. ----- et al. Malnutrition and mental development in rural Guatemala: Anapplied cross-cultural research study. In: Warren, N. (ed.), Advances inCross Cultural Psychology. New York, Academic Press, 1975, in press.

76. ----- et al. On malnutrition and mental development: Some preliminaryfindings. In: Chávez, A., H. Bourges, and S. Basta (eds.), Nutrition,Vol. 2. Switzerland, S. Karger Basel, 1975, pp. 315-21.

77. Lasky, R. E. The ability of six-year-olds, eight-year-olds, and adults toabstract visual patterns. Child Develop 45:626-32, 1974.

78. ----- et al. Age and sex discriminations in five and six month old infants.J Psychol 88:317-24, 1974.

79. ----- et al. Birthweight and psychomotor performance in rural Guatemala.Am J Dis Child 129:566-70, 1975.

54

80. ----- et al. VOT discrimination by four to six and a half month old infantsfrom Spanish environments. J Exp Child Psychol, 1975, in press.

81. Lechtig, A. et al. Influencia de la nutrición materna sobre el crecimientofetal en poblaciones rurales de Guatemala. II. Suplementación alimentaria.Arch Latinoamer Nutr 22:117-31.

82. ----- et al. Influencia de la nutrición materna sobre el crecimiento fetalen poblaciones rurales de Guatemala. 1. Aspectos dietéticos. Arch Latino-amer Nutr 22:101-15, 1972.

83. ----- et al. Fetal growth in rural populations of Guatemala. In: Proceed-ings of the Western Hemisphere Nutrition Congress ll, 1971. Mount Kisco,New York, Futura Publishing Company, 1972, pp. 342-43.

84. ----- et al. Morbilidad materna y crecimiento fetal en poblaciones ruralesde Guatemala. Arch Latinoamer Nutr 2-2:243-53.

85. ----- et al. Influencia de las características maternas sobre el crecimientofetal en poblaciones rurales de Guatemala. Arch Latinoamer Nutr 22:255-65.

86. ----- and L. J. Mata. Levels of C3 in newborns and mothers from differentecosystems. Rev Latinoamer Microbiol 14:73-76, 1972.

87. ------ and -----. IgM and C3 in serum of Peruvian mothers and cord blood oftheir infants. (Letter to the Editor). Arch Latinoamer Nutr 22:309-13, 1972.

88. ----- et al. Niveles de IgG, IgA, IgM y C3 en niños indígenas de Guatemaladurante los primeros seis meses de edad. Rev Latinoamer Microbiol 14:65-71,1972.

89. ----- et al. Influence of maternal nutrition on the characteristics of theplacenta and fetal growth. Paper presented at the Symposium on Effects ofInadequate Nutrition on Human Development from the Continental Meeting ofScience and Man, México, D.F., July 1973.

90. ----- et al. ll¡. Influencia de la nutrición materna sobre el crecimientoy desarrollo del niño hasta los 15 meses de edad. Paper presented at theX Congreso Centroamericano de Gineco-Obstetricia, Symposium on: "Factoresambientales que afectan la salud de la madre y del niño en Centro Américay sus implicaciones para la formulación de un programa materno-infantil".Guatemala, November 17-December 2, 1974.

91. ----- et al. Efectos de la nutrición sobre la lactancia materna y susimplicaciones de salud pública. Paper presented at the Mesa Redondaon "Lactancia Materna en Guatemala" of the XXV Congreso Nacional de Medi-cina, Guatemala, November 18-22, 1974.

92. ----- et al. A simple assessment of the risk of low birthweight to selectwomen for nutritional intervention. Paper presented at the Western Hemi-sphere Nutrition Congress, Research Forum on Applied Nutrition Programs,Bal Harbour, Miami Beach, August 19-22, 1974.

93. ----- et al. Indicadores simples para seleccionar madres con alto riesgode dar a luz niños con bajo peso al nacer. Paper presented at the MesaRedonda "Alto Riesgo Obstétrico", XXV Congreso Nacional de Medicina, Guate-mala, November 18-22, 1974.

55

94. ----- et al. Maternal nutrition and birthweight. In: Pediatría XIV, Vol. 5.Buenos Aires: Editorial Médica Panamericana, 1974, pp. 29-39.

95. ----- et al. Levels of immunoglobulin M (IgM) in cord blood of Latin Ameri-can newborns of low socioeconomic status. Ecol Food Nutr 3:171-78, 1974.

96. ----- Efectos de la nutrición materna durante el embarazo y la lactancia.Paper presented at the IX Congreso Nacional de Pediatría on "Factores queAfectan el Desarrollo Durante el Primer Año de Vida, Guatemala, March 13-15,1975.

97. ----- et al. Maternal nutrition and fetal growth in developing societies.Socioeconomic factors. Am J Dis Child 129:434-37, 1975.

98. ----- et al. Maternal nutrition and fetal growth in developing countries.Am J Dis Child 129:553-56, 1975.

99. ----- et al. Effect of improved nutrition during pregnancy and lactation ondevelopmental retardation and infant mortality. In: White, P. L. and N.Selvey (eds.), Proceedings of the Western Hemisphere Nutrition Congress IV,1974. Acton, Mass., Publishing Sciences Group Inc., 1975, pp. 117-25.

100. ----- et al. Effect of improved nutrition since conception on growthretardation up to three years of age and on infant mortality. In: Ab-stracts of Papers, Symposia and Free Communications of the Xth InternationalCongress of Nutrition. Kyoto, Japan, August 3-9, 1975, Abstract No. 342,PP. 34.

i01. ----- et al. Relación entre aspectos socioeconómicos y peso al nacer.Centro Latinoamericano de Perinatología y Desarrollo Humano (CLAP). Monte-video, Uruguay, 1975, in press.

102. ----- et al. Factores socioeconómicos y peso al nacer. Paper presentedat the Congreso Centroamericano de Sociología, Costa Rica, September 1975.

103. ----- et al. A simple assessment of the risk of low birthweight to selectwomen for nutritional intervention. Am J Obst Gynecol, 1975, in press.

104. ----- et al. Field indicators of high risk of infant death based on birthinformation. J Trop Pediat, 1975, in press.

105. ----- et al. Influencia de la nutrición materna sobre el peso al nacer.Centro Latinoamericano de Perinatología y Desarrollo Humano (CLAP), Monte-video, Uruguay, 1975, in press.

106. ----- et al. Effect of food supplementation during pregnancy on birth-weight. Pediatrics 56:508-20, 1975.

107. ----- et al. Influence of food supplementation during pregnancy on birth-weight in rural populations of Guatemala. In: Chávez, A., H. Bourges, andS. Basta (eds.), Nutrition, Vol. 2. Switzerland, S. Karger Basel, 1975,pp. 44-52.

108. ----- et al. Influence of maternal nutrition on birthweight. Am J ClinNutr, 1975, in press.

109. ----- et al. Effect of moderate maternal malnutrition on the placenta.Am J Obst Gynecol 123:191-201, 1975.

56

110. ----- et al. Usefulness of the urea/creatinine (U/C) ratio as indicatorof protein intake under field conditions. In: Abstracts of Papers, Symposiaand Free Communications of the Xth International Congress of Nutrition.Kyoto, Japan, August 3-9, 1975. Abstract No. 3407, p. 192.

111. Lester, B. M. The effect of malnutrition on autonomic reactivity to anauditory signal: some preliminary findings. (Abstract). Paper presentedat the Annual Meeting of the Society for Research in Child Development,Philadelphia, Pa., March 20-April 1, 1973.

112. ----- and R. E. Klein. The effect of stimulus familiarity on the conserva-tion performance of rural Guatemalan children. J Soc Psychol 90:197-205,1973.

113. ----- et al. Separation protest in Guatemalan infants: cross culturaland cognitive findings. Develop Psychol 10:79-85, 1974.

114. -----. The consequences of infantile malnutrition. In: Fitzgerald, H. E.and J. P. McKinney (eds.), Developmental Psychology: Studies in HumanDevelopment, Illinois, Dorsey Press, 1975, in press.

115. Malina, R. M. et al. Skinfold thicknesses at seven sites in rural GuatemalanLadino children,birth through seven years of age. Hum Biol 46:453-69, 1974.

116. ----- et al. Head and chest circumferences in rural Guatemalan Ladino chil-dren, birth to seven years of age. Am J Clin Nutr 28:1061-70, 1975.

117. Marshall, R. et al. Effectiveness of measles vaccine given simultaneouslywith DPT. J Trop Pediat 20:126-29, 1974.

118. Martorell, R. La natropometría como indicador del estado nutricional. Paperpresented at the XV Congreso Médico Centroamericano, San José, Costa Rica,December 4-8, 1973.

119. -----. Illness and incremental growth in young Guatemalan children. Doc-toral Dissertation, University of Washington, 1973.

120. ----- et al. Diarrheal diseases and incremental growth in Guatemalan chil-dren. Paper presented at the 42nd Annual Meetinq of the American Associationof Physical Anthropologv (AAPA), Dallas, Texas, April 1973.

121. ----- et al. Morbidity and physical growth in children from rural Guatemala.In: Pediatría, XIV, Vol. 5. Buenos Aires, Editorial Médica Panamericana,1974, PP. 177-86.

122. ----- et al. Normas antropométricas de crecimiento: Centroamericanas o depaíses desrrollados. Paper presented at the XVII Congreso Nacional dePediatría, Guatemala, February 9-16, 1974.

123. ----- et al. Diarrea y retardo en crecimiento físico. Paper presented atthe XLI Congreso Internacional de Americanistas, México, D.F., September 2-7,1974, in press.

124. -----. Morbilidad y crecimiento durante el destete. Paper presented at theIX Congreso Nacional de Pediatría on "Factores que Afectan el DesarrolloDurante el Primer Año de Vida", Guatemala, March 13-15, 1975.

57

125. ----- et al. The identification and evaluation of measurement variability inthe anthropometry of preschoolchildren. Am J Phys Anthropol, 1975, in press.

126. ----- et al. Underreporting in forthnightly recall morbidity surveys. J TropPediat, 1975, in press.

127. ----- et al. Acute morbidity and physical growth in rural Guatemalan chil-dren. Am J Dis Child, 1975, in press.

128. ----- et al. Normas antropométricas de crecimiento físico para países envías de desarrollo: Nacionales o internacionales. Bol Of Sanit Panam, 1975,in press.

129. ----- et al. Relación entre tenencia y acceso a la tierra y estado nutri-cional de niños pequeños. Paper presented at the Seminar on EstructuraSocial, Rural y Análisis Regional en Centro América y Panama, Costa Rica,September 22-27, 1975.

130. ----- et al. Nutrition and physical growth: Results from a feeding experi-ment in Guatemala. In: Abstracts of Papers, Symposia and Free Communicationsof the Xth International Congress of Nutrition. Kyoto, Japan, August 3-9,1975, Abstract No. 2507, pp. 154.

131. ----- et al. Upper arm anthropometric indicators of nutritional status.Am J Clin Nutr, 1975, in press.

132. ----- et al. Diarrheal diseases and growth retardation in preschool Guate-malan children. Am J Phys Anthropol, 1975, in press.

133. ----- et al. The head circumference/chest circumference ratio in mild-to-moderate protein-calorie malnutrition. J Trop Pediat, 1975, in press.

134. MejTa-Pirvaral, V. Características económicas y socioculturales de cuatroaldeas ladinas de Guatemala. Guatemala Indigena (Monograph), VII, No. 3,1972.

135. Mirón de, E. M. C. et al. IV. Indicadores sencillos de riesgo de bajo pesoal nacer en el área urbana de Guatemala. Paper presented at the X CongresoCentroamericano de Gineco-Obstetricia,Symposium on "Factores ambientales queafectan la salud de la madre y del niño en Centro América y sus implicacio-nes para la formulación de un programa materno-infantil". Guatemala,November 27-December 2, 1974.

136. Morrison, F. J. et al. Cognitive style in rural preschool Guatemalan chil-dren: A serendipitous finding. J Genet Psychol, 1975, in press.

137. Nerlove, S. B. et al. Natural indicators of cognitive development: anobservational study of rural Guatemalan children. Ethos 2:265-95, 1974.

138. ----- et al. Dimensions of "Listura" ("Smartness"): Community judgmentsof rural Guatemalan children. Paper presented at the Biennial Meeting ofthe Society for Research in Child Development. Denver, Colorado, April 1975.

139. Read, M. S. et al. Maternal malnutrition, birthweight, and child develop-ment. In: Nutrition, Growth, and Development. Mod Probl Pediat, Vol. 14,1975. Karger Basel, pp. 203-15.

140. Romney, A. K. et al. A normalization for correcting biased response data.Soc Sci Res 2:307-20, 1973.

58

141. Salomón, J. B. et al. Efectos de la nutrición e infecciones sobre el desar-rollo óseo de niños en una área rural de Guatemala. Arch Latinoamer Nutr22:417-49, 1972.

142. Sellers, M. J. et al. Developmental determinants of attention. A cross-cultural replication. Develop Psychol 6:185, 1972.

143. Teller, C. et al. Técnicas de recolleción de datos socioeconómicos: Con-sideraciones sobre la validéz y la confiabilidad de datos especialmentedifíciles de obtener en un área rural de Guatemala. Paper presented at theConference on "Recolección de Datos Socioeconómicos en Areas Rurales dePaíses en Desarrollo", México, D.F., November 17-24, 1974.

144. -----. Physical health status and health care utilization in the Texasborderlands. Paper presented at the Conference on the Contemporary Dilem-mas of the Mexican-United States Border, San Antonio, Texas, April 14-18,1975.

145. ----- et al. Effect of declines in infant and child mortality on fertilityand birthspacing: Preliminary results from retrospective and prospectivedata in four Guatemalan villages. Paper presented at the Latin AmericanSession of the Seminar on "Infant Mortality in Relation to the Level ofFertility", organized by the Committee of International Coordination ofNational Research Demography (CICRED), New Delhi, India, January 20-27,1975.

146. ----- et al. Patrones de emigración y su impacto sobre estado nutricional.Un análisis preliminar de familias en cuatro comunidades rurales de Guate-mala. Paper presented at the Seminar on "Estructura Social Rural y Aná-lisis Regional en Centro América y Panamá", Asociación Centroamericana deSociología, La Catalina, Heredia, Costa Rica, September 22-27, 1975.

147. Vuori, L. and P. Engle. Preschool tests of mental abilities in two LatinAmerican cultures: Guatemala and Colombia. Paper presented at the XVInteramerican Congress of Psychology, Bogotá, Colombia, December 14-19,1974.

148. Yarbrough, C. et al. Determining the biological age of the preschoolchildfrom a hand-wrist radiograph. Invest Radiol 8:233-43, 1973.

149. ----- et al. Influence of gestational nutrition on birthweight and otheroutcomes of pregnancy. Paper presented at the 1973 American Public HealthAssociation Meeting, San Francisco, California, November 1973.

150. ----- et al. Testing for mental development. In: Symposia of the SwedishNutrition Foundation, XII. Uppsala: Almquist and Wiksells, 1974, pp. 7-12.

151. ----- et al. Anthropometry as an index of nutritional status. In: Roche,A. F. and F. Falkner (eds.), Nutrition and Malnutrition. New York, PlenumPress, 1974, pp. 15-26.

152. ----- et al. Length and weight in rural Guatemalan Ladino children, birthto seven years of age. Am J Phys Anthropol 42:439-48, 1975.

59

Dr. Robert E. Klein, Division of Human Development, Instituteof Nutrition of Central America and Panama (INCAP), GuatemalaCity, Guatemala.

U.S. National Institute of Child Health and Human Development.

1965-1976.

60

OTHER DATA

Grantee:

Funded by:

Timetable:

CROSS CULTURAL STUDIES IN COGNITIVE DEVELOPMENT

PROBLEM

Cognitive development in humans is affected by many factors. Although generalpatterns of development may be found in all cultures, there are also striking culturaldifferences among children in various parts of the world. The principal objective ofthis investigation is the study of the relationship between cultural factors and cog-nitive development among children in diverse cultural settings. Although culture isconsidered a primary factor in this research, problems of health and nutrition arealso of central concern.

METHOD

Infants and children are being studied in two Indian villages of Guatemala andin the United States. A battery of tests was developed to assess memory and conceptualfunctions in preschool and school aged children, as well as in infants. Detailed be-havioral observations and ethnographic studies were also carried out and will play animportant role in the evaluation of cognitive development among these infants andchildren.

RESULTS TO DATE

Levels of cultural isolation were generally correlated with rates and patternsof cognitive development. Children in the United States showed the greatest improve-ment in performance between 6 and 9 years of age, most children reaching the ceilingof tests by age 10. In relatively modern and accessible Indian villages in Guatemala,the performance of children was 2 years behind that of United States children, withrapid cognitive growth between the ages of 8 and 10. On absolute levels of performance,children from more isolated and.traditional Indian villages were yet another 2-3 yearsbehind. Family characteristics and parental occupation were related to cognitive testperformance in all villages.

SIGNIFICANCE

This investigation is closely related to the longitudinal study of mental develop-ment and malnutrition, in that it provides information on patterns of cognitive develop-ment and their association to culture. This will help the design of educational programsbeing planned as part of a project of nutritional intervention aimed at these samevillages.

PUBLICATIONS

1. Irwin, M. et al. The problem of establishing validity in cross-culturalmeasurements. Ann N Y Acad Sc, in press.

61

2. Kagan, J. et al. A study in cognitive development. Unpublished manuscript.

3. Klein, R. E. et al. Cognitive test performance and indigenous conceptionsof intelligence. Unpublished manuscript.

OTHER DATA

Drs. Robert E. Klein and Jerome Kagan, Division of HumanDevelopment, Institute of Nutrition of Central America andPanama (INCAP), Guatemala City, Guatemala.

Grant Foundation.

1973-1976.

62

Grantee:

Funded by:

Timetable:

EVALUATION OF THE EFFECTS OF NUTRITION EDUCATIONON NUTRITIONAL STATUS

PROBLEM

Nutrition education has always been a part of programs to combat malnutrition.Its specific contribution to the betterment of nutritional status has been difficult toassess, however, because it has usually been integrated with other inputs such as foodaid or specially recruited personnel.

Not only is it important to try to define the role of nutrition education per se,but it is equally, if not more important, to make models of nutrition programs that willbe easily replicable in other regions where childhood malnutrition occurs.

Consequently, the need exists for devising a program in which the dominant inputis nutrition education provided as economically as possible, and having as its main con-tributors people indigenous to the region.

METHOD

A rural area, the Lambs River district of the Parish of Westmoreland, WesternJamaica, was selected as the study area, and a similar area, Darliston, as a control.

Socioeconomic and relevant anthropometric data were collected from both areasduring the first 6 months of the project. Such data have established the basic similari-ties between the two areas.

A program of nutrition education for the study area was planned for implementationby technically trained government personnel working in the field of public health, agri-cultural and home economics extension, primary school teaching, and community develop-ment. This was to be complemented by the use of indigenous village-level volunteers inthe role of home-visitors and "educators". However, all the educational inputs werenot put into effect as planned, because of the shortage or absence of supporting govern-ment technical officers, e.g., nurse, agricultural or community development officer.None of these inputs were deliberately made into the control area.

Anthropometric data on the under-5-year-old children; nutrition knowledge andpractices, especially in relation to the production and use of local foods; and breast-feeding practices were used as the objective parameters of change. Because of the stronghuman component of the project, some degree of subjective assessment was thought to be anecessary complement to the objective findings.

RESULTS TO DATE

The results showed that there was no significant change in the nutritional statusof children under-5 in either the study, or control area.

However, the following specific intermediate objectives were achieved:

63

-Community-wide awareness of a nutrition program and what it was doing.-Cultivation of more kitchen gardens.-Breast-feeding more widely practiced and for greater duration.-Successful introduction of multimixes in infant feeding.-Greater "nutrition orientation" in the choice of milk purchased.

SIGNIFICANCE

The project has demonstrated that indigenous nutrition education at the ruralcommunity level is feasible and can be effective but needs to be supported by technicalresource personnel for enhanced and sustained effect.

It also demonstrates that in situations where basic health, education, andsocial services are lacking, a low-cost volunteer service can be a useful agent ofchange in nutrition behavior.

Of no less significance is the realization that in the very poorest circumstancesa community is still capable of helping itself with appropriate guidance and stimula-tion, even though these are the very circumstances in which inputs from official sourcestend to be least dependable and least readily available.

It is also evident that nutrition education, as a single major input in circum-stances of extreme poverty, and unaccompanied by measures that will effectively leadto greater food availability, is unlikely in the short, medium, or long term to gener-ate convincingly any enduring change that will lead to significant improvement in nutri-tional status.

PUBLICATIONS

The report on this project has been published as a CFNI monograph under thetitle "Programme Planning and Evaluation in Community Nutrition Education".

OTHER DATA

Grantee: Caribbean Food and Nutrition Institute (CFNI)Kingston, Jamaica.

Funded by: U. K. Freedom from Hunger Campaign.


64

PROGRAM OF SOCIAL STUDIES

PROBLEM

Mainutrition is an exceedingly complex problem and is determined by a varietyof social, cultural, and economic factors. To date the social sciences, as a formaldiscipline, have participated relatively little in basic and applied research onproblems of malnutrition and on programs designed to ameliorate nutritional problemsin underdeveloped countries. The purpose of the activities carried out under the grantfrom the Ford Foundation is to develop the social sciences at INCAP in service of basicresearch, teaching, and intervention programs as they relate to nutritional problemsin the area.

METHOD

Three general areas of activities have characterized work to date. These includeanalyses of data currently available at INCAP and elsewhere, directed at examining so-cial, structural, and economic factors as they relate to the nutritional status of ruralpopulations in Central America. In addition, basic work has been carried out on thedevelopment of research methods and techniques and the design of nutritional inter-ventions and their evaluation. Third is the design and implementation of a generalmodel for the functional classification of nutritional status of populations in under-developed countries, on the basis of which food and nutrition programs can be rationallydeveloped.

RESULTS TO DATE

Because research under this grant is not structured around a specific hypothesis,results are necessarily more general. Basic work has been completed on the design andtesting of simple indicators of nutritional status and their application under fieldconditions, in service of a rapid and precise technique for generating functional clas-sifications of nutritional status. Analysis of land tenure patterns, family income,agricultural production, and work and migratory patterns have been conducted and theseanalyses form a basis for the design of interventions, which have as their objective,increases in family income and food availability.

A conference on research techniques and methodology was held in Guatemala andwas attended by social scientists from Mexico, Central America, and Panama. The con-ference resulted in the pooling of ideas, techniques, and methodologies to be usedtoward the general goals of this grant.

SIGNIFICANCE

All the activities under this grant relate directly or indirectly to the develop-ment of theory and methodology for the application of the social sciences to nutritionalproblems in developing countries.

65

PUBLICATIONS

1. Delgado, H. et al. A design for socioeconomic intervention programs inrural communities: health delivery as entry point and organizationalframework. Paper presented at the Symposium on The Biological andCultural Sources of Variability in Human Nutrition, Berkeley, California,December 3-5, 1975.

2. Martorell, R. et al. Relación entre tenencia y acceso a la tierra yestado nutricional de niños pequeños. Paper presented at the Seminariosobre Estructura Social-Rural, September 1975, Costa Rica.

3. Rawson, I. and V. Valverde. The etiology of malnutrition among preschoolchildren in rural Costa Rica. J Trop Pediat Environ ChId Hlth, in press.

4. Valverde, V. and 1. Rawson. Dietetic and anthropometric differencesbetween rural and urban children in a provincial region of Costa Rica.Ecol Food Nutr, in press.

5. ----- et al. Analisis del aporte calórico-proteínico de las dietas depoblaciones de bajo nivel socioecon6mico en Centroamérica. Existe unproblema de proteínas? Arch Latinoam Nutr, in press.

6. ----- et al. La deficiencia calórica en preescolares del área rural deCosta Rica. Arch Latinoam Nutr, in press.

7. ----- et al. Family land availability and nutritional status of youngGuatemalan children. Ecol Food Nutr, in press.

OTHER DATA

Grantee: Institute of Nutrition of Central America and Panama (INCAP),Guatemala City, Guatemala.

Funded by: Ford Foundation.


66

EPIDEMIOLOGIC AND METABOLIC STUDIES OF CHOLELITHIASIS IN CHILE

PROBLEM

Cholesterol gallstones are unusually frequent in the Chilean population, andthe complications of cholelithiasis constitute a major cause of morbidity and mortalityin this country.

The purposes of this investigation are (1) to study the incidence and naturalhistory of the disease, and the dietary habits of patients with cholelithiasis, and(2) to investigate in humans and experimental animals some of the metabolic disturbancesthat could play a role in cholesterol gallstone formation.

METHOD

(1) Epidemiologic studies are being performed in a sample of urban population.Subjects are submitted to cholecystography and clinical examination. Alimentary trendsare being studied in patients with cholelithiasis and their normal controls paired byage and sex.

(2) Metabolic studies are conducted on two groups of patients examined understandardized conditions: (a) young women, under 30 years of age, with gallstones anda functioning gallbladder and (b) women over 50 years of age with normal cholecysto-graphy.

In these patients, the composition of fasting gallbladder bile, pool and kineticsof cholic acid and total bile acid pool size, and the hourly secretion of biliary lipidsare analyzed.

(3) In addition, the biliary excretion of cholesterol, the dynamics of theenterohepatic circulation of bile salts, and the hepatic synthesis of bile acids arebeing studied in rats under different experimental conditions.

RESULTS TO DATE

I. Epidemiologic studies

Cholecystography was performed in 415 patients of the sample population. Ofthem, 239 were under 20 years of age. The frequency of cholelithiasis in this groupof young people was 5 percent.

In 176 individuals over 20 years of age, galIstones were detected in 54 percentof the women and in 20 percent of the men. About 17 percent of this adult populationhad already been cholecystectomized.

The natural history of the disease and the alimentary trends of the subjectswith and without galistones are being studied by blind methods, and results are notyet available.

67

II. Metabolic studies

(a) The composition of fasting gallbladder bile was studied in 25 women withgallstones and in 14 women with normal gallbladders.

(1) The relative percentage of biliary cholesterol was similar inboth groups.

(2) The relative percentage of biliary phospholipids was significantlylower in patients with cholelithiasis.

(3) The mean Cholesterol Saturation Index (CSI) was abnormally high inthe fasting bile of both groups: 120 ± 9 s.e. in patients withgallstones vs 113 ± 11.5 s.e. in patients with normal biliary tract.Moreover, there was a considerable overlapping for the individualvalues of both groups. Seven of the 25 women with cholelithiasishad an undersaturated bile. On the other hand, 8 patients withnormal cholecystography had a fasting bile oversaturated withcholesterol.

(4) The chromatographic analysis of the bile acids indicated thatdeoxicholic acid constituted 14.8 percent ± 2.2 s.e. of them innormal patients, and only 9.5 percent ± 1.4 s.e. in the groupof young women with gallstones (p less than 0.05). Both valueswere clearly lower than those reported from the bile of patientsor normal controls from Europe or the United States (18-30 percent).

(b) The pool and kinetics of the bile acids was studied in 7 patients withcholesterol gallstones and in 5 patients with normal gallbladders.

(1) The mean total bile acid pool was 2.3 g ± .3 s.e. in patients withcholelithiasis and 2.4 g ± .4 s.e. in the normal subjects. Thesevalues were not significantly different, and were similar to thosereported in normal controls from other western countries.

(2) Cholic acid pool size was similar in both groups, but cholic acidsynthesis was significantly different: 267 mg/day ± 36 s.e. inwomen with normal gallbladders vs 160 mg/day ± 22 s.e. in patientswith cholelithiasis (p less than 0.02).

(3) Hourly secretion of biliary lipids. The method to be used (Gastro-enterology 62:1200, 1972) has been settled but only four patientshave completed the study.

SIGNIFICANCE

Cholesterol galistones constitute a serious health problem in Chile. A betterunderstanding of the epidemiology of cholelithiasis and of the metabolic disturbancesthat explain the precipitation of biliary cholesterol is clearly needed. The searchand application of rational methods for the medical dissolution of gallstones andfor the prevention of the disease must be founded on this basic information.

The results obtained thus far provide valuable information on the frequencyof cholelithiasis. The population already included in the epidemiologic study con-stitutes a sizable sample for future work on the incidence and natural history of the

68

disease. Simultaneously, we are obtaining some insights into the metabolic disturbancesof cholesterol and bile acids that may contribute to the formation of gallstones.

PUBLICATIONS

1. Valdivieso, V. and M. C. Severin. Efecto de diferentes acidos biliaressobre la secreción de lipidos biliares en la rata. Rev Med Chile103:657-59, 1975.

OTHER DATA

Dr. Vicente Valdivieso, Departamento de Gastroenterología,Escuela de Medicina, Universidad Católica de Chile,Santiago, Chile.

Pan American Health Organization.

1974-1976.

69

Grantee:

Funded by:

Timetable:

DENTAL CARIES RESEARCH IN TWO COLOMBIAN COMMUNITIES

PROBLEM

The prevalence of dental caries is universal and has reached such proportionsthat in some areas of Latin America up to 99 percent of schoolchildren suffer from thisdisease. In recent years convincing proof has been adduced to show that caries is aninfectious disease and that certain strains of streptococcus have a causative role. Inthis context, the Department of Dental Science of the Royal College of Surgeons ofEngland has conducted investigations into the components of the oral flora associatedwith active caries and with the absence of caries. This work has been carried out onnonhuman primates in which caries, indistinguishable from that seen in human children,has been produced by infecting the animals with cariogenic streptococci and maintainingthem on a high sugár diet.

In Colombia a semiisolated community has been identified by scientists from theUniversity of Antioquia in which the prevalence of caries is totally at variancewith what would be expected by those who regard sugar consumption as the sole cause ofcaries. In this community sugar (in the form of panela) is a staple part of the diet.The hypothesis to be tested is that the relative freedom from caries in this communityis attributable to the absence of an infective agent capable of initiating tooth decay;or, alternatively, that this lower level of caries may result from the presence of afactor that neutralizes the carious attack resulting from the interactions of micro-organisms and sugar on the tooth surface by either enhancing the resistance of theenamel or modifying the cariogenic challenge.

METHOD

Two similar communities in Colombia were studied. In Heliconia there was a lowcaries prevalence and in Don Matias, which was otherwise comparable, the caries pre-valence was high. Two hundred children in each community were examined dentally anddivided into 7 to 10- and 10 to 14-year-old age groups. Samples of dental plaque andof saliva were obtained from each analysis. Follow-up samples have also been takenfor comparison with initial findings. Analyses of water from both communities arebeing undertaken.

RESULTS TO DATE

In the community with low caries, dental plaque was present in relatively smallamounts and, in some instances, it was difficult to gather sufficient quantity foranalysis. By contrast, plaque from the community with high caries was abundant andmore cohesive; it was also more difficult to suspend in bacteriologic media. Sub-stantially more Streptococcus mutans was found in plaque from the high caries townthan in that from Heliconia. Serotype C was prevalent in both communities, althoughthere appeared to be a relative abundance of the rarer serotype F. Plaque from eachtown could be distinguished by means of electron histochemistry. More intracellularpolysaccharides, and extracellular polysaccharides were present in plaque from thehigh caries town. Thus far significant differences have not been found in the com-position of saliva from the two towns. The capacity of plaque to form acid wasmeasured and the pH value reached after minutes' exposure to a sucrose solution was

70

lower in samples from the community exhibiting higher prevalence of caries. Further-more, the pH value of sugar solutions prepared in Heliconia water invariably remainedhigher than those prepared in Don Matias water following addition of plaque from eithersource. An unusual difference was observed in the physical nature of polysaccharideformed by a standard strain of S. mutans when grown on sucrose-containing media madeup with water from Heliconia, Don Matias, and London. When the culture medium was madeup with Heliconia water, the degree of adherence of the polysaccharide appeared to besubstantially less than when the media were prepared with water from the other twosources. This suggests that elements are present in Heliconia water that influencethe formation of polysaccharide from sucrose by S. mutans. Thus far it has been ascer-tained that Heliconia water is unusually hard and possesses an unusually high bufferingcapacity. It is particularly rich in magnesium and calcium. It has also been observedthat water from Heliconia can prevent the development of caries in vitro. It does notseem likely that these features alone could be completely responsible for the disparityin levels of caries. More information is required and is in the process of beingsought.

SIGNIFICANCE

The existence of a community enjoying relative freedom from dental caries isunremarkable, even though the prevalence of this disease in most urban populations hasapproached saturation. What is extremely unusual in the situation under investigation,is that the degree of freedom from caries occurs in a population whose diet is richin readily fermentable carbohydrate. Whatever factor is operating to reduce the cariesincidence in Heliconia is able to do so despite a dietary regimen that would normallybe classified as highly cariogenic. Furthermore, it is apparent that this populationis exposed to a cariostatic agent that is both safe and effective.

PUBLICATIONS

1. Bowen, W. H. et al. The microbiology and biochemistry of plaque anddrinking water from two communities with contrasting levels of cariesin Colombia, S.A. 21st Congress, European Organization for CariesResearch (ORCA), London 1974 (Abstract).

OTHER DATA

Grantee: Dr. George M. Gillespie, Principal Investigator, Departmentof Dental Health, Division of Family Health, Pan AmericanHealth Organization, Washington, D.C.; Dr. Bertram Cohen,Royal College of Surgeons of England, London, England; andDr. William H. Bowen, National Caries Program, NationalInstitute of Dental Research, Bethesda, Maryland, U.S.A.

Funded by: International Sugar Research Foundation and National Institutesof Health.


71

EPIDEMIOLOGY OF ALCOHOLISM IN LATIN AMERICA

PROBLEM

Alcoholism and alcohol abuse are thought to be serious public health problemsin many Latin American countries. However, the extent and distribution of these pro-blems in Latin America has not yet been adequately documented.

The present project seeks to: (1) conduct an epidemiologic study of drinkingpatterns and attitudes in six Latin American cities, using survey techniques; and(2) develop a research center in Costa Rica to carry out in-depth studies of a sociol-ogic, anthropologic, and clinical nature on alcoholism, and to devise and test preven-tion strategies.

METHOD

The epidemiologic study is being conducted in Sao Paulo, Santo Domingo, Caracas,Bogota, San José, and Lima, through the use of surveys of stratified population samplesof 1,200-1,500 adults in each city. Respondents are queried about their drinking pat-terns and their attitudes and beliefs about drinking and alcoholics.

The Center for Studies on Alcoholism (CESA), established in 1973 in Costa Ricawith the support of PAHO and the Government of Costa Rica, has been conducting 10-12research and prevention projects over the past 3 years, including a study of the alco-holic patient in the treatment system; research on contraband liquor production; a5-year follow-up study of heavy drinkers; a survey of drinking patterns in San José;and a case study of a small community's response to alcoholism.

RESULTS TO DATE

From 1972-1975, several national and international meetings on traffic accidentswere held in Latin America.

Several CESA research projects were completed and are currently being revisedfor publication. One of them, on education and training, contains data on the currentteaching of alcohol abuse in the public schools in Costa Rica, and a large-scale pro-gram and materials for alcohol education of grade-school children. This program willbe pilot tested from 1976-1979.

Also at CESA, an alcoholism library was established to serve Costa Rica andneighboring countries. CESA has also begun an information service and publication ofa newsletter on alcoholism for distribution to health professionals throughout LatinAmerica.

Results of the epidemiologic study are now being received and tabulated.

72

SIGNIFICANCE

The research will provide comparative prevalence and other important data forsix Latin American cities. It has also enabled PAHO, in collaboration with the CostaRican Government, to do further in-depth studies on the extent and nature of alcoholproblems and to provide information services to Latin American professionals working inthis field.

PUBLICATIONS

1. CESA. Programa Escolar de Prevención de Alcoholismo. San José, Costa Rica,1974.

2. -----. Manual para la Enseñanza de Alcohol y Alcoholismo. San José, CostaRica, 1975.

3. Escalante de Fonseca, A. Proyecto Area Metropolitana. Estudio de Patronesde la Ingestión de Alcohol. Report #1, Exploratory Study, 1975.

OTHER DATA

Grantee: Dr. Marilyn E. Katatsky, Principal Investigator, Division ofFamily Health, Pan American Health Organization, Washington, D.C.,U.S.A.

Funded by: U.S. National Institute on Alcohol Abuse and Alcoholism (USNIAAA).


73

PATHOPHYSIOLOGY OF ENDEMIC GOITER AND CRETINISM IN ECUADORAND THEIR PREVENTION WITH IODINATED OIL

PROBLEM

Well-documented studies on endemic cretinism have demonstrated that this dis-order is found in geographic association with endemic goiter, which in turn is relatedto chronic iodine deficiency. The expression, endemic cretinism, has traditionallybeen applied to subjects with obvious mental retardation and defects in hearing,speech, and walking.

In 1965, a survey of endemic goiter and endemic cretinism was made in eightrural villages of the Ecuadorean Andean Provinces most affected by the diseases.Diagnosis of cretinism took into account the mental deficiency of the subject. Inidentified cretins, language defects were interpreted as disorders related to mentalretardation in some cases, and to mental retardation plus deafness in others. Addi-tional findings in the survey were frequent dysrhythmia, dysarthria, and sharp re-tardation of language development. In view of these findings, a program was developedto study the role of iodine on language development, and to correct chronic iodinedeficiency by intramuscular administration of iodized oil. The study was based onthe principle that several factors influence speaking ability, such as the centralnervous system, hearing mechanisms, diction structure integrity, cognitive and emo-tional qualities, and favorable environmental stimuli.

METHOD

The study was conducted in Tocachi and La Esperanza, two isolated, entirelycomparable neighboring rural Andean communities. In March 1966, iodized oil wasinjected to every person in Tocachi. Details on this iodine supplementation programhave been reported elsewhere. All children born after March 1966 were studied chro-nologically according to Gesell scales for measuring neuromotor development, includinglanguage development. The children born in Tocachi were divided into two groups.The first group, G1, comprised children in whom correction of iodine deficiency occurredbetween the fourth and the seventh month of fetal life. The second group, G2, had chil-dren of mothers given iodized oil before conception. This classification was madebecause three periods during the embryogenesis were considered of importance in deter-mining the effects of iodine correction on language development: (a) during the first8 weeks of gestation, when the central nervous system begins to form and develop;(b) during the initiation of fetal synthesis of thyroid hormones, which occurs at aboutthe 12th week of intrauterine life; and (c) during the initiation of myelinization,which occurs at the 12th-16th week of gestation. The children born in the village ofLa Esperanza were the control group.

A total of 156 children, ranging from 4 to 8 years of age, received an adapta-tion of the Mayo Clinic language evaluation test. Results were intended to complementthe language development study that we had conducted using the Gesell scales, and toidentify and classify language disorders in the study children. Severe chronic iodinedeficiency, protein-calorie malnutrition, vitamin A deficiency, and high prevalence ofgoiter and cretinism are the most important characteristics of the Spanish-speakingcommunities of Tocachi and La Esperanza.

74

RESULTS TO DATE

In terms of evolutive global language development there was no differencesbetween Tocachi G1 and Tocachi G2. As a whole, the children of La Esperanza presentedmore severe retardation in language development than did the Tocachi groups. Never-theless, statistically, test-to-test comparisons and analysis of language developmentas a whole, showed no significant differences in the children from Tocachi and thecontrol group from La Esperanza (p 0.25).

When speech functions were compared, no differences were found between theTocachi G1 and La Esperanza's corresponding control group. There were differencesbetween the Tocachi G2 and La Esperanza's corresponding control group. But thesedifferences were not statistically significant. No differences in speech disorderswere found between Tocachi G1 and Tocachi G2. As a whole group, the Tocachi childrenshowed less delay in acquisition of word sounds than did the children of La Esperanza.Both in Tocachi and La Esperanza, the prevalence of defects related to rhythm andarticulation seemed to be higher than in normal populations. There were no cases ofmutism in Tocachi, but 4.54 percent of children of La Esperanza had the defect.

SIGNIFICANCE

In places where severe chronic iodine deficiency and protein calorie malnutritioncoexist, global language development is retarded and serious defects in speech are morefrequent.

In the study places mentioned above, correction of chronic 'iodine deficiencygave the following results:

(a) If correction occurred before conception or after the third month of intra-uterine life, it appeared to have no effect on global language development. This meansthat global language development is not related to fetal thyroid function.

(b) Correction of chronic iodine deficiency seemed to prevent speech disordersrelated to deafness. This would be caused by a fetal hypothyroidism, recently demon-strated in areas with severe iodine deficiency.

(c) Retarded global language development should be referred to protein-caloriemalnutrition and/or other causes, i.e., cultural deprivation.

PUBLICATIONS

1. Fierro-Benítez, R. et al. The role of iodine in intellectual developmentin an area of endemic goiter. In: Dunn, J. T. and G. A. Medeiros-Neto(eds.), Endemic Goiter and Cretinism: Continuing Threats to World Health,Washington, D.C., 1974, PAHO Scientific Publication No. 292, pp. 135-42.

2. ----- et al. Biopatología andina y nutrición. América Indígena 34:777-95,1974.

3. ----- et al. The role of iodine on intellectual deficiency in areas ofchronic iodine deficiency and protein-calorie malnutrition. In Xth Inter-national Congress of Nutrition, Kyoto, August 1975, No. 355.

75

4. ----- et al. The role of iodine on language development in areas of chroniciodine deficiency and protein-calorie malnutrition. In Xth InternationalCongress of Nutrition, Kyoto, August 1975, No. 3340.

5. -----. Prevención del bocio endémico en el Ecuador. En V Congreso Boli-viano de Endocrinología. Bogotá, Agosto, 1975.

6. Fierro-Benítez, R. et al. Deficiencia mental en las areas rurales de losAndes Ecuatorianos. En II Congreso Panamericano de Retardo Mental, Panamá,Agosto 1975.

7. Ramírez, I. and R. Fierro-Benítez. Ritmo circadiano e interrelación deTSH, T-3 y T-4 en el cretinismo endémico. En V Congreso Bolivariano deEndocrinología, Bogotá, Agosto 1975.

OTHER DATA

Grantee: Dr. Rodrigo Fierro-Benítez, Department of Radioisotopes,National Polytechnic School, Quito, Ecuador.

Funded by: Pan American Health Organization.


76

MULTIDISCIPLINARY STUDIES ON PRIMITIVE POPULATIONSIN LATIN AMERICA

PROBLEM

The primary purpose of these studies is to ascertain the population structure ofprimitive man as exemplified by the American Indian, and to define the selective pres-sures and the milieu within which that structure functioned. Secondary objectives are(1) to collect data on gene frequencies which, properly programmed for large-capacitycomputers, will provide new insights into the taxonomic relationships of variousIndian groups, and (2) to study changing patterns of disease, especially those witha genetic component, as the Indian makes the transition from the stone age to theatomic age.

METHOD

A multidisciplinary team, always preceded by an anthropologist thoroughlyfamiliar with the group under study, obtains genealogies; performs medical, dental,and anthropometric examinations; and collects blood, saliva, urine, and stool specimens.Each specimen of blood, saliva, and urine is typed in the laboratory with reference toa variety of genetic-marker systems, and studies are made of the antibodies, biochemicalconstituents, and parasites involved.

RESULTS TO DATE

Over the past 10 years we have published some 50 papers reporting on variousaspects of this program. The findings fall in two categories. On the one hand, wehave worked extensively on the genetic structure of unacculturated Indian populations,developing therefrom a number of arguments concerning the constraints under which manhas been operating during some three million years of human evolution. On the otherhand, we have pursued a number of aspects of Indian health, with particular referenceto how the biological pressures bearing on Indian populations differ from those bearingon civilized populations. Of particular interest at the moment is the matter of mineralbalance. An entirely unexpected finding is evidence that mutation rates among Indianpopulations may be higher than anticipated. A vigorous effort is in progress to con-firm this finding.

SIGNIFICANCE

Primitive man is rapidly disappearing. These studies are designed to provide

additional insights into the circumstances under which human evolution occurs. Theyalso help to explain some of the biomedical aspects of modern man, who in the geneticsense, has only recently left the primitive state.

It seems almost certain that during the next several decades, modern man willattempt to extend his activities into many parts of the tropics still relatively un-touched. If he does this in such a way as to leave the tropics relatively untouched--and this seems necessary if the tropics are to survive as an exploitable ecosystem--then

77

he will inevitably be subjected to many of the same biomedical pressures which weare studying in the Indian. Thus a knowledge of these pressures could be of valueto modern man in the tropics.

PUBLICATIONS

1. Bloom, A. D. et al. Chromosomal breakage in leukocytes of South AmericanIndians. Cytogenet Cell Genet 12:175-86, 1973.

2. Gershowitz, H. et al. The genetic structure of a tribal population, theYanomama Indians. II. Eleven blood-group systems and the ABH-Le secretortraits. Ann Hum Genet, Lond 35:261-69, 1972.

3. Hecker, L. H. et al. Heavy metal levels in acculturated and unacculturatedpopulations. Arch Environ Health 29:181-85, 1974.

4. Li, F. H. F. and J. V. Neel. A simulation of the fate of a mutant geneof neutral selective value in a primitive population. In: B. Dyke andJ. W. MacCluer (eds.), Computer Simulation in Human Population Studies.New York, Academic Press, pp. 221-40, 1974.

5. MacCluer, J. W. et al. Demographic structure of a primitive population:a simulation. Am J Phys Anthropol 35:193-207, 1971.

6. Neel, J. V. The genetic structure of a tribal population, the YanomamaIndians. 1. Introduction. Ann Hum Genet, Lond 35:255-59, 1972.

7. ----- and R. H. Ward.Yanomama Indians. VI.with the Makiritare).

The genetic structure of a tribal population, theAnalysis by F-statistics (including a comparisonGenetics 72:639-66, 1972.

8. ----- et al. Studies on the Yanomama Indians. In: Human Genetics,Proc. IV Int. Cong. Hum Genet., Paris. Amsterdam, Excerpta Medica,pp. 96-111, 1972.

9. -----. "Private" genetic variants and the frequency of mutation amongSouth American Indians. Proc Nat Acad Sci 70:3311-315, 1973.

10. -----. Control of disease among Amerindians in cultural transition.PAHO Bull 8:205-11, 1974.

11. -----. Control de las enfermedades de los Amerindios en transicióncultural. Bol Of Sanit Panam 76:478-85, 1974.

12. ----- etIndians.based on

al. The genetic structure of a tribal population, the YanomamaX. Agreement between representations of village distances

different sets of characteristics. Am J Hum Genet 26:281-303, 1974.

13. -----. A note on congenital defects in two unacculturated Indian tribes.In: Janerich, D. T., R. G. Skalko, and 1. H. Porter (eds.), CongenitalDefects, New Directions in Research. New York, Academic Press, pp. 3-15,1974.

14. ----- and K. M. Weiss.Yanomama Indians. XII.42:25-51, 1975.

The genetic structure of a tribal population, theBiodemographic studies. Am J Phys Anthropol

78

15. Oliver, W. J. and J. V. Neel. Evidence of urinary tract disease in aprimitive society. Proc., XIII International Congress of Pediatrics,Vienna, 1971. Vienna, Verlag der Wiener Medizinischen Akademie,pp. 107-12, 1971.

16. ----- et al. Blood pressure, and sodium related hormones in the YanomamaIndians, a "no-salt" culture. Circulation 52:146-51, 1975.

17. Rocha, F. J. et al. A comparison of gene frequency and anthropometricdistance matrices in seven villages of four Indian tribes. Hum Biol46:295-310, 1974.

18. Rothhammer, F. and R. S. Spielman. Anthropometric variation in theAymará: genetic, geographic, and topographic contribution. Am J HumGenet 24:271-380, 1972.

19. ----- et al. The genetic structure of a tribal population, the YanomamaIndians. VIII. Dermatoglyphic differences among villages. Am Hum Genet25:152-66, 1973.

20. Salzano, F. M. et al. Serum proteins, hemoglobins and erythrocyte enzymesof Brazilian Cayapó Indians. Hum Biol 44:443-58, 1972.

21. ----- et al. Blood groups and H-Lea salivary secretion of Brazilian CayapóIndians. Am J Phys Anthropol 36:417-26, 1972.

22. Spielmann, R. S. et al. The genetic structure of a tribal population,the Yanomama Indians. VII. Anthropometric differences among Yanomamavillages. Am J Phys Anthropol 39:461-79, 1973.

23. -----. Do the natives all look alike? Size and shape components ofanthropometric differences among Yanomama Indian villages. Am Naturalist107:694-708, 1973.

24. ----- et al. Regional linquistic and genetic differences among YanomamaIndians. Science 184:637-44, 1974.

25. Tanis, R. E. et al. Albumin Yanomama-2 a 'private' polymorphism of serumalbumin. Ann Hum Genet, Lond 38:179-90, 1974.

26. Ward, R. H. The genetic structure of a tribal population, the YanomamaIndians. V. Comparisons of a series of genetic networks. Ann Hum Genet,Lond 36:21-43, 1972.

27. ----- et al. The genetic structure of a tribal population, the YanomamaIndians. XI. Gene frequencies for 10 blood groups and the ABH-Le secretortraits in the Yanomama and their neighbors; the uniqueness of the tribe.Am J Hum Genet 27:1-30, 1975.

28. Weitkamp, L. R. and J. V. Neel. The genetic structure of a tribal population,the Yanomama Indians. IV. Eleven erythrocyte enzymes and summary of proteinvariants. Ann Hum Genet, Lond 35:433-44, 1972.

29. ----- et al. The genetic structure of a tribal population, the YanomamaIndians. III. Seven serum protein systems. Ann Hum Genet, Lond 35:271-79,1972.

79

30. ----- et al. Human serum albumin: twenty-three genetic variants andtheir population distribution. Ann Hum Genet, Lond 36:381-92, 1973.

OTHER DATA

Dr. James V. Neel, Department of Human Genetics, The Universityof Michigan Medical School, Ann Arbor, Michigan, U.S.A.

National Science Foundation.

1975-1977.

80

Grantee:

Funded by:

Timetable:

CLINICAL AND GENETIC INVESTIGATIONS ON THE ALPORT SYNDROME(URUGUAYAN KINDRED)

PROBLEM

An infrequent hereditary disease, the Alport syndrome, has spread to the Uruguayanterritory and bordering countries, involving about 1,200 persons, the largest familialdissemination known in the world.

The syndrome is characterized by a kidney dysfunction, eye lesions, and deafness.The disease is regularly transmitted through affected females but it is fatal to affectedmales before the ages of 22 to 25 years. The hereditary transmission mechanism is stillunknown.

The first stage of the study of this family was initiated early in 1958 andlasted about 12 years. The purpose of the current investigation is: (1) to reexaminethe present status of the disease and determine its prevalence, morbility, mortality,and the evolution of the kidney, eye, and ear lesions; (2) to record the genealogicchanges occurred, and thus to estimate the degree of consanguinity and effect of geneticisolation and gene migration in the area; (3) to carry out a detailed cytogenetic studyof the patients, carriers, and healthy members of the family, using the latest techniqueson chromosome banding to detect any minor chromosome aberrations that could be relatedto the condition; and (4) to establish the genetic mechanism operating in the trans-mission of the disease.

METHOD

A detailed genealogic investigation is performed to detect newly affected groupsin the area. Family members are examined by clinicians in several regions of the coun-try. After routine clinical testing, some patients are selected for detailed kidney,ear, and eye studies. The incidence of various anatomic changes related to the syndromeare recorded for statistical interpretation and to estimate the evolution of charac-teristic signs of the disease after 15 years of study.

Peripheral blood from selected patients is cultured for cytogenetical studies.Several banding procedures (C-banding according to Arrighi and Hsu, 1971; G-bandingaccording to Drets and Shaw, 1971; Q-banding according to Caspersson and Zech, 1969;and R-banding according to Dutrillaux and Lejeune, 1971) are used systematically ineach case. A number of them will also be densitometrically analyzed with an analoguedensitometer (Drets and Seuanez, 1974) and with a Zeiss photometrical apparatus. Theseprocedures will be used to determine if a chromosomal aberration is involved in theproduction of the syndrome, and will give an insight into the genetic mechanism of thedisease. Moreover, a family planning service will be organized in the area to assistaffected families so as to reduce consanguinity incidence.

RESULTS TO DATE

More than two hundred affected family members have already been examined. Dataon these patients are pooled for further processing. The results of the clinical testsare still incomplete since the projected equipment for this research just arrived a fewweeks ago. Since this disease is characterized by a triad of symptoms, patients areexamined systematically for alterations in the three organs iñvolved.

81

Of 234 patients reexamined, the following malformations were found: kidney,vision, and hearing: 83; kidney and vision: 65; kidney and hearing: 10; vision andhearing: 4; kidney: 43; vision: 2; hearing: 1; chromosome karyotyping: 26. Patientsand members of the family are also examined odontologically and associated malforma-tions recorded.

SIGNIFICANCE

The research provides information on the incidence of the Alport syndrome inUruguay and neighboring countries. The genealogic study, clinical tests, and chromo-somal analysis will help to understand better the underlying genetic mechanisms in-volved in the disease. The complete revision of the problem will permit better familyplanning and genetic counseling, thus preventing the further dissemination of the dis-ease in this area.

PUBLICATIONS

1. Purriel, P. et al. Familial hereditary nephropathy (Alport's syndrome).Am J Med 49:753-73, 1970.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Máximo E. Drets, Division of Human Cytogenetics, Institutode Investigación de Ciencias Biológicas, Montevideo, Uruguay.


1975 - continuing.

82

SOCIODEMOGRAPHIC AND BIOLOGICAL DETERMINANTS OFNATURAL FERTILITY IN GUATEMALAN VILLAGES

PROBLEM

Since the start of the study, basic demographic data have been collected in theform of bi-annual censuses (1967, 1969, 1971, 1973, 1975). Moreover, through bi-weeklyhome visits, data have been collected on the timing and incidence of various reproduc-tive events such as births, conceptions, spontaneous abortions, menstruation, migration,and changes in family composition. A three-year study of the socioeconomic and biodemo-graphic determinants of birthspacing, lactation, and infant development was started atINCAP and reliable instruments to obtain data on postconception cyclic events developed.

The overall objective of this study is the analysis of the demographic, social,and biological determinants of natural fertility in four rural villages of easternGuatemala: Santo Domingo, Conacaste, Esp7ritu Santo, and San Juan.

METHOD

For this study, data collected in the 1969-1976 longitudinal study by INCAP willbe analyzed. Three areas of investigation are planned: (1) Basic demographic pro-cesses; (2) Fertility and intermediate fertility variables; and (3) Nutrition and fer-tility.

RESULTS TO DATE

Preliminary results showed that improved nutrition prolonged the duration oflactation and reduced the duration of postpartum amenorrhea. No evidence was found ofbehavioral compensation mechanisms operating consistently in response to events affect-ing length of birth intervals. A biodemographic and socioeconomic model of familydecision-making about birth interval is being developed.

SIGNIFICANCE

This study has focused on detailed, accurate measurements of various reproductiveevents. Because of the length of the observation period (7 years) and the absence ofcontraception in the study population, sociobiological determinants of fertility havebeen investigated in greater depth than has previously been possible.

Although some indirect evidence of the impact of nutritional status on fertilityis available, present knowledge of this process is poor. The present study has under-taken to specify and analyze in detail the various links between malnutrition and fecun-dity impairments. If such a correlation exists, findings will be of importance to in-terpret and predict fertility trends in populations where nutrition is changing, and torecommend appropriate nutrition programs.

83

PUBLICATIONS

None thus far.

OTHER DATA

Drs. Robert E. Klein, Hernán Delgado, and Charles Teller, Divisionof Human Development, Institute of Nutrition of Central Americaand Panama (INCAP), Guatemala City, Guatemala, and Dr. JohnBoongaarts, Population Council, New York, New York, U.S.A.

Population Council.

1975 - 1976.

84

Grantee:

Funded by:

Timetable:

SOCIOLOGICAL, ECONOMIC, AND PSYCHOLOGICAL DETERMINANTSOF BIRTH SPACING IN GUATEMALA

PROBLEM

Human fertility is determined by a combination of biological, behavioral andsociostructural factors that are highly interrelated. Of special concern are theintermediate fertility variables of lactation, fetal and infant mortality, and con-traception and how these variables are affected by economic, sociopsychological, andsociocultural factors in the family and the community.

Three of the specific aims of the study are to determine: (1) If the economicutility or affective value of children influence parent's birth timing and spacing,or the number of children they want; (2) If the female opportunity costs and its per-ceptions influence length of lactation and birth intervals; and (3) If the probabilityof child death and its perception create compensation mechanisms that affect birthspacing.

METHOD

The study compares couples within and between four rural communities (totalpop. 3,500) in a northeastern department of Guatemala City and a small urban town(pop. 5,000) south of Guatemala City. Prospective data on lactation, postpartumamenorrhea, conception, and pregnancy outcomes have been gathered since 1969 in thefour villages, as part of another longitudinal study, and since June 1974 in the town,as part of a selection process in the first year of this study.

A major part of this study has been the preparation of seven new questionnairesthat combine retrospective, cross-sectional, and prospective time factors. Thesequestionnaires are:

(1) Female Retrospective Pregnancy and Life History.

(2) Male Retrospective Economic and Social Life History.

(3) Family Agricultural Production, Income, and Wealth.

(4) Community Economic and Social Data.

(5) Social Stimulation and Modernism.

(6) Attitudes and Expectations of Parents Towards Their Children andToward Contraception.

(7) Time Budget of Daily Domestic and Economic Activities.

RESULTS TO DATE

The data gathered are being thoroughly edited and tested for their reliabilityand validity. Preliminary results indicate, however, important differences between the

85

villages and the town. The duration of lactation is shorter, the use of contraceptionis more widespread, and the length of birth intervals are more varied in the town thanin the villages. A reliable scale of modern outlook has been developed in which atti-tudes towards family size are a component. These attitudes are related to nationalknowledge, contact with mass media, and community participation. The economic utilityof the child seems higher in the villages than in the town, and the female opportunitycosts higher in the town than in the villages. Infant and child mortality has declinedsignificantly in the villages in the past 10 years.

SIGNIFICANCE

In most of the Third World, infant mortality and length of lactation are declin-ing while fertility remains high and population growth rates increase. At the sametime, social change and public health interventions are taking place. Their effecton birth spacing, on the one hand, and the effect of changed birth intervals on healthand nutrition on the other, are little known and rarely considered.

From the public health point of view, longer birth intervals may be advisable,but the resulting possible disadvantages--from the point of view of the socioeconomicand sociopsychological condition of the family--should also be known. This study isdeveloping valuable research instruments that permits the acquisition of such knowledge.

One possible direct application of the results might be the ability to identify,in modernizing environments, couples from whom shortening of their children's birthintervais might be detrimental, so that appropriate countermeasures can be taken.

PUBLICATIONS

1. Martorell, R. et al. Relación entre tenencia y acceso a la tierra yestado nutricional de niños pequeños. Paper presented at the Seminariosobre Estructura Social-Rural, September 1975, Costa Rica.

2. Teller, C. et al. Effect of declines in infant and child mortality onfertility and birthspacing. Preliminary results from retrospective andprospective data in four Guatemalan villages. Report on CICRED Seminaron Infant Mortality in Relation to.the Level of Fertility, Bangkok,Thailand, May 1975, in press.

3. ----- et al. Patrones de emigración y su impacto sobre estado nutricional.Un análisis preliminar de familias en cuatro comunidades rurales de Guatemala.Paper presented at the Seminar on Estructura Social-Rural y Análisis Re-gional en Centro América y Panama, Asociación Centroamericana de Sociología,La Catalina, Heredia, Costa Rica, 22-27 September 1975.

4. ----- et al. Técnicas de recolección de datos socioeconómicos: con-sideraciones sobre la validéz y la confiabilidad de datos especialmentedifíciles de obtener en un área rural de Guatemala. Paper presented atthe Conference on: Recolección de datos socioeconómicos en areas ruralesde países en desarrollo. México, D.F., November 17-24, 1974.

86

Dr. Robert E. Klein, Division of Human Development, Instituteof Nutrition of Central America and Panama (INCAP), GuatemalaCity, Guatemala.

Rockefeller Foundation.

1973-1976.

OTHER DATA

Grantee:

Funded by:

Timetable:

EFFECTS OF IMMUNIZATION WITH HUMAN CHORIONICSOMATOMAMMOTROPIN (HCS) ON REPRODUCTION

PROBLEM

There are several reports on the interruption of gestation, embryonic or fetalresorption and an increase in the neonatal mortality of laboratory animals actively orpassively immunized with HCS.

It has been postulated that the antigens responsible for such disorders of repro-duction are embryonic or fetal proteins that contaminate the commercial preparations ofthis hormone.

The aim of this research is to test the effects on reproduction of an impurecommercial HCS preparation and a highly purified hormonal preparation, to determine ifthe hormonal protein is the antigen responsible for those effects.

METHOD

(1) Obtention of a highly purified preparation of HCS

Briefly, the isolation of HCS was achieved through: (a) homogenization of washedplacentas in pH 4.5 buffer; (b) extraction of the residue with pH 8.6 buffer; (c) preci-pitation at pH 4.5; (d) dialyzation and lyophylization of supernatant; (e) fractionationof soluble material at pH 8.6 by exclusion chromatography on Sephadex G-100; (f) frac-tionation by ion exchange chromatography on DEAE-Cellulose of the HCS fraction; and(g) final purification by exclusion chromatography on Sephadex G-75.

(2) Purification of an impure commercial HCS

The final steps (f and g above) of the isolation method was applied to purify acommercial preparation of HCS.

(3) Obtention of anti-HCS serum in rabbits

Adult male New Zealand rabbits were used to produce antisera to the highlypurified HCS preparation, and also to the impure commercial hormone and a purifiedpreparation obtained from the same commercial hormone. Antibody titers were deter-mined by passive hemagglutination. Only those animals that exhibited titers of 1/5,000or more were used.

(4) Effects of immunization on reproduction

Adult female rats of known fertility and normal cycling were passively immunizedwith the three anti-HCS sera produced. Active immunization with the three preparationswill be also performed.

RESULTS TO DATE

The amount of highly purified HCS necessary to perform the immunization proposedhas been obtained. Also the commercial preparation of HCS has been purified.

88

The production of the antiserum against the three HCS preparations has beenachieved.

SIGNIFICANCE

The knowledge of the immunologic factors that can affect human fertility are ofspecial clinical interest. For instance, it is a well-known fact that habitual abortersmay have antibodies against placental proteins. An experimental approach to this humanpathology is immunization with placental protein hormones that are available as highlypurified proteins.

PUBLICATIONS

1. Castellano, M. A. et al. The effects on reproduction of the immunizationwith HCS. In preparation.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Miguel A. Castellano, Laboratory of Protein Hormones, LatinAmerican Center of Perinatology and Human Development, Montevideo,Uruguay.

Programa Latinoamericano de Investigaciones en Reproducci6nHumana (PLAMIRH).

1975 - continuing.

89

ISOLATION, CHARACTERIZATION, AND ELUCIDATION OF THE BIOLOGICAL FUNCTIONOF A NEW HUMAN PLACENTAL HORMONE

PROBLEM

There is evidence that the human placenta contains a substance that possesses aremarkable uterotropic activity.

Whether other known hormones possibly contributing to this activity are presentin the placental preparation, has not been elucidated, however.

A series of experiments was performed with the following purposes: (1) to obtaina pure preparation of the uterotropic substance; (2) to study the possible contaminationof this preparation with other known uterotropic hormones; (3) to characterize chemi-cally, physically, and biologically the substance responsible for such activity.

METHOD

Acetic acid extract of human term placenta was fractionated by pH and salt pre-cipitation and by exclusion chromatography on Sephadex G-75 column. A partially purifiedfraction (F-11) possessing uterotropic activity in immature and young mice was obtained.

The possible presence of estrogens in this active preparation was investigated.Three different extraction procedures effective in the liberation of estrogens tightlybound to proteins, were employed.

F-11 was also submitted to the action of protein denaturating agents (heat,8M urea) and of specific proteolytic enzymes (trypsin, alpha-chymotrypsin,and pronase).

The relationship between this biologically active substance and HCG was studiedby comparative immuno-double diffusion technique and two agglutination inhibition tests.

The biological characterization of the substance was achieved by injection ofsolutions of F-11 to immature female mice and to young intact and spayed mice.

RESULTS TO DATE

No estrogen was extracted from the placental preparation, which strongly sug-gests that the active substance is not of steroid nature. Moreover, the treatment ofF-ll with denaturating agents and proteolytic enzymes, completely destroyed the utero-tropic activity, thus showing that the active substance is of protein nature.

Results of the immunological studies showed that this protein is neither immuno-logically related to human chorionic gonadotropin (HCG) hormone, nor contaminated byit.

Precocious sexual maturity, ovarian weight increase, and stimulation of the sexaccessory organs in impuber female mice was induced by F-11. The administration of F-11to spayed mice did not produce any increase in their uterine weight. This finding in-dicated that the uterotropic activity might be due to stimulation of the female gonad.

90

SIGNIFICANCE

The purification and biological characterization of a new placental hormone opensan important field of investigation and could be a significant contribution to theknowledge of the endocrine control of reproduction and gestation.

PUBLICATIONS

1. Brovetto-Cruz, J. et al. Evidencias sobre la existencia de una nuevaproteína placentaria activa. In: 49 Congreso Argentino de Endocrinologíay Metabolismo, Parana, Entre Ríos, República Argentina, September 1975,pp. 38-40.

2. ----- et al. Chemical and biological characterization of an active sub-stance of the human placenta. Sent for -publication to Eur J Obstet GynecolReprod Biol, 1975.

3. Castellano, M. A. et al. Biological and immunological studies of a pla-cental gonadotropic protein. Sent for publication to Eur J Obstet GynecolReprod Biol, 1975.

4. Sardi-Valverde, Y. et al. Attempted estrogen extraction from human placentaluterotropic preparations. IRCS Medical Sciences: Biochem Endocrinol ReprodObstet Gynecol 3:622, 1975.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Jorge Brovetto-Cruz, Laboratory of Protein Hormones, LatinAmerican Center of Perinatology and Human Development, Montevideo,Uruguay.

Programa Latinoamericano de Investigaciones en Reproducci6nHumana (PLAMIRH).

1975 - continuing.

91

RELATIONSHIP OF OXYTOCIN AND OVARIAN CONTRACTILITY IN THE RAT

PROBLEM

Many studies have been performed to investigate the role of the ovarian smoothmuscle in reproduction. The participation of ovarian contractility in the process ofovulation and particularly in follicular rupture and extrusion of the ova has been sug-gested. Pharmacological studies showed that a-adrenergic agents, prostaglandin F2a,and oxytocin, among others, have a stimulating effect on ovarian smooth muscle. Theaction of oxytocin has been studied mainly in the periovulatory stages such as proestrusor late proestrus. It has been proved that the action of oxytocin on the uterus andoviduct depends on the hormonal status of the animal. However, the hormonal influenceon the action of oxytocin has not been thoroughly approached regarding the ovary.

In this project, the effect of the peptide on rat ovarian contractility in vitroand in vivo in all stages of the estrous cycle is studied. The specific ovarian uptakeof oxytocin is also being investigated.

METHOD

The experiments were carried out in white female rats (Wistar), 4 to 5 monthsold.

The outline of experimental design is as follows:

(1) To record spontaneous contractility of the rat ovary in vitro as well asin vivo.

(2) To assess the action of oxytocin on rat ovarian contractility in vitroand in vivo.

(3) To assess the action of oxytocin antisera on the spontaneous and oxytocin-induced contractility of the rat, both in vitro and in vivo.

(4) To measure the specific uptake of oxytocin by the ovary in the rat; forthis purpose 3H-oxytocin and 125I-oxytocin were used.

All these studies were performed for every stage of the estrous cycle, i.e.,proestrous, diestrous I, and diestrous II.

For in-vitro studies, tension was recorded isometrically by a strain gauge.Intraovarian pressure was recorded in in-vivo experiments.

RESULTS TO DATE

(1) In-vitro experiments

A total of 88 experiments were-carried out, i.e., both ovaries of 44 rats werestudied. Spontaneous motility was observed in 100 percent of the ovaries, confirmingthat this occurs in all stages of the estrous cycle. Administration of oxytocin to the

92

bath stimulated the contractility in one or both ovaries in 29 of the 44 animais studied(66 percent).

Regarding sensitivity of the responding organs, the threshold dose was 0.5 mU/mlin 4.9 percent of the ovaries, and 5 mU/ml in 73.2 percent. The remaining 22 percent ofthe ovaries only responded to 25 mU/ml. No significant differences were observed in theamount of positive responses among groups of ovaries in the different cycle stages(X2=0.78). The responsiveness of right ovaries with respect to left was very similar.

(2) In-vivo experiments

Intraovarian pressure was recorded in 35 rats; both ovaries of each animal wereassayed. Concerning spontaneous motility the results were the same that for the in-vitro studies, i.e., all the ovaries showed spontaneous contractions. An increase ofintraovarian pressure was observed in 20 of the animals when oxytocin was administeredto them. Statistical analysis showed a significantly greater number of positivl re-sponses in animals in proestrous when compared with any other phase of cycle (X =9.20).

(3) Action of oxytocin antisera on ovarian contractility

The study is still in progress. Investigations of the specific uptake of thepeptide by the ovary are at present in preliminary stages and do not allow reportingof results.

SIGNIFICANCE

The in-vitro results would suggest that the hormonal environment does not in-fluence the action of oxytocin on the ovarian smooth muscle.

Nevertheless in the in-vivo experiments the sensitivity of the ovaries tooxytocin is higher during proestrous than in the other stages of estrous cycle. Theovary withdrawn from its natural medium (ovary in vitro) for several hours may sufferbiochemical modifications that could explain the differences observed in the in-vivostudies.

It has been suggested that oxytocin might play a role in the process of ovula-tion through an increase of ovarian muscular activity. From our results (stressingthe in-vivo studies) it seems that ovarian muscle fibers are influenced by the sexualhormones to be more sensitive to oxytocin in the preovulatory phase. It should beinteresting to measure plasma oxytocin levels in the periovulatory period, to throwmore light on the participation of the peptide on ovulation.

PUBLICATIONS

1. Roca, R. et al. Effects of oxytocin on rat ovarian contractility in vitroduring estrous cycle. Submitted for publication to Biol Reprod.

2. ----- et al. Effects of oxytocin on rat ovarian contractility in vivoduring estrous cycle. Submitted for publication to J Reprod Fertil.

93

Dr. Ricardo Roca, Laboratory of Neurohypophysial Hormones,Latin American Center of Perinatology and Human Development,Montevideo, Uruguay.

World Health Organization.

1974-1975.

94

OTHER DATA

Grantee:

Funded by:

Timetable:

EVALUATION OF MONITORING IN LABOR INDUCTION

PROBLEM

Two remarkable factors arise from a critical analysis of published reports onlabor induction: (a) incomplete information on perinatal results; and (b) insufficientdata on possible advantages or disadvantages of continuous electronic monitoring inthose cases.

The aim of this study is to evaluate critically the possible benefits of contin-uous maternal and fetal electronic monitoring in high-risk patients being induced.

To accomplish this objective, two study groups controlled clinically and bymonitoring were compared.

METHOD

Perinatal data about 400 inductions of labor made with continuous oxytocin in-fusion, were collected. Eighty percent of the patients were considered as having high-risk pregnancies. Data were collected in specially designed precoded files, and theinformation transcribed on Hollerith computer cards.

From this information, 2 groups of 117 inductions were matched through electro-mechanical selection (by IBM 082 classifier). One of the groups was controlled by usualclinical method, and the other by monitoring. The patients in the two groups had simi-lar gestational age, fetal presentation, maternal and/or fetal pathology, parity, ripen-ing of the uterine cervix, and status of the ovular membranes at the beginning of theinduction. All inductions were controlled and assisted by specialists with similarbackground.

The two groups were compared for total duration of induction, cesarean sectionrate, and perinatal outcome (proportion of intrapartum fetal deaths, Apgar scores oflive-born babies, morbidity, and neonatal deaths in the first week of life).

RESULTS TO DATE

(1) The total duration of labor induction in vaginal deliveries was significantlyshorter in monitored than in clinically controlled patients, whether they had a ripeor unripe uterine cervix at the start of oxytocin infusion.

(2) Cesarean sections were performed more frequently--but not statisticallysignificantly--in monitored than in clinically controlled gravidas.

(3) The two fetal deaths that occurred during induction in the whole studywere found in the clinically controlled patients.

(4) The proportion of depressed newborns at the fifth minute of life, and theproportion of neonates with abnormal neurological exam--at 48-72 hours from delivery--were greater in clinically controlled inductions than in those monitored electronically.

(5) No accidents occurred by using electronic monitoring systems for trainedpersonnel.

95

SIGNIFICANCE

Because of the absence of accidents with continuous electronic monitoring oflabor inductions, and the advantages of the technique (shorter induction and betterperinatal outcome) the use of the method is recommended to improve the management ofinductions in high-risk pregnancies.

PUBLICATIONS

1. Cuadro, J. C. El control clínico y el monitoreo electrónico en las in-ducciones del parto. Resultados perinatales. Doctoral Thesis presentedto the School of Medicine, University of the Republic, Montevideo, Uruguay.Printed and distributed by CLAP. Publication CLAP 630, 1975.

2. ----- and R. Belitzky. El monitoreo electrónico en las inducciones delparto. Presented to the Uruguayan Society of Obstetrics and Gynecology,August 1975.

3. ----- et al. Fetal monitoring in inductions of labor. To be submittedfor publication in J Perinat Med.

4. Gonzalez-Doldan, P. et al. Resultados obtenidos en inducciones monitori-zadas y no monitorizadas. Proceedings of the XIV Argentinian Congress ofObstetrics and Gynecology, October 1973, in press.

OTHER DATA

Grantee: Drs, J. C. Cuadro and Rubén Belitzky, Latin American Center ofPerinatology and Human Development, Montevideo, Uruguay.



96

EFFECTS OF MEMBRANE RUPTURE ON LABOR PROGRESSAND ON CEPHALIC MOLDING

PROBLEM

The objectives of this study are:

(1) To learn about the influence of ovular membranes and other fetomaternalfactors on: (a) the duration of the different stages of labor, (b) the evolution andtype of termination of labor, and (c) the condition of the newborn and the molding ofits head.

(2) To establish criteria of normality for the variables included in this study.

(3) To stress the importance of critical judgement of obstetricians and neo-natologists about the possible harmful effect of some medical conducts or actions rou-tinely taken in the management of labor.

(4) To promote the participation in this project of a great number of maternaland child health centers in Latin American countries, with the aim of achieving a wideexperience, discussion, and unified criteria regarding the management of normal labor.

METHOD

(1) Clinical material

Study patients had to fulfill the following requirements:

(a) They suffered no known disease and had a normal pregnancy.

(b) They were nulli- and multiparas without cephalopelvic disproportion andwith a normal pelvis.

(c) They had between 266 and 294 days of amenorrhea.

(d) They carried live fetuses in cephalic presentation.

(e) They started labor spontaneously (without any medication).

(f) They had intact membranes at the start of the study.

(g) They had a cervical dilatation greater than 3 cm and less than 5 cm at thetime they entered the study.

(2) Design

In 50 percent of the labors, early amniotomy was performed when cervical dilata-tion was between 3 cm and 5 cm.

In the other half of the cases, membranes were kept intact until the beginningof the late second stage, and whenever possible until birth.

97

Each procedure was performed alternatively (for example, one patient underwentearly membrane rupture, the next, a late rupture, and so on). Choice of the first pro-cedure in each center was made in an aleatory way. Under no circumstances was thedecision maintained if it was contraindicated for a given patient.

Following membrane rupture, patients were kept in a horizontal position but wereallowed to change their decubitus as often as they wished.

The patients were not given any drug until the onset of labor. Those who hadprecise medical indications to take a given drug, were also recorded and the data ob-tained interpreted in a given section of this study.

(3) Control of labor and the newborn

The variables selected to assess the progress of labor and the condition of thenewborn were recorded in a coded file history. This coded file was compiled accordingto an instruction manual covering all the variables.

(4) Analysis

The coherence of the information received from the collaborating centers werechecked and data processed by electronic computation and statistically treated.

RESULTS TO DATE

Twenty-two maternity hospitals of 11 Latin American countries have joined theresearch program. So far, these 22 centers have provided 1,413 clinical protocols.

Results of the protocols presented in our third Progress Report revealed:

(1) Phase of labor at which membranes rupture spontaneously

In the group of labors in which amniotomy was not performed, 66 percent of laborsreached full cervical dilatation with intact membranes, and in 12 percent these werestill intact at delivery. The higher frequency of spontaneous rupture (34 percent) oc-curs at full dilatation.

The proportion of labors reaching delivery with intact membranes is greater inmultiparas than in nulliparas.

Therefore intrapartum amniotomy is an artificial maneuver which alters thephysiological timing of spontaneous rupture.

(2) Duration of labor

Early amniotomy shortens in about 28 percent of the patients the median of theduration from the beginning of labor until the end of the first stage. Similar differ-ences between both groups are maintained when the sample is broken down according toparity.

It should be emphasized that acceleration of labor is not necessarily beneficialfor the fetus and that it may have undesirable effects. The duration of the secondstage is not influenced by the status of the membranes.

When the umbilical cord is around the fetal neck, this causes the second stageto last longer.

98

(3) Cephalic molding of the neonate

In labors with early rupture of membranes, the incidence of caput succedaneum,and bone disalignment is greater.

With early amniotomy, one of every 3 neonates has caput succedaneum, whereaswhen membranes are allowed to rupture spontaneously, only one of every 7 newborns hascaput.

Of every 100 newborns with early amniotomy during labor, 74 show cranial bonedisalignment. When membranes are not artificially ruptured, 65 of every 100 newbornshave disalignment.

One of every 3 neonates with early amniotomy shows simultaneously caput succe-daneum and cranial disalignment. When membranes remain intact, only one of every 9newborns shows both signs of mechanical trauma simultaneously.

Cephalic molding is influenced by parity. With early amniotomy there is a sig-nificantly greater proportion of caput in the first and second neonates than in thefollowing ones. When membranes remained intact until 10 cm of cervical dilatation,this incidence was significantly greater in the first child as compared with the follow-ing neonates. When membranes were still intact at the end of labor, the effect of parityon the production of caput succedaneum tends to disappear.

With early amniotomy, there is a significant association between neonatal weightand incidence of caput succedaneum. This was not found with intact membranes.

Intact membranes during labor reduce the production of caput succedaneum, cephal-hematomas, and cranial bone disalignment. They also diminish the effect of parity andfetal weight on the production of caput succedaneum.

(4) Progress of labor and delivery

Only 4 percent of the total population did not progress normally. In the groupof early amniotomy, the incidence of abnormal progress was significantly greater.

The fact that the great majority of labors progress normally without any drugadministration is hereby stressed.

Of the total study population, 10 percent of labors ended artificially. About9 percent of the deliveries were done through forceps, 0.8 percent with vacuum extractor,and 0.3 percent through cesarean sections.

These data coming from a low perinatal risk population, seem ideal taking intoaccount the low morbidity and mortality recorded.

(5) Neonatal mortality and morbidity

In the series of 1,413 labors of low perinatal risk which were studied, neonatalmortality in the first 48 hours of life, was 0.71 per 1,000.

In the whole sample, 99 percent of the neonates were vigorous at the fifth minuteof life. Only 1 percent of the neurological exams at 48 hours of life was abnormal.No association was found between the status of membranes, and Apgar scores, and the re-sult of the neurological exam at 48 hours of life.

99

SIGNIFICANCE

This study reveals the real effect of early rupture of membranes, or of nonrupture,on the progress of labor and on cephalic molding of the neonate.

Though this fact is still being discussed, there already exists some evidence thatthe intrapartum amniotomy may be harmful to the newborn.

The information collected has never before been obtained, since most obstetriciansroutinely perform amniotomy before cervical dilatation reaches 6 cm, to shorten the dura-tion of labor.

In addition, this investigation has provided other important information as fol-lows:

(1) Because of its "perinatal" objective, it has involved the teamwork of obste-tricians and neonatologists.

(2) It has required intrapartum and neonatal control, which is feasible andefficient.

(3) It has taught the obstetrician to observe the physiologic phenomena of laborand has prevented unnecessary minor interventions (use of analgesics or oxytocic drugs).

(4) It has tended to improve intrapartum and neonatal care.

(5) It has permitted the collection of data on the physiology of labor inseveral Latin American countries with different socioeconomic, ethnic, and culturalconditions.

Finally, this research leads to the description of normal characteristics of themost important parameters in the physiology of labor.

PUBLICATIONS

1. Caldeyro-Barcia, R. et al. Adverse perinatal effects of early amniotomyin labor. In: Gluck, L. (ed.), Modern Perinatal Medicine. Year BookMedical Publishers, Chicago, Illinois, Chapter 32, pp. 431-49, 1974.

2. Schwarcz, R. L. et al. Latin American collaborative study in the influenceof membranes upon labor and the newborn. First Progress Report presentedto the Pan American Health Organization and to the collaborating groups.Latin American Center of Perinatology and Human Development, Montevideo,January 1973.

3. -----. Latin American collaborative study in the influence of membranesupon labor and the newborn. Second Progress Report presented to the PanAmerican Health Organization and the collaborating groups. Latin AmericanCenter of Perinatology and Human Development, Montevideo, June 1973.

4. ----- et al. Conservative management of labor. Proc. VII World Congr.Obstet. Gynec., Moscow, August 1973. In: Persianinov, L. S., T. V.Cherwakova, and J. Presl (eds.), Recent Progress in Obstetrics andGynaecology. Excerpta Medica, Amsterdam, pp. 105-26, 1974.

100

5. ----- et al. La rotura precoz de las membranas ovulares y sus efectossobre el parto y el neonato. Publicación Científica del Centro Latino-americano de Perinatología y Desarrollo Humano. OPS/OMS, No. 595,Montevideo, 1974.

OTHER DATA

Dr. Ricardo L. Schwarcz, Latin American Center of Perinatologyand Human Development, Montevideo, Uruguay.


1972-1975.

101

Grantee:

Funded by:

Timetable:

INTRAUTERINE GROWTH CHARTS OF A NORMAL POPULATION AT SEA LEVEL

PROBLEM

Since there are many factors that influence human intrauterine growth, the

elaboration of national and even institutional intrauterine growth charts has been re-commended.

The aims of the project are:

(1) To determine what are the normal intrauterine growth patterns for the new-born population of the maternity of the Hospital de Clínicas.

(2) To study the influence of maternal pathologic conditions--such as diabetes,toxemia, or bleeding during pregnancy--on fetal growth.

METHOD

A population of 2,017 neonates was studied, 1,805 of these born from healthymothers. In order to assess normal patterns, the following were excluded from thestudy:

(1) Neonates born from mothers with pathologic condition;

(2) Twins;

(3) Babies with malformations;

(4) Babies with uncertain gestational age at birth.

All the babies were examined by a neonatologist. Weight and height were measuredin the first minutes of life. The error of measurement was evaluated. Precoded fileswere used to obtain information.

Data obtained were transferred to punched cards and then were electronicallyprocessed by computer.

Percentiles 10, 50, 90, means, and SD, for weight, length, and ponderal indexwere calculated for each week of amenorrhea. The curves of each parameter were smoothedby using a polynomial regression and were obtained directly by an IBM plotter.

RESULTS TO DATE

(1) Weight and gestational age

The weight of the newborns increased until the 40th week of gestation and thenremained stable. The mean weight at 40 weeks was 3,290 g.

Males always weighed more than females but the differences were significant onlyin weeks 38 and 40. The average weight of females at 40 weeks was 150 g less than thatof males.

102

(2) Weight and gestational age of babies born from diabetic and hypertensivemothers

The weight of 23 percent of the infants of diabetic mothers was above the 90thpercentile of the normal distribution.

On the other hand, 19 percent of the newborns of toxemic mothers had values belowthe lOth percentile of the same curve.

Newborns of mothers who had bleeding during pregnancy, behaved similarly to thenormal ones.

(3) Length and gestational age

Length increased continuously from 33 to 43 weeks of gestation. The averagelength at 40 weeks was 49.1 cm.

The average values of length for males were greater than for females: the dif-ference was significant at 38, 40, 41, and 43 weeks.

The distribution of the length of newborns of diabetic and hypertensive motherswas not different from that of normal newborns.

(4) Ponderal Index

The Ponderal Index (weight x 100) increased from the 33rd until the 39th weekand then stabilized. The average value at 40 weeks was 2.77.

SIGNIFICANCE

The local pattern of intrauterine growth is of practical use in each hospital orservice. It allows planned organization of health services according to the needs, andindicates the risk of mortality in each particular case.

PUBLICATIONS

1. Curbelo, V. et al. Fetal growth and mortality of newborns of diabeticmothers. Internal presentation at CLAP, 1975.

2. ----- et al. Normal intrauterine growth. Sent for publication to ArchPediat Uruguay.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Violeta Curbelo, Latin American Center of Perinatology andHuman Development (CLAP), Montevideo, Uruguay.

Pan American Health Organization/World Health Organization.

1974-1975.

103

GROWTH AND DEVELOPMENT OF LOW-BIRTHWEIGHT NEWBORNS

PROBLEM

There is no agreement about norms of postnatal growth and development of low-birthweight newborns, in comparison with those of babies born with normal weight.Under-weight newborns essentially comprise: (a) those born prematurely but withadequate intrauterine growth, and (b) those born at term but with insufficient weight,because of factors affecting their normal intrauterine growth.

The aim of this study is to establish, by means of a follow-up program, the nor-mal patterns of both groups during the first 2 years of life and their possible dif-ferences with a control group of full term and size newborns.

METHOD

Three groups of infants were studied: group A had 48 full-term, normal sizenewborns; group B had 40 preterm newborns with adequate weight for their gestationalage; and group C had 24 full-term infants with low wei:ght for their gestational age.

All study newborns had the following characteristics: they were born duringthe same period, they resided in the same geographic area, their mothers had receivedthe same medical care during labor and delivery, and they lived in similar socioeco-nomic and environmental conditions.

Follow-up consisted of periodic registration of clinical examinations, anthro-pometric measurements, and psychological tests.

Data collection was done in especially precoded file and information transcribedto Hollerith punched cards. Data processing was achieved through electronic computersystems that took into account individual values as well as rates of increment.

RESULTS TO DATE

In the group A, postnatal growth patterns (weight, height, and cephalic peri-meter) were different from those of previous local patterns.

Group B showed significant differences with group A up to 1 year of postnatalage; differences disappeared at 18 and 24 months of life. The same was true of groupsA and C.

The speed rate of growth was higher during the first year of life than duringthe second one; the lower the weight at birth, the higher the speed of growth duringthe first year.

When age corrections were made, no differences were found between the groups,as far as the time when they began to walk and the results of Gesell tests.

104

SIGNIFICANCE

Results achieved showed that the speed of growth of low-birthweight infants ishigher than that of full-term normal size infants during the first year of life.

However, if a nourishing diet is supplied, no differences of growth and develop-ment patterns are expected at the 2-year period of observation,

PUBLICATIONS

1. Martell, M. et al. Speed of growth from birth up to the second year of life.To be submitted to J Perinat Med.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Miguel Martell, Hospital de Clinicas, Latin American Centerof Perinatology and Human Development, Montevideo, Uruguay,


1973-1976.

105

PERINATAL FACTORS AFFECTING CHILD DEVELOPMENT

PROBLEM

Some studies have been made of the possible effects of acute intrapartum fetaldistress on the neuropsychological development of humans and animals.

Experimental perinatal asphyxia performed in monkeys showed an evident deleteriouseffect on the central nervous system with detectable organic lesions. Similar resultswere achieved by analyzing the history of children with cerebral palsy.

Though low Apgar scores have been correlated to abnormal neurological exams inthe first year of life, the association is not clear during the fourth year.

The aim of this study is to learn which of the factors surrounding delivery arecorrelated with periods of perinatal hypoxia, as well as their possible effects on theneurologic and psychological development of infants from delivery to school age.

METHOD

This study uses the perinatal data on a series of newborns followed since 1962from birth to school age by a multidisciplinary team. Maternal data, pregnancy factors,characteristics of labor and delivery (continuous recordings of amniotic pressure andfetal heart rate), and biochemical information on the blood of the fetus and newbornare recorded for each study patient.

Follow-up includes somatic, neurologic, psychologic, and EEG testing, as wellas a study of environmental factors.

To date, the study encompasses 230 patients. Pertinent information is collectedin special coded files and is being prepared for automatic computer analysis.

RESULTS TO DATE

Recent preliminary results from a sample of 84 children over 5 years of ageshowed that:

(1) Infants born with neonatal depression (Apgar score below 7) had a high per-centage of neurologic abnormalities at 72 hours of life (especially alterations of thetonus).

(2) There was a significant association between severe neonatal depression(Apgar score below 4) and the incidence of abnormal EEG exams.

(3) There was a statistical association between severe neonatal depression(Apgar score below 4) and the incidence of apyretic convulsions.

(4) Better performances (but not statistically significant) in psychologictests (Gessell, W.I.S.C., Bender), were seen in healthy than in depressed newborns.

106

The preliminary results of data processing are being interpreted and a programfor automatic discriminant analysis is being developed.

SIGNIFICANCE

This research has shown that factors, such as bírthweight, gestational age, ortreatments, are also implicated in child development.

The ultimate goal of the study is to determine the significance of hypoxia itselfand its interaction with the other main factors (prematurity, fetal malnutrition, andothers).

The final results achieved from this study could be the scientific basis toidentify special high-risk groups of children and to establish for them standards ofcare.

PUBLICATIONS

1. Ruggia, R. and D. Fonseca. Perinatal hypoxia: results of follow-up study.In preparation.

2. -----. Diagn6stico Precoz del Retardo Mental. Proceedings of the IICongreso Panamericano de Retardo Mental, Panamá, Agosto 29, 1975, :n press.

OTHER DATA

Grantee:

Funded by:

Timetable:

Drs. Daniel Fonseca and Raúl Ruggia, Latin American Center ofPerinatology and Human Development, Montevideo, Uruguay.


1974-1976.

107

ANTIBACTERIAL DEFENSES IN THE NEWBORN

PROBLEM

It is well accepted that newborn infants are more vulnerable to infection bysome agents than are older infants. There is a particular proclivity to infection withgram-negative bacilli of the enterobacteriaceae and pseudomonas families. Becauseopsonic antibodies for these organisms are mainly of the IgM class, poor phagocytosisof nonopsonized bacteria probably contributes to this susceptibility. The influencesof maternal mainutrition and intrauterine infection on these defense mechanisms arenot known. Infection of the fetus results in elevation of the IgM levels in cordserum. There have been no studíes to date to determine whether the IgM present in cordsera in Guatemalan village newborns can function as an opsonin. Bactericidal functionof cord blood leukocytes from infants in developing countries is not known and thepossible influence of prematurity and/or intrauterine growth retardation cannot there-fore be evaluated.

METHOD

Assessment of leukocyte function is by two methods. They monitor critical stepsof the antibacterial role of the neutrophil: Stimulation of hexose monophosphate shunt(HMS) activity during phagocytosis, and intracellular killing of a manitol and coagulasepositive, penicillinase producing Staphylococcus aureus and a serum-resistant strainof Escherichia coli. Opsonizing antibody against the two-test organisms is also deter-mined.

RESULTS TO DATE

Intracellular bactericidal studies using bacteria preopsonized with pooled AB Rh+Guatemalan adult sera have not revealed any difference in bacterial killing by cordbloods from infants with birthweight greater than 2,500 g. Bactericidal activity ofcord blood from low birthweight infants, in contrast, was enhanced by preopsonization.The cord sera obtained have been preserved at -700C for quantitative assay of opsoninsduring the forthcoming year.

Metabolic activity during phagocytosis of polystyrene latex particles was de-pressed in samples from low birthweight infants compared to those from high birthweightinfants. In no sample, however, was there a "CGD-like" absolute failure to increasethe decarboxylation of carbon 1 of glucose via the hexose monophosphate shunt pathwayduring phagocytosis.

SIGNIFICANCE

The results obtained by this research, will provide data that will help to explainthe antibacterial defenses of newborns.

108

PUBLICATIONS

None thus far.

OTHER DATA

Dr. Juan José Urrutia, Division of Environmental Biology,Institute of Nutrition of Central America and Panama (INCAP),Guatemala City, Guatemala; and Dr. Gerald T. Keusch, MountSinai School of Medicine, New York, New York, U.S.A.

U.S. National Institutes of Health (NIH).

1974-1976.

109

Grantee:

Funded by:

Timetable:

FACTORS AFFECTING PERINATAL MORTALITY AND MORBIDITY IN DIABETICS

PROBLEM

The way to improve perinatal results in the management of diabetic pregnanciesis still an unsolved problem at present.

The main objectives of the study are:

(1) To determine during pregnancy, labor, and the neonatal period, which factorsare related to the high perinatal mortality and morbidity.

(2) To minimize perinatal mortality and morbidity by using special standards ofcare for pregnancy, labor, and the neonate.

METHOD

The project is being carried out by a multidisciplinary team, composed of diabet-ologists, obstetricians, and neonatologists.

Special studies were performed during pregnancy such as the oxytocin stress test(Pose test), amniotic fluid analysis, and the PAH test, in order to evaluate fetal con-dition and fetal maturity. Most of the spontaneous and induced labors were controlledby monitoring.

The condition of the newborns was evaluated at birth by Apgar scores, and severalsomatic and neurologic examinations were done in the first week of life.

Data on each case were collected on coded files for computer analysis.

Based on these studies, special standards of care for diabetic pregnancy weredeveloped and tested.

RESULTS TO DATE

From the preliminary processing of data on 180 diabetic gravidas, an approachwas agreed on to determine the factors influencing the incidence of fetal and neonataldeath. Based on these facts, special standards of care for the diabetic mother and hernewborn infant were prepared and approved.

New standards require the best metabolic control of the mother and the delay ofinterruption of pregnancy until term. Thus, the usual risk of prematurity is avoided.

At present, the establishment of this policy showed a transient fall in morbidityand mortality.

110

SIGNIFICANCE

Diabetes is quite frequently found in Latin American countries (0.5 to 3.0 per-cent of the population). When diabetes is present, perinatal mortality is about 100to 200 per 1,000 live newborns.

Knowledge of the most important factors related to mortality and morbidity willhelp in preventive measures.

Effective standards of care for the diabetic mother and her newborn may be widelyadopted.

PUBLICATIONS

1. Bayce, M. et al. Morbidity of the newborn infant of the diabetic mother.Proceedings of the IV Argentinian Congress of Endocrinology, p. 61, 1975.

2. Belitzky, R. et al. Factors affecting perinatal mortality in diabetics,Proceedings of the IV Argentinian Congress of Endocrinology, p. 56, 1975.

3. ----- et al. New tendency in the management of diabetic pregnancies, inpreparation. To be presented at the Symposium of Perinatology, EighthWorld Congress of Obstetrics and Gynecology, Mexico, 1976.

4. Pose, S. V. et al. Perinatal standards of care for diabetic mothers andtheir newborns. CLAP Publication No. 574, 1974.

5. ----- et al. Fetal tolerance test to uterine contractions in diabetics.Proceedings of the IV Argentinian Congress of Endocrinology, p. 58, 1975.

6. ----- and R. Belitzky. Fetal condition and fetal maturity in diabetic preg-nancies. Proceedings of the Eighth National Congress of Medicine, Montevideo1974, in press.

OTHER DATA

Grantee: Drs. S. V. Pose and R. Belitzky, Latin American Center ofPerinatology and Human Development (CLAP), and ClínicaGinecnlógica "B", Montevideo, Uruguay.



111

IMPLEMENTATION OF A SIMPLE SYSTEM FOR COLLECTINGAND PROCESSING PERINATAL DATA

PROBLEM

Knowledge of the main characteristics of the maternal-fetoneonatal populationcared for in the obstetric department of the Hospital de Clínicas, as well as theperiodic evaluation of perinatal data, are essential for an efficient supervision andeconomic use of resources.

At the moment, many Latin American institutions have no system for collectingand processing perinatal data.

The aim of this project is to develop and test a simple method for the collectionof this information and for automated analysis by using different levels of complexity.

Ultimately, perinatal statistics will be improved and local research promotedthrough availability of coded files, instruction manuals, control schemes, and computerprograms.

METHOD

A multidisciplinary team prepared a carefully selected list of 89 questionson maternal characteristics (somatic examínation and obstetric history), evolution ofthe pregnancy (pathology), information on labor and delivery, and evolution of theneonate during the first weeks of life.

A special coded file was prepared in which data collected from hospital patients'medical charts were recorded in one sheet, for transcription in only one Hollerith com-puter card. It took only 2 to 3 minutes to obtain the complete information requestedfor a case. A simple method for periodic control of data collected, as well as anautomatic program to detect errors were developed.

RESULTS TO DATE

By using the above methods, perinatal data were collected from 3,000 consecutivebirths at the Department of Obstetrics "B" of the Hospital de Clínicas.

This information was available for processing by using automated, mechanical,or electronic procedures.

Computer programs are already established for a complete perinatal report.Practical monthly or annual perinatal reports are being prepared.

The team is preparing several computer programs for various institutions.

112

SIGNIFICANCE

A better knowledge of patients' characteristics, and interpretation of perinatalresults, easily show the main problems on which to focus attention. From time to timestandards of care are evaluated in relation to perinatal mortality and morbidity. Thispermits logical alterations of the program and the management of human and materialresources according to local needs.

A simple and economical system for collecting and processing perinatal data wouldbe widely accepted and adopted.

PUBLICATIONS

1. Béjar, R. et al. Análisis perinatal de la población de recién nacidos vivos.Hospital de Clínicas, 1972. Arch Pediat de Uruguay 46:103-16, 1975.

2. ----- et al. Instructivo de la Codificación de Datos de la Historia ClínicaPerinatal Codificada. Publicación del Centro Latinoamericano de Perinato-logía y Desarrollo Humano, CLAP No. 56.

3. ----- et al. Sistema para Archivo de Datos Perinatales y Análisis Automá-tico de los Mismos. Publicación del Centro Latinoamericano de Perinatologíay Desarrollo Humano, CLAP No. 653.

4. Capurro, H. et al. Informe estadístico perinatal del año 1973. Publicacióndel Centro Latinoamericano de Perinatología y Desarrollo Humano, CLAP No. 527.

OTHER DATA

Grantee: Drs. Raul Béjar and Ruben Belitzky, Latin American Center ofPerinatology and Human Development, Montevideo, Uruguay.



113

MECHANISMS OF ANAPHYLACTIC REACTIONS

PROBLEM

A great number of studies have been made to understand the release mechanism ofpharmacological mediators and their role in anaphylaxis. Many of these studies havebeen made with the use of agents that interfere specifically with certain enzyme orenzyme systems. In this regard recent reports have strongly suggested that the ana-phylactic release of mediators is regulated by the intracellular level of cyclic3'-5'-adenosine monophosphate (C-AMP). The importance of the different mediators haveusually been evaluated by observing the effect of their specific antagonists on anaphylac-tic reactions. In most of these studies, passive cutaneous anaphylactic (PCA) reactionshave been used as an in-vivo model of anaphylaxis in many species.

The purpose of this study is to determine the effect on PCA reactions inducedin mice with IgG1 or IgE of antagonists of histamine and serotonin and of various anti-anaphylactic drugs, some of which are known to change the level of intracellular C-AMP.In addition, based on the observation that mouse IgE can sensitize rat mast cells, acomparative study was made of PCA reactions induced in mice and rats with mouse IgE.

METHOD

Mice were doubly sensitized with IgGl and IgE, whereas rats were sensitized withmouse IgE only. Each species was then divided into two or more groups. One of thegroups served as control and the other received injections of the drug or combinationof drugs to be tested. Immediately afterwards, or at a determined time after theadministration of the drugs, the animals were challenged, the PCA lesions measured,and the differences between the areas of the lesions of the control and experimentalgroups statistically analyzed.

RESULTS TO DATE

Antihistamines produced a partial inhibition of PCA reactions induced in micewith IgGl or IgE, and in rats with mouse IgE. Antiserotonin inhibited PCA reactionsinduced in rats with mouse IgE, but had no effect on PCA reactions induced in micewith IgG1 or IgE. The simultaneous use of antihistamine and antiserotonin resultedin a total inhibition of PCA reactions induced in mice with IgGl and in an intensebut not total inhibition of PCA reaction due to IgE; PCA reactions induced in ratswith mouse IgE were totally inhibited.

Compounds known to change the intracellular level of C-AMP were found to havelittle or no effect on PCA reactions induced in mice with either IgGl or IgE. However,they produced a complete or very intense inhibition of PCA reactions induced in ratswith mouse IgE. Diethylcarbamazine or disodium cromoglycate were also very effectiveinhibitors of rat PCA reactions induced with mouse IgE, but they had no effect on PCAreactions induced in mice with this same antibody or with IgG1. Thus, although theyshared common mediators released from the same type of target cell sensitized withthe same type of antibody, PCA reactions induced in mice and rats with mouse IgE weredifferently affected by most of the drugs tested.

114

SIGNIFICANCE

This research helped understand the cellular and pharmacological mechanismsinvolved in the anaphylactic phenomena and provided a basis for explanation of atopicreactions in humans.

PUBLICATIONS

1. Braga, F. and 1. Mota. Homologous passive cutaneous anaphylaxis (PCA)in mice and heterologous PCA induced in rats with mouse IgE. Immunology,in press.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Ivan Mota, PAHO/WHO Immunology Research and TrainingCenter, Instituto Butantan, Sao Paulo, Brazil.


1974-1975.

115

REGULATORY MECHANISM OF REAGINIC ANTIBODIES

PROBLEM

Humoral response against some antigens appears to be under the regulatoryinfluence of a T-cell population. This regulatory influence not only affects theproduction of IgM and IgG but seems to act on the formation of antibodies of theIgE class. Depletion of T cells in rats by adult thymectomy, splenectomy,or admin-istration of antithymocyte serum has been shown to result in an enhanced and pro-longed homocytotropic antibody (IgE) formation. The present investigation wasundertaken to extend the above work to the intact mouse and to study the regulatorymechanisms involved in the production of the two types of homocytotropic antibodies,IgE and IgG, found in this animal.

METHOD

Immunization was performed on inbred A/SN and BALB/c mice of either sex,weighing 18-22 g and skin tests applied to 4- to 6-week old male and female A/SNand BALB/c mice weighing 10, 15, and 18 g and to male Wístar rats, weighing between120 and 200 g. DNP-Ascaris and ovalbumin were used as antigen. Normal, thymecto-mized, splenectomized, and antithymocyte-serum treated or sublethally irradiatedmice were immunized using aluminum hydroxide as adjuvant, and their production ofIgE and IgGl antibodies observed. The antibody production was assayed by passivecutaneous anaphylaxis.

RESULTS TO DATE

Thymectomy inhibited IgE formation in adult mice but did not affect IgGlproduction. Splenectomy had no effect on either IgE or IgGl production. A singledose of rabbit antithymocyte serum (ATS) given 8 days after immunization inhibitedthe formation of IgE.

Sublethal irradiation given before immunization enhanced IgE formation andpartially suppressed IgG1 production; applied after immunization, it caused an en-hancement of IgE production that was inversely proportional to the interval elapsedbetween the two procedures. On the other hand, the IgGl antibody production wasfairly resistant to the same treatment. The results suggest a clear-cut separationbetween the mechanisms regulating IgE and IgGl production in mice.

SlGNIFICANCE

Elucidation of the regulatory mechanisms involved in the homocytotropicantibody production, mainly of the IgE class, would allow a more objective analysisof the allergic conditions in man, thus providing a more appropriate therapeuticapproach to them.

116

PUBLICATIONS

1. Macedo, Sahihi et al. Control of IgE and IgGl antibody productionin mice. Cell Immunol (submitted for publication).

OTHER DATA

Dr. Ivan Mota, PAHO/WHO Immunology Research andCenter, Instituto Butantan, Sao Paulo, Brazil.

Tra in ing


1974-1975.

117

Grantee:

Funded by:

Timetable:

ADJUVANT EFFECT OF SYNTHETIC DOUBLE-STRANDED RIBONUCLEIC ACIDS

PROBLEM

The adjuvant effect in guinea pigs of some substance isolated from Bordetellapertussis suspensions has been recently studied. It was observed that RNA preparationscould elicit the production of IgE antibody in a high percentage of the animals. Sincethe adjuvant property of RNA seems to be due to double-stranded RNA molecules, theeffect on IgE production in guinea pigs of a synthetic double-stranded RNA in whichcontamination with lipopolysaccharides (LPS) could be ruled out was studied. Poly-nucleotides, which share many of the biological properties of LPS, were chosen forthis purpose and their effect on the production of IgE by guinea pigs was comparedwith that exerted by LPS.

METHOD

The polynucleotides were purchased as lyophilized polyriboinosinic acid (poly-I)and polyribocytidylic acid (poly-C) (Miles Laboratory, Kankakee, Illinois) andstored at -200C. Doses of complexes are expressed as the total weight of the poly-nucleotides contained in the complexes. LPS was obtained from B. pertussis cells orfrom Salmonella typhosa. Guinea pigs were inoculated intraperitoneally with 10 pgovalbumin (Oa) plus 0.5, 1, or 2 mg/kg poly-IC, or 20 ig/kg LPS. Controls wereinoculated with Oa alone or with Oa plus 1 mg/kg poly-I or poly-C. The animals werebled at different times after immunization by cardiac puncture or by puncture of theophthalmic plexus and the blood was allowed to clot in an ice bath. The serum wasseparated by centrifugation in a refrigerated centrifuge. Serum samples were usedas soon as possible or were kept at -200C when necessary. Antibody activity wasassayed by passive cutaneous anaphylaxis (PCA) using a short sensitization period(24 hours) for IgGl antibody, and a long sensitization period (10 days) for IgE anti-body.

RESULTS TO DATE

Poly-IC was very effective in inducing the synthesis of IgG1 and IgE when atleast 1 mg/kg of the polymer was injected with antigen, but was without effect whenonly 0.5 mg/kg was used. When 2 mg/kg was used, an apparently more persistent anti-body response was induced. The level of IgE antibody produced by the animals stimu-lated with poly-IC was, however, always very low. Poly-IC had an adjuvant effectwhen injected 24 hours after the antigen, but had no effect when injected either 48hours after or 24 hours before the antigen.

The adjuvant properties of LPS, which induces an early preferential productionof IgE in guinea pigs, were compared to those of poly-IC. Results showed that thePCA level of the animals stimulated with LPS was considerably higher than that of theanimals stimulated with poly-IC.

118

S4GNIFICANCE

This research helped understand the mode of action of adjuvants and the mechanismsthat lead to the preferential production of reaginic antibody.

PUBLICATIONS

1. Mota, I. and A. Perini. The effect of a synthetic double-stranded RNA onIgGl and IgE production by guinea pigs. A comparative study with lipo-polysaccharide. Immunology 29:319, 1975.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Ivan Mota, PAHO/WHO Immunology Research and TrainingCenter, Instituto Butantan, Sao Paulo, Brazil.


1974-1975.

119

STUDY AND QUANTITATION OF BODY FLUIDS BY MEANS OF IMMUNONEPHELOMETRY

PROBLEM

Immunochemical analysis of proteins has enormous potential for modern medicine.However, the time-consuming assays usually performed with gel diffusion techniquesneed to be improved and expanded to the more than 100 plasma proteins described to date.

Immunoneplielometry appears to be an effective technique for the immunochemicalanalysis of proteins, when the enhancing effect of polymers is added to the antigen-antibody reaction.

The purposes of this project are to study the methodologic variables of theimmune reaction when a polymer is added to the liquid-free medium; to understand betterthe mechanism of the polymer enhancement; and to develop a simple manual immunonephelo-metric technique to analyze the most important body fluid proteins.

METHOD

Experiments were conducted with single radial immunodiffusion (SRID) and immuno-nephelometry.

(1) Single radial immunodiffusion

The effect of polyethylene glycol 6000 (PEG) on SRID was studied for serum IgG,IgM, IgA, albumin, and haptoglobin, with a view to determining the PEG concentrationthat produces optimal enhancement of the immune reaction. The polymer (O to 6 9/g00 ml)was incorporated to the agar matrix. Precision, sensitivity, and accuracy of the testwere assessed and results of 60-70 serum samples tested by SRID with and without PEG,compared.

(2) Immunonephelometry

A manual fluorimeter (Farrand Optical Co.) was used as a nephelometer. Thespecific antiserum was diluted with PEG (4 to 6 g/100 ml) and mixed with aliquots ofserum in a total volume of 1.0 ml. The light scattered by the immune complexes wasread after 10 min in the apparatus at a 90 angle and 360 nm.

Methodologic variables were studied, like stability of the antiserum-PEG reagent,characterization of proteins that precipated when antiserum was mixed with polymer,nephelometric response of antigen-antibody complexes of different molecular weight,and effect of PEG plus electrolyte solutions on the immune reaction.

RESULTS TO DATE

The enhancing effect of PEG on the antigen-antibody reaction in liquid-freemedium allowed us to develop an automated continuous-flow immunonephelometric methodfor the specific quantitation of human urinary albumin. The speed of the method was60 samples/hr; the sensitivity was 1 mg/liter; and the day-to-day variations were3.8 percent CV. A comparative study of 176 samples tested by standard SRID showed

120

a correlation coefficient of 0.994. Later an automated method was developed for beta2-microglobulin, a low molecular weight urinary protein, very important in assessing pro-teinurias of tubular origin. The latter method has a sensitivity of 0.1 mg/liter andpermitted the processing of 50 samples/hr.

A high-speed (150-210) samples/hr immunonephelometric continuous-flow system wasalso tested. Fibrinogen and albumin were used as examples of application. Immunonephe-lometry was shown to be more precise, fast, and reliable than gel diffusion techniques,and with equal accuracy.

In conjunction with this study, and in collaboration with LKB Instruments ofSweden, an LKB 7400 calculating absorptiometer was adapted into a nephelometer and adiscrete immunonephelometrical system was developed. The system is capable of speedsof 400 samples/hr for serum albumin, with an overall precision of 5 percent CV, and asensitivity of 0.1 mg/liter.

Manual immunonephelometric assay of proteins with use of polymer enhancement wasalso performed using a Turner fluorimeter and human fibrinogen, albumin, haptoglobin,and IgG, as examples to illustrate the principle; but the number of proteins should beexpanded.

Many factors remain unstudied, especially methodologic variables of the antigen-antibody reaction when polymer is present in the medium. The mechanism of action of PEGis unclear and more basic research is needed to clear this point.

SIGNIFICANCE

To be able to quantitate specifically, by means of manual and (or) automated immu-nonephelometry, the more than 100 plasma proteins known will represent a significantprogress in today's medicine. The development of these powerful techniques for the assayof proteins moieties will permit clinicians and researchers to study in detail the patho-physiology and molecular biology of an increasing number of body fluid proteins, whosebiological functions are not clearly understood.

PUBLICATIONS

1. Hellsing, K. and J. Lizana. Immunochemical analysis of proteins using anew discrete nephelometric system. Z Klin Chem Klin Biochem 12:245, 1974.

2. Lizana, J. and K. Hellsing. Automated immunoprecipitin determination ofalbumin in urine using polymer enhancement. Clin Chem 19:673, 1973.

3. ----- and -----. Polymer enhancement of the automated nephelometricanalysis, as illustrated by determination of urinary albumin. Clin Chem20:245-50, 1974.

4. ----- and -----. Manual immunonephelometric assay of proteins, with useof polymer enhancement. Clin Chem 20:1181-86, 1974.

5. ----- et al. Automated determination of beta2-microglobulin in urine.Clin Chem 20:894, 1974.

6. ----- et al. Electronic timing of a high-speed immunonephelometric con-tinuous-flow system. Clin Chem 21:762-65, 1975.

121

7. ----- and K. Hellsing. Analysis of proteins in clinical medicine:specific immunological assays using nephelometry. Rev Med Chile103:260-66, 1975.

OTHER DATA

Dr. Jorge Lizana, Instituto de Bioquímica Clínica, UniversidadAustral, Valdivia, Chile.


1975 - continuing.

122

Grantee:

Funded by:

Timetable:

EPIDEMIOLOGIC STUDY OF MYOCARDIAL ISCHEMIA, HIGH-DENSITYLIPOPROTEIN AND OTHER CORONARY RISK-FACTORS IN TRINIDAD

PROBLEM

The purpose of this study is (1) to establish the prevalence of electrocardio-graphic myocardial ischemia in Trinidadian middle-aged men according to ethnic group,and to relate it to the distribution of the major coronary risk-factors in the commu-nity (plasma lipoproteins, blood glucose, blood pressure. adiposity, level of habitualphysical activity, tobacco smoking); and (2) to explore further the hypothesis thatplasma high-density lipoprotein promotes cholesterol transport from extra-hepatic tis-sues to the liver for excretion, and thereby retards the atherosclerotic process.

METHOD

(1) A privately censused urban population will be stratified according to 10-year-age group and ethnic group (people descended from immigrants of either African orIndian origin) and 800 men aged 40-60 years identified by random sampling.

(2) Blood samples will be drawn in the fasted state for serum lipoproteins andblood glucose. After preliminary lipoprotein precipitation, prepared samples of serawill be freighted to London (Department of Chemical Pathology, St. Thomas Hospital) forcholesterol and triglyceride estimation.

(3) Electrocardiography will be performed before and during submaximal progres-sive test exercise. Oxygen consumption will be monitored during work. Other measure-ments include anthropometry and casual blood pressure. Smoking habits and alcoholconsumption will be recorded.

RESULTS TO DATE

None thus far.

SIGNIFICANCE

The results will provide estimates of the levels of the major plasma lipoproteinsand prevalence of myocardial ischemia in the Trinidadian male community. Thus the studywill test the validity of earlier reports of a higher prevalence of coronary heart dis-ease in men of East Indian origin as compared with those of African descent. By exam-ining the relationships of high-density lipoprotein to electrocardiographic ischemia andcoronary risk (as assessed by risk-factor status) the investigation will examine in moredetail the hypothesis that high-density lipoprotein is an antiatherogenic agent.

PUBLICATIONS

None thus far.

123

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. G. J. Miller, Caribbean Epidemiology Centre, Port of Spain,Trinidad.

U. K. Medical Research Council.

1976-1979.

124

CONTROL MECHANISMS OF DNA SYNTHESIS IN HUMANLYMPHOCYTES AND LEUKEMIC CELLS

PROBLEM

The purpose of this research is to study the mechanisms of cell division incancer. The focus will be on DNA replication, because disordered replication ofgenetic material might be one of the crucial events that determine whether a cellbecomes cancerous or not. The sequence of events that control the DNA replicationin a normal cell was first established. Later, the manner in which this sequence isaltered in cancer cells was explored.

The main questions to be answered are: When a malignant process in the initialstage is detected, are the cells from different patients affected similarly or indifferent steps of the sequence. Second, is the initial alteration:maintained through-out the disease, or do new alterations appear? The answer to these questions willobviously have important therapeutic implications.

METHOD

The program involves the study of DNA synthesis in human lymphocytes, bothnormal and leukemic. For this purpose, blood samples will be obtained from healthyhuman volunteers and from untreated leukemic patients. Their lymphocytes will bepurified by passage through nylon fibers and Ficoll-Hypaque gradients.

After purification, several aspects of DNA synthesis will be studied: DNAsynthesis by whole cells and purified nuclei; analysis of DNA polymerases; role ofthe cell membrane in cellular division; and the effect of specific drugs.

RESULTS TO DATE

We have studied the DNA polymerases of stimulated and nonstimulated lymphocytes.The cytoplasm from nonstimulated lymphocytes contain two enzymatic activities (Cln andClin), whereas the nuclei contain only one demonstrable activity (Nin). The cytoplasmfrom stimulated lymphocytes has only one enzymatic activity' (CIs), whereas the nucleicontain two activities, a minor peak (Nis), and a major one (Nlis).

Some properties of the Nin enzyme, isolated from nuclei of nonstimulated lympho-cytes, were studied. Some of its properties suggest that this enzyme could act in DNArepair.

Analysis of DNA polymerases in several cases of acute lymphoblastic leukemiademonstrated that in one case, the NII leukemic enzyme considerably differed from thenormal one, suggesting a possible role of this enzyme in neoblastic transformation.

SIGNIFICANCE

This project, besides giving information about the mechanisms of control of DNAsynthesis and cell division in normal and cancerous human cells, could also be important

125

from the point of view of its clinical application. If different alterations could bedetected in individual cancer patients, a more rational treatment could be used in eachcase.

PUBLICATIONS

1. Franze de Fernandez, M. T. et al. New properties of the DNA polymerase-Bisolated from nonstimulated normal human lymphocytes. Biochem BiophysRes Commun 65:1409-17, 1975.

2. Fridlender, B. et al. DNA polymerases from nonstimulated and phyto-hemagglutinin stimulated normal human lymphocytes. Biochem BiophysRes Commun 60:983-89, 1974.

3. Mordoh, J. A modified nuclear DNA polymerase. Biochem Biophys Res Commun67:888-94, 1975.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. José Mordoh, Instituto de Investigaciones Bioquímicas,"Fundación Campomar", Buenos Aires, Argentina.

Pan American Health Organization and Consejo Nacional deInvestigaciones Científicas y Técnicas (Argentina).

1974-1976.

126

LATIN AMERICAN CANCER RESEARCH INFORMATION PROJECT

PROBLEM

The Pan American Health Organization is establishing an information system de-signed to monitor the current status of cancer programs and cancer research in LatinAmerica. This Latin American Cancer Information Project (LACRIP) will be integratedto the International Cancer Research Data Bank (ICRDB) Program of the National CancerInstitute (NCI).

The objectives of the project are:

(1) To identify in Latin America the major institutions and centers devoted

to cancer research, diagnosis, treatment, rehabilitation, and related activities.

(2) To locate, collect, select, and disseminate published cancer research arti-cles; abstracts of papers presented at meetings; and descriptions of ongoing cancerresearch projects in Latin America, and to contribute these documents to the ICRDB

Program.

(3) To facilitate mechanisms of communication that will lead to identifyingareas for collaborative cancer research as well as to achieving comprehensive programsof cancer control in Latin America.

METHOD

(1) Pan American Health Organization Country Representatives will provide anupdated country-by-country list of institutions and centers devoted to cancer research,diagnosis, treatment, rehabilitation, and related activities.

The countries participating in the program, so far, are Argentina, Brazil, Chile,Colombia, Costa Rica, Mexico, Peru, and Venezuela. Others will progressively be in-cluded. A coordinator is appointed through PAHO in each of these countries, to assistin the identification of cancer institutions and centers. The coordinators' institu-tions serve as focal points for the collection of data about cancer research projectsto be supplied to the ICROB Program.

(2) PAHO Regional Library of Medicine and the Health Sciences (PAHO/RLM), lo-cated in Sao Paulo, Brazil, will help develop and implement mechanisms for identifying,collecting, and supplying published articles dealing with cancer research to the ICRDBProgram and for the provision of ICRDB services to scientists in Latin America.

RESULTS TO DATE

Information on the major institutions and centers devoted to cancer research,diagnosis, treatment, rehabilitation and related activities in Latin America was pro-vided to the International Union Against Cancer (UICC) for inclusion in their Inter-national Directory.

127

Key individuals have been selected as coordinators in eight major Latin Americancountries, to set forth the objectives of the project. Coordinators in other countrieswill be progressively appointed in the future.

PAHO keeps close contact and collaboration with ICRDB, the Current Cancer Re-search Project Analysis Center (CCRESPAC), in regard to the recommended procedures forcollecting data within each country or area. Each coordinator sends a project descrip-tion to PAHO for review and delivery to CCRESPAC. The latter institution acknowledgesreceipt of the descriptions directly to the investigators and keeps in contact with thecoordinators for annual updating of current projects. The coordinator is kept informedby CCRESPAC of the tentative schedules of technical bulletin publications in each sub-ject of cancer research.

Sixty forms with ongoing cancer research projects were received from the coordi-nators in 1975 and sent on to ICRDB.

A Working Group was convened at RLM in Sao Paulo, in November 1975, with theparticipation of the coordinators, NCI, and PAHO representatives.

SIGNIFICANCE

PAHO's Latin American Cancer Research Information Program is an important stepin the identification of the resources available in the Region for cancer research andtreatment. This information will make it possible to determine rationally potentialareas of cooperation and will facilitate the design of control programs and the trans-fer of technology.

PUBLICATIONS

1. Pan American Health Organization. Working Group on the Latin AmericanCancer Research Information Project. Final Report. PAHO Int Doc D-DC/NCD,Rev. 2, January 1976.

OTHER DATA

Grantee: Dr. Jorge Litvak, Department of Noncommunicable Diseases,Division of Disease Control, Pan American Health Organization,Washington, D.C., U.S.A.

Funded by: U.S. National Cancer Institute.


128

SPOROZOITE-INDUCED IMMUNITY IN MAMMALIAN MALARIA

PROBLEM

The aim of thís research is to develop in a simian system a vaccination procedurethat will provide basic knowledge for the development of a malaria vaccine for man. Theresearch is based on: (a) earlier vaccination attempts in rodents (close to 100 per-cent protection in mice repeatedly immunized with irradiated sporozoites); (b) prelimi-nary results on malarial vaccination in rhesus monkeys; and (c) pilot vaccinationattempts in volunteers, resulting in the protection of three individuals against sporo-zoite-induced Plasmodium falciparum and two against P. vivax malaria, upon theirrepeated exposures to infected irradiated moaquitoes.

Recent findings in the P. cynomolgi-rhesus system showed that only some of theimmunized animals become totally protected. This protection occurred after multipleboosters, given over a long period of immunization. In view of these results, and be-cause of the relatively short-lived protection observed in sporozoite-immunized humans,the possibility of potentiating sporozoite-induced protection by the use of certainadjuvants was explored.

We also intend to establish further baseline data on: (a) sporozoite immuniza-tion in the P. cynomolgi-rhesus system; (b) standardization of parameters for themonitoring of the development of this immune response; and (c) immunization using asimian malaria, characterized by greater analogies to P. falciparum infections in man,namely P. knowlesi in rhesus monkeys.

METHOD

The immunization of rhesus monkeys will follow the approaches below:(a) injection of a large initial sporozoite inoculum followed by repeated boostersover a period of several months; (b) immunization by the bite of infected, irradiatedmosquitoes; (c) administration of Corynebacterium parvum, a potent RES stimulant.These studies include determination of protective immunity and antisporozoite anti-body levels, by establishing both the serum neutralizing activity (SNA) and thecircumsporozoite (CSP) reaction of the sera of these animals.

The mechanism of the effect of these antibodies on the sporozoites is alsobeing investigated at the ultrastructural level. Furthermore, a new method to detectantisporozoite antibodies under field conditions making use of preserved parasitesand fluorescein conjugated antiglobulin is being standardized.

A number of adjuvants will first be screened in rodent malaria and the mostpromising ones assayed in sporozoite-immunized monkeys.

Immunization of rhesus monkeys with sporozoites of P. knowlesi will follow theexperimental design used for P. cynomolgi.

129

RESULTS TO DATE

Work has been completed on a number of aspects related to the immunogenicity andinfectivity of sporozoites of P. cynomolgi and the specificity of CSP precipitationantigen(s) of human and simian malarias (See Research in Progress 1974, p. 166). A con-siderable amount of data was also obtained on the effects of different dosages andschedules of sporozoite administration on the degree of protection and CSP antibodytiters obtained in the immunized rhesus monkeys, and the in-vitro sporozoite neutralizingactivity of their sera. Initial data have been obtained on some of the other researchobjectives, namely antibody induced ultrastructural changes of malarial sporozoites;in-vitro growth of Plasmodia in insect tissue culture; and blast transformation inducedby plasmodial antigen.

Immunization schedule and its relation to protection, CSP, and sporozoiteneutralizing antibodies. Under present conditions of immunization, extensive or totalprotection of the rhesus has been obtained only after a prolonged period of immuniza-tion and upon administration of multiple sporozoite doses. Certain approaches whichappeared to promote a more effective immune response are presently being pursued.The results on serum mediated sporozoite neutralization, in this system, have so farcorrelated well with protection against challenge.

Ultrastructural aspects of antibody-sporozoite interaction (in press, J Immunol,1976). Transmission electronmicroscopy revealed the presence of a prominent surfacecoat surrounding the outer membrane of sporozoites incubated in immune but not in nor-mal serum. Immunoglobulin was shown to participate in the formation of this surfacedeposit. In contrast to the CSP reaction, coat formation also occurred on the surfaceof metabolically inactive parasites.

Ookinete development in vitro. (Res Note, Parasitology, December 1975, inpress).

Blast transformation induced by malarial antigen. Lymphocytes of the peripheralblood of infected and control rhesus monkeys were cultivated to determine their responseto a soluble malarial extract and phytohemagglutinin (PHA). Soluble malarial extractsproduced a stimulating effect on the blastogenesis of lymphocytes from previously sen-sitized, and to a lesser extent, of lymphocytes of normal animals. P. cynomolgi infec-tions also had a suppressive effect on PHA-induced blast transformation, an effect foundto be serum mediated.

SIGNIFICANCE

Studies on sporozoite-induced immunity in simian malaria are necessary to attemptto develop a vaccination procedure against human malaria and to understand the mechanismof host resistance to malarial infections.

It seems certain that much of the background knowledge necessary for the estab-lishment of a successful vaccine against human malaria will have to be derived fromfurther studies of other mammalian malaria systems. This certainly is true for thesimian malarias which present the greatest analogy with the human host-parasite rela-tionship.

On the basis of the present evidence, there appears to be a need for enhancingthe immune response to both systems by the use of adjuvants, higher immunizing doses,use of cleaner antigen preparations, and/or different schedules of immunization. Thepurpose of the present work is to obtain further information on these basic aspects,and complete our previous observations on simian malaria vaccination.

130

PUBL I.CATIONS

1. Krettli, Antoniana U. and Ruth Nussenzweig. Depletion of T and B lymphocytesduring malarial infections. Cell Immunol 13:440-46, 1974.

2. ----- et al. Complement alterations in rodent malaria. Am J Trop Med Hyg,January 1976, in press.

3. Nussensweig, Ruth S. and David Chen. Some characteristics of the immuneresponse to sporozoites of simian and human malaria. PAHO Bull 8:198-204,1974.

4. ----- and -----. The antibody response to sporozoites of simian and humanmalaria parasites: its stage and species specificity and strain cross-reactivity. Bull WHO 50:293-97, 1974.

5. Shapiro, Martin et al. Evaluation of a method for in-vitro ookinetedevelopment of the rodent malarial parasite, Plasmodium berghei. J Parasitol(Research Note), December 1975, in press.

6. Spitalny, George L., Rivera-Ortiz, Carmen-lnes, and R. S. Nussenzweig;Plasmodium berghei: role of the spleen in sporozoite-induced immunity tomalaria. Exp Parasitol, in press, 1976.

OTHER DATA

Grantee: Dr. Ruth S. Nussenzweig, Department of Preventive Medicine,New York University Medical School, New York, New York, U.S.A.

Funded by: Pan American Health Organization, World Health Organization,National Institutes of Health/U.S. Public Health Service,and U.S. Agency for International Development.


131

INTERRELATIONSHIP OF TRYPANOSOMA CRUZI INFECTION AND CHRONICCHAGAS' CARDIOMYOPATHY IN A RURAL POPULATION

PROBLEM

The general objective of this study is to determine the causal relationship ofT. cruzi infection to morbidity. The presence of T. cruzi infection will be ascertainedby selected indicators of infection in a defined rural population and in hospitalizedpatients with Chagas' disease (e.g., serologic and immunologic tests for T. cruzi, xeno-diagnosis, blood cultures, and microscopic examination of blood and cerebral spinalfluid). These indices will be correlated with indicators of disease (e.g., electro-cardiographic abnormalities, history and physical examination, ECG monitoring, chestX-ray, and characteristics of immunological response). The role of other factors intransmission (e.g., house construction, household composition, entomological factors,infection in domestic and peridomestic animals, and maternal and congenital infection)will be investigated. Field epidemiologic methods applicable to other endemic areaswill be developed.

METHOD

A population of over 1,000 individuals located in Castro Alves, 200 km west ofSalvador, Bahia, has served as a defined rural population. The area has been mappedand a complete census taken each year. A base line clinical study of the entirepopulation was completed in 1974, which included history, physical examination, electro-cardiography, and collection of stool, urine, and blood specimens. A well equippedlaboratory, established with the assistance of a grant from PAHO, is located in theFaculty of Medicine of the Federal University of Bahia in Salvador with capabilitiesfor serologic testing--Complement Fixation (CF) and Immunofluorescent Antibody (IFA)--blood culture of T. cruzi, xenodiagnosis, and special immunological studies. Inaddition to the studies described here the facilities have also been useful to otherBrazilian investigators working on related projects. All data collected has beencoded and analyzed with computer assistance in Salvador and Boston.

RESULTS TO DATE

The initial emphasis in the study was on the establishment of a replicablemethodology. Field procedures for clinical examination and specimen collection weredeveloped. Following intensive evaluation, the CF test, with a standard microtitertechnique, was selected as the standard reference procedure to assess reactivity toa T. cruzi antigen. The IFA test is now also available. An abbreviated 5 lead ECGtracing with a modification of the Minnesota Code having an amplified classificationfor conduction defects and arrhythmias was developed and shown to be useful for popu-lation based studies of Chagas! disease.

In the rural study area of Castro Alves, a high rate of seropositivity to T. cruziwas found. There was a gradual rise in the rate to over 60 percent positive in indi-viduals between 20 and 55 years of age, followed by a considerable decline in the olderage groups. The geometric mean titer in the CF reaction did not decline in the olderage groups, suggesting that the decline in prevalence may be explained by an increasedmortality among the seropositive individuals. The presence in a house of a seropositive

132

child under age 5 was shown to be a good indicator of a high rate of seropositivity inother members of the household. Children of seropositive mothers converted to sero-positivity at an earlier age than did others, and there was evidence that the earlyconversion was not solely due to increased exposure.

The type of house construction was found to relate directly to the probabilityof finding triatomines in the house and also to the rate of seropositivity of thoseliving in the house. Individuals living in houses of a mid-stick construction were ata much greater risk of being seropositive than those in houses of brick construction.This association was particularly striking for children and those living in brickconstruction appeared to be protected.

Individuals who were seropositive had a much higher rate of ECG abnormalitiesthan did those who were seronegative. The most common ECG abnormalities associatedwith seropositivity were ventricular extrasystoles and ventricular conduction defects(VCD). In young adult males aged 25-44, VCD and/or arrhythmias were 7 times morefrequent in the seropositive than the seronegative individuals. Further, seropositivemales had about twice the rate of abnormalities than did seronegative females. Amongseropositive individuals the median age of those with VCD is about 15 years youngerthan those with ventricular extrasystoles, which may be a reflection of the naturalprogression of Chagas' disease.

Preliminary results from other aspects of the study include the associationof persistent T. cruzi parasitemia and chronic heart disease, the finding of T. cruziin the cerebral spinal fluid in patients with acute Chagas' disease even without signsor symptoms of CNS abnormalities, and the detection of congenital transmission ofT. cruzi in the offspring of women identified as being serologically positive forT. cruzi before delivery.

SIGNIFICANCE

This study has established a reproducible methodology for quantification of theimportant morbidity associated with T. cruzi infection in rural areas. It providesan understanding of the natural history of T. cruzi infection and identifies otherfactors that contribute to the risk of infection and disease. Through this work itshould be possible to develop more effective means of preventive intervention bothat the individual and at the community level.

PUBLICATIONS

1. Hoff, R. Recent advances in cell-mediated immunity to Trypanosoma cruzi.In: Pan American Health Organization International Symposium on NewApproaches in American Tryppnosomiasis Research, Belo Horizonte, Brazil,18-22 March 1975.

2. Jones, T. C. et al. A technique for isolating and concentrating micro-filariae from peripheral blood by gradient centrifugation. Trans R SocTrop Med Hyg 69:243-46, 1975.

3. Mott, K. E. et al. The relationship of Trypanosoma cruzi seropositivityto electrocardiographic abnormalities within a rural community in north-eastern Brazil. Presented at Ann Meet Soc Trop Med Hyg, November, 1975,New Orleans.

133

4. ----- et al. The epidemiology and household distribution of serologicreactivity to Trypanosoma cruzi. Submitted to Am J Trop Med Hyg.

5. ----- et al. Electrocardiographic classification for studies of Chagas'disease in populations. In preparation for Bull WHO.

OTHER DATA

Grantee: Drs. Kenneth E. Mott, Rodney Hoff, J. Stauffer Lehman, Jr.,Richard H. Morrow, Jr., and Thomas H. Weller, Department ofTropical Public Health, Harvard School of Public Health,Boston, Massachusetts, in collaboration with Drs. ArmenioCosta Guimaraes, José Pericles Esteves, Celso Mario de AraujoPugliese, Mrs. Celina Marques Bittencourt, and Eduardo Mota(ICOMI Fellow), Federal University of Bahia, and with Dr. ItaloSherlock and T. M. Muniz, Instituto Nacional de Endemias Rurais(FIOCRUZ).

Funded by: The Wellcome Trust, Pan American Health Organization, InstitutoNacional de Endemias Rurais (FIOCRUZ), Federal University ofBahia, and Harvard School of Public Health.


134

NATURAL HISTORY OF SCHISTOSOMIASIS MANSONIIN A RURAL POPULATION OF NORTHEASTERN BRAZIL

PROBLEM

To date, few attempts have been made at measuring the impact of schistosomiasison defined populations. Our broad objective has been the creation of a longitudinalstudy of schistosomiasis morbidity in a highly endemic area of rural Brazil. Quantita-tive aspects of infection and disease will be measured and correlated, new hypothesesconcerning morbid sequelae will be tested, and field epidemiologic methods reproduciblein other endemic areas will be developed.

METHOD

A rural population of 1,000 individuals located 200 km west of Salvador, Bahia,has been surveyed by census, qualitative stool examination, and venous blood sample.A substudy of the prevalence of renal disease in 300 individuals included physicalexamination, urine analysis, urine culture, quantitative urine protein, and quantitativestool egg count (Bell's method). The clinical history, the influence of intercurrentinfections (including Chagas' disease), and the significance of the quantitative eggoutput pattern by age as related to morbidity have been tested.

RESULTS TO DATE

The prevalence of S. mansoni infection in Castro Alves increased more rapidlybetween 1 to 4 and 5 to 9 year age groups and the maximum increase in fecal egg countswas observed between the 5 to 9 and 10 to 14 year age groups. Highest counts wereobserved in the 10 to 14 year age group and maximum prevalence (100 percent) was inthe 20 to 24 year age group. In the study group nearly 50 percent of the total fecaloutput was accounted for by 6 percent of individuals who had a mean age of 12.6 years.In children under 15 years of age, the frequency of hepatomegaly and splenomegalyvaried directly with the egg count. In adults, neither splenomegaly nor hepatomegalycould be directly related to infection per se.

SIGNIFICANCE

This collaborative effort has permitted the development of field methodologyand the location of a suitable rural population for cross-sectional and longitudinalstudies of schistosomiasis morbidity. Epidemiologic, clinical, and parasitologichypotheses may be tested and the influence of multiple variables on schistosomiasismorbidity analyzed by computers within Brazil.

PUBLICATIONS

1. Lehman, J. S. et al. The association of schistosomiasis mansoni andproteinuria in an endemic area. Am J Trop Med Hyg 24:616-18, 1975.

135

2. ----- et al. The intensity and effects of Schistosoma mansoni infectionwithin a rural community in northeastern Brazil. Am J Trop Med Hyg 25:1975,

OTHER DATA

Grantee:

Funded by:

Timetable:

Drs. J. Stauffer Lehman, Jr., Kenneth E. Mott, Richard H.Morrow, Jr., Rodney Hoff, and Thomas H. Weller, Department ofTropical Public Health, Harvard School of Public Health,Boston, Massachusetts, in collaboration with the Departmentof Preventive Medicine, Federal University of Bahia, and theInstituto Nacional de Endemias Rurais (FIOCRUZ).

The Wellcome Trust, Pan American Health Organization, InstitutoNacional de Endemias Rurais (FIOCRUZ), Federal University ofBahia, and Harvard School of Public Health.

1973-1977.

136

HUMAN FILARIAS AND THEIR VECTORS IN TRINIDAD

PROBLEM

The purpose of this research is to investigate the prevalence and distributionof Wuchereria bancrofti and its vectors, as well as its interaction with Mansonellaozzardi and its vectors.

METHOD

(a) Blood samples are collected from individuals in selected communities andexamined for microfilariae.

(b) Blood samples from cases of hydrocele are collected and surveys conductedin areas of residence of individuals with microfilaremia.

(c) Selected individuals with microfilariae are examined in detail.

(d) Biting insects from houses of positive individuals are collected anddissected to find infective larvae. Once vectors are determined, their biology isstudied in detail.

RESULTS TO DATE

A preliminary day blood smear survey of four northern coastal villages in 1974showed that 16 percent of the population examined (192 out of 1,203) were positivefor microfilariae of M. ozzardi. Nineteen cases of W. bancrofti were detected.

Dissections of house-resting Culex fatigans mosquitoes have yielded infectionsof W. bancrofti in 2 out of 17 specimens.

SIGNIFICANCE

The epidemiology of W. bancrofti and M. ozzardi in Trinidad is not known. Thepresent study will provide base-line data upon which treatment of the former infectioncan be based, and, if necessary, of the latter also. An understanding of the biologyof the vectors may provide information upon which a program of control can be estab-lished.

PUBLICATIONS

None thus far.

137

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Patrick J. S. Hamilton, Director, Caribbean EpidemiologyCentre, Port of Spain, Trinidad.

U.K. Medical Research Council.

1976-1977.

138

KABOWRA FLY (SIMULIIDAE) STUDIES IN GUYANA

PROBLEM

The purpose of this research is to study the Simuliidae in the Rupununi savannahsof Guyana in relationship to their nuisance potential in the economic development ofthe interior and their possible involvement in disease transmission.

METHOD

Collection of Simuliidae for 15 min at 24 fixed stations at Lethem and its vicin-ity at different seasons. Collection of larvae for taxonomic studies and distributionof biting species. Collection of adults for 5 min at hourly interval between 7:00 a.m.to 7:00 p.m. to determine diurnal activity of the flies.

RESULTS TO DATE

Two species, Simulium incrustatum and S. sanguineum were found biting. A bitingrate of up to 652 flies/man/hour was established for S. incrustatum at one station(No. 9) and 380 man/hour for S. sanguineum at another station (No. 20). Peak bitingrates appeared at the height of the rainy seasons.

S. sanguineum appear to have two main biting peaks in the early morning between7:25 and 8:25 and late afternoon at 6:25 p.m. with an intermediary peak at noon. Peakbiting periods were not established for S. incrustatum.

SIGNIFICANCE

Any control measures would have to be based on a reasonable knowledge of thehabits and ecology of the nuisance species. The present study provides a standardizedmethod of assessing fly density so that the numbers of flies biting at different timesof the year may be compared. The study will also provide base-line data against whichthe effects of any control scheme can be compared.

There is a recent report of a focus of onchocerciasis in the nearby AmazonasState of Brazil where the vector is S. amazonicum. Onchocerciasis also exists in theVenezuelan Amazon region and the possibility of its existence in the Rupununi areas ofGuyana must be considered.

PUBLICATIONS

None thus far.

139

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Patrick J. S. Hamilton. Director, Caribbean EpidemiologyCentre, Port of Spain, Trinidad.

U.K. Medical Research Council.

1975 - continuing.

140

STUDIES OF ARTHROPOD-BORNE VIRUSES

PROBLEM

Arboviruses, including Venezuelan encephalitis, St. Louis encephalitis, anddengue viruses have caused extensive epidemics in the Western Hemisphere in recentyears. Eastern and western encephalitis viruses and other arboviruses of unknownpotential for causing disease in humans and their domestic animals are also widelydistributed in North, Middle, and South America.

The present investigations have the following purposes: (1) to study theecology, geographic distribution, and importance to man and domestic animals ofVenezuelan encephalitis (VE) virus and several other recently discovered arboviruses;(2) to search for enzootic refugia of equine-virulent strains of VE virus; (3) todetermine the factors involved in virus movement from one area to another; and (4) toidentify the factors that may affect the virulence of geographic isolates of VE virusstrains.

METHOD

The program involves monitoring of moist tropical habitats for arbovirus activitythrough the use of sentinel animals (equines and Syrian hamsters), periodic sampling ofsera from resident equine and other domestic and wild vertebrate populations to searchfor antibody development indicating virus transmission, and through attempted isola-tions of viruses from tissues of mosquito vectors or wild vertebrates. Experimentalinoculations of viruses are utilized to determine the potential of a given species ofvertebrate to act as an amplifying host or as a transoort agent for arboviruses, andto determine the virulence of isolated strains of VE viruses for equines.

RESULTS TO DATE

Ecologic studies of VE virus at a marsh habitat near the epicenter of the 1969outbreak in Guatemala revealed that the virus was enzootic there. Venezuelan ence-phalitis virus was isolated in focal habitats yearly during 1968-1973 from sentinelhamsters exposed during rainy seasons (40 strains), or dry seasons (1 strain) and frommosquitoes. Culex mosquitoes yielded 20 strains of VE virus and Mansonia and Aedesone each. Culex (Melanoconion) and Aedes taeniorhynchus were most prevalent near themarsh.

Five strains of group C arbovirus (one identified as Nepuyo) were recoveredfrom sentinel hamsters during 1968-1970 and one strain of Nepuyo virus was isolatedfrom the blood of a person with a febrile illness during 1972. Two strains of Patoisgroup arbovirus came from Culex mosquitoes during 1970.

During wet seasons, sentinel horses became infected by VE virus in a temporallyand geographically progressive manner inland from this enzootic marsh focus; theseobservations were compatible with movement of VE virus from the marsh habitat duringsome wet seasons. Mechanisms of virus movement from the marsh are currently unknown,but bats are under study as a possibility. The potential for birds to act in the inter-continental transport of arboviruses is understudy.

141

The only epizootic activity of VE virus discovered in Central America during1970-1975 occurred in Nicaragua between April and June 1972. Several hundred horsesdied, and neutralizing antibody, like that engendered by epizootic virus, was foundin two young, unvaccinated horses. Whether this virus activity represented persistenceof epizootic VE virus or reintroduction of virus, is unknown.

Seventy-four strains of VE virus recovered from sentinel hamsters or mosquitoesat enzootic habitats in Guatemala during a 2-year period following the 1969 epidemic-equine epizootic were examined for their abilities to produce small plaques in VeroAfrican green monkey kidney cell cultures, like isolates obtained during the epizootic.One strain, recovered from a sentinel hamster in late October 1969 at an enzootichabitat, had growth and antigenic and equine-virulence properties like epizootic virus.None of the other 73 strains produced uniformly small plaques, but 31 formed a fewsmall plaques among large ones. Virions from small plaques of five strains were clonedtwice in Vero cell cultures. One of three cloned strains produced encephalitis anddeath in one of four horses with histopathologic lesions of encephalitis and normalhematopoietic tissues. Another strain produced encephalitis with recovery in one oftwo horses. Virus from blood of the horse that died produced only small Vero plaques.Thus these small Vero plaque clones resembled Central American enzootic strains of VEvirus in Hl and equine-virulence tests, and the small Vero plaque characteristic wasnot a satisfactory marker for consistently isolating equine-virulent, epizootic VEvirions.

SIGNIFICANCE

This research provides information on arboviruses, espec'ially Venezuelan enceph-alitis virus in tropical America, and it evaluates the role of wild vertebrates anddomestic animals in the ecologies of arboviruses. Complete understanding of the naturalcycles of arboviruses will make way for intelligent approaches to the prevention andcontrol of epidemics caused by these agents.

PUBLICATIONS

1. Dickerman, R. W. and C. M. Bonacorsa. Venezuelan equine encephalomyelitisviral infection of newly-hatched chickens and embryonated eggs. Am J VetRes 36:1231-33, 1975.

2. ----- and C. D. Powers. Primary immunoglobulin response of herons to in-fection with Venezuelan encephalitis virus. Infect Immun 11:303-08, 1975.

3. Schaffer, P. A. and W. F. Scherer. Growth of Venezuelan encephalitisvirus and disappearance of Coxsackie A, Rous sarcoma, herpes simplex andvaccinia viruses following inoculation of Aedes aegypti and other mosquitoes.J Med Entomol 11:189-96, 1974.

4. Scherer, W. F. et al. Ecologic studies of Venezuelan encephalitis virus inPeru during 1970-1971. Am J Epidemiol 101:347-55, 1975.

5. ----- and K. Anderson. Antigenic and biologic characteristics of Venezuelanencephalitis virus strains including a possible new subtype, isolated fromthe Amazon region of Peru in 1971. Am J Epidemiol 101:356-61, 1975.

6. ----- et al. The first isolations of eastern encephalitis, group C andGuama group arboviruses from the Peruvian Amazon region of western SouthAmerica. PAHO Bull 9:19-26, 1975. (In Spanish, Bol of Sanit Panam78:485-93, 1975).

142

Drs. William F. Scherer and Robert W. Dickerman, CornellUniversity Medical College, New York, New York, U.S.A.

U.S. Army Medical Research and Development Command andNational Institute of Allergy and Infectious Diseases ofthe United States Public Health Service, Washington, D.C.,U.S.A.

1961 - continuing.

143

OTHER DATA

Grantee:

Funded by:

Timetable:

EARLY BIOPSY DIAGNOSIS OF RABIES

PROBLEM

Present knowledge of rabies is inadequate, largely because in-vivo diagnosticmethods do not exist. Traditional methods for the diagnosis of the disease use onlybrain tissue and require the death of the individual.

A clinical diagnosis of rabies can be confirmed early in the course of thedisease using biopsy specimens of skin sectioned while in a frozen state, and the re-sulting "frozen section" stained with high quality fluorescein-labeled antirabiesglobulin. The sections are examined by ultraviolet microscopy. This technique hasbeen used on dogs, cats, foxes, skunks, raccoons, cattle, monkeys, insectivorousbats, mongooses, and mice, as well as humans. All specimens are processed on a blindbasis to assure objectivity. Most past experience is with tissues acquired at thetime of death. Some tissues were obtained several days before death occurred andbefore a specific and confirmed diagnosis was made by other means.

Rabies virus antigen was detected in biopsy and necropsy specimens of skintissue in a number of human cases--a few from the United States, and most obtained inMexico City. The technique is a good tool to study the pathogenesis of rabies. Thepresence of rabies virus antigen in the skin of mice that have recovered from rabiesis being determined. The reliability and applicability of the technique need carefulassessment.

METHOD

The reliability of this biopsy technique in human or animal cases will be eval-

uated using positive and negative tissues processed on a blind basis, to learn iftraditional diagnostic methods can be circumvented and if the brain-skin correlationcan approach 100 percent. Diagnostic biopsy services will be furnished and all tissuesreceived from bona fide sources will be processed and reported. All the animal casespossible will be accumulated soon after onset or even perhaps in anticipation of onsetfrom Mexico City and from the U.S. Public Health Service, Rocky Mountain Laboratory,Hamilton, Montana. An index of reliability to the biopsy technique in early rabies inthe dog will be established and used also to see if the onset of rabies can be predicted,as in the mouse. The predicted onset is important when there is a serious human exposureinvolved and the dog can still be available. The study of the skin of mice that haverecovered from rabies for days, months, or even 1 or 2 years, is being finalized.

RESULTS TO DATE

The technique has been used to diagnose rabies in humans, dogs, cattle, cats,

mongooses, bats, mice, rhesus monkeys, foxes, skunks, raccoons, and horses. Even thoughthe different species present somewhat different problems in processing and reading,continued experience shows the technique to be highly reliable. It seems likely thata reliable, confirmed diagnosis of clinical rabies, at any stage following onset, can

be made by a biopsy procedure. An in-vivo diagnostic technique will produce fundamentalchanges in the perspective of the morbidity-mortality ratio in rabies.

144

Cases have been diagnosed on their day of onset. Previous experience in miceshows that onset may actually be predicted up to 5 days in advance of symptoms. Thereliability of this prediction is important in view of the serious exposure to humansand the need to retain the animal in quarantine for observation. Indeed, it seemsplausible that the virus appears in the nerve fibers of face skin at about the sametime it appears in the salivary glands, by centrifugal spread from the brain. Thepossibility that the same biopsy procedure can be used to diagnose other viral en-cephalitides of animals and humans (e.g., Venezuelan encephalitis, herpes) is beingexplored.

Evidence of latent infections is easily apparent. Thus, we find virus antigenin the skin of normal animals and in mice that have long recovered from acute rabies.

SIGNIFICANCE

(1) Early diagnosis of clinical rabies in man (or animais) for purposes ofdetermining treatment plans.

(2) Evaluation of the biting animal, and retaining it for quarantine andfurther observation.

(3) Advancing the knowledge of the ecology and morbidity-mortality ratio ofrabies.

(4) The technique may be applicable for diagnosis of other virus infectionsof the brain (e.g., herpes, Venezuelan encephalitis, western encephalitis, LaCrosse,and others) for early specific diagnosis.

PUBLICATIONS

1. Blenden, Donald C. Diagnosis of rabies by immunofluorescent stainingof frozen sections of skin: A preliminary report. J Am Vet Med Assoc161(11):14 95-1501, 1972.

2. -----. Diagnosis of rabies in various species by immunofluorescentstaining of skin. J Am Vet Med Assoc 165:735, 1974.

3. Fuh, T. H. and D. C. Blenden. Biopsy technique for the early clinicaldiagnosis of rabies. Bact Proc, 1976, p. 176.

OTHER DATA

Grantee: Dr. Donald C. Blenden, College of Veterinary Medicine,University of Missouri, Columbia, Missouri, U.S.A.



145

EPIDEMIOLOGY OF RABIES

PROBLEM

The purpose of this investigation is:

In Grenada: To continue and modify the rabies research and surveillance programundertaken by the Grenada Government and the U. K. Medical Research Council.

(1) To study mongooses by: (a) making population estimates to evaluate proba-ble reinvasion of areas previously "cleared" of mongooses by poison; (b) trapping about1,000 mongooses each year throughout the island; (c) maintaining records of mongooseattacks on man and animals and their outcome; (d) examining mongooses brought in afterattacking for the presence of rabies; (e) estimating the prevalence in (a) and (b) aboveof rabies virus and rabies serum neutralizing antibodies (RSNA); and (f) comparing natu-ral and ERA vaccine induced immunity estimated by the titer and duration of RSNA, andthe response to challenge with wild virus.

(2) To continue records of all humans receiving postexposure treatment.

(3) To act as a resource to the Government of Grenada in relation to domesticanimal/livestock vaccination.

In Trinidad and Tobago: To initiate a program in collaboration with the TrinidadVeterinary Diagnostic Laboratory, to estimate the proportion of bat sera with RSNA inselected samples of a variety of species from different localities.

In the Caribbean: To act as a resource in relation to rabies. In particularby (a) testing antibody levels after preexposure treatment of at-risk-groups, e.g.,veterinary laboratory staff; and (b) providing diagnostic facilities, if requested.

METHOD

(1) Grid trapping of mongooses for population estimates and effectiveness ofcontrol after poisoning.

(2) Trapping of mongooses for rabies surveillance in wildlife.

(3) Collating and reporting all incidence involving attacking mongooses andabnormal behavior in domestic animals/livestock.

(41 Examination of serum samples from trapped mongooses for persistence andtiter of naturally acquired rabies serum neutralizing antibody (RSNA).

(5) Vaccination of mongooses with ERA vaccine and measuring antibody response.

(6) Rabies virus challenge in mongooses with and without detectable RSNA.

RESULTS TO DATE

None thus far.

146

SIGNIFICANCE

In Trinidad, where overt rabies is mainly vampire bat/livestock transmitted,the results will provide estimates of the proportion of vampire and other bats withRSNA and their involvement in rabies transmission. The program will provide resourcefacilities for other areas in the Caribbean, if needed.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. C. O. R. Everard, Caribbean Epidemiology Centre, Port ofSpain, Trinidad.


1976-1978.

147

EPIDEMIOLOGY OF LEPTOSPIROSIS IN TRINIDAD AND GRENADA

PROBLEM

The purpose of this research is to study leptospirosis in three defined areasand to estimate:

(1) The prevalence of leptospiral infection in selected human groups;

(2) The proportion of infected animals in selected groups of peridomesticand wild rodents, domestic animals, mongooses, marsupials, and bats;

(3) The distribution of leptospiral serogroups in: (a) the populations in (1)and (2), and (b) certain selected groups at high risk e.g., canefield/agricultural workers; and to examine

(4) The relationship between the serogroups/types found in man with (a) thosefound in animals; (b) human occupation; and (c) the environment, e.g.,season.

METHOD

(1) Attempt isolation of leptospira from patients with febrile illness compati-ble with a diagnosis of leptospirosis in hospital and outpatient clinics.

(2) Examine survey sera for leptospira antibody in the general population andin special at-risk-groups, such as sugarcane workers and abattoir employees.

(3) Attempt isolation of leptospira from wild and peridomestic rats, mongooses,opossums, and bats trapped in a variety of habitats, and also from stray dogs and otheranimals.

(4) Examine sera from animals in (3) above for leptospira antibody.

RESULTS TO DATE

None thus far.

SIGNIFICANCE

This pilot study will be closely associated with national health and veterinaryprograms, and should allow evaluation of the importance of leptospirosis in the areasinvestigated and provide data for subsequent incidence and quantitative transmissionstudies, if warranted, and possible control measures.

PUBLICATIONS

None thus far.

148

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. C. O. R. Everard, Caribbean Epidemiology Centre, Port ofSpain, Trinidad.


1976-1978.

149

DIARRHEA IN A CLOSED COMMUNITY

PROBLEM

The girl's ward of the St. Ann's Mental Hospital, Port of Spain, houses 48 pa-tients, aged 4-22 years, who are mentally retarded or ill and, in some cases, physicallyhandicapped. The standard of hygiene is expectedly low and in addition to periodic out-breaks of illness, there is a chronic problem of diarrhea on the ward. A study was be-gun in 1975 to investigate the occurrence of diarrhea and the microbial agents or otherfactors that might be associated with it.

METHOD

Records were kept of the occurrence of diarrhea, vomitting, and elevated tempera-ture. The standard of personal hygiene was noted. An assessment of the nutritionalstatus was done by the Caribbean Food and Nutrition Institute, using height, weight, andskinfold thickness as indices. Hemoglobin levels were measured.

Three stool specimens from each patient, taken at weekly intervals, were examinedfor the presence of (a) enteric bacterial pathogens, using standard media; (b) fecalparasites, using direct-examination and the formol-ether concentration technique (Tri-nidad Public Health Laboratory); (c) viruses, using tissue cultures and mice; (d) "rota"or "orbi" viruses using the electron microscope (EM).

RESULTS TO DATE

During the first 30 weeks of the study there were on average 5.7 cases of diar-rhea per day (range 0-26). The children most severely affected were young, sufferedsome degree of mainutrition and were unable to take care of their personal hygiene.Salmonella group D, Shigella dysenteriae, flexner, and boyd, and enteropathogenic E.coli were cultured from the stools of 22 children (45 percent). Trichuris trichiurainfestation was present in all but one child, Strongyloids stercoralis in 24 (50 per-cent), and Giardia lamblia, hookworm, and ascaris in 10-17 percent. Adeno-, echo- andcoxsackieviruses were isolated from 63 percent of the patients. Shigella sp., E. coli,strongyloides, and Giardia lamblia were present most often in those with a high fre-quency of diarrhea while Salmonella, hookworm, and viruses did not show this association.No "rota" or '"orbi" viruses were observed by EM.

SIGNIFICANCE

This study concerns an area of microbiological investigation that has been neg-lected in the region.

The project continues with attempts to reduce the prevalence of factors thatmay be contributing to the ill health of the patients. Dietary supplements are beingused and the heavy helminth infestations are being treated with mebendazole. Successof this treatment is to be monitored by quantitative methods.

150

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Caribbean Epidemiology Centre (CAREC).


1976-1978.

151

INFECTIOUS DISEASES EPIDEMIOLOGY IN SOUTH AMERICAN INDIAN POPULATIONS

PROBLEM

Previous studies have concentrated on the vaccination of virgin soil Indianpopulations against measles and rubella. The reactions to the measles live virusvaccine included more than usual fever. The reaction to live virus rubella vaccinewas minimal. The present study aims at (1) collecting sera from these vaccinees todetermine the height and durability of their immune response; (2) following anothergroup receiving measles vaccine in a location where a control group of the Caucasianrace can be observed in parallel, so as to determine better whether the difference isgenetically based; and (3) determining the patterns of Treponema, Clostridia, Myco-bacteria and various virus disease epidemiologies in the primitive Brazilian tribes.

METHOD

In Brazil

Serum specimens were collected from four isolated tribes in Brazil with definedhistories of measles and rubella immunization to determine antibody stability in wildand vaccine-induced immunity, in the absence of booster stimuli.

Specimens and skin tests were used to determine the pattern of infection withtetanus, tuberculosis, treponema, and several viruses in these primitive communities.

Children born since our last visit were vaccinated against measles and DPT.

In Chile

Schwartz further attenuated measles vaccine was also given to 92 Indian andChileno children with negative immunization histories. Daily temperature recordingswere made during the reaction period. Sera were collected to check previous immunestatus and whole bloods and sera were obtained from 150 other children living in thesame remote area to test their immunity and to determine the degree of racial mixing.

Height, weight, arm circumference, and skin fold measurements were taken onboth groups as indicators of nutritional status.

RESULTS TO DATE

In Brazil

(1) Rubella

(a) Naturally acquired antibody was retained in undiminished titer overthe 4- to 12-year postinfection period.

(b) Vaccine-induced antibody titers 2-1/2 years after the rubella vaccineRA27/3 was administered were comparable to naturally acquired levels.

(c) The vaccine caused no more illness in a virgin soil population than incosmopolitan groups.

152

(d) There was little, if any, secondary spread.

(2) Measles

Measles vaccine-induced antibody titers were maintained undiminished but at lowerlevels than in naturally immune subjects.

(3) Influenza

A population with antibody only to AH2N2 flu in 1972 had a broad response in 1974.

(4) BK Polyoma

Brazilian Indian tribes represent almost the only known groups free of this virus.

(5) Tetanus

C. tetani has been found in soil from the Indian villages although the inhabit-ants had no tetanus antibody.

(6) Tuberculosis

One-third of the susceptible persons in one tribe converted to a PPD-positivestatus in 30 months.

(7) Treponema

High rates of antitreponemal antibody are not accompanied by comparable anti-gonococcal rates. Medical examination revealed only one individual with a skin lesionthat might have been caused by Treponema. Serological studies to determine FTA-ABSconversion rates are in progress.

(8) Spirochetal Infections

A very high incidence of treponemal antibody had been found in adults of threeKayapó tribes by means of both the VDRL and FTA-ABS tests. In contrast to the Kayapó,very few Tiriyo and none of 10 Ewarhoyana or 34 Parakanan were positive. In pairedsera collected 27 to 30 months apart, positive tests were generally reproducible but2 percent of the Xikrin and 43 percent of the Mekranoti who had negative tests ini-tially, converted to positive tests.

Sixty-seven serum specimens collected in the southern part of Tahiti 2 yearsafter the peak of a yaws epidemic were tested for comparison: 25 percent of the serawere positive, and the youngest patient was 6 years old. This pattern was similar tothat in the Kayapó.

If the treponemal infection were acquired venereally from non-Indians, gonorrheamay have come with it. One hundred Mekranoti specimens were tested for gonococcal piliantibody. Only two were positive.

The FTA reaction might be a nonspecific cross with some other spirochete. Tenspecimens from each of the tribes Mekranoti, Xikrin, Kuben Kran Kegn, Parakanan, Tiriyo,and Mapuche were tested for Leptospira by agglutination. Positive specimens were foundin each tribe. No correlation with the treponemal tests was found either on the basisof tribe or individual specimens. Species specific tests are in progress.

The same serum specimens were tested for Borrelia. Positive reactions werefound in the Mapuche, but not in the Brazilian Indians.

153

(9) Malaria

Twenty-five serum specimens from each of the Mekranoti and Xikrin tribes have beentested for antibody to three plasmodial species using a fluorescence antibody test. Posi-tive reactions were found against all three species in both tribes. In the Xikrin, allthree antibodies were found in more than 90 percent of the specimens. In the Mekranoti,Plasmodium falciparum with 72 percent positive reactions was more prevalent than theother plasmodial species. We have already reported that 42 percent of the Xikrin and6 percent of the Mekranoti had splenomegaly. The two indices of malaria incidence thusconcur in indicating more infection in the Xikrin. However, by standards based on Afri-can data one would expect that the high rate of serological positivity would have beenassociated with immunity and lower spleen rates in adults. The fact that this was notthe case may be genetically determined.

(10) Amebiasis

Other tests indicate antibody to E. histolytica in 92 percent of the Mekranotibut only 24 percent of the Xikrin.

(11) Nutritional Status

Data were collected on height, weight, skin fold thickness, and arm circumference.Hair root specimens were collected for measurement and sera were available for albumindeterminations. These tests have now been completed and the data analyzed for theMekranoti and Xikrin.

In terms of average stature and weight the two tribes are near the top of therange for South American Indians but below the norms for North American whites. Theweight of infants under 1 year seems to be quite low, but with increasing age they gainedor held their position relative to cosmopolitan norms.

In weight for height, the Kayapó generally compared well with North American norms.Men were a little below it and women a little above. The Mekranoti were a little heavierthan the Xikrin. The few individuals who fell much below this norm were generally knownto be affected by malaria or tuberculosis.

Again, mid-arm bone and muscle circumference measurements compared favorably withNorth American norms for all ages. Two-thirds of the adult women were above the U.S.median for this parameter. Individual values correlated with weight for height.

Serum albumin levels were higher in the Mekranoti than in the Xikrin (4.42 vs4.20 g/100 ml). But neither value was significantly below the U.S. mean of 4.26 (TenState Nutrition Survey). Both Kayapó levels were above that of any New Guinea group.

Hair roots diameters were the only measurements that suggested frequent malnutri-tion. Twenty-two percent of the Mekranoti and 63 percent of the Xikrin fell in the cate-gory of moderate protein-calorie malnutrition. Age and sex differences were not recog-nized in this measurement; however, in our series, roots from children (106 pm) weresmaller than from adults (137 pm) and roots from females (118 pm) were smaller than frommales (124 pm). The Xikrin roots were also smaller (105 pm) than the Mekranoti's (133pm). The proportion of actively growing hairs was normal in all population subsets.

In sum, there appeared to be a little malnutrition associated with chronic infec-tions but, if the hair roots data are discounted, the general level was very good. Thiswork is in manuscript form preparatory for publication.

154

In Chile

(1) Adequate temperature records were obtained on 32 susceptible vaccinatedchildren. These showed 1.070C elevation in 12 Chilenos; 0.610C elevation in 14 Mapuche;and 0.73 0C elevation in 6 mestizo.

(2) Serology indicates that 25 percent of the school population is susceptibleto measles. This would be an easier group to work with than the younger children.

(3) Growth rates and fat-free arm circumference do not differ between the racialgroups.

SIGNIFICANCE

The rubella studies are the first in which it has been possible to study vaccinereactions in a certified fully susceptible population in the absence of booster expo-sures.

The other studies give a picture of infectious disease in primitive populationsthat is more complete and freer of outside influence than any published heretofore.They also point up health problems that may have accounted for the demise of many pre-viously contacted tribes.

The measles study in Chile was too small to be conclusive because bad weatherinterfered with data collection. It suggested either (1) that the enhanced suscepti-bility observed previously in Brazilian Indians was due to lack of cross protectiveimmunity resulting from other infections or, (2) if the effect was genetic, that selec-tion for resistance may occur in a few hundred years with minimal introduction of newgenetic traits from other races.

Medical and immunological services were provided in the areas visited.

The spirochetal studies have increased the probability that the Kayapó are in-fected with a relatively harmless treponeme. This may mean they have an immunity tomore virulent strains and suggests a potential prophylactic for other populations.

The malarial studies indicate a focus of very high endemicity. They suggestfurther that standard criteria for estimating endemicity may not be applicable toSouth America.

The nutritional studies indicate that the measles vaccine reactions are notattributable to malnutrition and that the Kayapo economy provides an adequate sus-tenance.

PUBLICATIONS

1. Black, F. L. et al. Evidence for persistence of infectious agents inisolated human populations. Am J Epidemiol 100:230-50, 1974.

2. -----. Infectious diseases in primitive societies. Science 187:515-18, 1975.

3. -----. Poliomyelitis in isolated populations. JAMA 232:486, 1975.

4. ----- et al. Rubella vaccine reactions and antibody persistence after vac-cine and wild virus induced immunization. J Infec Dis, 1976, in press.

155

5. ----- et al. Nutritional status of the Brazilian Kayapó Indians. In manu-script.

6. Brown, P. et al. Seroepidemiology of human papovaviruses: discovery ofvirgin populations and some unusual patterns of antibody prevalence amongremote peoples of the world. Am J Epidemiol, in press.

7. Napiorkowski, P. and F. L. Black. Influenza A in an isolated population inthe Amazon. Lancet 2:1390, 1974.

8. Salzano, F. M. et al. Blood groups, serum proteins, and hemoglobins ofBrazilian Tiriyo Indians. Hum Biol 46:81-87, 1974.

OTHER DATA

Grantee: Dr. Francis L. Black, Department of Epidemiology and PublicHealth, Yale University School of Medicine, New Haven,Connecticut, U.S.A.

Funded by: Pan American Health Organization and U.S. Public Health Service.


156

CELLULAR STUDIES ON RHEUMATIC AND GLOMERULONEPHRITISPATIENTS IN TRINIDAD

PROBLEM

In Trinidad there is a high incidence of streptococcal infections and, coincidentwith this, a relatively large number of cases of the poststreptococcal sequelae, rheu-matic fever, and acute glomerulonephritis. Thus is presented an opportunity to studyand compare the pathogenetic mechanisms of both diseases.

Although there is a fairly high background incidence of acute poststreptococcalglomerulonephritis in Trinidad, this disease has been known to occur in epidemics asso-ciated with a particular M-type of the group A streptococcus. There is no known familiarincidence of this disease but, rather, it seems more directly related to a particularstrain of streptococcus infecting members of a community. Rheumatic fever, on the otherhand, tends to occur in families and maintains a fairly constant rate in unchangingsocioeconomic conditions. It has been suggested from the humoral immune response tostreptococcal antigens, that rheumatic fever may be due to an abnormal immune responseto streptococcal infections. Two major questions are being asked regarding the sus-ceptibility to and pathogenesis of rheumatic fever: (1) Is there an abnormal immune re-sponse to the streptococcus, i.e., an abnormal immune response gene, in susceptibleindividuals? (2) Does lymphocyte sensitization to streptococcal antigens play a rolein the pathogenesis? A comparison of these parameters in rheumatics with nephritis isof interest because of the apparently different patterns of susceptibility and patho-genesis.

METHOD

Besides the nephritic and rheumatic patients, a control group is being studied,comprising age- and sex-matched individuals with no history of streptococcal infectionor of either of the sequelae. Clinical laboratory tests are performed on all thesepatients including ESR, CRP ASO titer, complement levels and anti-heart antibodies.

Two parameters are being employed to determine the degree of sensitization oflymphocytes from nephritic, rheumatic, and control individuals to isolated cell walland membrane antigens from the group A streptococcus. These are: (a) the directleukocyte migration inhibition techniques, and (b) blastogenesis as measured by 4C-thymidic incorporation by lymphocytes.

The genetic studies are being carried out on families of rheumatic and nephriticindividuals as well as control families. Histocompatibility typing (HL-A) is beingdone on lymphocytes from all available members of approximately 30 families from eachgroup.

Lymphocytes from members of HL-A typed selected families are being compared forreactivity to those of other members in the mixed lymphocyte reaction (MLR). Also,rheumatics are being compared to other rheumatics-in the MLR and nephritics to ne-phritics,

157

RESULTS TO DATE

Results to date indicate that patients with acute rheumatic fever have a lightenedcellular response to group A streptococcal membrane antigens when compared to both con-trol subjects and acute poststreptococcal nephritic patients. Preliminary data indicatethat this response may be dependent on the streptococcal strain used in the test system.There was no apparent difference between rheumatic and control patients when antigensfrom a strain isolated from a skin infection were used. "Throat" strain membrane anti-gens gave a statistically significant response in rheumatic fever patients alone.

Though the reasons for this difference in membrane reactivity are at present un-known, these findings suggest that certain strains of group A streptococci are morelikely to be associated with rheumatic fever than others. This observation is beingfurther investigated using antigens from other "throat" and "skin" strains isolated inTrinidad.

HL-A typing is nearing completion but the results have not yet been analyzed.Preliminary screening of the results suggest that there is an increased incidence ofshared HL-A antigens in parents of rheumatics and that rheumatics themselves have fewertypable antigens than expected. The MLR studies are now in progress.

SIGNIFICANCE

Rheumatic fever and acute glomerulonephritis are important health problems inmany countries of the world. Despite the fact that the association of these diseaseswith infection with the streptococcus has been known for many years, the pathogenesisof these diseases has not been elucidated. The finding of a high degree of lymphocytesensitization to group A streptococcal membrane antigens in rheumatics suggests thatdelayed hypersensitivity may play an important role in its pathogenesis. The asso-ciation of this reactivity with only the "throat" strain may indicate that only certaingroup A streptococci are responsible for the development of rheumatic fever.

Although the data from the genetic studies have not yet been analyzed, theobjective is to be able to determine whether or not an individual is susceptible torheumatic fever and then to prevent the occurrence of the first attack by penicillinprophylaxis.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. John B. Zabriskie, Department of Microbiology and Immunology,Rockefeller University, New York, New York, U.S.A.

Funded by: American Heart Association and Pan American Health Organization.


158

HEPATITIS B IN TRINIDAD

PROBLEM

With the availability of rapid methods of testing for hepatitis B surface antigens(HBsAg), it is possible to differentiate hepatitis B (serum) from hepatitis A (infectious).

HBsAg has been found in the blood of healthy carriers who form a higher propor-tion of the population in certain tropical countries, particularly in Africa, than intemperate countries. It has also been associated with acute and chronic viral hepatitis.

In the Caribbean, cases of infectious and serum hepatitis are commonly reportedon clinical evidence. Study of HBsAg infection in this area is needed for surveillance.The area is of particular interest because of its ethnic heterogeneity, tropical andecologic diversity, and the large numbers of visitors from temperate areas.

The purpose of the study is to determine the prevalence of HBsAg carriers amongselected Caribbean residents and the role of hepatitis B infection in liver and otherdiseases.

METHOD

The study is being carried out in the English-speaking Caribbean and involves thecollection of sera from (a) healthy residents; (b) patients with liver diseases; (c) pa-tients attending venereal disease clinics; (d) persons whose occupation brings them intocontact with blood or its products; (e) household contacts of those found to have hepa-titis B infections; and (f) retarded children.

Antigen was detected by counter-immune electrophoresis; positive sera and se-lected sera from all groups were examined by radio-immune assay (RIA). Antibodies weretested for by passive hemagglutination.

RESULTS TO DATE

A total of 1,416 sera from Trinidad residents have been tested with the follow-ing results:

No antigen was detected in the sera of 1,011 healthy persons, but 22 patientswith liver disease were positive as were 2 patients with Hansen's disease and one labo-ratory technician. As expected, RIA detected more antigen-positive sera. Antibodywas detected in all groups but not the laboratory technician's. In the group of healthypeople antibody presence was not associated with reports of earlier episodes of jaun-dice.

A follow-up of antigen-positive persons has been started. Eight persons withliver disfunction and eight healthy carriers detected by the blood bank of the Port ofSpain general hospital were checked after intervals of 2 or more months, half werestill positive, and 3 had developed low-level antibody.

Household contacts were also studied. One of 53 contacts was antigen negativeand 9 were HBsAg positive.

159

SIGNIFICANCE

This pilot study indicates a low level of HBsAg carriage among healthyTrinidadians and among some of the groups that are reported to be high-frequencygroups in other areas of the world. It also shows that nearly one fifth of the in-fectious hepatitis cases examined were associated with HBsAg. It appears that con-siderable transmission of the antigen takes place without the development of carriersand that a great majority of infections are subclinical.

The prevalence of antigenemia needs to be determined in other areas in theCaribbean and this study underlines the need for the introduction of simple and cheapHBsAg screening techniques for diagnosis and blood banks throughout the region.In addition, there is a need for a test which will identify hepatitis B which isthought to be endemic and a significant cause of morbidity throughout the region.

PUBLICATIONS

A communication entitied "Hepatitis B in Trinidad" was presented at the20th Scientific Meeting of the Commonwealth Caribbean Medical ResearchCouncil.

OTHER DATA

Grantee: Dr. Leslie Spence, Department of Medical Microbiology,University of Toronto, Ontario, Canada; and Dr. Miles C.Williams, Caribbean Epidemiology Centre, Port of Spain,Trinidad.



160

ORGANIZATION AND DEVELOPMENT OF A MULTIDISCIPLINARY

RESEARCH PROGRAM IN THE AMAZON AREA

PROBLEM

The opening of the Trans-Amazon Highway presented an opportunity to carry outresearch on the incidence of infectious diseases in a vast area of virgin forest. TheHighway construction force and settlers are exposed to a variety of ecologic environ-ments, and will almost certainly come into contact with certain zoonotic agents such asarboviruses and Leishmania. In addition, about 80 percent of the colonists come fromother parts of tne country, which creates the possibility that other infectious diseases,e.g., schistosomiasis, and their vectors may be introduced into areas of the Amazonvalley where they have been previously unknown. The main purposes of this research areto study infectious diseases enzootic in the Highway area which may be of importance toman, as well as those which are extraneous to the area but can be introduced by man.Disease agents, vectors, and reservoirs have been studied in the area for years, butlittle quantitative data are available to assess the health burden on the population.A multidisciplinary research program has been developed to obtain the required quantita-tive data on the relative importance of various diseases and to study factors relatingto the distribution, behavior, and general ecology of vectors and reservoirs.

METHOD

A field team is collecting clinical material from humans and is trapping animalsand arthropods along the Highway.

Epidemiology

An active surveillance program consisting of the collection of serum, whole blood,and clinical and environmental information, every 6 months and during acute illness froma stratified random sample of 2,300 persons living along the Highway is being conductednear the cities of Marabá and Altamira. Information is also gathered locally from theFundaçao Serviços de Saúde Publica (FSESP) hospitals, Superintendencia de Campanhas deSaúde Publica (SUCAM), health posts, and private physicians.

Entomology

Routine surveillance in the same areas covered by the epidemiology program isconducted at sites selected to provide comparative data on hematophagous insect densitiesby macrohabitat with special emphasis on studying the temporal and spatial distributionof these insects. Specimens collected in the field are identified in the laboratory andprocessed for pathogen identification and taxonomic purposes.

Mammology

Live trapping is routinely conducted at two sites each in the Marabá and Altamiraareas. Animals are trapped and hunted in various macrohabitats to document speciesabundance by habitat. Serum, whole blood, slides, organs and ecto- and endoparasitesare processed in the laboratory.

161

Laboratory

Laboratory examinations (performed in Belém by the Evandro Chagas Institute,Project 4311, or in Washington, D.C. at the Walter Reed Army Institute of Research) in-clude isolation of viruses in mice and tissue cultures, serological tests for viruses,bacteria, rickettsia, parasites and mycoses, in-vitro chloroquine sensitivity testing,and microscopic examination for blood and fecal parasites.

RESULTS TO DATE

Examinations for toxoplasma antibodies performed on 333 paired sera showed a 62.2percent seropositivity of the original and 54.8 percent of the 6-month sample. Therewere 44 fourfold increases in titer and 18 fourfold decreases. Examinations for lepto-spirosis showed 14.1 percent positive on the original sample, 19.1 percent on the 6-month sample, 43 (12.8 percent) fourfold increases in titer and 35 (10.4 percent) four-fold decreases. All 335 examinations for brucellosis were negative. For group A arbo-viruses, there were 73 (12.4 percent) of 589 sera from the original visit with a titerof ' 1:20, and 63 (13 percent) of 485 from the 6-month visit. There were 11 conversionsfrom negative to positive with 4 of these being a fourfold increase. For group B arbo-viruses, there were 224 (38.2 percent) of 587 sera from the original visit with a titerof k 1:20 and 174 (36.0 percent) of 484 from the 6-month visit. There were 30 conver-sions from negative to positive with 9 of these being a fourfold increase. For otherarboviruses, there were 55 (9.4 percent) of 596 sera from the original visit with a titerof ' 1:20 and 44 (9.1 percent) of 484 from the 6-month visit. The greatest number ofreactions were to Guaroa and to Itupiranga viruses. There were 17 conversions from nega-tive to positive, but none were fourfold increases. The first case of chloroquine re-sistant falciparum malaria in Para State was documented by in-vitro testing. Strongevidence exists that transmission of malaria by previously unknown secondary vectorsoccurs along the Highway. An epidemic investigation in Santarém incriminated Culicoidesparaensis and/or Culex pipiens quinquefasciatus as the vector of Oropouche. Plagueserology on 264 mammal sera were negative. Six hundred mammal blood smears Were nega-tive for plasmodia and treponema but slides from five marsupials and one rodent werepositive for microfilariae.

SIGNIFICANCE

Surveillance programs

These programs will provide much needed quantitative population-based data on thedistribution of disease agents in humans, insect vectors, and vertebrate hosts. Thesedata can be used to direct further research to more specific areas and to assist in theplanning of health services in the area.

Malaria

The existence of falciparum malaria resistant to chloroquine in the State ofPará and the transmission of malaria by unknown secondary, perhaps exophilic vectors,will have a large impact on treatment and eradication programs in the area.

Arboviruses

Aside from yellow fever, Oropouche is the major epidemic arboviral disease of theAmazon region. Discovery of the vector(s) and reservoir(s) will facilitate control ofepidemics that sometimes affect up to 40 percent of urban populations. Information on

162

the endemic activities of 19 arboviruses will aid in assessing their importance anddevising control measures.

PUBLICATIONS

1. Dixon, K. E. et al. Malaria along the Trans-Amazon Highway of Brazil.Presented at the annual meeting of Am Soc Trop Med Hyg, New Orleans, La.,U.S.A., 10-14 November 1975.

2. Lovelace, J. K. and M. A. P. Moraes. Toxoplasmosis among the Ticuna Indiansin the State of Amazonas Brazil. Presented at the annual meeting of Am SocTrop Med Hyg, New Orleans, La., U.S.A., 10-14 November 1975.

OTHER DATA

Grantee: Dr. Mauricio Martins da Silva (Project Coordinator), Departmentof ReSearch Development and Coordination, Division of HumanResources and Research, Pan American Health Organization,Washington, D.C., U.S.A., and Dr. Kenneth E. Dixon (ProjectManager), Evandro Chagas Institute, Belém, Pará, Brazil.

Funded by: U.S. Army Medical Research and Development Command.


163

CARIBBEAN EPIDEMIOLOGY CENTRE

PROBLEM

Following a request from the Government of Trinidad and Tobago and the CaribbeanHealth Ministers' Conference, a special team from the Pan American Health Organizationassessed the problems of epidemiological surveillance in the Caribbean and recommendedthat a surveillance system for communicable and noncommunicable diseases within theCaribbean area be developed. In addition, the team recognized the need for training andupgrading of surveillance and laboratory support services, as well as for the encourage-ment of research in diseases prevalent in the area.

METHOD

The Caribbean Epidemiology Centre (CAREC) was established on 1 January 1975 andincorporated the Trinidad Regional Virus Laboratory, which was transferred, completewith its premises and assets, from the University of the West Indies to the Pan AmericanHealth Organization (PAHO). The functions of CAREC were divided into four categories:

(1) Surveillance

(a) To serve as a specialized technical resource in the field of com-municable diseases and to cooperate in the programs being developedby the governments.

(b) To achieve the reduction of mortality and morbidity associated withcommunicable diseases in the area.

(c) To act as a center for epidemiological surveillance for all countriesin the Caribbean.

(d) To assist or advise governments on the development of effectivesurveillance.

(e) To assist and advise governments, by providing visiting staff expertin surveillance, diagnosis, and control of communicable diseases.

(2) Laboratories

(a) To assess resources and needs of laboratories within the area andassist in their development.

(b) To promote collaborative relations with laboratories that may servethe area.

(c) To provide selective diagnostic laboratory services and facilitiesneeded for surveillance.

(d) To maintain facilities for-the investigation of selected animalviruses.

164

(3) Training

(a) To collaborate closely with universities of the area, particularlyschools of medicine and agriculture, the Commonwealth CaribbeanMedical Research Council (CCMRC), and the Secretariat of theCaribbean Health Ministers' Conference (CHMC).

(b) To provide training in epidemiological surveillance, laboratorydiagnosis, and their field applications, for personnel at variouslevels in health and other related services.

(4) Research

(a) To carry out laboratory and field research on disease problems inthe Caribbean.

(b) To provide facilities for visiting scientists.

(c) To study virus diseases and their ecology.

RESULTS TO DATE

The staff and buildings of the Trinidad Regional Virus Laboratory (TRVL) weretaken over on 1 January 1975 and an administrative staff structure was established.During its first year of operation, the staff engaged in expanding the surveillancefacilities and consolidating and reorienting the Laboratory from pure arbovirus re-search to a back-up facility for surveillance. A surveillance unit was establishedwith one Medical Officer from PAHO and one Medical Officer and one Sanitarian secondedby the Center for Disease Control (CDC), Atlanta. The surveillance unit published amonthly CAREC Surveillance Report, which carries digest of statistics from the Carib-bean, as well as articles on topics of particular significance to the area. Circula-tion of this Report is now over 1,000 copies per month and the number of participatingterritories has risen from 7 to over 20, including a number of Central American coun-tries.

The surveillance unit has also been involved in direct aid to territories in theinvestigation of such outbreaks as typhoid fever in Grenada, parathion poisoning inJamaica, lead poisoning in Barbados, and fish poisoning in Bermuda. Consultation re-quests have come from many of the territories and this work is expanding. At the re-quest of the Centre, PAHO's weekly communicable disease report has been modified toinclude gastroenteritis and malnutrition.

Individual governments have been advised on the development of surveillanceactivities and particular emphasis has been put on a pilot project in Grenada usingmedical auxiliaries, and on the development of new scheduling for the notification ofdisease in Trinidad and Tobago and Jamaica. Partly as a result of work by the Centre,the Government of Jamaica is introducing new notification legislation. The Centre isworking closely with the CHMC Secretariat in the design of new legislation for diseasenotification for the area.

The epidemiological and the laboratory staff of the Centre have paid regularvisits throughout the area and a program of regular visits, 2 to 3 times a year, hasbeen instituted. A major problem has been encountered in introducing good communica-tions and a regular transport of laboratory specimens between the territories.

In December 1975 CAREC organized the first introductory workshop for surveil-lance in the Caribbean, in which all but three territories surrounding the Caribbean

165

participated. It is intended that it will be an annual event initially supported byfunds from CDC.

In February and March 1975, a survey of the surveillance and laboratory facilitiesin the territories was carried out. Detailed recommendations were discussed with ChiefMedical Officers, and CAREC has now supplied many items of equipment for the upgradingof laboratory and surveillance activities with funds provided from a special grant tothe Caribbean Community Secretariat (CARICOM) from CDC. These items include calculators,typewriters, filing cabinets, and simple graphic material. CAREC has also provided sim-ple laboratory emergency supplies. Proficiency testing has been introduced into thelaboratory in Trinidad and will shortly be introduced to other territories starting withBarbados. This is carried out in association with CDC.

Collaborative relationships have been established with the laboratories in con-tributing countries but there is still a long way to go in getting efficient transferof specimens and it is hoped that in 1976-1977, this will be established routinely.CAREC is actively cooperating with CDC, the U.K. Public Health Laboratory Service, theVeterans Administration in Puerto Rico, the laboratories in Guyana, the Public HealthLaboratories in Surinam and Curacao, and further contacts are being developed with thelaboratories of Central America.

In laboratory test services, the Centre has been developing its expertise on awide range, particularly with reference to gastroenteritis, hepatitis, arbovirology,and serology. CAREC is a WHO cooperating center for influenza. Detailed diagnosticfacilities are available in dengue, leptospirosis, poliomyelitis, and rabies. A de-tailed study of the antibody levels for poliomyelitis and other immunizable diseasesthroughout the Caribbean has started in Trinidad and Tobago and Bermuda.

The research facilities for rabies and hepatitis are being developed, and newlinks have recently been formed with Cuba for associated developments. Joint taskforces with veterinarians have been set up in Trinidad and a collaborative plan for theinvestigation of leptospirosis and rabies is now operating in Trinidad and Tobago. Ac-tive surveillance for yellow fever is monitored by a multidisciplinary group represent-ing human, veterinarian, agricultural, and other interests.

The Centre developed a training program based on the results of a survey. Work-shops were held for epidemiologists on epidemiology, surveillance, and sexually trans-mitted diseases. Courses for laboratory workers have been held in cholera, entericorganisms, and parasitology in association with CDC and with the London School of Hy-giene and Tropical Medicine. CAREC intends to follow-up those trained, by proficiencytesting and regular visits by the staff. To facilitate training, a special laboratorywas built at the Centre with funds from CDC. The Centre also developed programs forin-service training in surveillance in Jamaica, Belize, Trinidad, Grenada, and furthersessions are planned for the other territories. Special emphasis has been placed ontraining of medical auxiliaries and determined efforts to report symptom complexes arebeginning to bear fruit.

CAREC's staff have been closely associated with the University of the West In-dies in training postgraduates for the Diplomas in Public Health and Community Healthin Epidemiology in 1975 and in 1976. The Centre has been asked to carry out under-graduate training in microbiology starting in 1977. In addition, close liaison isestablished with the University in Guyana. Students not only from the University ofthe West Indies but from elsewhere are being welcomed in the postgraduate and under-graduate fields to carry out elective and high degree courses.

The library at the Centre is being expanded to meet the new role and a librar-ian has recently been appointed.

166

CAREC developed new techniques in quantitative parasitology, rabies, poliomyelitis,hepatitis, and microtechniques for serology. Current research carried out in associationwith the Medical Research Council of the United Kingdom (MRC) includes rabies in mon-gooses, leptospirosis, streptococcal disease, filariases, and Kabowra fly (in Guyana).The Centre's role in noncommunicable diseases will be expanded with the introduction ofa research project on ischemic heart disease to be carried out in association with MRC.New studies on diabetic gangrene are also proposed in association with a PAHO workinggroup on diabetes in the Caribbean.

SIGNIFICANCE

By providing surveillance and laboratory facilities for the area, the CaribbeanEpidemiology Centre is raising the awareness of the need for surveillance and its appli-cation.

The general level of laboratory services for surveillance is being slowly raised.

A satisfactory advisory and research facility supplying prompt diagnosis is beingbuilt-up both in epidemiology and laboratory services. However, considerable effortsare still required to provide an adequate transport system and proper awareness of thepotential of the Centre among the doctors and laboratory staff of the territories.

The Centre is still developing, but it is encouraging that over the last year anincreasing number of territories have shown an interest in its work and more local staffare being trained to participate actively in the surveillance and laboratory programs.

Importance is attached to the development of projects presented for support tointernational agencies so as to widen the basis of the scientific work of the Centre,while maintaining a realistic approach to its priorities and not spreading its effortstoo thinly, and to avoid duplication of research within the area. It is of particularimportance therefore, for CAREC to establish and maintain good relations with othercenters of microbiological research and service in the area to produce a rational ap-proach to the measurement and control of communicable and noncommunicable disease.

PUBLICATIONS

Monthly CAREC Surveillance Report.

OTHER DATA

Grantee: Dr. Patrick J. S. Hamilton, Director, Caribbean EpidemiologyCentre, Port of Spain, Trinidad.

Funded by: Pan American Health Organization, U.K. Medical Research Council,and U.S. Center for Disease Control.


167

SPHERULIN AND COCCIDIOIDIN: CROSS-REACTIONS IN DERMAL SENSITIVITYTO HISTOPLASMIN AND PARACOCCIDIOIDIN

PROBLEM

Until recently coccidioidin was the only antigenic preparation available fordetecting delayed dermal sensitivity induced by an experience with Coccidioidesimmitis. It is prepared from autolysates of the mycelial phase (saprophytic) of thefungus. A more sensitive reagent, spherulin, was developed in 1969 from the spherulephase (parasitic) of the organism. Use of spherulin showed that coccidioidin failedto detect approximately 30 percent of individuals specifically sensitive to C. immitis.However, the potential of spherulin to detect cross-sensitivity induced by Histoplasmacapsulatum was unknown. This information was considered to be germane because of thecapacity of coccidioidin to detect a histoplasmal experience. Accordingly, both re-agents as well as paracoccidioidin were compared simultaneously in 365 Colombiansoldiers from areas endemic for histoplasmosis but not for coccidioidomycosis.

METHOD

Study population. The study group comprised 368 Colombian soldiers, aged 18to 25, from two regiments stationed in Medellin. The men of one regiment came fromthe State of Caldas and those from the second were from the State of Tolima. Bothstates are endemic for histoplasmosis. Three individuals who had visited Colombia'sone area known to be endemic for coccidioidomycosis, Guajirajin the northeast of thecountry, were excluded from the study.

Design. All subjects were tested initially with only the standard strengths ofthe antigens, using both forearms. Readings were made at 24 and 48 hours. Indurationgreater than 5 x 5 mm at either reading was scored as positive. At 48 hours, 99 histo-plasmin-positive and 107 histoplasmin-negative individuals were given second strengthcoccidioidin and spherulin. None of these subjects had reacted to the standardstrength coccidioidal reagents. Reactions were recorded as before at 24 and 48 hours.Chi-square analysis was used to determine the significance of difference betweenreactor ratios.

RESULTS TO DATE

The rate of histoplasmin sensitivity was high, approximately 27 percent. Theoverall rates obtained with first-strength spherulin and coccidioidin were, however,low and ranged between 0.3 and 0.8 percent. These values translated into rates of 2and 3 percent among histoplasmin reactors compared to rates of 0.4 and 1.1 percentamong histoplasmin-negative subjects. Reactor ratios to the two Coccidioides-derivedreagents were not significantly different.

The second-strength reagents clearly cross-reacted in some histoplasmin-sensitive individuals, however. Among 99 such subjects, there were 8 reactors and4 of these reacted to both antigens, 3 to spherulin only, and 1 to coccidioidin only.The response rates elicited by the two second-strength reagents, 7.1 percent and5.1 percent, were not significantly different. It was, however, significant that

168

reactors to both coccidioidal antigens came only from the histoplasmin-positive group;none was found among the 107 histoplasmin-negative subjects tested.

Of 25 paracoccidioidin reactors, 24 were found in the histoplasmin-positivegroup, compared to 1 in the histoplasmin-negative group. Cross-reaction by para-coccidioidin in histoplasmin-sensitive individuals is known.

SIGNIFICANCE

Because the coccidioidin skin test is frequently positive in subjects sensitizedby H. capsulatum, we considered it particularly important to determine if the moresensitive spherulin reagent similarly cross-reacts in such subjects. At standardstrength both reagents detected extremely low cross-reactor ratios. At 10-times thestandard dose, however, cross-detection of histoplasmin sensitivity was apparent;both antigens cross-reacted comparably in 5-7 percent of histoplasmin-sensitivesubjects only.

It thus appears that despite spherulin's greater sensitivity than that of cocci-dioidin, in detecting an experience with C. immitis, the reagent will not give higher"false-positive" results attributable to cross-antigenicity between C. immitis andH. capsulatum than will coccidioidin.

PUBLICATIONS

1. Deresinski, S. C. et al. Use of spherulin in clinical coccidioidomycosis.International Conference on Lung Diseases, Montreal, P.Q., Canada, May 18-21.(Am Thor Soc), Abstract, Am J Resp Dis 111:916, 1975.

2. Levine, H. B. and G. M. Scalarone. Properties of spherulin, a skin testreagent in coccidioidomycosis. In: Pan American Health OrganizationProceedings of the Third International Symposium on the Mycoses.August 27-29, 1974, Sao Paulo, Brazil, pp. 101-09.

3. ----- et al. Spherulin and coccidioidin: cross-reactions in dermalsensitivity to histoplasmin and paracoccidioidin. Am J Epidemiol 101:512-16, 1975.

4. Scalarone, G. M. et al. Spherulin as a complement-fixing antigen in humancoccidioidomycosis. Am Rev Resp Dis 110:324-28, 1974.

5. Stevens, D. A. et al. Dermal sensitivity to different doses of spherulinand coccidioidin. Chest 65:530-33, 1974.

6. ----- et al. Spherulin in clinical coccidioidomycosis. Comparison withcoccidioidin. Chest 68:697-702, 1975.

OTHER DATA

Grantee: Dr. Hillel B. Levine, Medical Microbiology Department, NavalBiomedical Research Laboratory, University of California, Schoolof Public Health, Naval Supply Center, Oakland, California, U.S.A.

169

Funded by:

Timetable:


1976.

170

STUDIES ON THE DEVELOPMENT OF SEROLOGIC ANTIGEN(S) FORPARACOCCIDIOIDES BRASILIENSIS YEASTS GROWN IN VITRO

PROBLEM

One of the several drawbacks to a more adequate study of paracoccidioidomycosis--both the infection and the disease--is the absence of a reproducible and/or characterizedantigen for use in serologic tests from either yeast or mycelial phase of Paracoccidioi-des brasiliensis. Using the amino acids delineated in the amino-peptidases profileof P. brasiliensis yeasts, a simple, completely synthetic liquid medium was developed,which supported luxuriant growth of these yeasts at 370C within 24-48 hours after trans-fer from the maintaining medium, brain-heart infusion agar supplemented with 0.1 percentcystine and 1 percent glucose.

The present investigations in North America have the following purposes: (1) tostudy the yeasts antigens of P. brasiliensis prepared in the synthetic medium; (2) toselect the strain of P. brasiliensis with greatest reactivity (antigenicity) persistingduríng cultivation exclusively in synthetic medium; (3) to compare the hyperimmune seradeveloped in rabbits with sera from proved cases in humans.

METHOD

The proposed study involves (a) growing yeasts of P. brasiliensis in the syntheticmedium; (b) preparing antigens--whole cell homogenates, cytoplasmic and culture filtrate;(c) immunizing rabbits with these antigens and collecting their sera for antibody detec-tion; and (d) characterizing antibodies produced by the antigens. Yeasts are grown inthe synthetic liquid medium with continuous gyratory motion at 370C for various hoursand removed by centrifugation. Celis are broken by Braun MSK homogenizer. Cytoplasmicantigen (soluble fraction of whole cell homogenate) is obtained by centrifugation ofthe homogenates. Culture filtrate is dialyzed (vs water) and concentrated by hollowfibers. The nitrogen and carbohydrate contents of the cytoplasmic and culture filtratefractions are determined by the Lowrey and phenol-sulfuric acid methods, respectively.Hyperimmune sera are developed in rabbits. Such sera are used to evaluate and to char-acterize the immunological reactivities of the antigens (soluble fractions) by means ofimmunodiffusion and crossed immunoelectrophoresis.

RESULTS TO DATE

Experimental results to date indicate that:

(1) P. brasiliensis yeasts produce the best cytoplasmic and culture filtrateantigens in the liquid synthetic medium at 120 hours of growth at 370C.

(2) Cytoplasmic antigen contains components found in whole cell homogenate,cell wall, and culture filtrate.

(3) Cytoplasmic antigen shows many specific and distinct precipitin bands withpooled homologous rabbit antisera and with one proved human case serum.

171

(4) Serum from a proved human case of paracoccidioidomycosis, as well as rabbitsera hyperimmunized with cytoplasmic antigen, show cross reaction with cytoplasmicantigens of Histoplasma capsulatum yeasts and Blastomyces dermatitidis yeasts but notwith cytoplasmic antigens of yeast and mycelial phases of Candida albicans.

(5) The cytoplasmic antigen of P. brasiliensis yeasts contains at least 27components as demonstrated by crossed immunoelectrophoresis against its pooled homol-ogous hyperimmune rabbit sera.

SIGNIFICANCE

The research provides information on the antigenic composition of P. brasiliensisyeasts grown in the synthetic medium. The cytoplasmic antigen produced in the syntheticmedium is reproducible, economical, and specific to its homologous sera from hyper-immuned rabbits and one proved human case serum.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. Charlotte C. Campbell, Department of Medical Sciences,Southern Ill:inois UnJversity, School of Medicine, Springfield,Illinois, U.S.A.



172

PRODUCTION OF A LATEX-PARACOCCIDIOIDIN REAGENT FOR THESEROLOGIC DIAGNOSIS OF PARACOCCIDIOIDOMYCOSIS

PROBLEM

Serologic tests are of great value in the diagnosis of paracoccidioidomycosis.Two standard procedures, agar gel immunodiffusion and complement fixation, are currentlyused. Unluckily, however, few laboratories in the endemic areas of Latin America areperforming such tests, probably because of inadequate facilities or lack of trained per-sonnel. A latex reaction would have the advantage of being extremely simple to perform,even in the most restricted laboratory facilities.

The present investigation has the following purposes:

(1) To develop a latex-paracoccidioidin reagent that will permit the detectionof anti-P. brasiliensis antibodies in sera.

(2) To determine the sensitivity, specificity, and reliability of the test.

(3) To correlate results of the latex test with those of the standard serologicprocedures used in the diagnosis of paracoccidioidomycosis.

(4) To evaluate on the basis of the results obtained, the convenience of dis-tributing the reagent to laboratories within the endemic area.

METHOD

The project involves a series of steps: (1) preparation of a large batch ofconcentrated paracoccidioidin antigens (yeast and mycelial) from P. brasiliensis;(2) obtention of the latex-paracoccidioidin reagents (yeast and mycelial); (3) deter-mination of the optimal conditions for the sensibilization procedure; (4) developmentof the latex-paracoccidioidin test; (5) determination of the specificity and sensi-tivity of the test using known human sera from patients with paracoccidioidomycosis,histoplasmosis, tuberculosis, as well as from normal individuals; and (6) determinationof the stability of the reagent.

RESULTS TO DATE

A reactive latex-yeast paracoccidioidin reagent has been obtained. Varioustrials were made before reaching this point as the pH of the buffer solution and thesensitization of the particles had to be worked out before obtaining a reagent suitablefor titration. Once this was accomplished, the conditions for the slide latex testwere defined using known paracoccidioidomycosis sera as well as sera from normal con-trols.

To date, 50 serum samples from patients with proved paracoccidioidomycosis havebeen studied. Approximately half of the specimens were collected at diagnosis. Thetest was positive in 78.0 percent of the patients in this group and in 60.0 percent ofthose bled during treatment. A large percentage of the patients had titers of 1:64 orabove with the latex reagent.

173

Cross-reactions, in the range of 30.0 percent have been noticed in patients withhistoplasmosis at the initial 1:8 serum dilution; with additional dilutions (1:16, 1:32),cross-reactions tend to disappear. Emphasis is being placed on quantification to deter-mine significance of the titers.

Analysis of 100 serum samples from tuberculous patients yielded 3 positivespecimens only. Analysis of 100 serum samples from blood bank donors gave 5 positivesamples at the 1:8 dilution. Data submitted here constitute preliminary results onlyas a larger number of tests involving all types of sera is planned for the near future.Trials with the latex-mycelial paracoccidioidin are being studied at the same time.

SIGNIFICANCE

This research will provide useful information on a simple laboratory test forthe diagnosis of paracoccidioidomycosis. From the results obtained thus far, the testprovides adequate screening of the patients in the active stage of the disease.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Angela Restrepo-Moreno, Facultad de Medicina, Universidadde Antioquia, Medellin, Colombia.


1974-1976.

174

STUDY OF CROSS-REACTIVITY AMONG FUNGAL SKIN-TEST ANTIGENS

PROBLEM

The primary purpose of this study is to determine the degree of cross-reactivitythat may be expected among fungal skin-test antigens derived from Paracoccidioidesbrasiliensis, Histoplasma capsulatum, and Sporotrichum schenckii in endemic zones ofCentral and South America, where the respective homologous diseases are prevalent. Asecondary objective is to determine the incidence of specific reactivity to each anti-gen in the different geographic areas included in the study.

METHOD

A large sample of the normal population, comprising all age groups, was simul-taneously skin-tested with three antigenic fractions derived respectively from Para-coccidioides brasiliensis, Histoplasma capsulatum, and Sporotrichum schenckii. Thefractions were selected on the basis of sensitivity studies previously carried out inpatients with active fungal disease. The testing was done by various collaboratinginvestigators working and domiciled in selected geographic areas of the followingcountries: Brazil, Colombia, Costa Rica, El Salvador, Guatemala, Panama, and Venezuela.Testing was actually begun in November 1973 and was continued until an adequate andcomparable number of individuals had been tested in each geographic area. The data wasthen analyzed to determine the degree of cross-reactivity, if any, among the three an-tigens utilized in the study.

RESULTS TO DATE

To date 1,022 persons have been tested in El Salvador, 393 in Venezuela, and94 in Guatemala. Results of skin testing in Brazil and Panama have not yet been re-ceived. In El Salvador, of 952 persons who had negative reactions to either polyblasto-M (antigen from P. brasiliensis) or polysporo-Y (antigen from S. schenckii) 124 or 13percent gave positive reactions to histoplasmin. This figure represents the miínimumlevel of specific histoplasmin sensitivity among the group tested. If there is nocross-reactivity between histoplasmin, polyblasto-M, or polysporo-Y, then one wouldalso expect approximately 13 percent reactivity to histoplasmin among the 70 personswho gave positive reactions to either polyblasto-M or polysporo-Y. Actually, 83percent of these 70 persons had positive reactions to histoplasmin and 66 of these 70persons had positive reactions to polyblasto-M, indicating a high degree of cross-reactivity between the latter two antigens. This finding is reinforced by the factthat among 842 persons who reacted negatively to histoplasmin, only 12 or 1.4 percentgave positive reactions to polyblasto-M. Among 180 positive reactors to histoplasmin,58 or 32 percent gave positive reactions to polyblasto-M antigen. The incidence ofreactivity to polysporo-Y was quite low, and was essentially the same among bothpositive and negative reactors to histoplasmin, as well as among positive and negativereactors to polyblasto-M. This finding indicates that there is no significant amountof cross-reactivity between polysporo-Y and either histoplasmin or polyblasto-M. Thedata obtained in Venezuela and Guatemala are essentially similar to those observed in.El Salvador with respect to the degree of cross-reactivity between histoplasmin andpolyblasto-M.

175

SIGNIFICANCE

To assess the true public health importance of mycotic diseases in Central andSouth America it is necessary first to ascertain the prevalence and distribution ofeach disease. This will entail the mapping of endemic areas by means of skin-testsurveys. For meaningful interpretations, it is essential that the degree of speci-ficity of the antigens employed in such surveys be known, since an antigen that cross-reacts widely with other fungal diseases prevalent in the surveyed area would have verylimited usefulness.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. John D. Schneidau, Jr., Department of Microbiology andImmunology, Tulane University School of Medicine, New Orleans,Louisiana, U.S.A.


1973-1976.

176

USEFULNESS OF THE INDIRECT IMMUNOFLUORESCENCE TESTIN LUNG ASPERGILLOSIS

PROBLEM

A review of the literature on the diagnostic value of the indirect immunofluo-rescence test in lung aspergillosis shows lack of agreement about its sensibility andspecificity.

The purpose of this study is to compare the results of the serologíc testscommonly performed in lung aspergillosis with those obtained with the indírect immuno-fluorescence technique.

METHOD

Studies were made of serum samples from 51 patients with mycologically confirmedlung aspergillosis; 14 patients with paracoccidioidomycosis, 11 patients with histo-plasmosis, 10 patients with tuberculosis, 7 patients with systemic candidiasis, 6patients with fibrosis of the lung, 3 patients with coccidioidomycosis, 1 patient withpulmonary aspergilloma by Aspergillus flavus, and 60 healthy controls. Complement fixa-tion, agar gel immunodiffusion, and agarose immunoelectrophoresis tests were carriedout with a metabolic antigen of Aspergillus fumigatus prepared according to the methodof Negroni et al. These tests were performed following the techniques of the PanAmerican Health Organization (complement fixation), Negroni et al (gel immunodiffusion),Negroni and Costa (immunoelectrophoresis), and Drouhet et al (indirect immunofluores-cence). The mycelium was fixed by heating and serum dilutions from 1:20 to 1:640 inphosphate buffered solution (pH 7.2) were examined. A positive and a negative controlserum were studied in each group of reactions. Rabbit antihuman globulin serum labeledwith fluorescein isotiocinate from the Pasteur Institute of Paris, diluted 1:100, wasemployed.

RESULTS TO DATE

The 60 healthy controls had negative reactions with the gel immunodiffusion,complement fixation, and immunoelectrophoresis tests. The indirect immunofluorescencetechnique yielded 9 false positive reactions (8 with a titer of 1:20 and 1 with atiter of 1:40). The complement fixation, the immunodiffusion, and the immunoelectro-phoresis tests were negative in all the patients with lung diseases, except one patientwith chronic disseminated histoplasmosis. The latter patient had a positive complementfixation test with a 1:8 serum dilution. The results of the indirect immunofluorescencetest in patients with pulmonary diseases were as follows:

Diseases No. of Patients Positive Reactions Titers

Paracoccidioidomycosis 14 3 1:160 - 1:20Histoplasmosis 11 7 1:320 - 1:20Systemic candidiasis 7 4 1:640 - 1:20Coccidioidomycosis 3 - -Tuberculosis 10 3 1:160 - 1:40

177

Pulmonary fibrosis 6 1 1:20Aspergilloma by Aspergillus

flavus 1 1 1:40

The complement fixation tests were positive in all but 1 of the patient withconfirmed aspergillosis (titers from 1:8 to 1:1024). The gel immunodiffusion testswere positive in all the patients (titers from undiluted sera to 1:32). The agaroseimmunoelectrophoresis technique showed a precipitation arc in 50 of the aspergillosissera. One to seven arcs were observed with the metabolic antigen. The immunofluores-cence tests gave positive results in 49 of 51 cases of aspergillosis (titer from 1:20to 1:640; 35 of them were positive with the serum dilutions 1:80 or above).

SIGNIFICANCE

The gel immunodiffusion, immunoelectrophoresis and complement fixation testsshowed a high degree of sensitivity and specificity in the serologic diagnosis of pul-monary aspergillosis. The indirect immunofluorescence test does not seem to offer anyadvantage over the other serologic techniques employed in this disease, being lessspecific and no more sensitive than the others. Cross-reactions between aspergillosisand other mycoses, especially histoplasmosis and systemic candidiasis, were observedwith this serologic method.

PUBLICATIONS

1. Drouhet, E;, cited by Biguet, J. et al. Le diagnostique biologiquedes aspergilloses. Torax (Montevideo) 20:33-47, 1971.

2. ----- et al. Valeur de l'immunoprécipitation et de ltimmunofluorescenceindirecte dans les aspergilloses bronchopulmonaires. Ann Inst Pasteur123:379-95, 1972.

3. Joung, R. C. and J. E. Bennett. Invasive aspergillosis (absence ofdetectable antibody response). Am Rev Resp Dis 104:710-16, 1971,

4. Negroni, R. et al. Estudio comparativo de las reacciones serológicasquantitativas con un antigeno metabólico de Aspergillus fumigatus.Mycopatologia 48:275-87, 1972.

5. ---- and M. R. 1. de ElTas Costa. Inmunoelectroforesis en gel de agarosay precipitación en tubo en el estudio serol6gico de la aspergillosis.Rev Asoc Arg Microbiol 6:1-6, 1974.

6. Pan American Health Organization. Manual of standardized serodiagnosticprocedures for systemic mycoses. Part II: Complement fixation test.Pan American Health Organization, Washington, D.C., 1972.

7. Thomas, A. et al. Diagnostique sérologique de l'aspergillose par immuno-fluorescence sur coupes de reins de lapins inoculas avec Aspergillusfumigatus. Bull Ass Dipl Microbiol Farm Pharm Nancy 115:27-37, 1969.

8. Warnock, D. W. and G. F. Eldred. Immunoglobulin classes of antibodiesto Aspergillus fumigatus in patients with pulmonary aspergillosis.Sabouraudia 13:204-08, 1975.

178

Dr. Ricardo Negroni, Department of Microbiology and Parasitology,School of Medicine, University of Buenos Aires, Buenos Aires,Argentina.


1975.

179

OTHER DATA

Grantee:

Funded by:

Timetable:

DETERMINATION OF SERUM ANTIBODIES TO ASPERGILLUSIN THE MARACAIBO POPULATION

PROBLEM

The purpose of this study is to determine (a) whether universal antibody responseto Aspergillus can be found among healthy persons, patients with collagen diseases, andpatients with pulmonary aspergillosis in Maracaibo; and (b) whether there is a corre-lation between the presence of serum precipitin antibodies and skin-sensitizing anti-bodies to Aspergillus sp., with clinical findings, in patients consulting for respiratoryproblems, and the presence or absence of true bronchopulmonary aspergillosis.

METHOD

Four different groups of subjects were studied: 200 attending an outpatientclinic; 100 attending the outpatient asthma and allergy clinic; 100 attending the out-patient chest clinic; and 100 randomly chosen from a healthy population surveyed forToxoplasma antibodies in a previous epidemiologic study.

Microimmunodiffusion tests for Aspergillus precipitins were done followingKaufman's recommendations (Manual of Standardized Serodiagnostic Procedures forSystemic Mycoses) and using a 1/8-in plexiglass matrix with a 17-7 well pattern forall 400 patients' sera and 100 control sera. Aspergillus fumigatus, A. Niger, andA. flavus antigens were prepared by concentration of acetone precipitated culturefiltrates from 5-week old Sabouraud dextrose broth cultures. Proper controls forantigens and for antisera for Aspergillus antigens were supplied by the Center forDisease Control's Bureau of Laboratories.

In addition, sputum or bronchial washing cultures, aspergillus antigen skintests, and aspergillus precipitin tests were performed on 11 hospitalized patientswith clinical diagnoses of aspergillosis, aspergillomas, or pulmonary mycosis, and156 sputum cultures were done on nonhospitalized patients to investigate mycotic flora.

RESULTS TO DATE

Thirty-one percent of the patients and 30 percent of the controls showed pre-cipitin antibodies in their sera. Of the total patient population, 22.6 percent hadskin-sensitizing antibodies to Aspergillus sp., but the incidence was higher amongthe patients with respiratory problems and allergic conditions. No true allergicbronchopulmonary aspergillosis cases were detected despite the relative high incidenceof precipitin antibodies to Aspergillus sp. in Maracaibo.

In the group of 11 hospitalized patients, 7 grew Aspergillus sp., in the sputumcultures but none in the bronchial washings. Of the 156 sputum cultures done to in-vestigate mycotic flora, 20 percent grew Aspergillus sp., 15 percent grew Cladosporiumsp., and 10 percent grew Penicillium sp., and, as expected, 60 percent grew yeast sp.

180

SIGNIFICANCE

The RAST assay for measuring IgE antibodies to Aspergillus should be thetechnique to use for the diagnosis of allergic bronchopulmonary aspergillosis. Thisconclusion was arrived at in view of the reported evidence that elevated IgE antibodyto A. fumigatus is present in most patients with allergic bronchopulmonary aspergillosisbut not in patients with a variety of pulmonary conditions whose sera contain A. fumi-gatus precipitins.

PUBLICATIONS

1. Arbesman, C. E. et al. IgE antibodies in sera of patients with allergicbronchopulmonary aspergillosis. Clin Aller 4:349-58, 1974.

2. Bardana, E. J. et al. The primary interaction of antibody to componentsof aspergilli. II. Antibodies in sera from normal persons and frompatients with aspergillosis. J Aller Clin Immunol 50:222-34, 1972.

3. Casas Rincón, G and H. Méndez Romero. Estudio de los hongos atmosféricosde la ciudad de Maracaibo (study on the atmospheric fungi of the city ofMaracaibo). Kasmera 3:89-109, 1969.

4. Coleman, R. M. and L. Kaufman. Use of the immunodiffusion test in theserodiagnosis of aspergillosis. Appl Microbiol 23:301-08, 1972.

5. Meyer, R. D. et al. Aspergillosis complicating neoplastic diseases.Am J Med 54:6-15, 1973.

6. Serrano, H. Aeroalergenos y alergias respiratorias en Maracaibo(aeroallergens and respiratory allergies in Maracaibo). Kasmera:4:107-270, 1973.

7. -----. Estudio sobre la incidencia de anticuerpos séricos para Toxo-plasma en la población de Maracaibo y un pueblo rural del Estado Zulia(Venezuela) y comparación de tres métodos serológicos distintos (studyon the incidence of serum antibodies to Toxoplasma among the Maracaibopopulation and a rural town of the Zulia State (Venezuela) and comparisonof three different serology techniques). Kasmera 5:75-101, 1974.

8. Spiro, R. G. Analysis of sugars found in glycoproteins. In: Neufeld,E. F. and V. Ginsburg (eds.), Methods in Enzymology, vol. 8, pp. 3-26.Academic Press, New York, 1966.

OTHER DATA

Grantee: Dr. Guillermo Casas Rincón, Mycology Section of the Microbiologyand Tropical Medicine Department, University of Zulia Schoolof Medicine, Maracaibo, VeneZuela.



181

EXPERIMENTAL TAXONOMIC STUDIES OF CERTAIN HYPHOMYCETESPATHOGENIC TO MAN

PROBLEM

The taxonomy of the dermatophytic hyphomycetous fungi is quite confused,primarily because of limited and often obscure data available from light microscopicand culturing studies alone. There is a real need for the assimilation of informationon these fungi derived from both conventional and experimental systematic approaches,clarification of taxonomic problems, and presentation of a working classification ofthese microorganisms.

These investigations of dermatophytic fungi have the following purposes:(1) to compare the cultural features of pathogenic hyphomycetes when grown axenicallyon artificial substrate; (2) to determine the different modes of conidiogenesis(asexual reproductive cycles) in these fungi and utilize these data in the clarifi-cation of generic concepts; (3) to establish a working classification of dermato-phytes based primarily on cultural and developmental characters.

METHOD

The fungi to be examined include Phialophora, Exophiala, Epidermophyton,Microsporum, Trichophyton, Chrysosporium, Arthroderma, Ctenomyces, Nannizzia, andother related genera. Cultural studies will involve comparative analyses of growthrate and gross morphological features of these fungi grown on different media(potato dextrose, malt extract, corn meal, pablum cereal,and Sabouraud 's agar) atvarious temperatures. In addition, variations in the above media (carbon source,pH, glucose content, etc.) will be used to test the degree of polymorphism of thefungi. The microorganisms will also be tested for their growth potential in thepresence of urea, ability to utilize starch, and ability to utilize keratin.

Light microscopic studies will involve: (1) morphological examinations offixed material obtained from the various cultural procedures outlined above;(2) time-lapse analyses of conidium and conidiogenous cell development. For thislatter procedure, the fungi are grown in a specially-designed culture chamber.

Ultrastructural examinations include transmission and scanning electronmicroscopy and freeze-etching. Emphasis is placed on correlating results of theseultrastructural studies with the data obtained from light microscopy.

RESULTS TO DATE

Developmental and ultrastructural investigations of Sporothrix schenckii,Exophiala salmonis, Geotrichum candidum, and Microsporum canis were examined usinglight and scanning electron microscopy. The fungi, grown in pure culture on potatodextrose agar under defined conditions, demonstrated four different kinds of coni-diogenesis. The taxonomic value of these developmental characters was assessed.

One of the basic modes of conidiogenesis in imperfect fungi is demonstratedby Geotrichum candidum and this developmental process was extensively studied using

182

ultrastructural and light microscopic techniques. A consideration of other species ofimperfect fungi demonstrating similar mechanisms of conidiogenesis was explored.

Revision of the generic concepts of Phialophora, Rhinocladiella, Torula,Sporotrichum, Exophiala, Hormodendrum, and Fonsecaea has been completed, basedprimarily on new taxonomic data derived from developmental and ultrastructural in-vestigations of these fungi.

SIGNIFICANCE

The aim of the project is to present clear definitions of conidium and coni-diogenous cell development in the dermatophytic fungi, solve taxonomic problems withthe aid of new data and thus revise the systematics of this important group of humanpathogens. At the same time, an attempt will be made to establish a functional orworking classification which will be useful to clinical microbiologists. In thisregard, all data derived from this project will be compiled in a "Laboratory Manualfor Practical Identification of Dermatophytic Fungi", which is being prepared incooperation with research personnel at the Center for Disease Control, Atlanta,Georgia.

PUBLICATIONS

1. Cole, G. T. A preparatory technique for examination of imperfect fungiby scanning electron microscopy. Cytobios 12:115-21, 1975.

2. -----. The thallic mode of conidiogenesis in the fungi imperfecti.Can J Botany 53: 1975, in press

3. -----. Conidiogenesis in pathogenic hyphomycetes. 1. Sporothrix,Exophiala, Geotrichum, and Microsporum. Sabouraudia 13:76-93, 1976.

4. -----. Application of scanning electron microscopy to developmentaland taxonomic studies of the fungi imperfecti. Proc IITRI Symp,Toronto, 1976, in press .

5. ----- et al. Developmental and taxonomic considerations of the blackyeasts, in preparation.

OTHER DATA

Grantee: Dr. Garry T. Cole, Department of Botany, University of Texas,Austin, Texas, U.S.A.

Funded by: U.S. Army Medical Research and Development Command andPan American Health Organization.


183

KERATINOPHILIC FUNGI AND ADIASPIROMYCOSIS IN ONTARIO

PROBLEM

The purpose of this project is to expand studies on the distribution and mor-phology of keratinophilic (geophilic) fungi in Ontario soils and to continue the searchfor the pulmonary fungus Emmonsia in the wild rodent and small mammal population inOntario.

METHOD

(1) Soil samples were collected from May to November from a variety of regionsand soil sites in Ontario. Keratinophilic fungi were isolated using a hair-baitingtechnique. Pure cultures of these fungí were maintained on a variety of nutrient mediaand at temperatures varying from 220-370C. Slide cultures were used to study the mor-phology of individual isolates.

(2) Traps were set daily from May through October in a variety of locationssuch as cultivated and fallow fields; wooded areas; river banks; garbage dumps andother rural and urban sites. The lungs of trapped rodents were removed and examinationswere made of stained sections for the adiaspores of Emmonsia.

RESULTS TO DATE

Keratinophilic Fungi

The areas surveyed during the past year included (a) the Niagara Escarpment;(b) the Muskoka Lakes District; (c) regions adjacent to the east shore of Lake Huronas far north as Tobermory.

Soil samples taken from various sites in these regions yielded new strains ofTrichophyton terrestre, Microsporum, and Chrysosporium spp. Amongst these fungi were11 new, thermotolerant strains which grew at 370C. These included 4 strains of T.terrestre, 3 strains of Chrysosporium, and 4 strains of Microsporum. One of the thermo-tolerant strains of T. terrestre (#1-73) had been obtained from a grassy lawn on theoutskirts of North Bay, the northernmost region from which such a strain has thus farbeen isolated.

One strain of T. terrestre and three strains of Chrysosporium did not produceconidia at 370C while one strain of Microsporum cookei lost its pigment at this tem-perature. When subcultures of these fungi were incubated at 22 C they once again pro-duced conidia and/or pigment.

There does not appear to be any direct relationship between the pH of the soilssampled in Ontario and the fungi isolated from these samples. For example, species ofTrichophyton (e.g., T. ajelloi and T. terrestre) and Chrysosporium were isolated alone,or in combination with other geophilic fungi, from acid or from alkaline soils (ph <6.6 to > 7.2). Furthermore, all the soil surveys conducted to the end of 1975 failedto reveal a definite pattern for the distribution of keratinophilic fungi throughoutOntario. Thus, many strains of Trichophyton, Microsporum, and Chrysosporium could befound in all the regions and soil types surveyed from the north shore of the Great Lakesand as far north as Sudbury.

184

One strain of T. terrestre and one strain of Microsporum were also isolated fromseveral soil samples taken in a horse pasture adjacent to the Royal Botanical Gardens inNassau, The Bahamas. Both of these strains are thermotolerant and produce conidia whengrown at 370C. These strains appear to be the southernmost isolated to date that willgrow at 370C.

Additional soil surveys in Ontario for keratinophilic fungi will resume duringthe summer of 1976.

Adiaspiromycosis in the Wild Rodent Population of Ontario

Lungs of the wild rodents Peromyscus and Microtus, which had been trapped through-out southwestern Ontario during 1975, were not found to be infected with the adiasporesof Emmonsia spp. In contrast to previous experience with adiaspiromycosis in Albertaand Saskatchewan there does not appear to be any evidence, as yet, for this pulmonary in-fection in Ontario.

SIGNIFICANCE

(1) Several strains of Trichophyton terrestre isolated from lesions in humansresiding in southwestern Ontario were reported on in a previous publication. Thesestrains not only elicited reactions in guinea pigs but were also able to grow at 370C.More thermotolerant strains of geophilic fungi including Microsporum cookei and Chryso-sporium have now been isolated. The ability of these fungi to grow at 370C strengthensthe argument that some geophilic fungi could "invade or infect" animals and/or humansdebilitated by some underlying primary disease or infection. Further animal studieswith these strains will be made.

(2) It is noteworthy that, in contrast to the positive results obtained in thewestern provinces of Canada, no evidence of adiaspiromycosis in the rodent populationof Ontario was found in the past 15 years of study. Several cases of this infectionhave now been found in humans living in France, U.S.S.R., and Guatemala. It is, there-fore, important that the search for this infection be continued in the rodent populationof Ontario as well as in lung sections taken from patients with "atypical" respiratoryinfections and other pulmonary conditions.

PUBLICATIONS

1. Bakerspigel, A. The keratinophilic fungi of Ontario. Mycopathol MycolAppl 53:1-12, 1974.

OTHER DATA

Grantee: Dr. A. Bakerspigel, Department of Clinical Microbiology,Victoria Hospital, London, Ontario, Canada.



185

CAUSAL AGENTS OF GRANULOMAS AND MYCETOMAS WHICH FORM GREY COLONIES

PROBLEM

A mycologist who isolates a grey cottonous mold from a black grain mycetoma, forexample, is today almost completely lacking in guidelines to identify it. A rationalbasis for treatment and for specific identification of the parasites needs thus to becreated or consolidated.

The causes of uncertainty and confusion come principally from the existence oftwo akin species: Madurella grisea (Mackinnon, Ferrada, and Montemayor, 1949), andPyrenochaeta romeroi (Borelli, 1959). It is believed, however, that other species,producing both mycetomas and granulomas, do exist, and must be differentiated fromthose mentioned above.

The purpose of this study is to identify which and how many fungi among the greyspecies, are capable of producing granulomatous and mycetomatous lesions in man.

METHOD

All available strains of grey molds isolated from human granulomatous or myceto-matous lesions, are gathered and compared as to their morphologic properties in vivoand in vitro and to their pathogenicity. Special attention is given to the grain struc-ture in vivo and to thermophilic characteristics in vitro. Inoculations into mice andother laboratory animal species are performed.

RESULTS TO DATE

(1) Cotypes of Madurella grisea were obtained, studied, and compared. Thesespecies have changed very little, if at all, since their original description. Theyare sterile molds forming grey colonies that are not identical, because one of themis folding, whereas the other is flat. Their thermophily is similar to that of thestrains currently attributed to the species: for example, they do not grow at 370C.Their pathogenicity in mice, hamster, and guinea-pigs (testicles) is nil.

(2) Nine strains, freshly isolated from human foot mycetomata with black grainsin Central and South America showed similar characteristics. Their colonies, gentlyfolding at first, became flat later and were sterile all the time. Their pathogenicitywas found to be almost nil. The strains were provisionally retained within this taxon(Madurella grisea).

(3) One strain, which was isolated from a granulomatous lesion in the ankle ofa North American resident and which produced grey mold colonies, was found to growup to 370-38°C and to produce several pycnidia and pycnidioconidia. These findingsconfirmed its belonging to a new species of the genus Plenodomus. Its pathogenicityin mice (peritoneum), hamster (peritoneum), and guinea pigs (testicles) is almost non-existent. From the original folding colony, flat variants are born all the time.

(4) One strain, isolated in Paris from a black-grain foot mycetoma in an Afri-can-born man, produced grey folding colonies, with a fair number of pycnidia that

186

justified its belonging to the same genus Plenodomus. It was different from the pre-ceding one. Its pathogenicity is rare; its thermophily goes up to 380C.

(5) One strain of grey folding mold (with less folding or flat variants), iso-lated from a black grain mycetoma in Venezuela, was found to produce rather abundantpycnidia that warranted its position among members of the genus Pyrenochaeta. Its patho-genicity is almost nil. The flat variants are indistinguishable from usual colonies ofMadurella grisea, being sterile and isothermophilic.

(6) One strain of a very slowly growing grey mold, isolated from a black grainfoot mycetoma in Lara, is producing scarce and irregular aleuria on reptant hyphae thatare reminiscent of those by Pyrenochaeta romeroi. Compact, immature bodies similar toPyrenochaeta pycnidia are also produced. The pathogenicity of the strain is almost nil.Its thermophily ranges below 370C. Its grains in vivo are similar to those of M. griseaon the one hand, and to those of P. romeroi on the other. The taxonomic position ofthis fungus (probably new) has not yet been determined.

(7) One strain of a slowly growing, silvery grey, gently folding mold has beenisolated from a black grain foot mycetoma in Lara. Its thermophily ranges well below370C and its experimental pathogenicity is almost nil. It has produced, however, numer-ous pycnidia that support its belonging to the genus Chaetosphaeronema.

SIGNIFICANCE

(1) The position of the cotypes of Madurella grisea Mackinnon, Ferrada, andMontemayor, could not be moved to a more definitive location through the study of theircultural and pathogenetic properties. Nine additional strains, freshly isolated fromhuman mycetomata, have been (at least provisionally) attributed to the same taxon.

(2) Three strains of grey molds, isolated from black grain mycetomata, and onestrain, isolated from a human granuloma, have been judged to belong to new species.

(3) One species was attributed to the genus Chaetosphaeronema larense.

(4) New ideas are needed in order to define properly the pathogenicity of theseoligothermophilic species. Perhaps human volunteers would serve this investigation butthey are not easily found.

PUBLICATIONS

1. Borelli, D. and R. Zamora. Chaetosphaeronema larense, nova species, agentede micetoma. Bol Soc Venez Dermatol, VI (No. 58), Mayo 1973.

OTHER DATA

Grantee: Dr. Dante Borelli, Universidad Central de Venezuela, Caracas,Venezuela.

Funded by: Pan American Health Organization and U.S. Army Medical Researchand Development Command.


187

DEEP MYCOSES IN PATIENTS SHOWING ABNORMAL CHEST X-RAYS

PROBLEM

Cases of all known deep mycoses have been diagnosed in Venezuela and partialinformation about the geographic distribution of some of these diseases has been derivedthrough large surveys with intracutaneous tests. We still do not know, however, theactual frequency or the significance of these deep mycoses in the country.

Patients from many places of Venezuela come to the Hospital Vargas of Caracas,and about 22,000 adults receive there a 70-mm chest fluorophotography yearly. Thosewho show any abnormality receive full size chest X-rays. The purpose of our study is todetermine how many abnormal X-ray findings in the latter group of patients can be attri-buted to fungi.

METHOD

Each patient with abnormal chest X-rays is submitted to complement fixation andimmunodiffusion tests for histoplasmosis, coccidioidomycosis, and paracoccidioidomycosis,and to intracutaneous tests with histoplasmin, coccidioidin, and paracoccidioidin. Inaddition, direct examination and culture of sputum is performed if the patient expecto-rates. Results are discussed and tabulated during the monthly staff meeting.

RESULTS TO DATE

These studies, which were to start on January 1975, were to have covered a largenumber of patients for that year. However, because of unforeseen problems in the X-rayand pneumatology departments, the work, as planned, could not be accomplished. The studyof 70 patients with abnormal X-ray findings of the chest was, however, completed fromJuly to December. These patients had pulmonary mycoses: 1 histoplasmosis, 1 paracoccid-ioidomycosis, and 1 cryptococcosis.

This limited study permitted the adjustment of the laboratory methods and to trainthe personnel to be involved in the full project. It is hoped that significant progresswill be made during 1976.

SIGNIFICANCE

The research will provide information on the frequency of pulmonary mycoses ina general hospital of Caracas. Results will help determine the importance of fungal in-fections in chest pathology, in Venezuela.

PUBLICATIONS

None thus far.

188

OTHER DATA

Grantee: Dr. Humberto Campins, Departamento de Patología Clínica,(Sección Micología Médica), Instituto de Patología, HospitalVargas, Caracas, Venezuela.



189

FERNANDEZ AND MITSUDA REACTIONS IN LEPROSY PATIENTSUSING HUMAN AND ARMADILLO ANTIGENS

PROBLEM

The objective of the study was to determine the effectiveness of leprosy bacilliharvested from lepromas of infected armadillos when used as the antigen in the humanlepromin test. This was done by comparing the delayed hypersensitivity skin reactionsto two types of lepromin; that prepared from leprosy bacilli from lepromas of humanleprosy patients, and leprosy bacilli from lepromas of armadillos infected with humanleprosy.

METHOD

One hundred and twelve patients of the Mahaica Hospital (government hospitalfor leprosy patients of Guyana) voluntarily participated in a double-blind studyusing previously coded human and armadillo lepromin antigens. The human lepromin wasprepared from cutaneous material obtained from Venezuelan cases of untreated leprosy.The armadillo lepromin antigen was obtained from an armadillo (Dasypus novemcinctus)from New Iberia, Louisiana. This animal had been inoculated 30 months before withmaterial from a human lepromatous case from Paramaribo, Surinam. Histopathologicspecimens of the infected animal were diagnostic of lepromatous leprosy.

Simultaneous intradermal infections of the coded antigens into each arm wasdone and the Fernandez and Mitsuda reactions were read and recorded by the same ob-servers. Biopsies were obtained from the intradermal site of reaction of both typesof lepromin in five patients.

RESULTS TO DATE

The overall results of this study indicate that in each patient tested, thelepromin prepared from the lesions of infected armadillos stimulate a delayed hyper-sensitivity reaction similar to that initiated by lepromin prepared from the lesionsof infected patients. In 32.3 percent of the patients tested there was no differencein the size of the induration reaction but in 52.7 percent of them there was a largerreaction to the armadillo lepromin than to the human lepromin. In 15 percent of thecases the reaction to the armadillo was smaller. With the significant informationcompiled in this study it is concluded that lepromin prepared from bacilli obtainedfrom infected armadillos is as effective as that prepared from the lesions of leprosypatients.

SIGNIFICANCE

Since 1919 when Kensuke Mitsuda developed the prototype of lepromin as it isusually made, there has been no standardization of lepromin because of the difficultyin obtaining sufficient quantities of the leprosy bacilli. However, with the dis-covery of the nine-banded armadillo as the first natural animal model to developdisseminated leprosy, a source of sufficient bacilli in large quantities is now

190

available for this standardized lepromin. It then becomes necessary to demonstrate thatthese bacilli can be effectively used in the lepromin tests for humans and to establishsome baseline findings of the type of skin reaction to expect.

PUBLICATIONS

1. Millar, J. W. et al. Comparison in leprosy patients of Fernandez andMitsuda reactions, using human and armadillo antigens. Accepted forpublication in Int J Leprosy.

OTHER DATA

Dr. Jack W. Millar, Department of Epidemiology and EnvironmentalHealth, George Washington University School of Medicine andHealth Sciences, Washington, D.C., U.S.A.

Pan American Health Organization and George WashingtonUniversity School of Medicine and Health Sciences.

1974.

191

Grantee:

Funded by:

Timetable:

MYCOBACTERIA AND POOR HOUSING IN LEPROGENOUS FOCI

PROBLEM

Leprosy, despite efforts made in many countries to decrease or eradicate it inthe world, is still a serious public health problem.

Many publications have noted the problem of poor housing in leprogenous foci,and it has been observed that the prevalence of leprosy tends to decline because ofmodifications in the conditions of the dwelling and a decrease in arthropoda which onceabounded in houses. Nevertheless, new cases continue to appear.

Because of this persistence of new cases, it is important to continue ecologicstudies in leprogenous foci to compare such places to those where the prevalence ofthe diseases is nil or very low, in order to confirm the hypothesis of the relationshipbetween leprosy and bad housing in special climatologic conditions.

The above problems are closely related to the continuation of studies on thein-vitro culture of Mycobacterium leprae and M. leprae murium and efforts to obtainexperimental leprosy in hamsters and armadillos. These aspects are part of this pro-ject, which has been divided as follows:

(1) Ecologic aspects of poor housing in leprogenous foci.

(2) Inoculation of experimental animals.

(3) M. leprae and M. leprae murium culture trials.

METHOD

(1) Ecologic aspects of bad housing in leprogenous foci

The material used was obtained by sweeping the rooms superficially, lookingdirectly for arthropoda or organic residues. Cultures are made from this materialin order to determine the presence of fungi, green algae, or bacteria. Material isalso obtained from areas where the prevalence of leprosy is very low or nil to com-pare such places to leprogenous foci. In addition, a study of the examined rooms'characteristics is carried out in both areas.

(2) Inoculation of hamsters and armadillos

Hamsters were inoculated with BCG or any other autoclaved mycobacteria, simul-taneously with, or 10 days or 1 month later, intraperitoneal administration of M.leprae and M. leprae murium. After 4 to 6 months the animals are necropsied, lesionsare observed, and cultures and new serial passages are made.

Armadillos were inoculated subcutaneously, intramuscularly, and orally.

(3) M. leprae and M. leprae murium culture trials

For the first trials, lepromas and mucus from untreated patients were used; forthe second ones, the Pasteur murine leprosy strain which had been maintained in mice

192

since 1954 was used. They are processed under different temperatures, conditions, andculture media.

RESULTS TO DATE

(1) Earlier results (see Research in Progress 1974) have been fully verifiedat the San Gil Laboratory of Colombia, and basic techniques developed for the isolationof mycobacteria from floor sweepings in lepers' dwellings, and for their control insupposedly uncontaminated homes.

(2) Inoculation of hamsters with M. leprae or M. leprae murium is continuing.So far, two new forms of granulomatosis of hamsters inoculated with M. leprae have beenobtained. Inoculation of armadillos (Dasypus novemcinctus and the l1-banded Cabassouscentralis) is also continuing. Very recently, C. centralis has been successfully in-fected experimentally. On 18 March 1976, granulomatous nodules on the abdomen of theanimal were found to be positive for acid fast bacilli at the site of two separateinjections. Ear smears were negative, as were smears taken from the nasal mucous mem-branes. This is the first time the armadillo has been infected in the laboratory inColombia. Cabassous centralis is the fourth type of armadillo to be infected experi-mentally. The first one was the Dasypus novemcinctus in 1971, followed by D. sabanicolaand Chaetophractus villosus. In addition, bacilloscopies and cultures for environmentalmycobacteria in lymph nodes have been made from animals unable to survive in captivity.

(3) Attempts to cultivate M. leprae and M. leprae murium are still inconclusive.

PUBLICATIONS

1. Muñoz Rivas, G. Estado actual de nuestras investigaciones en relación a latransmisión de la lepra a los animales. Conferencia en la Academia Nacionalde Medicina. Temas Médicos, Tomo 11, pp. 15-30, 1970.

2. -----. Posibles fenómenos de transformación en micobacteria (Nota Previa).Rev Invest Salud Púb (Mexico), vol. 30.

3. -----. Notes on the transmission of M. leprae to laboratory animals.VII Iberolatinamerican Dermatology Congress, Caracas, 12-16 December 1971,unpublished document.

4. -----. Ambient Mycobacteria in Colombian Armadillos. Leprosy Subgroup,National Institute for Special Health Programs, Bogotá, 1973, unpublisheddocument.

5. -----. Granulomatosis of hamsters inoculated with M. leprae and M. lepraemurium.

OTHER DATA

Grantee: Dr. Guillermo Muñoz Rivas, Head of the Leprosy Subgroup, NationalInstitute for Special Health Programs, Bogotá, Colombia.

Funded by: Pan American Health Organization/World Health Organization.


193

EXPERIMENTAL INFECTION OF THE ARMADILLO WITH MYCOBACTERIUM ULCERANS

PROBLEM

The primary objective of this project is to determine if the nine-banded arma-dillo (Dasypus novemcinctus, L.) is an ideal animal model for studying the clinical andhistopathologic development of Mycobacterium ulcerans infection. The long-range sec-ondary objectives are (1) to determine in Central and South America, the prevalence ofM. ulcerans infection, (2) to define and purify the toxic principle of M. ulceransfollowing which an attempt at developing an antitoxin for treatment and prophylaxis will,be made, (3) to determine risk factors as have been partially defined in Africa and NewGuinea (i.e., increased susceptibility of riverine people, migrant people, and peopleliving in thatched dwellings on or near poorly drained land), and (4) to develop bettertechniques for screening exposed subjects resulting in earlier treatment essential topreventing the late sequelae of contraction deformity, lymphedema, and amputation.

METHOD

Armadillos, already adapted to laboratory life, have been inoculated at the GulfSouth Research Laboratory, New Iberia, La. These were inoculated at eight differentsites on the skin of the abdomen. Starting at the end of 4 weeks postinoculation andcontinuing every 2 weeks thereafter, a complete inoculation site was removed surgicallyfor histologic and histochemical examination. If ulcers developed, at least one wasleft untouched to determine the ultimate and natural course of the infection in thearmadillo. These serially excised specimens, taken before ulceration and contaminationwith surface organisms, reveal the spread of M. ulcerans and its effect on the adjacenttissues. When these studies are completed and a thorough knowledge of the infectionis gained, studies will be designed to determine: (1) optimum therapeutic treatment,and (2) practical prophylactic measures. These experiments would include the develop-ment and testing of antitoxin and antibodies against M. ulcerans. Blood and serum willbe collected with biopsies to determine the armadillos' immunologic response as reflectedby cellular and serum-protein profiles during the course of the infection. Lymphoidtissues will be taken and studied to determine the armadillos' cell-mediated responseduring infection. Histochemical and electron microscopy studies will also be done alongwith routine bacteriologic studies.

RESULTS TO DATE

Thirty-four armadillos were inoculated to determine the effect of route of inocu-lation (intravenous, intraperitoneal, intradermal), dose, different strains of M. ul-cerans, source of inoculum (isolated from infected mice foot pads and grown in Dubosmedia), ageof the animal, and environmental temperature. Animals inoculated by routesother than intradermal do not develop the typical ulcerated lesion. Five months afterinoculation the animals died and were edematous with evidence of hemorrhage. With highdoses of bacteria (10 -1011) inoculated intradermally, small preulcerative nodules de-veloped and receded, erythematous areas appeared on the thighs, and animals becameedematous and died. The source of inoculum, different strains of M. ulcerans, age ofanimals, and environmental temperatures had no effect on the course of infection.

194

SIGNIFICANCE

This research will, (1) create an awareness of this disease in the Americas andlead to a determination of the scope of this infection through more accurate and com-plete case finding, and (2) in endemic areas in the Americas or elsewhere, lead to amore thorough understanding of the disease and thence to better control, better pre-ventive measures, and to earlier and more effective treatment.

PUBLICATIONS

1. Hockmeyer, W. T. et al. Characterization of the toxin of Mycobacteriumulcerans. Bacteriol Proc 75:21, 1975.

2. Krieg, R. E. et al. The toxin of Mycobacterium ulcerans. Production andeffects in guinea pig skin. Arch Dermatol 110:788-893, 1974.

3. ----- et al. The toxin of Mycobacterium ulcerans. Production and effectsin guinea pig skin. Laboratory Investigation 30:32 (Abstract), 1974.

4. ----- et al. The toxin of Mycobacterium ulcerans. Production and effectsin guinea pig skin and mouse fibroblast cells. Bacteriol Proc 74:90, 1974.

5. - et al. Experimental infection of the armadillo with Mycobacteriumulcerans. Bacteriol Proc 75:24, 1975.

6. ----- et al. Hyperbaric oxygenation of Mycobacterium ulcerans infection.Bacteriol Proc 75:4, 1975.

7. ----- et al. Treatment of Mycobacterium ulcerans infection by hyperbaricoxygenation. Aviat Space Environ Med 46(10):1241-45, 1975.

8. ----- et al. Absence of an antibiotic effect of Mycobacterium ulcerans.Int J Leprosy., in press.

9. Walsh, G. P. et al. Mycobacterium ulcerans infection of armadillos(Dasypus novemcinctus, L.). Bacteriol Proc 74:90, 1974.

OTHER DATA

Grantee: Or. Richard E. Krieg, Chief, Bacteriology Branch, Armed ForcesInstitute of Pathology, Washington, D.C., U.S.A.

Funded by: U.S. Army Medical Research and Development Command andPan American Health Organization.


195

CONTRIBUTION TO THE STUDY OF THE ARMADILLO MODEL

PROBLEM

Once the experimental transmission of leprosy to the armadillo Dasypus sabani-cola was obtained, the objectives of the project were: (a) to increase the number ofinoculated animals; (b) to establish animal-to-animal passages; (c) to study the basicimmunology of Dasypus sabanicola; and (d) to introduce the Dasypus sabanicola as a newlaboratory animal.

METHOD

One hundred three armadillos D. sabanicola were inoculated, 60 of them directly,with human material from lepromatous leprosy lesions at a bacillary concentration whichvaried between 107- 108 acid-fast bacteria per milliliter. Each animal received 0.5-1ml intradermally and endovenously. The rest of the animals (43) were inoculated withmaterial from experimental lesions produced in the first group. Thus, a first animal-to-animal passage was established.

Major lymphoid organs were evaluated in paraffin-embedded sections stained withhematoxylin-eosin. Blood was taken by cardiac puncture. Complement activity was as-sayed in vitro. Animals were sensitized with ovalbumin (OA), in complete Freund'sadjuvant; serum was tested for antibodies by double diffusion and precipitation in agar.Skin tests were done with OA and PPD tuberculin and read at 12, 25, and 48 hours. Ani-mais were also sensitized with tuberculin; blood samples were taken to study antigeninduced lymphocyte transformation and the presence of antibodies by passive hemaggluti-nation. Sensitization to dinitrochlorobenzene (DNCB) was also studied. Some of theanimals were sensitized with M. leprae in incomplete Freund's adjuvant, and skin testedwith M. leprae.

Lymphocyte transformation was evaluated by culturing these cells, after separat-ing them from circulating blood, with the various antigens; after an adequate number ofdays, according to the antigen, titriated thymidine was added and 18 hours later, thesamples were read in a Packard tri-carb liquid scintillation spectrometer.

RESULTS TO DATE

Thirty-four armadillos (33 percent) developed lesions after inoculation and 6had systemic lesions, with involvement of the skin, subcutis, lymph nodes, lungs, liver,spleen, muscle, testicles, and central nervous system. Of the 6 animals that developedsystemic lesions, three had heavy secondary tissue infection caused by gram-negativeand gram-positive bacteria. The secondary tissue infection may have occurred becausethese animals were sacrificed when their disease was extremely advanced.

The initial lesions of the first animal-to-animal passage appeared approximatelybetween 4 to 6 months, that is earlier than did the lesions in animals inoculated di-rectly from human material. The mycobacteria obtained in both instances were identi-fied as M. leprae through the Mitsuda test and the pyridine test.

D. sabanicola was found to have a typical mammalian distribution of lymphoidcells in thymus, spleen, lymph nodes, and blood. Their complement was active in

196

bactericidal, protozoan immobilization, and hemolytic systems. Blood lymphocytes re-sponded to phytohemagglutinin, and pokeweed mytogen. Sensitization with ovalbumin incomplete Freund's adjuvant resulted in the production of circulating precipitins;strong Arthus' reactions were detectable in the sensitized animals. Responses of cell-mediated immunity to DNCB and to M. tuberculosis were very discrete. Heat-killed M.leprae elicited granulomatous reactions characterized by microscopic necrosis but with-out abundant lymphocytic infiltration; skin tests and lymphocytic transformation weregenerally negative in the animals inoculated with M. leprae. The armadillo D. sabani-cola was introduced as a new laboratory animal because it is easy to handle, has a lowbody weight (average between 1.5 - 1.7 kg), adapts easily to captivity, and can possi-bly reproduce in captivity. This last aspect is still being studied.

In initial experiments, it has been possible to transmit human onchocerciasisto D. sabanicola, inoculating infective larvae from S. metallicum raised in the labora-tory and infected on human cases of onchocerciasis.

SIGNIFICANCE

Transmission of human leprosy to the armadillo Dasypus sabanicola will providelarge numbers of M. leprae for laboratory research and adequate supplies of materialsto study leprosy from the epidemiologic and control viewpoints. Another importantaspect of the project is the study of this animal as a model for therapeutical researchin leprosy, and the study of the mechanisms of production of the disease.

The initial success obtained with the transmission of human onchocerciasis tothe armadillo D. sabanicola opens the possibility that an experimental model of thedisease could be developed in this animal.

PUBLICATIONS

1. Campo-Aasen, I. and J. Convit. The histochemistry of armadillo skin.J Anat 120:337-48, 1975.

2. Convit, J. and M. E. Pinardi. Leprosy: confirmation in the armadillo.Science 184:1191-92, 1974.

3. ----- and -----. Preparación de un antígeno soluble a partir de Myco-bacterium leprae para pruebas de tipo tuberculínico. Acta Cient Venez25:51, 1974.

4. ----- and -----. Inoculación de Mycobacterium leprae en dos especies dearmadillo: Dasypus sabanicola y Dasypus novemcinctus. Acta Cient Venez25:51, 1974.

5. ----- and E. Storrs. ThIe pygmy armadillo (Dasypus sabanicola) as a newresearch animal. 26th Annual Session American Association for LaboratoryAnimal Science. Boston, November 16-21, 1975.

6. ----- et al. Estudio comparativo de Fernández y Mitsuda reacciones en enfermostuberculosos de Venezuela y Chile. Acta Cient Venez 26:74, 1975.

7. ----- et al. Tests with three antigens in leprosy-endemic and nonendemicareas. Bull WHO 52:1975.

197

8. ----- et al. Specificity of the 48-hour reaction to Mitsuda antigen.Bull WHO 52, 1975.

9. Monz6n, H. et al. Estudio comparativo entre lepromina preparada de materialhumano y lepromina preparada de material de armadillo. Acta Cient Venez26:74, 1975.

10. Ulrich, M. and J. Convit. Caracteristicas inmunológicas del armadillo.Dasypus sabanicola. Acta Cient Venez 25:51-52, 1974.

11. ----- and -----. Transformación linfocitaria inducida por Mycobacteriumleprae de origen humano y de armadillo. Acta Cient Venez 26:75, 1975.

OTHER DATA

Grantee: Dr. Jacinto Convit, PAHO/WHO International Center for Trainingand Research in Leprosy and Related Diseases, Caracas, Venezuela.



198

IMPROVEMENT OF TECHNIQUES FOR FOOT-AND-MOUTH DISEASE (FMD) RESEARCH

PROBLEM

Most virologic test systems and procedures are applicable to FMD virus researchprovided they are adapted to local conditions.

Routine tests for circulating and local neutralizing antibodies for the assayof large numbers of samples are required to study population immunity. Some of thesetests have been used successfully for many years at the Center, but research is underway to develop or expand further these techniques. Moreover, the interpretation ofroutine test results is sometimes difficult to estimate population immunity.

More research is required to update and/or develop new diagnostic proceduresthat facilitate or speed up the identification of virus strains or of antibodies.

METHOD

The project includes the following tests for the detection of local and circu-lating antibody: (a) microneutralization; (b) plaque reduction neutralization;(c) hemagglutination; (d) immunoradial diffusion; and (3) immunohistochemical assay.

Emphasis is placed on the preparation of purified and standardized referenceantibody and virus preparations to calibrate the test reagents, particularly for radialdiffusion tests and fluorescent or immunoperoxidase studies.

The establishment of a collection of sera from animals with a well-documenteddisease or infection history for common use is receiving priority, so that new testsystems can be compared and evaluated. The classes of antibodies active in each testmust be determined. A critical statistical analysis of each of the test systems willbe made. Once the necessary hardware becomes available, computer programs can bewritten for the processing of the raw test results. The value of the virus infectionassociated (VIA) tests for mass screening of serum samples to determine viral activi-ties in the livestock population will be further investigated.

A review will be made of the methods for the detection of small quantities ofvirus in field samples with regard to collection, storage, and shipping of samples andisolation techniques. Many of the collection and isolation techniques are still veryempirical and undoubtedly can be improved considerably.

The effect of emulsification and type of emulsion with TTE for the liberationof antibody-bound virus will be further investigated.

RESULTS TO DATE

The main modification of described techniques for microneutralization was theuse of preformed monolayers of IBRS-2 cells. HEPES buffer was used to stabilize thepH. The standard error of the test and the influence of the dose of virus were deter-mined. Test results were found to be reproducible under routine test conditions.

Several changes in the plaque reduction neutralization test were made. Topsor bottoms of glass Petri dishes in groups of six or four in aluminum-covered trays

199

were generally as convenient as disposable multidishes but provided the additionaladvantage that ordinary glass Petri dishes can be used repeatedly.

Gum overlay was introduced instead of agar. Several locally available vegetablegums were tested. Karaia gum was selected for general use because it is readily avail-able at low cost. Plaquing efficiency was found to be as good or better than those ofagar, with plaques countable at 24 hours. The system allows for large numbers of titra-tions and plaque reduction neutralization tests.

A comparison of the preliminary results obtained from 32 sera assayed by thepassive hemagglutination test and the mouse protection test suggests that the passivehemagglutination test is very sensitive. Comparisons with the mouse protection testare being made.

The stability at -70°C of a sheep red blood cell (SRBC) and A24 virus conjugatewere tested. Portions of 10 percent suspension of the SRBC conjugate in a glycine/Tris/phosphate buffer pH 7.5 were snap frozen in a dry-ice acetone bath and kept at-70 C. Micropassive hemagglutination tests were performed periodically. After 3months, false-positive results occurred because of autoagglutination. The test thusbecame rather unreliable even though the autoagglutination could be prevented by re-peated washings of the conjugate with phosphate buffered saline. Therefore, the use ofthe conjugate beyond 3 months was not recommended.

The following experiments are in progress on diagnostic methods:

(1) Preparation of hemolytic serum with forssman antigen.

(2) Improvement of the preparation of guinea pig hyperimmune serum.

(3) Application of electroendosmosis for FMD virus.

(4) Comparison of double diffusion and radial diffusion test.

(5) Simplification of the preparation and concentration of 140S, 12S, and VIAantigens of FMD virus.

(6) Purification of VIA antigen by physical-chemical methods and quantitationby electroimmunodiffusion.

(7) Immunoelectrophoresis of VIA antigen.

SIGNIFICANCE

Development of renewed laboratory techniques will create new opportunities in thefield of foot-and-mouth disease research.

PUBLICATIONS

1. Brugh, M. Jr. Influence of serum restriction on foot-and-mouth diseasevirus infections in cell cultures. I. Enhanced viral susceptibility.Zentralblat Vet Med B 22:285-94, 1975.

2. -----. Influence of serum restriction on foot-and-mouth disease virusinfections in cell cultures. II. Viral replication. Zentralblat Vet MedB 22:295-301, 1975.

200

3. Cunha, R. G. et al. El uso de los ratones lactantes en la evaluación delos anticuerpos contra el virus de la fiebre aftosa y su significacióninmunológica. Gac Vet 19:243-67, 1957.

4. ----- and U. M. Serrao. Investigación del poder profiláctico y curativodel suero antiaftoso en ratones blancos. Gac Vet 22:230-39, 1960.

5. -----. Epreuve de protection par le vaccin anti-aphteux chez les jeunessouris adultes. Bull Off Int Epizoot 53:676-78, 1960.

6. -----. Demonstration of immune response to foot-and-mouth disease vaccinein protection test in young adult mice. Proc Soc Exp Biol Med 103:700-03,1960. Em espanhol: Publ. N' 2 del Centro Panamericano de Fiebre Aftosa,julio, 7 p., 1960.

7. -----. Comparison of serum protection tests in guinea pigs and mice forfoot-and-mouth disease antibody evaluation. Can J Comp Ped 27:42-44, 1963.Em espanhol: Cuad Centr Panam Fieb Aft 1:1-5, 1963.

8. ----- and M. N. Honigman. A comparison of serum tests in mice for thedetection of foot-and-mouth disease antibody. Am J Vet Res 24:371-75, 1963.

9. Gaggero, A. C. and P. Sutmoller. The use of serum and blood dried onblott-ing paper in the detection of foot-and-mouth disease antibody. Br VetJ 121:509-14, 1965.

10. Gomes, 1. and A. Vieira. Effect of polyionic compounds on the plating effi-ciency of some strains of vesicular stomatitis virus. Arch Ges Virusforsch34:223-31, 1971.

OTHER DATA

Grantee: Drs. Eduardo R. Centeno, Albino A. Fernandez, Antonio Vieira,and Maria Elma V. Ferreira, Pan American Foot-and-Mouth DiseaseCenter, Rio de Janeiro, Brazil.



201

PATHOGENESIS, TRANSMISSION, AND RESERVOIR OF FOOT-AND-MOUTH DISEASE VIRUS (FMDV)

PROBLEM

Studies on virus-animal interaction serve to add to the scientific basis of con-

trol measures, vaccine development, and vaccine potency testing. The project includespathogenetic studies of FMD, using different routes of exposure with virulent and atten-

uated strains of FMDV. This research involves determination of portals of entry, estab-

blishment of growth curves at sites of virus replication, and studies of the clinical

response (fever, development of lesions, viremia patterns), of the pathologic changes

and of systemic and local antibody development, including the characterization of the

antibody.

The specific objectives of the studies of the virus-host interaction at the herd

level are to provide better understanding of transmission, dissemination, and reservoirsof the virus and to determine the effects of vaccination and control measures on the

spread and persistence of FMDV.

The role of wildlife in the epidemiology of FMD in South America is, as yetpoorly understood. It is of practical importance to determine if wildlife can be re-sponsible for the spread of FMD or for maintaining the virus during interepidemic

periods and whether there are other vectors of FMDV than those normally incriminated.

METHOD

Studies focus on: (a) the virus and the cell; (b) the virus and the animal; and

(c) the virus in relation to the population.

Immunohistochemical techniques are used to visualize the formation of FMD viraland virus infection associated (VIA) antigens during the growth cycle of the

virus in cell cultures. Differences will be determined between virulent, attenuated,and "carrier" virus strains.

The studies on virus-animal relationships will involve both laboratory and fieldexperimentation as follows:

(1) determination of the portal of entry of the virus after different routes

of exposure;

(2) establishment of growth curves in the upper respiratory tract or other

sites where virus multiplication is found to occur, using different routes of exposureand different strains of FMDV;

(3) establishments of viremia patterns;

(4) determination of the clinical response (fever, development of lesions), and

the pathologic changes that occur;

(5) establishment of patterns of systemic and local antibody development in-cluding antibody characterization.

The studies of virus in the population will comprise:

202

(1) surveillance of the area for spread and persistence of virus;

(2) experimental transmission of FMDV in controlled groups;

(3) determination of the role of wildlife by infection trials in the laboratoryand antibody studies in the field.

In these surveys, various techniques such as virus isolation, neutralizing andVIA antibody tests, and tests for local antibodies will be used.

RESULTS TO DATE

Use of VIA antigen for the detection of FMDV infections

VIA antigen proved to be very useful in a serologic survey in Paraguay, RoraimaTerritory of Brazil, and Guyana to detect the presence of infection.

Use of VIA tests in Bagé for the detection of viral activity

A total of more than 3,200 sera were tested for the presence of VIA antibodies.The results showed that virus activity in the population disappeared during the 4-yearperiod in which the Center's oil adjuvanted vaccine was used. In 1972, 6 cattle werepositive. In 1973 and 1974 the number of positive cattle were 2 and 1, respectively.All cattle were negative in 1975.

Intranasal inoculation of capibaras with types 01 and A?4

Preliminary results indicate that capibaras may not be susceptible to FMD. Novirus growth or antibodies could be demonstrated after intranasal inoculation of FMDV,type 07. The development of epithelial oral lesions in some animals before and aftervirus exposure,which very closely resembled those of FMD,complicated the experimentalwork. Virus could not be isolated from these lesions and since similar lesions wereobserved in deer at the Plum Island Animal Disease Center, it is believed they may haveresulted from repeated injections of Rompun used to sedate the animals for examination.

Pharyngeal virus growth after intradermal lingual (IDL) inoculation of cattle

The IDL route of virus inoculation is used in vaccine potency assays either asa titration method (Lucam method) or as a standard dose (classical challenge method).With the modified Lucam method the appearance of lesions on one or more feet is con-sidered generalization, and this observation is taken into account when estimating thevaccine potency.

Two unvaccinated and four vaccinated cattle were inoculated in the tongue epi-thelium with FMDV.

The unvaccinated cattle developed severe tongue lesions at 2 days postinoculationfollowed by vesicles on the 4 feet.

Virus was isolated from the blood of one of these steers at 6 hours postinocula-tion and a relatively high titer viremia was present in both steers at 12 hours post-inoculation. The relatively long pad period of pharyngeal growth probably points to alow level of initial infection of the pharyngeal area. The tongue epithelium was grosslynormal at 12 hours postinoculation and relatively high levels of virus release are re-quired for the maintenance of a viremia of approximately 2 log plaque forming units permilliliter of blood. The early viremia in the two unvaccinated cattle may indicate thatthere are sites of virus production other than the pharynx.

203

SIGNIFICANCE

Disease control measures require a sound scientific basis. The results of thisproject will provide further knowledge on virus-host interaction and will show bettermeans to deal with the disease.

PUBLICATIONS

1. Alonso, F. A. and V. M. Fernandes. Experimental inoculation of sheep withfoot-and-mouth disease virus. Bull Off Int Epizoot 73:507-20, 1970.

2. ----- et al. The use of virus-infection-associated antigen (VIA) in thedetection of cattle exposed to FMD virus. Bol Cent Panam Fieb Aft 17-18,in press.

3. Augé de Mello, P. et al. Further information on the survival of modifiedfoot-and-mouth disease virus in cattle. Bull Off Int Epizoot 73:489-505,1970.

4. Cunha, R. G. et al. Experimental mixed infection of cattle with foot-and-mouth disease viruses. Am J Vet Res 19:78-83, 1958.

5. Federer, K. E. Susceptibility of the Agouti (Dasyprocta aguti) to foot-and-mouth disease virus. Zentralbl Vet Med B 16:847-54, 1969.

6. Fernandes, M. V. El aire y la transmisión de la fiebre aftosa. Bol CentPanam Fieb Aft 9:1-7, 1973.

7. Gomes, 1. et al. Foot-and-mouth disease circulating antibodies in conva-lescent cattle. Bull Off Int Epizoot 77:731-41, 1972.

8. Honigman, M. N. et al. Persistencia en terneros de la inmunidad postvacunalcontra el virus aftoso. Bol Cent Panam Fieb Aft 2:12-20, 1971.

9. Rosenberg, F. J. and P. Augé de Mello. Portadores de virus aftoso. Procesoterminal de la infección o eslabón intermedio en la cadena epidemiológicade la enfermedad? Bol Cent Panam Fieb 13-16:50-60, 1974.

10. ----- et al. Prevalence of antibodies against foot-and-mouth disease virus-infection-associated antigen (VIA) in cattle of the Paraguayan Chaco.Bol Cent Panam Fieb Aft 19-20, in press.

11. Sutmoller, P. and A. C. Gaggero. Foot-and-mouth disease carriers. Vet Rec77:968-69, 1965. Em Espanhol: Cuad Cent Panam Fieb Aft 3:217-18, 1965.

12. ----- et al. Infectivity for cattle and pigs of three strains of foot-and-mouth disease virus isolated from carrier cattle. Am J Vet Res 18:101-05,1967.

OTHER DATA

Grantee: Drs. Paul Sutmoller, Paulo A. de Mello, Ivo Gomes, and AntonioVieira, Pan American Foot-and-Mouth Disease Center, Rio de Janeiro,Brazil.

204

Funded by:

Timetable:


1975 - continuing.

205

INVESTIGATION OF FMD VIRUS IN MILK AND MILK PRODUCTS

PROBLEM

Milk-borne transmission of foot-and-mouth disease virus (FMDV) was recognizedmost notably during the 1967-1968 epizootic in England. Consequently, researchers inthe United'States examined the survival of FMDV in milk and dairy products, includingcasein. They found that FMDV survived in whole milk of infected cows after heatingat 720C for 5 min and in skim milk after heating for 2 min at the same temperature.Evaporation of the whole milk samples after heating at 720C for 3 min did not completelyinactivate the virus. Casein obtained by acidulation of skim milk with HC1 to pH 4.6were negative for FMDV when assayed in cell cultures. However, by cattle inoculation,it was shown that FMDV could survive this casein production process. These laboratorydata suggested that a portion of the FMDV infectivity in infected cow's milk is heat-and acid-stable, or this unique resistance is provided by one or more milk components.

These findings require a new appreciation of the risk involved in the interna-tional trade of milk products.

The project is designed to determine the risk of the spread of FMDV from endemicareas to FMD-free areas by milk and milk products. Its specific objectives are:

(1) determining the amount of virus arriving at the industrial processingplants in endemic areas;

(2) assessing survival of FMDV during the processing of milk products underindustrial conditions.

(3) estimating the risk of introducing FMD into free areas by such products,if the final products are found to be infectious.

METHOD

(1) Infectivity levelsof milk from infected farms and amounts of virus arriv-ing at dairy cooperatives and processing plants are determined.

At the farm, one or more infected herds are located. From these, a compositemilk sample is taken every 3 days. Daily milk samples are taken from approximately5 cows at risk until 2 weeks after appearance of clinical signs of the disease.

At the cooperative and processing plant, weekly samples are collected from allsuppliers regardless of whether or not they have reported clinical disease.

(2) Samples for the isolation of infectious FMDV are taken at the normal sam-pling points in the production process so as not to interfere with the regular opera-tions of the plant. At these points, samples are routinely obtained in order todetermine pH, bacterial content, and dry matter.

(3) If final products are still found to contain infectious FMDV, furtherexperimentation on these products will involve exposure of susceptible animals to them.The results of these studies will be evaluated in terms of the final use of the pro-duct and the possibilities of altering production conditions so as to guarantee itssafety.

206

The following investigations are also carried out:

(1) Kinetics of FMDV inactivation under the conditions at which milk arrives atthe processing plant (pH 6.3-6.6, 10°C). This will be done to determine what happensto the virus under the conditions occurring during the transportation of the raw milkfrom the cooperatives to the processing plant. Results will also serve to determinethe choice of strains to be used in controlled experiments.

(2) The role of antibodies in milk with regard to reduction of levels of in-fectivity.

(3) Determination of most suitable methods for the detection of minimal amountsof FMDV in milk or milk products.

RESULTS TO DATE

None thus far.

SIGNIFICANCE

Recent results of studies on the survival of FMDV in milk and milk products mayseriously jeopardize international trade of milk products. Realistic assessment ofthe risk involved may prevent eventual restriction of the marketing of such productsand prevent serious economic consequences for the exporting or importing countries.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Drs. Paul Sutmoller, Felix Rosenberg, Vicente Astudillo, andHernán Málaga, Pan American Foot-and-Mouth Disease Center,Rio de Janeiro, Brazil.


1975-1978.

207

SELECTION OF STRAINS FOR FOOT-AND-MOUTH DISEASE VACCINE PRODUCTION

PROBLEM

The immunogenic spectrum of strains used for vaccine preparation is of greatimportance for the production of a high potency vaccine that provides protection againsta variety of field strains. Large differences between strains have been observed inthis respect, and selection of the most suitable strains involves extensive, continuouslaboratory and animal experimentation.

METHOD

The method used is determination of the serologic and immunologic relationshipsof virus strains by complement fixation (CF) and cross immunization tests in cattle.Cattle are immunized with inactivated vaccines and their immunity is assessed by themouse protection and neutralization tests in cell culture using the virus strainsemerging in the field. Pools of strain-specific bovine and guinea pig antisera arebeing established.

RESULTS TO DATE

(1) Selection of a type O straín--immunologic coverage

The results of vaccine studies in cattle indicated that there would be no advan-tage in substituting 01 Campos or 01 Pacheco strains for 07 Brazil/71.

(2) Cross protection between type 01 strains and between C3 strains

There was a close relationship by complement fixation between the three strainsof subtype 01, but the C3 strains were quite different serologically.

There was little difference in protection of the cattle between the O1 strainsCampos and Sao Paulo or between the C3 strains Resende and Indaial.

The 01 Caseros strain proved to be inferior to the other two 01 strains. It wasnoted that the CF relationship did not indicate such differences.

(3) Selection of a type C strain--immunologic coverage

The following strains of foot-and-mouth disease virus type C were studied todetermine their serologic relationship and to select the best strain for vaccine pro-duction:

(a) C1 GC LMR

(b) C2 997 LMR

(c) C Resende (Brazil/55)

(d) C4 Tierra del Fuego (Argentina/66)

(e) C5 Argentina/69

(f) C2 Pando (Uruguay/45)

208

(g) C3 Indaial (Brazil/71)

(h) C Sao José dos Campos (Brazil/72)

(i) C3 Paraguay/69

(j) C Chaco (Paraguay/74)

A close relationship was established between the present field strains C Indaialand C Paraguay and the Center's reference C Resende strain.

Cross immunity tests in cattle indicated that the reference C virus (strainResende) would provide adequate immunologic coverage for the new strains.

The C Indaial vaccine produced good protection against the C Resende strain andthe homologous virus but gave less protection against the Paraguayan strain.

The C Paraguay strain did not prove to be a particularly good antigen and didnot protect well against the C Resende or C Indaial strains.

Cross immunity experiments are in progress using strains C3 Resende, C5 Argentina,C2 Pando, C Sao José dos Campos, C3 Indaial, and C Chaco.

SIGNIFICANCE

Strains of a given subtype of FMDV may be different enough antigenically so thatvaccines prepared from those strains do not provide adequate protection for anotherstrain of the same subtype. Such strains are emerging regularly and require continuousresearch to identify and characterize those of FMD field outbreaks and to compare themto the strains used for vaccine production and vaccine potency tests. For the successof the FMD campaigns, such a continuous research program is essential to determine intime if a change of vaccine production strain is required.

PUBLICATIONS

1. Alonso Fernandez, A. et al. Serological and immunological relationshipof FMD viruses, type C in South America. Paper presented at the meetingof the research group held at Brescia from 23-26 September 1975.

2. Lumca, F. et al. Etude immunologique des sous-types du virus aphteux.Bull Off Int Epizoot 75:1-20, 1971.

OTHER DATA

Grantee: Drs. Albino A. Fernandez, Daniel Abaracon, Magnus S. Sondahl,and Eduardo R. Centeno, Pan American Foot-and-Mouth DiseaseCenter, Rio de Janeiro, Brazil.



209

PRODUCTION, PURIFICATION, CONCENTRATION, AND TREATMENT OF FOOT-AND-MOUTHDISEASE VIRUS (FMDV) ANTIGEN FOR VACCINE PRODUCTION

PROBLEM

In the near future, the main method of antigen production will be from cellcultures. Continued research is needed to determine optimal conditions and methodsfor the growth of cells in monolayer or suspension cultures and for virus productionin those systems.

The virus suspensions employed in vaccine preparations contain unwanted cellu-lar proteins, which require purification of the virus suspension before use in vaccineformulation. This purification process also permits better physical determination ofthe antigenic mass and will hopefully lead to more standardized products.

Means to stabilize the antigens must be investigated further to prevent loss ofimmunogenicity during vaccine formulation and storage.

METHOD

(1) Pilot scale production of FMD antigens used for vaccine production;

(2) Optimization of all conditions of cell growth and virus production andconcentration.

(3) Utilization of a combination of physicochemical procedures including pre-cipitation of the virus with polyethylene-glycol, inorganic absorbents such as aluminumhydroxide or barium sulphate, and Sepharose column chromatography for the purificationof the antigen; and

(4) Determination of the antigen treatment which influences the immunogenicityor shelf-life of the vaccines.

RESULTS TO DATE

The pilot plant has been put into full operation, and several improvements ofproduction methods have been investigated, including optimum conditions for cell growthand virus production. It was found that high yields of antigen could be obtained withcells adapted to suspension culture when grown in the roller bottle system, as wellas in medium-sized tanks. High-quality vaccines have been prepared with these antigens.Concentration and purification of antigens also received careful attention.

(a) Use of inorganic absorbents and chromatography for the purification ofFMDV A24: batches of 2.3 ml of virus suspension type A24 with CF titers of 1/15,800are produced.

(b) Antigen production of BHK celis infected with FMDV using different buffercombinations.

For the growth of virus in cell cultures used in the production of foot-and-mouth disease vaccine, pH control is critical because of the instability of the virus

210

under acid conditions. With different initial pH with the use of organic buffers, noappreciable differences were found after 20 hours of incubation of the cultures. Also,virus production as measured by CF or infectivity was not influenced by the final pH.Thus, no influence of the various buffers tested was detectable under the conditionsof the experiment. A different range of NaHCO3 concentration in the medium was tested.With 3 mM of NaHCO 3, it is impossible to maintain the initial pH, and a pH of 8 hasan adverse effect on the cells. Virus production is independent of the pH in the rangeof 7.1 to 7.6.

(c) Merthiolate is commonly used in commercial inactivated FMD vaccines. Dif-ferences between the stability of the three antigens in the presence of merthiolatewere observed. Type O was stable for as long as 12 months after the preparation ofthe vaccines. The immunogenicity of types A and C was unfavorably influenced by mer-thiolate, particularly after a 12-month storage period of the vaccines. The additionof EDTA resulted in loss of the immunogenicity of the A and C antigens.

SIGNIFICANCE

Control of FMD in South America is based on the systematic vaccination of cattle.Improved antigen production will be one of the major factors determining the successof the anti-FMD campaigns now in progress in the Western Hemisphere.

When purity of the antigen is increased, vaccines can be controlled more effi-ciently and will be less likely to produce allergic reactions.

Stability of the antigen during storage is another important factor in theefficacy of vaccines.

PUBLICATIONS

1. Abaracon, D. Algunos ensayos sobre producción de vacunas antiaftosas in-activadas, a partir de virus multiplicado en conejos neonatos. Bol CentPanam Fieb Aft13-16:30-49, 1974.

2. Abreu Martins, I. de. Producción económica de virus aftoso para fabricaciónde vacunas por el método Frenkel. Cienc Vet, México 5:489-96, 1960. Publ.N. 12 del Centro Panamericano de Fiebre Aftosa, junio, 10 p., 1961.

3. -----. Experiencias preliminares de cultivo de amostras nacionais de vírusde febre aftosa pelo método de Frenkel. Trabalho apresentado ao 7? CongBras Vet, Recife, 1957. Publ. N? 23 do Centro Panamericano de Febre Aftosa,maio 1962, 15 p., 1962.

4. -----. Vacunas antiaftosas hidróxido-saponinadas inactivadas por el formol.Bol Cent Panam Fieb Aft 1:1-19, 1971.

5. Cunha, R. G. et al. Studies on rabbit-adapted foot-and-mouth disease virus.II. Immunogenicity. Am J Vet Res 20:138-44, 1959.

6. Dacorso Filho, P. and R. G. Cunha. Lesoes observadas em coelhos recém-nascidos inoculados com amostras de tres tipos de vírus de febre aftosa.Rev Brasil 14:343-58, 1954.

211

7. Norambuena, M. A. et al. Vacuna contra la fiebre aftosa preparada de viruscultivado en células renales. Bull Off Int Epizoot 57:618-19, 1962.

8. Nottebohn, A. et al. Examen antigénique d'un vaccin anti-aphteux inactivé(type "A" Vallée) préparé avec du virus cultivé sur des cellules BHK 21.Bull Off Int Epizoot 61:919-33, 1964.

OTHER DATA

Drs. Daniel Abaracon, Homero V. Giacometti, Eduardo R. Centeno,and Julio Mesquita, Pan American Foot-and-Mouth Disease Center,Rio de Janeiro, Brazil


1953 - continuing.

212

Grantee:

Funded by:

Timetable:

FOOT-AND-MOUTH DISEASE VIRUS (FMDV) INACTIVATION

PROBLEM

In large-scale vaccine preparation, the virus usually is inactivated by chemicalmeans. Research must provide methods of inactivation which insure a safe vaccine,while not damaging the antigen, and which are active on large volumes of virus suspen-sion in a reasonable amount of time. The whole inactivation process must be fairlysimple and safe.

METHOD

The method used consists of treatment of FMDV with chemicals that inactivatethe virus according to first order kinetics. Satisfactory treatments will be appliedto the formulation of experimental vaccines. These vaccines are first to be screenedin guinea pigs and, if promising, further tested in cattle.

RESULTS TO DATE

(1) Inactivation of FMDV with binary ethylenimine (BEI)

From the many substances studied for the inactivation of FMDV, only the aziri-dine compound N-acetylethylenimine (AEI) is now used to some extent in vaccine produc-tion. It is a safer inactivant than formaldehyde since inactivation proceeds as a firstorder reaction, but has the disadvantage of being unstable at room temperature.

Ethylenimine (El) was shown to give a similar inactivation reaction to AEI withFMDV and to be stable when stored at room temperature.

Ethylenimine was prepared in dilute form from 2-bromoethylamine in alkaline solu-tion, (binary ethylenimine), in order to reduce its toxicity. Studies of the inactiva-tion of FMDV by BEl showed that it was equally suitable as an inactivant as pure El.

(2) Immunogenicity of FMD vaccines inactivated with BEI

In cattle, the immunogenicity of foot-and-mouth disease vaccines inactivatedwith BEl and adjuvanted with either aluminum hydroxide or incomplete Freund's adjuvantwas compared to that of identical vaccines inactivated with AEI. No differences werefound in the levels of immunity induced by vaccines of the same adjuvant type.

Binary ethylenimine is easy to prepare and the preparation is considerably lesstoxic than AEI. These properties make it a more desirable inactivant and will facili-tate its application in industrial vaccine production.

(3) Inactivation of viruses in serum

The inactivation of six virus strains from three different groups of viruseswith 0.001 M BEI at 370C proceeded at the same rate in either bovine serum or cellculture medium. The inactivant did not impair the growth-promoting capacity of bovineserum used in cell culture nor did it affect the biological activity of guinea pighyperimmune serum.

213

SIGNIFICANCE

FMD campaigns now in progress in South America require a vaccine with a highdegree of safety such as those prepared with AEI.

BEl being easier to prepare, and being considerably less toxic than AEI, appearsvery attractive as an inactivant for industrial vaccine production.

BEI can be used for the inactivation of viruses in biological materials, whichfacilitates international exchange of test reagents. BEI can also be used to inactivatethe contaminant viruses that might be present in serum for cell culture.

PUBLICATIONS

1. Bahnemann, H. G. Empleo de la etilenimina y la propilenimina como agentesinactivantes del virus de la fiebre aftosa. (Nota previa). Bol Cent PanamFieb Aft 7:41, 1972.

2. -----. The inactivation of foot-and-mouth disease virus by ethylenimineand prophylenimine. Zentralblat Vet Med B 20:356-60, 1973.

3. ----- et al. Immunogenicity in cattle of foot-and-mouth disease vaccinesinactivated with binary ethylenimine. Bull Off Int Epizoot 81:1335-43, 1974.

4. -----. Binary ethylenimine as an inactivant for foot-and-mouth diseasevirus and its application for vaccine production. Arch Virol 47:47-56, 1975.

OTHER DATA

Grantee: Drs. Hanz Bahnemann, Daniel Abaracon, and Homero V. Giocometti,Pan American Foot-and-Mouth Disease Center, Rio de Janeiro,Brazil.



214

ADJUVANTS FOR FOOT-AND-MOUTH DISEASE (FMD) VACCINES

PROBLEM

Control of FMD in South America is largely based on the systematic vaccinationof cattle. The vaccines are produced from inactivated FMD virus with aluminum hydro-xide as adjuvant. Immunity is often enhanced by the addition of saponin. However,these vaccines induce only a short-lasting immunity requiring repeated vaccinations at4-month intervals.

Vaccines containing mineral oil as adjuvant have been shown to produce highlevels of immunity of long duration. Problems related to the production of such vac-cines on an industrial scale are being investigated.

The use of commercially available saponin in FMD vaccines has sometimes led tolocal or generalized reactions. Purification of saponin to use the active fractiononly reduces these side effects and has great practical value.

Pigs play an important role in the epidemiology of FMD. They are difficult toprotect with the current inactivated vaccines and have a high population turnover,which make maintenance of a sufficient population immunity difficult. The disease inpigs may have a severe economic impact because of the large aggregations of these ani-mals in certain areas. Once a reasonable level of FMD control in the cattle populationis reached, FMD in pigs is likely to become an important problem. Experimental oiladjuvanted AEI-inactivated FMD vaccines have been shown to induce excellent levels ofneutralizing antibodies in pigs. However, field application of that product cannot, asyet,be recommended because of tissue reactions at the inoculation site and in the re-gional lymph nodes. Therefore, one of the aims of this project is to develop a vaccinefor pigs with acceptable low levels of undesired side effects.

METHOD

Emulsions of mineral oil (Marcol 52) and Arlacel A and inactivated virus suspen-sions are being prepared on a pilot plant scale. Parameters to be studied are physicalproperties of the emulsion such as conductivity and viscosity in relation to vaccine im-munogenicity. Different oils and emulsifiers are to be tested and incorporated in vac-cine for screening in guinea pigs. Promising products will be tested in cattle.

Other adjuvants will be tested, again first in guinea pigs, and eventually byvaccination of cattle.

Toxicity and the enhancement of immune response of different batches of saponinis to be tested in cattle.

Mineral oil or other adjuvants will be used to prepare experimental vaccines forpigs. Other improvements to be considered in combination with suitable adjuvants are:(a) reduction of the dose of vaccine, for instance, by virus concentration and purifica-tion; and (b) using different vaccine application techniques.

215

RESULTS TO DATE

(1) Oil adjuvant vaccine production in large volume and field test in cattle

No differences were observed in the response of cattle vaccinated with emulsionmade with the original bench top emulsifier or with vaccines prepared with the pilotplant emulsification tank.

(2) Immune response of cattle injected with FMD virus A24 conjugated withsheep red blood cells

The conjugate of FMD virus A24 with sheep red blood cells and oil emulsifiedwith adjuvant induced the formation of passive hemagglutinating and protective anti-bodies.

(3) Selection of saponin for inactivated vaccines

Batches of commercially available saponin vary greatly in their ability to enhancethe immune response of FMD vaccine and in their toxicity. The three batches tested didnot, however, differ in their effect on the immune response, and all produced only amoderate local reaction.

SIGNIFICANCE

Increase in the time interval between vaccinations will substantially reduce thecosts of FMD vaccination campaigns in South America.

Such increases, as well as a more solid immunity of the vaccinated cattle popu-lation, depend greatly on the contributions made by this research project.

No effective vaccines are available to immunize pigs. The right combination ofantigen, adjuvant, dose, and route of application is likely to provide the answer.

PUBLICATIONS

1. Callis, J. J. et al. Foot-and-mouth disease virus. Immunization. Proc19th World Veterinary Congress, Mexico City (1971). Published in 1972.

2. Maes, R. and M. V. Fernandes. Viability of foot-and-mouth disease virusin oil emulsions. Proc Soc Exp Biol Med 132:447-49, 1969.

3. U.S. Department of Agriculture and Pan American Health Organization. Foot-and-mouth disease oil adjuvant vaccines. I. Vaccination and revaccinationof cattle, sheep, and pigs. Collaborative research Plum Island AnimalDisease Center and Pan American Foot-and-Mouth Disease Center. Bol CentPanam Fieb Aft, in press.

4. ----- and -----. Foot-and-mouth disease oil adjuvant vaccines. II. Furtherstudies on duration of immunity in vaccinated cattle and pigs. Collabora-tive research Plum Island Animal Disease Center and Pan American Foot-and-Mouth Disease Center. Bol Cent Panam Fieb Aft, in press.

216

OTHER DATA

Grantee:

Funded by:

Timetable:

Drs. Daniel Abaracon, Homero V. Giacometti, Paulo A. de Mello,and Julio Mesquita, Pan American Foot-and-Mouth Disease Center,Rio de Janeiro, Brazil.


1967 - continuing.

217

FIELD APPLICATION OF INACTIVATED FOOT-AND-MOUTH DISEASEOIL ADJUVANTED VACCINE

PROBLEM

Studies at the Plum Island Animal Disease Laboratory (PIADL) and the Pan AmericanFoot-and-Mouth Disease Center (PAFMDC) demonstrated that under laboratory conditionscattle could be adequately protected against foot-and-mouth disease (FMD) with vaccinesprepared from FMD virus inactivated with N-acetylethylenimine (AEI) and adjuvanted withmineral oil. In view of these favorable results a long-term program was implemented tostudy the use of such vaccines under controlled field conditions.

It is the specific aim of this project to determine the best vaccination sched-ules, eventual side effects, or other problems that may arise from the large-scale useof oil vaccines.

The first site selected for these studies was the Ministry of Agriculture's"Cinco Cruzes" Experimental Station in Bagé, in the state of Rio Grande do Sul, southernBrazil, located about 400 km south of the state capital, P¿rto Alegre. This farm hasapproximately 3,000 cattle heads.

Further expansion of the vaccination program to other farms and areas will de-pend on the results of these first trials in Bagé.

METHOD

Field trials will be carried out with comparable groups of cattle to compare con-ventionally prepared inactivated aluminum gel vaccines and those containing oil adju-vants. The antigen source will be similar in both vaccines.

Duration of immunity will be studied after the vaccination of young cattle at6-month intervals followed by yearly vaccinations of cattle older than 2 years. Thevaccination schedule will be modified according to the results.

The effect of the vaccine will be measured by its influence on the disease inthe vaccinated herd, by antibody assay, and by a survey of virus persistence in thecattle population.

Observations will be made on the influence of vaccination on milk production,vaccine lesions, or hypersensitivity reactions.

RESULTS TO DATE

Vaccination of young cattle: comparison of oil and Al-gel adjuvants

The serum protection test results of a group of 5-to 7-month old cattle vacci-nated and revaccinated under controlled field conditions with oil adjuvanted foot-and-mouth disease virus vaccine were compared with those of cattle vaccinated with aluminum-hydroxide vaccine. The same AEI inactivated antigens were used in both vaccines. Themean expected percentage of protection showed that the oil-adjuvanted vaccines produceda higher and longer lasting protection than did the aluminum-gel vaccines. The immune

218

response at revaccination with the oil vaccine 6 months later produced a booster effectand induced persisting high levels of protection.

Study of population immunity after vaccination of young cattle

A bimonthly serum survey of the calf population of nearly 300 animais vaccinatedwith the oil vaccine at 6-month intervals showed that under field conditions this typeof vaccine produced high levels of long-lasting protection. Six months after adminis-tration of oil vaccines, at least 70 percent of the young cattle were protected. Theselevels of protection persisted for up to 9 months for types O and C, and 12 months fortype A.

A choice of virus strains to be used for the preparation of the vaccine is im-portant.

Vaccination of adult cattle

Twenty adult cattle were vaccinated and revaccinated at six-month intervals.Another group of 20 cattle was revaccinated only once. Revaccination of both groupsproduced an excellent long-lasting immunity, and early vaccination may be feasiblewith oil adjuvanted vaccines.

The yearly vaccination program at the Bagé farm covered 3,000 cattle and producedexcellent immune response over a 4-year period.

Routes of application

No differences in immune response were observed using different inoculationroutes for the oil adjuvanted vaccine.

Effect of oil adjuvanted vaccines on animal health and milk and meat production

No ill effects of the vaccine were observed. Repeatedly vaccinated cattle wereslaughtered and their carcasses passed the requirements for export meat inspection.

No ill effects of simultaneous rabies and FMD vaccinations were detected.

SIGNIFICANCE

Present vaccines only protect for a short period of time and must be administeredat 4-month intervals to be effective. This frequent vaccination is costly and requiresmuch effort in vaccine production, administration, and control.

This project will indicate how to reduce significantly the amount of vaccine andvaccinations needed, which will result in a considerable saving.

PUBLICATIONS

1. Augé de Mello, P. et al. Field application of inactivated oil adjuvantedfoot-and-mouth disease virus vaccine: vaccination and revaccination ofyoung cattle. Submitted to Appl Microbiol.

219

OTHER DATA

Grantee:

Funded by:

Timetable:

Drs. Paulo A. de Mello, Vicente Astudillo, and Ivo Gomes, PanAmerican Foot-and-Mouth Disease Center, Rio de Janeiro, Brazil.


1972 - continuing.

220

MODIFIED LIVE FOOT-AND-MOUTH DISEASE VIRUS VACCINES

PROBLEM

The development of modified live virus FMD vaccines has been one of the majorefforts of the Center. Even though at the present time this type of vaccine is re-stricted, partly because of limitations established by the beef importing countries,the Center is continuing research to explore its potential use under certain conditions.One of the objections to modified live virus vaccine has been that strains attenuatedfor cattle may still remain pathogenic for pigs. Methods to shorten the relativelylong time needed for attenuation and the use of selected clones as vaccine strain arebeing investigated in Venezuela.

Local defense mechanisms can be stimulated by the application of live attenuatedFMD virus in the upper respiratory tract.

Local defense mechanisms at the portal of entry of the virus will probably havemore effect in controlling the spread of the virus in a population than will conven-tional vaccination alone, and will be a valuable adjunct to other measures such asquarantine or slaughter of potentially exposed contact animals.

Experiments should be done to determine whether massive intranasal administrationof modified strains can replace virulent field strains.

METHOD

Strains of FMD virus are attenuated by passage in embryonated eggs and by selec-tion of clones in BHK cell cultures. Viremia in cattle is also used as a marker forstrains that multiplied well in the animal. Such strains supposedly induce high levelsof protection and, in the absence of lesions, are studied as potential vaccine strains.

Oil adjuvant is incorporated to the vaccine to increase immunogenicity and pro-bably decrease pathogenicity. Dose response studies are made in cattle to study theoptimum amount of antigen per vaccine dose. Whether viral multiplication is essentialfor the induction of immunity is also determined.

Growth curves of attenuated strains after intranasal inoculation of cattle areestablished, followed by studies of local and circulating antibodies and challengeof immunity by intranasal or control exposure.

Intranasal inoculation of attenuated strains are tried in outbreak situationsto determine if the vaccine strain will replace the wild virulent virus.

RESULTS TO DATE

(1) Pathogenicity and immunogenicity of types O, A, and C.

Selection of strains with a low pathogenicity but with a high immunogenicity wascontinued,using plaque selection techniques. Some of these strains were used success-fully in Venezuela in routine vaccine production.

221

(2) Field use of trivalent vaccine without adjuvant, and with oil or aluminumgel adjuvant

The response of modified live virus was improved by the incorporation of mineraloil in the vaccine. The number of cattle with post-vaccinal lesions was also reduceddrastically, probably because of the slower release of the virus from the inoculationsite.

(3) Vaccination of cattle with live attenuated and inactivated viruses inthree dose levels and adjuvanted with oil

In both vaccines, dilution of the antigen resulted in gradual decrease of theimmune response.

The immune response of the live vaccine was longer lasting than that of the in-activated vaccine.

Live virus vaccine induced anti-VIA antibodies, indicating that at least in somecattle the virus multiplied.

The irregular response with live vaccine in the 1:250 dilution was probablycaused by individual differences in virus multiplication.

(4) Vaccination by aerosolized modified live virus

Preliminary experiments indicated that the virus replicated and induced a solidprotection in the vaccinated cattle.

(5) Pharyngeal growth of attenuated strain A24 in cattle after intranasalinstillation and subsequent development of local neutralizing activityand circulating antibodies

In six inoculated cattle, the infectious process was initiated normally as withvirulent strains, but interference with the "normal" growth occurred in most instances,resulting in abortive growth and in absence of viremia and of clinical signs. Therewas no relationship between the levels of circulating antibody before exposure and thevirus growth patterns. Stimulation of circulating antibodies did not always occur.

Four steers similarly inoculated but without previous exposure to the modifiedvirus had pharyngeal virus growth within 6 hours, starting viremia between 24 and 30hours, and severe oral and foot lesions of FMD at 48 hours after exposure.

In a further experiment, 8 cattle at the Jardim experimental farm were inoculatedintranasally with 107-7 plaque-forming units (PFU) of modified virus type A24.

None of the cattle developed clinical signs.

Two steers had intermittent virus growth. ihe other 6 steers showed a more orless continuous virus growth until the end of the observation period (72 hours).

Virus was not detected in any of the blood samples.

It can be concluded that the MLV A2 4 strain at the passage level used replicateswell when inoculated intranasally. Growth in several cattle was, however, abortiveand titers were lower than could be expected with virulent virus. Viremia appears tobe a rare occurrence since no virus could be detected in the blood of any of the 13cattle used in the experiments. Also, in many instances, there was no serum conver-sion. By the plaque reduction neutralization test, specific neutralizing activitycould be demonstrated in the pharyngeal mucus.

222

(6) Intranasal exposure of cattle with virulent FMD virus A24 followingintranasal inoculation of modified live virus

Intranasal inoculation of modified live virus strain A24 showed an inhibitoryeffect on subsequent growth of virulent homologous virus inoculated by the same route.

No viremia could be detected and no clinical signs of FMD developed even thoughtwo of the steers did not have detectable circulating antibodies at the time of expo-sure to the virulent virus. Also, one of these steers did not show serum conversionafter the virulent virus infection.

SIGNIFICANCE

Modified live virus vaccines are inexpensive to prepare and should afford a highdegree of protection.

It can be expected that the substitution of the wild virus population by atten-uated virus and the development of local defense mechanisms will favorably change theepidemiologic characteristics of the disease. Moreover, these studies will help toclarify the basic virus-host relationships in virus infections and to determine thechanges that occur in the virus population during the attenuation process.

PUBLICATIONS

1. Augé de Mello, P. et al. Supervivencia en bovinos del virus modificado dela fiebre aftosa. 5° Congr Panam Med Vet Zootec, Caracas, Memorias 1:58-68,1966, and Bull Off Int Epizoot 65:2091-06, 1966.

2. ----- et al. Adaptagao do subtipo A17 do virus da febre aftosa a ovosembrionados de galinha. Arq Inst Biol, S. Paulo 35:99-105, 1968.

3. Bernal, C. et al. Estudio sobre la modificación de una cepa de virus defiebre aftosa (tipo "A" Vallée) y su utilización como vacuna. 5? CongrPanam Med Vet Zootec, Caracas. Memorias 1:42-58, 1966.

4. Crandell, R. A. and I. Gomes. Plaque morphology of some South Americanstrains of foot-and-mouth disease virus and the effects of polyionic com-pounds on plaque formation. Arch Ges Virusforsch 30:137-46, 1970.

5. ----- and -----. Further studies on markers of foot-and-mouth diseasevirus. Bull Off Int Epizoot 73:521-33, 1970.

6. Cunha, R. G. and E. A. Eichhorn. Influence of cortisone on susceptibilityof adult mice to foot-and-mouth disease virus. Am J Vet Res 15:149-51, 1954.

7. ----- and -----. Studies on rabbit-adapted foot-and-mouth disease virus.1. Propagation and pathogenicity. Am J Vet Res 20:133-37, 1959.

8. ----- and M. G. Guerreiro. Différences dans le pouvoir pathogenedu virus lapinisé chez divers hotes. Bull Off Int Epizoot 53:596-98, 19b0.

9. ----- et al. Etude immunologique de deux souches modifiées du virus aphteuxde type "O" Vallée. Bull Off Int Epizoot 57:717-32, 1962.

223

10. -----. Quelques aspects de l'application du vaccin anti-aphteux 3 virusvivant attenué. Bull Off Int Epizoot 57:733-47, 1962. Em espanhol:Publ. N' 28 del Centro Panamericano de Fiebre Aftosa, junio, 15 p., 1962.

11. ----- et al. Studies on a rabbit-adapted foot-and-mouth disease virusof type C Waldmann. 1. Adaptation and immunogenicity. Can J Comp Med28:288-96, 1964.

12. ----- et al. Vaccination bivalente au moyen de deux souches modifiéesde virus aphteux. Bull Off Int Epizoot 61:801-18, 1964.

13. Goic, R. Campaña antiaftosa con vacuna de virus vivo modificado enVenezuela. Bol Cent Salud Pec, Quito 3:31-34, 1963.

14. Gomes, 1. and R. A. Crandell. Marker studies of foot-and-mouth diseasevirus. Bull Off Int Epizoot 71:835-46, 1969.

15. Henderson, W. M. and R. G. Cunha. Research on the development of a modifiedlive virus vaccine in foot-and-mouth disease. 16° Congr Mund Vet, Madrid,2:395-96, 1959.

16. ----- and -----. Le controle de la production des vaccins anti-aphteux 3virus vivant atténué. Symp Int Virol Vét, Lyon 101-07, 1962. Em espanhol:Publ. N' 29 del Centro Panamericano de Fiebre Aftosa, junio, 6 p., 1962.

17. Maes, R. Attenuation of foot-and-mouth disease virus by chemical means.Arch Ges Virusforsch 29:63-76, 1970.

18. ----- and J. Mesquita. High mutation rate of foot-and-mouth disease virustreated with hydroxylamine. Arch Ges Virusforsch 29:77-82, 1970.

19. -----. Investigations of the attenuation induced in foot-and-mouth diseasevirus by a chemical mutagen. Arch Ges Virusforsch 37:19-33, 1972.

20. Palacios Garcia, C. A. et al. Avianización del virus de la fiebre aftosatipo "0" Vallée, cepa Lara de Venezuela. Bol Inst Invest Vet 12:3-27, 1960.

21. -----. Investigaciones realizadas con los virus atenuados de la fiebreaftosa de los tipos "0" y "A" Vallée en Venezuela. Bull Off Int Epizoot61:757-800, 1964.

22. ----- et al. Live foot-and-mouth disease vaccines, with special referenceto South America. Publ. N? 39 del Centro Panamericano de Fiebre Aftosa,1966.

23. ----- et al. Attenuated vaccines against foot-and-mouth disease using type0 virus of the high-passage Lara strain. Proc 5th Panam Congr Vet MedZootech, Caracas 1:29-42, 1966, and Vet Bull, Weybridge 38:4517, 1968.

24. -----. Estudios sobre vacunas de virus vivo contra la fiebre aftosa. RevVet Venez 24:241-77, 1969.

25. Quiroz, R. et al. Factors associated with anaphylactic reactions to chickenembryo foot-and-mouth disease vaccine and flury rabies vaccine in cattle ofVenezuela. Am J Vet Res 25:1627-34, 1964. Em espanhol: Cuad Cent PanamFieb Aft 3:97-106, 19-6.

26. Zahran, G. E. D. Foot-and-mouth disease virus. I. Propagation of 3 immu-nologic types of virus in chicks. Am J Vet Res 22:518-26, 1961.

224

27. -----. Foot-and-mouth disease virus. II. Propagation and modificationof 3 immunologic types of virus in embryonating chicken eggs. Am J Vet Res22:527-32, 1961.

OTHER DATA

Drs. Carlos Bernal, Paul Sutmoller, Paulo A. de Mello, andKleise F. Costa, Pan American Foot-and-Mouth Disease Center,Rio de Janeiro, Brazil.


1956 - continuing.

225

Grantee:

Funded by:

Timetable:

QUALITY CONTROL OF FOOT-AND-MOUTH DISEASE (FMD) VACCINE

PROBLEM

Vaccine control is one of the major problems facing anti-FMD campaign managersin South America. In many instances potency testing of FMD vaccines in cattle hasproved impractical. Quality testing during the vaccine production process has been,at best, spotty. Furthermore, the changing concepts of FMD virus pathogenesis arehardly reflected in current vaccine potency testing.

METHOD

Emphasis is placed on the development of practical methods to monitor vaccineproduction at all stages, starting with the selection of virus strains, and continuinguntil the finished product is injected into the animal. Research on reproducible testsystems in small laboratory animals, such as guinea pigs, will continue, and attemptswill be made to relate the results to resistance against the disease under conditionscomparable to those encountered in the field.

RESULTS TO DATE

Correlation of serum neutralization titer and immunity of cattle after intra-dermal lingual (IDL) inoculation

Sera from cattle used in vaccine control in Argentina, Rio Grande do Sul, Brazil,and the Center were classified according to neutralization test results obtained at theCenter.

Results showed difficulty in finding a suitable standard to reflect the immunestatus of cattle. Even though all serum neutralization tests were performed at theCenter, great differences were observed in the neutralization titer/protection response.The problem appears to be related more to the challenge of the immunity in the cattlethan to the results of the neutralization tests.

Correlation of mouse protection index and immunity of cattle after IDLinoculation

A protection test in suckling mice for the assay of FMD antibodies was describedin detail by Cunha and collaborators. The results of this test are expressed as amouse protection index (MPI). These same authors found that cattie could be classifiedaccording to their immune status as follows: an MPI of 1.0 or less would normally in-dicate no resistance to the virus, whereas cattle with a MPI > 2.0 serum would, for themost part, be protected. The mouse protection test has been extensively used by theCenter, and much information has been accumulated with sera obtained from immunized ornonimmunized and recovered cattle that were subsequently exposed to the homologousvirus.

An attempt was made to investigate further the relationship between the resis-tance of cattle upon exposure to FMD virus and the MPI.

There is a significant correlation between MPI and cattle protection at challenge(r = 0.91, p < 0.05). However, with the inactivated vaccines as well as with the

226

modified live virus vaccines, more animals developed foot lesions with types O virus

than with the two other types of virus. No differences were demonstrated between in-activated or modified live virus vaccines, but a slightly higher MPI was required forinactivated vaccine to protect an equal percentage of cattle against foot lesions.

In the lower range (MPI = 0.0-1.0) nearly 30 percent of the cattle were pro-tected by the vaccine versus 2 percent of the unvaccinated cattle. This effect waseven more pronounced in the 1.1-2.0 MPI range, in which approximately 70 percent of thevaccinated cattle were protected with an MPI < 2. Only 5 percent of the cattle withMPI > 2 developed foot lesions but this figure is mainly determined by the large number

of cattle with an MPI > 3.

Under different challenge conditions (type of cattle, strains of virus, andothers), a somewhat different relationship between MPI and protection may be expected.In addition, protection as defined by the prevention of foot lesions after tongue in-oculation may be quite different from virus exposure under field conditions.

Protective dose in guinea pigs

The techniques of this test were established and standardized. Comparison of

the test results with those obtained in cattle are in progress.

Influence of vaccination routine, challenge period, and saponin on the immuneresponse of guinea pigs

Intramuscular inoculation of aluminum-gel vaccine gave a better immune response

than did subcutaneous inoculation. Guinea pigs should not be challenged before 21 dayspostvaccination; at 30 days, protection was still increasing. Best results were ob-tained with 0.15 mg of saponin per vaccination dose of aluminum-gel vaccine when usedsubcutaneously. Control of vaccine containing 0.4 mg or more of saponin was not possi-ble because of severe inflammatory reactions.

Pharyngeal growth of virus after IDL inoculation

Results indicated that virus replication in the pharynx was quite independentof the amount of circulating antibodies. This replication and viremia occurred severalhours before the development of any visible lesions at the site of inoculation on thetongue.

SIGNIFICANCE

Vaccine control is of crucial importance to South America. Monitoring methodsboth during the production process and the inoculation of the final vaccine are requiredto control eventually all FMD vaccines produced for campaigns now in progress.

PUBLICATIONS

1. Alonso Fernandez, A. et al. Control de vacunas antiaftosa. Relación entreel índice K (modificado) y los indices de sero-protección y seroneutraliza-ción. Bol Cent Panam Fieb Aft 6:1-16, 1972.

2. Gomes, I. and V. Astudillo. Foot-and-mouth disease: evaluation of mouseprotection test results in relation to cattle immunity. Bol Cent PanamFieb Aft, in press.

227

3. Henderson, W. M. et al. Le controle des vaccins anti-aphteux 3 virusinactivés. Solutions adoptées en Amérique du Sud, spécialement en RépubliqueArgentine. Symp Int Virol Vet, Lyon, 1962, pp. 71-88. Em espanhol: Publ.N' 30 del Centro Panamericano de Fiebre Aftosa, junio, 16 p., 1962.

OTHER DATA

Drs. Albino A. Fernandez, Ivo Gomes, Antonio Vieira, and Maria V.Ferreira, Pan American Foot-and-Mouth Disease Center, Rio deJaneiro, Brazil.


1967 - continuing.

228

Grantee:

Funded by:

Timetable:

DIAGNOSIS OF VESICULAR DISEASES OF LIVESTOCK

PROBLEM

Vesicular diseases (foot-and-mouth disease, vesicular stomatitis, swine exan-thema, and swine vesicular disease) are highly contagious and have similar symptoms,which makes rapid laboratory diagnosis essential to insure efficient control or eradi-cation.

The foot-and-mouth disease (FMD) virus is very unstable as proved by the exis-tence of 61 different subtypes. Continuous typing and subtyping of field virus isessential, especially for countries with disease control programs based on preventiveimmunization, to insure that appropriate strains are used for the production and con-trol of vaccines.

In addition to the antigenic study, the classification of a new subtype requiresthe determination of the immunologic relationships between field viruses of epidemiol-ogic significance and those used in vaccine production.

A standardized methodology and appropriate reagents are essential to obtainreproducible results for the subtyping and classification of a virus. For this purposean updated strain bank must be maintained.

In some areas of the Americas, vesicular stomatitis (VS) exists but FMD doesnot. Therefore, a prompt diagnosis is necessary to insure the success of eradicationmeasures in the event of an FMD outbreak.

Colombia, Ecuador, Peru, and Venezuela, which are affected by both VS and FMD,have FMD control programs. In these countries, differential diagnosis is a doublenecessity.

Recently the Pan American Foot-and-Mouth Disease Center demonstrated the exis-tence of subtypes of the Indiana VS virus in Argentina and Brazil, where the diseasehad never been diagnosed before. This finding points to the need for differentialdiagnosis, subtyping, and when necessary, classification of strains.

METHOD

Differential diagnosis, typing, subtyping, and serologic classification of FMDvirus are achieved by complement fixation (CF) tests and, if necessary, by a combina-tion of passages of the suspected viral agents in cell cultures, suckling, and adultmice.

The immunologic relationships are determined by the mouse protection test, sero-neutralization in tissue cultures, the Index C in guinea pigs, and the modified Index Kin cattle.

Differential diagnosis, typing, and subtyping of VS virus is done by passagesin tissue culture and in suckling or adult mice, and by CF. The virus is classifiedby determination of the serologic relationships using the CF test. Diagnosis will alsobe made by neutralization and CF techniques.

229

RESULTS TO DATE

Between 1952 and 1973, about 10,000 specimens of FMD field virus from SouthAmerica were typed and subtyped. During that period, 20 different subtypes were iden-tified:

Type 0 Subtypes 01 and 08

Type A Subtypes Ajo, A13, A16, A17, A18, A19, A24'

A25' A26' A27' A29' A30' A31' A32

Type C Subtypes C2, C3, C4, C5

With the exception of 01, A10, and C2, all were new subtypes in the World Refer-ence Laboratory's international classification.

In 1972 and 1973, subtypes 01, A24, A26, A2 7, A32, C2, C3, and C5, were identifiedin the field in South America, and the countries affected by one or more of these subtypeswere Argentina, Bolivia, Brazil, Chile, Colombia, Ecuador, Guyana, Paraguay, Peru,Uruguay, and Venezuela.

All the South American countries affected by FMD have national diagnostic labora-tories and are able to classify a new subtype.

Between 1952 and 1973, more than 1,000 specimens of VS virus from Central andSouth America were assayed. During the same period the Pan American Foot-and-Mouth Dis-sease Center demonstrated the existence of subtypes of the Indiana VS virus.

Subtype Indiana 1 is the strain designated as the Indiana reference strain. Boththis virus and the New Jersey one are present in the United States, Mexico, CentralAmerica, Panama, Venezuela, Colombia, Ecuador, and Peru. Subtype Indiana 2 is repre-sented by the Salto strain, identified in Argentina in 1963. The same subtype was iso-lated in Brazil in 1965 and 1966. This subtype also includes the Cocal or Trinidadstrain. Subtype Indiana 3 was identified in Alagoas, Brazil, in 1964.

Virus of subtypes Indiana 2 and 3 have not been isolated since 1966. The Saltoand Alagoas strains are characterized by the fact that they only affect the equine spe-cies. Subtype Indiana 3 produced plaques smaller than the New Jersey and the othersubtypes of the Indiana type strain when cultivated in monolayers of BHK celis coveredwith agar. These plaques were increased in size with DEAE dextran and completely in-hibited with dextran sulfate.

So far, no subtypes of the New Jersey strain have been identified.

During 1975, 240 epithelium samples were submitted for type or subtype identifi-cations from South American countries. Also, 425 serum samples were received fromBrazil, Colombia, Bolivia, Guyana, and South America to test for the presence of VIAantibodies.

In 1975 also, 291 epithelium samples were received from Central American coun-tries, Panama, and the Caribbean. VS virus was identified in 163 of those samples.

The'following experimental work was initiated in 1975:

(1) Serologic classification of the FMD strains A Ecuador/75, A Sabana/74(Colombia), A San Martin/75 (Argentina), A Sao Paulo/75 (Brazil), A Uruguaiana/74(Brazil), and A Alegrete/75 (Brazil) and cross immunization of these strains with theCenter's A24 Cruzeiro reference strain.

230

(2) Serologic classification of FMD virus A22 Iraq, with inactivated antigen.

(3) Selection of an FMD virus A strain with broad immunogenic spectrum.

(4) Serologic classification of FMD virus C Chaco/74 (Paraguay), C Bolivia/74,and C Santa Fé (Argentina).

(5) Serologic relationship between VS virus strains New Jersey, Indiana 1, andIndiana 2, before and after inactivation with BEI. The strains maintained their CFcapacity and the same parentage after treatment with BEI, but were completely inacti-vated. Thus, similarly treated diagnostic reagents will be safe to be shipped to areaswhere these agents do not exist.

(6) Influence of chloroform on the antigenicity and infectivity of VS virus.The CF capacity was not affected by challenge but the viruses lost their infectivity.

SIGNIFICANCE

Typing and subtyping of vesicular disease field samples usually are carried outby national diagnostic laboratories, but if any doubt arises about the identity of thestrain the Center acts as the Reference Center for the Americas. The Central Americancountries and Panama do not have vesicular disease typing facilities, and all samplesare forwarded to the Center for diagnosis.

This close collaboration of the countries and the Pan American Foot-and-MouthDisease Center has made it possible to identify the FMD virus subtypes present in thefield, to make a rapid diagnosis of VS, and to take the appropriate preventive measureswithout delay.

The projects are of great assistance to the countries with anti-FMD campaignsand to those free of FMD, but which may have VS.

PUBLICATIONS

1. Alonso Fernandez, A et al. Comparación inmunológica y serológica de dossubtipos del virus aftoso tipo C Waldemann. Bol Cent Panam Fieb Aft 4:9-20,1971-

2. ----- et al. Diagnóstico y referencia en la fiebre aftosa. Bol Cent PanamFieb Aft 11:1-12, 1973.

3. Cunha, R. G. et al. Differentiation between foot-and-mouth disease andvesicular stomatitis viruses by means of mouse inoculation. Am J Vet Res16:472-80, 1955.

4. ----- and A. Vieira. Investigación sobre la relación inmunológica entre losvirus de fiebre aftosa y Coxsackie. Bull Off Int Epizoot 63:505-14, 1965,and Cuad Cent Panam Fieb Aft 3:189-94, 1965.

5. Eichhorn, E. A. Laboratory diagnosis of foot-and-mouth disease. Proc 15thInt Vet Congr, Stockholm 1:187-91, 1953.

6. Federer, K. E. et al. Identification d'un nouveau sous-type A de virusaphteux. Bull Off Int Epizoot 57:1171-90, 1962, and Cuad Cent Panam FiebAft 1:1-15, 1963.

231

7. ----- et al. Identificación de un nuevo subtipo "O" del virus de lafiebre aftosa en Sud-América. Bull Off Int Epizoot 61:1563-78, 1964.

8. ----- et al. Vesicular stomatitis virus: the relationship between somestrains of the Indiana serotype. Res Vet Sci 8:103-17, 1967.

9. ----- et al. Développement d'un nouveau sous-type du virus de laaphteuse par passages en séries sur bovins partiellement immuns.apresentado ao simpósio sobre variantes do virus da febre aftosa,1967.

fievreTrabalhoLyon,

10. -----. Diagnóstico de laboratorio de la fiebre aftosa.J. C. and E. R. Castro (eds.), 5th Conf Simp Plen CongrMontevideo, Uruguay, pp. 171-74, 1971.

Grantee:

In: Bacigalupi,Latinoam Microbiol,

Drs. Albino A. Fernandez, Magnus S. Sondahl, Ivo Gomes, andEduardo R. Centeno, Pan American Foot-and-Mouth Disease Center,Rio de Janeiro, Brazil.


1957 - continuing.

232

OTHER DATA

Funded by:

Timetable:

THE MANAGEMENT OF MOUSE AND GUINEA PIG COLONIESUNDER TROPICAL AND SUBTROPICAL CONDITIONS

PROBLEM

There are well-developed methods for the management of small animal colonies forlaboratory use in several of the industrialized countries. In most tropical and sub-tropical developing nations, these techniques cannot, however, be applied directly, butmust be modified-according to local conditions. Although problems vary from one local-ity to another, they generally include lack of commercially available equipment andrations, extremes of temperature, lack of trained professional and subprofessional per-sonnel, and lack of diagnostic capabilities for laboratory animal diseases.

The aim of this project is to determine the minimum standards and the most use-ful practices for the management of mouse and guinea pig colonies to produce a regularsupply of laboratory animals of uniformly good quality.

METHOD

(1) Determination of adequate facilities and equipment

(a) Buildings. Although methods of construction of conventional buildingsvary from one area to another, certain basic requirements must be met to build satis-factory housing of animal colonies. The incorporation of these requirements into localconstruction practices by the most economical methods will be studied.

(b) Equipment. Problems to be solved concern cages, cage racks, water,feed dispensers, and others. The types of equipment vary tremendously. In order todevelop good equipment at a reasonable price, standardized requirements should be for-mulated.

(2) Rations

Industrialized countries use expensive ingredients in their commercial animalrations which less developed areas cannot afford. Efforts will therefore be made todetermine which locally available materials can be incorporated into the production ofadequate rations.

(3) Environmental control

It is well documented that temperature and humidity play an important role inthe health and reproduction capability of laboratory animals. For reliable year-roundproduction of animals, artificial control of the environment must be maintained to someextent in all areas, and to a great degree when extremes of temperatures occur. Theproject will thus investigate the degree and types of environmental control and theconstruction methods and equipment necessary,given local supplies and climate.

(4) Diseases

Methods applicable under prevailing conditions for the detection and preventionof diseases that can affect research results must be developed. The use of special dis-ease preventive equipment, such as filter-top cages, must be assessed under local con-ditions. A serologic screening for the identification of murine viruses will be made,

233

and the value of therapeutic measures for disease prevention and control will beevaluated.

(5) Breeding scheme

Studies on and evaluation of various breeding systems must be made to determinethe most economical system for the production of animals. This investigation will in-clude genetic control (inbred or non-inbred), cage population densities, cleaningschedules, and other aspects.

RESULTS TO DATE

(1) Mouse colony

The breeding systems used for the production of mice have been completely revised

to make them as cost-effective as possible for our particular supply demands. This hasallowed a reduction of 20,000 mice in the total colony size and has nearly doubled thenumber of litters of suckling mice available for laboratory use (January-June 1974,

9,772 litters; April-September 1975, 17,796 litters). This has also resulted in savingsof over $500 per month on feed costs alone.

The arrival of nearly 2,000 surplus stainless steel cages from the United Stateshas permitted the elimination of wooden cages in the laboratory. These stainless cagesalso permitted better conformation to recommended cage population densities, and drama-tically reduced the odor in the colony rooms associated with the use of wooden cages.

Consolidation of the mouse colony has freed two rooms that will be used forguinea pigs instead of the anticipated expensive construction and remodelling.

(2) Guinea pig colony

Minimal improvements only have been made in the guinea pig colony because offinancial shortages. On the whole, over the past 3 years, there has been a drop in

production of about 2/3 during the months of extreme summer heat. This caused fluc-tuations in the number of animals that can be supplied for laboratory use on a monthlybasis.

The only solution to this problem is environmental control by the use of airconditioned rooms, which will require the purchase of new tiered type caging to makemore efficient use of floor space.

SIGNIFICANCE

Research on diseases of man and animals greatly depend on the availability of agood supply of healthy laboratory animals.

At the Pan American Foot-and-Mouth Disease Center, mice are used for virus andantibody assays, and guinea pigs for the testing of vaccine potency and for the pre-paration of diagnostic antisera. The project will enable the Center to improve themanagement of its colonies and thus expand its service to the countries of the WesternHemisphere. Through an intensive training program at the Center, the results of thisproject are made immediately available to the national laboratories engaged in thediagnosis of vesicular diseases and/or foot-and-mouth disease vaccine control.

234

PUBLICATIONS

1. Smith, A. W. The Mouse. In: Centro Panamericano de Fiebre Aftosa.de Monografias Científicas y Técnicas No. 3, Rio de Janeiro, 1974.

Serie

2. Russell, R. J. and P. W. Schilling. The Rabbit. In: Centro Panamericanode Fiebre Aftosa. Serie de Monografias Científicas y Técnicas No. 4, Riode Janeiro, 1974.

OTHER DATA

Dr. Paul W. Schilling, Pan American Foot-and-Mouth DiseaseCenter, Rio de Janeiro, Brazil.


1973 - continuing.

235

Grantee:

Funded by:

Timetable:

DETERMINATION OF RABIES ANTIBODIES BY COUNTERIMMUNOELECTROPHORESIS

PROBLEM

The counterimmunoelectrophoresis (CIE) has received, in recent years, increasingattention for serology studies of several parasitic and viral diseases. Its value andpractical usefulness for the detection of rabies antibodies should be compared and eval-uated with the serum-neutralization (SN) test widely used in rabies.

METHOD

The sensitivity of the tests has been determined by the study of sera from vacci-nated persons or animals. To evaluate the specificity of the methods, sera from personsor animals with no previous history of exposure to rabies virus or vaccine, were used.

RESULTS TO DATE

Two hundred sera from vaccinated and nonvaccinated persons and animals have beenused to evaluate the sensitivity and specificity of the CIE in comparison with the SNtest for rabies antibodies detection. The high sensitivity and specificity of CIE test,the short time involved in obtaining a result and the observation that all positiverabies sera in the present study were also positive to SN test may favor its use as ascreening test when large numbers of sera have to be examined.

SIGNIFICANCE

This research provides evidence that CIE is an economic, rapid, and specific testfor the detection of rabies antibodies, which could be of practical importance.

PUBLICATION

1. Díaz, A. M. O. and V. M. Varela-Díaz. Detection of rabies antibodies bycounterimmunoelectrophoresis. Manuscript in preparation.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Ana María O. de Díaz, Pan American Zoonoses Center, RamosMejía, Buenos Aires Province, Argentina.

Pan American Health Organization/World Health Organization,United Nations Development Program, and the Governments ofthe Americas.

1975.

236

ANTIGENIC DIFFERENCES AMONG RABIES VIRUS STRAINS

PROBLEM

Several parameters have been employed to differentiate rabies virus strains sincethis is of potential importance in the characterization of vaccine strains and fieldvariants.

The counterimmunoelectrophoresis (CIE) test has been found to be of value in theidentification of antigenically different hepatitis and influenza viruses and for thetyping of adenovirus strains.

The present study was conducted to determine whether antigenic differences couldbe demonstrated between three rabies virus strains and the standard challenge virus(CVS) by the CIE test.

METHOD

Four rabies virus strains were used in this study: (a) the standard challengevirus (CVS); (b) 91-virus, a fixed rabies virus of human origin; (c) Apipé-virus, anisolate from the saliva of a vampire bat (Desmodus rotundus); and (d) Mangosta-virus,an isolate from a mangoose. Each of these strains were propagated in rabbit's brains.Monovalent antisera to four of these strains were prepared in rabbits.

The crossed serum neutralization (SN) test was performed with the monovalentantísera and the four rabies virus strains. On the basis of these titers, the anti-genic differences between virus strains (r value) were determined by the formula ofArchetti and Horsfall.

Rabies viruses were purified by centrifugation and Ecteola cellulose chromato-graphy and concentrated by ethylene glycol for use as antigen in CE test and for ab-sorption of antisera.

Aliquots of CVS antisera were absorbed with purified 91, Apipé and Mangosta ra-bies virus strains, respectively. Antisera to the latter three strains were similarlyabsorbed with CVS.

Crossed CE tests were performed with the absorbed antisera against homologousand heterologous strains. A positive CE test consisted in the detection of one or moreprecipitation bands.

RESULTS TO DATE

The present study has shown that absorption of CVS antisera with Mangosta orApipé rabies virus strains did not remove antibody activity to the standard CVS chal-lenge virus in CE tests. Similarly, antisera to each of these "street" viruses re-tained antibody activity to the corresponding homologous strains when absorbed withCVS virus. These findings may be explained in terms of the presence of different anti-genic determinants in the CVS virus and the two "street" rabies virus strains.

237

Failure of CVS or 91 antisera to reveal antibody activity to either strainsafter absorption with the heterologous strain indicates that no antigenic differencescould be detected between these two strains.

An r value of 1, obtained in crossed SN tests employing CVS and 91 strain-specific antisera, is indicative of an identity reaction between the two fixed rabiesvirus strains. The r values obtained for similar SN testing of CVS, Apipé and Mangostarabies virus strains, are suggestive of antigenic differences between the "street"virus strains and the CVS fixed virus strains.

The results obtained in crossed SN test and CE test are compatible.

SIGNIFICANCE

Using the counterimmunoelectrophoresis test, two "street" rabies virus strains(Mangosta and Apipé) revealed antigens not detected in the standard challenge virus(VS). No antigenic differences, however, were found between CVS and another fixed ra-bies virus strains (strain 91) by this method. Whether these differences are relatedto qualitative or quantitative variations in the antigenic structure of rabies virusstrains during their adaptation to a susceptible host, is the subject of interest inour laboratory.

PUBLICATIONS

1. Díaz, A. M. O. and V. M. Varela-Díaz. Studies on antigenic differencesamong rabies virus strains. Submitted for publication to Ann MicrobiolInst Pasteur (Paris).

OTHER DATA

Grantee:

Funded by:

Timetable:



1974-1976.

238

BIOLOGICAL CHARACTERISTICS OF RABIES VIRUS STRAINS

PROBLEM

Several parameters have been employed to differentiate rabies virus strains.These have included differences in incubation periods, viral pathogenicity, neutraliza-tion kinetics, and separation of viral components. Another response of host cells tovirus infection is the redistribution and release of hydrolytic enzymes. Díaz et alstudied enzymatic levels of lysosomal enzymes in tissue culture cells infected with twocell-culture-adapted fixed strains of rabies virus. The present study was designed tostudy the patterns of lysosomal enzymes activity when tissue culture cells are infectedwith noncell-culture-adapted strains of fixed and "street" strains of rabies virus.

METHOD

Two noncell-culture-adapted fixed rabies viruses--challenge virus standard (CVS)and 91 virus (human origin)--and two noncell-culture-adapted "street" rabies virus--Apipé virus (vampire bat origin) and Mangosta virus (Mongoose origin) were used.

A cell-culture-adapted fixed rabies virus--challenge virus standard (CVS)--wasincluded in the study.

Flasks seeded with BHK-21 celis were infected with the virus described above.At 1-day intervals after infection, fluids from one inoculated and one control flaskswere decanted and centifuged. These cell-free fluids were examined for lactic dehydro-genase activity (LDH) and virus titration. Cell monolayers were washed, removed, andcentrifuged. The pellets were lysated and examined for B-glucuronidase activity andvirus titration.

Total cell counts were made of each flask, and enzyme units were calculated fora constant number of cells.

RESULTS TO DATE

The results obtained to date show that cells infected with noncell-adapted-fixedrabies virus (CVS and 91) exhibited distinctive patterns of LDH activity when comparedwith noncell-adapted "street" rabies virus strains.

It was not possible to detect B-glucuronidase in cell lysates infected with CVS-cell adapted rabies virus strains.

Whereas infectivity titers and enzyme activity increase after infection withcell-culture-adapted CVS strains, activation of lysosomal enzymes seems to be inde-pendent of virus replication in cells infected with noncell-adapted rabies virusstrains.

SIGNIFICANCE

Some progress has been achieved in determining rabies virus strains differences.Since available methods are restricted to strains adapted to cell culture, the study of

239

lysosomal enzyme activation patterns of rabies viruses may prove useful to differentiatethem from other rhabdoviruses. This would be of interest for typing of viruses belong-ing to this group.

PUBLICATIONS

None thus far.

PUBLICATIONS

Grantee:

Funded by:

Timetable:



1974-1976.

240

RABIES VACCINE OF TISSUE CULTURE ORIGIN

PROBLEM

The lack of enough dog vaccine is one of the main problems encountered in thecontrol of rabies in Latin America. Although suckling mouse brain and chick embryovaccines are very effective to control rabies, the countries of the Region have beenunable to produce all the vaccine they need. Tissue culture vaccines could be thesolution for this problem if a technology were developed and provided to the countries.

Several cell culture vaccines have been produced, as attenuated or inactivatedvirus preparations. Most of these vaccines protected all the dogs challenged 1 yearafter vaccination; but none protected the animals 3 years after vaccination. Attenu-ated vaccines protected better than inactivated ones.

Rabies virus propagates in a great variety of tissue culture systems, but thebest viral yield is obtained in BHK21 C13 cells. The production of attenuated vaccinewith these cells has, however, raised objections because of their carcinogenic proper-ties for hamsters and, by inference, for human beings and other animals. Inactivationmight eliminate the carcinogenic properties. Inactivated foot-and-mouth disease vaccineproduced in BHK cells are of worldwide use.

The purpose of this project is to develop a cell-culture inactivated vaccine withan antigenic value of 3 by the NIH potency test, which is supposed to protect dogs for3 years.

METHOD

Studies include:

(1) The growth kinetic on BHK cells of several rabies virus strains to deter-mine the best growth conditions.

(2) Several inactivating agents to choose the one producing the best results.

(3) The development of an effective stabilizer, as well as the use of cellscapable of growing in media simpler than that used for BHK cells.

(4) Duration of immunity in different animal species.

RESULTS TO DATE

The replication of seven rabies virus strains (CVS, HEP, PV, ERA, WIRAB, CPZ,and Bolivar) in BHK cells and the inactivation dynamics of these strains by B-propio-lactone (BPL), acethyl-ethylenimine (AEI) and ethylenimine (El) were studied.

The seven strains reached the peak of virus production 3-5 days after inocula-tion of the cell culture; PV yielded the highest virus titer (109PFU/ml). Seven toeight log of virus were inactivated by BPL in 0.5 hour, AEI in 3.0 hours, and El in 1.0hour. Most of the vaccine lots prepared with the different strains and inactivatingagents passed a modified NIH potency test. The vaccines prepared with the PV strain

241

had consistently higher antigenic values (equal or better than 4) than did the other6 strains. This difference was highly significant F6. 12 = 59.8) but there were no sta-tistical differences among the antigenic values of the vaccine lots prepared with thethree inactivating agents. Lots of lyophilized and liquid vaccine stored at 40C main-tained potency for over 1 year.

Ten dogs vaccinated with a vaccine prepared with the PV strain and inactivatedwith El, developed a good antibody response and resisted challenge 60 days after vacci-nation, with 7 of the 8 nonvaccinated controls dying of rabies.

SIGNIFICANCE

The development of a vaccine that could be produced on a large scale without thelimitations of the currently used methods would help to overcome the shortage of thisvaccine for canine rabies control programs in Latin America. Furthermore, it couldpossibly help to develop the "know-how" of a tissue culture vaccine for human use.

PUBLICATIONS

1. Larghi, O. P. et al. Desarrollo de una vacuna antirrábica en cultivoscelulares. Resultados preliminares. In: III Jornadas Argentinas deMicrobiología. Resúmenes de Comunicaciones Libres. Tucumán, Argentina,1974, p. 8.

2. ----- et al. Vacuna antirrábica de uso animal producida en cultivoscelulares. In: VI Congreso Latinoamericano y I Venezolano de Micro-biología. Resúmenes. Caracas, Venezuela, 1974, p. 37.

OTHER DATA

Grantee: Dr. O. P. Larghi, Pan American Zoonoses Center, Ramos Mejía,Buenos Aires Province, Argentina.

Funded by: Pan American Health Organization/World Health Organization,United Nations Development Program, and the Governments ofthe Americas.


242

COMPARATIVE EFFECT OF ADJUVANTS IN SUCKLING MOUSE BRAINRABIES VACCINE FOR BOVINE USE

PROBLEM

More than 19 million bovines are exposed to rabies in the areas of Latin Americawhere hematophagous bats (Desmodus rotundus) are present. The economic losses causedby rabies are considerable.

Rabies vaccine production for bovine immunization is not sufficient because ofthe volume produced (6 million doses/year) and because of its quality.

Suckling mouse brain (SMB) vaccine is widely used for immunization of humans anddogs. The vaccine has also been tested in bovines with satisfactory results. A betterand longer response is obtained if adjuvants are added to the vaccine.

The present trial was designed to study comparatively the immunogenic value forcattle of SMB rabies vaccine using two different adjuvants.

METHOD

Vaccines

A lot of 8 percent SMB vaccine, inactivated with UV light, was prepared. Halfof this suspension was absorbed with an equal volume of 2 percent Al (OH)3 gel; theother half was emulsified with an equal volume of an oil adjuvant. The final concentra-tion of SMB in both vaccines was therefore 4 percent. The antigenic value of the vac-cines was determined by the NIH modified test in guinea pigs and mice.

Experiment No. 1

Vaccination of bovines: Three different doses were used: 10, 5, and 2.5 ml(400, 200, 100 mg of SMB), given subcutaneously to each of five 2-year old bovines pre-viously shown to have no rabies neutralizing antibodies. Blood samples for antibodydetermination were taken 7 and 25 days after vaccination and 10 and 90 days afterchallenge.

Challenge

On day 30 after vaccination, all animals and 6 controls received intramuscularlyin the neck, 5 ml of an inoculum containing 132,500 MIC LD50.

Experiment No. 2

A group of 48 heads of cattle were vaccinated (24 with SMB-V + Al (OH)3; 24 withSMB-V + oil adjuvant). Each animal received 5 ml subcutaneously. Blood samples forantibody determination were taken every 6 months and 30 days after challenge.

Challenge

Two years after vaccination all the animals and 8 controls received intramuscu-larly in the neck 5 ml of an inoculum containing 85,000 MIC LD50 of DR-19 rabies virus.

243

RESULTS TO DATE

Experiment No. 1

This experiment was designed to find the best immunizing dose of SMB vaccine plusadjuvant for bovine use. The results obtained indicated that cattle vaccinated with SMBvaccine with either oil adjuvant or with aluminum hydroxide were fully resistant tochallenge 30 days after immunization.

Experiment No. 2

In order to study whether protection similar to that of experiment No. 1 couldbe obtained after 2 years, 2 groups of 24 bovines each, whose sera were negative forrabies neutralizing antibodies, received either the oil or the Al (OH)3 vaccine. Theresults of the challenge indicate that both groups of animals were equally protected.Protection was 96 percent in both groups. Mortality in the control group was 63 percent.

SIGNIFICANCE

This study provides evidence that SMB vaccine combined with either oil adjuvantor aluminum hydroxide could be used successfully for bovine immunization. However,these vaccines have to be tested under field conditions.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. Ana Maria O. de Díaz, Pan American Zoonoses Center, RamosMejía, Buenos Aires Province, Argentina.

Funded by: Pan American Health Organization, Instituto Nacional deTecnología Agropecuaria, Argentina.


EVALUATION OF SEROLOGICAL TESTS USED IN THEDIAGNOSIS OF HUMAN BRUCELLOSIS

PROBLEM

Brucellosis is widespread in Latin America. The greatest incidence of humancases is seen in countries with a high prevalence of caprine brucellosis, such asArgentina, Mexico, and Peru. In each of these countries, over 1,000 human cases arereported annually.

In the majority of countries of the Region there is a lack of uniformity in thediagnostic methods used in human brucellosis, and there are no uniform criteria forthe interpretation of results. Most reagents used in the Region do not comply with re-quisite standards, and are the source of frequent diagnostic errors.

In general, new diagnostic techniques are studied in a limited number of patients;some techniques, such as the card test have been shown to be promising in acute humanbrucellosis but it would be convenient to evaluate this test in chronic disease, wherethe usefulness of the test might be much greater.

Argentina could offer an opportunity for a more extensive evaluation and com-parison of the different techniques. The Hospital Muñiz of Buenos Aires treats approxi-mately 120 brucellosis cases annually.

The objectives of this research are: (a) to define the relative value of thedifferent serological tests for acute and chronic human brucellosis, through a studyof a significant number of patients; (b) to establish the evolution of the differentantibodies in acute and subclinical infection; (c) to determine the activity of differ-ent immunoglobulins for the different serological tests; and (d) to prepare a documentbased on the results of this research containing recommended procedures for diagnosisof human brucellosis in the Region.

METHOD

Subjects

The studies are carried out in the following groups:

(a) Workers and employees of various departments of a packing plant. This groupis subdivided into recently employed staff and staff of long-standing employment at theplant who eventually show a clinical history compatible with brucellosis.

(b) Hospital groups. These groups are made up of patients at the HospitalMuñiz of Buenos Aires, and are subdivided into previously untreated patients who showpositive serological and clinical evidence, previously treated patients who show posi-tive serological and clinical evidence, and patients in whom brucellosis is suspectedon the basis of history and clinical examination.

Clinical examination

A register is kept of the history and clinical data of each patient.

245

In those patients in whom disease has been diagnosed by medical criteria, com-plementary examinations of radiologic, cardiologic, hepatic, and psychiatric nature arecarried out.

Bacteriological examination

In patients with acute clinical picture, or in chronic cases when justified,periodic hemoculture will be carried out in an attempt to confirm the disease throughBrucella isolation. Strains isolated will be typed by conventional methods, and, whennecessary, by the oxidative metabolism technique.

Serological examination

In all cases, 20 ml of blood is taken for serology as well as for immunoglobulinstudies. Sera are separated and kept frozen at -700C.

The samples will be examined by the following tests: (a) plate agglutinationtest; (b) tube agglutination test; (c) 2-mercaptoethanol; (d) rivanol; (e) bufferedBrucella antigen (modified card test); (f) complement fixation test; and (g) Coombs(antiglobulin test).

In all patients of the various groups, an initial serological test is carriedout. After an interval of 15 to 30 days, a new serological examination is carried outon those who are negative. In those patients with positive serology or hemoculture,serological examinations are repeated periodically.

In patients receiving treatment a serological examination is performed on theday treatment is begun, and later periodically.

In the staff recently appointed to the packing plant blood is taken, as soon asthey are employed, for reference purposes.

RESULTS TO DATE

Clinical and serological examinations have been carried out in 450 hospitalpatients. A total of 741 serum samples were processed by seven different diagnostictests.

Recently, the survey of workers and employees of various departments of a pack-ing plant (Frigorífico Lisandro de la Torre) was begun. The first group of sera hasbeen collected and is being analyzed.

SIGNIFICANCE

This project will provide information leading to the improvement and uniformityof diagnostic methods in the Region. It will also help to evaluate methods based onthe study of a large number of cases. The study will contribute information on theproduction and evolution'of immunoglobulins in patients with acute and chronic bru-cellosis and in treated and untreated individuals.

PUBLICATIONS

None thus far.

246

Dr. Raúl Casas Olascoaga, Pan American Zoonoses Center, RamosMejía, and Dr. Arno Turovetzky, Cátedra de Enfermedades In-fecciosas, Facultad de Medicina, Hospital Muñiz, Buenos AiresProvince, Argentina.

Pan American Health Organization/World Health Organization,United Nations Development Program, and the Governments of theAmericas.

1974-1976.

247

OTHER DATA

Grantee:

Funded by:

Timetable:

DEMONSTRATION PROJECT OF BOVINE BRUCELLOSIS ERADICATIONIN LARGE DAIRY FARMS OF ARGENTINA

PROBLEM

In 1959, the Pan American Zoonoses Center received a request from the NationalZoonoses Commission to set up a demonstration project for bovine brucellosis eradica-tion on a long-term basis, which would not require the immediate slaughter of reactors.

The study was therefore developed to investigate the possibility of eradicatingbovine brucellosis under conditions of management and facilities found in some farms ofArgentina; to train fellows from different member countries in diagnostic techniquesand in control and eradication procedures; and to evaluate the most appropriate proce-dures for control and eradication purposes.

The project was started on two large dairy farms of the Buenos Aires Province.One of the farms "Los Cerrillos" consists of 27 milking units; the other "La Media Luna"has 23 units. The farms keep good individual and production records.

The initial test carried out at "Los Cerrillos" in 1960 disclosed 14 percentpositive and 11 percent dubious reactors. Since 1969, the vaccination age of calveswith strain 19 vaccine was reduced to 3 months and monitoring of infection was insti-tuted by the milk-ring test.

The following livestock were used in "Los Cerrillos": 2,481 cows for production;1,222 dry cows; 1,000 pregnant heifers; and 1,200 heifers to be mated.

METHOD

All female calves 3-month old were vaccinated with strain 19 vaccine. The dairyunits were under continuous surveillance by the Brucella ring test (BRT). Every animal(production and dry cows) of each BRT-positive dairy unit was bled and examined sero-logically by the screening card and agglutination plate tests. Any serum reacting tothe card and/or plate test was tested by the SAT and complement fixation tests. Thereactors were isolated in a quarantine unit, together with TB reactors, or sacrificedat the slaughter house.

From time to time milk and tissue samples were collected to determine if virulentB abortus infection was still present in the herds.

The infected units were periodically retested to remove all reactors.

All pregnant heifers were tested after disclosure of pregnancy, and the reactorsremoved from the herd.

RESULTS TO DATE

Of 2,210 cow sera examined in 1974 at "Los Cerrillos" by the plate agglutinationand the buffered Brucella antigen tests, 6 (0.27 percent) were found positive and 2(0.09 percent) suspicious reactors. Eight hundred and eighty one pregnant heifers were

248

serologically examined and one reactor was found. In 1974, using BRT, 2 of 27 (7.4 per-cent) dairy units were found infected. Blood-testing of all the animals belonging tothe BRT-positive units disclosed four reactors; three isolates of Brucella abortus(biotype 1) were obtained from the milk. Subsequently, BRT was repeated periodicallyin the 27 dairy units, and some of the farms were found infected. Every animal of theBRT-positive units was bled and checked serologically; 15 reactors were detected among1,218 cows, and 10 among 1,254 pregnant heifers examined.

A retest survey of 1,000 cows from 5 infected dairy units is now underway.

SIGNIFICANCE

The results obtained so far show that the combination of calfhood vaccination andseparation of reactors, may lead to the eradication of bovine brucellosis. This low-cost procedure for the farmer might be applicable elsewhere in Latin America, where man-agement and facilities are adequate.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Raúl Casas Olascoaga, Pan American Zoonoses Center, RamosMejía, Buenos Aires Province, Argentina.


1974 - continuing.

249

PATHOGENESIS OF RAM EPIDIDYMITIS CAUSED BY BRUCELLA OVISEXPERIMENTAL STUDIES IN LABORATORY ANIMALS

PROBLEM

Ram epididymitis is an infection of sheep caused by Brucella ovis. During thecourse of the disease,microorganisms may be isolated from liver, kidney, spleen, lymphnodes, epididymis, testicle, and semen. Clinically, the infection is predominantlycharacterized by its primary epididymal localization and secondary testicular involve-ment (epididymitis and orchitis).

The object of this project is to study the pathologic and immunologic factorresponsible for the selective localization of the microorganisms in the male genitaltract.

METHOD

Research consists of three stages:

(1) Experimental inoculation of several laboratory animal species with a B. ovisisolate from ram semen to determine the species most susceptible to the infection thatmay be a useful experimental model of the natural disease.

(2) The host species indicated above are studied in terms of the lesions pro-duced and the localization of the microorganisms and its antigens. These studies areperformed using light, fluorescent, and electron microscopy.

(3) Immunopathologic studies of the experimental infection in the laboratorymodel of the disease.

RESULTS TO DATE

(1) The findings of the first stage (completed) have been published. Fromresults of serologic, bacteriologic, and pathologic studies, it was concluded that themost appropriate species for the study of epididymitis caused by Brucella ovis is thegerbil (Meriones unguiculatus).

(2) The second stage of this project is partially completed. A total of 64gerbils were inoculated. Serologic, bacteriologic, and histologic studies are con-cluded. Attempts to detect antigens using immunofluorescent techniques did not pro-vide satisfactory results. Samples of spleen, liver, testicle, and epididymis weretaken from studies with electron microscope, which are in progress.

(3) The third stage is under way. Guinea pigs were sensitized with extractsof epididymis and spermatozoa for pathologic studies. Allergic orchitis and epididy-mitis to spermatozoa were observed.

250

SIGNIFICANCE

Knowledge of the pathogenesis of experimental B. ovis epididymitis may be ofimportance in the eventual development of disease prevention in rams. Data on thedamage to other organs by this organism is equally important. This information mayalso be of value in understanding the immunopathologic aspects of similar diseasescaused in man and animals by other etiologic agents.

PUBLICATIONS

1. Cuba-Caparó, A. and D. M. Myers. Pathogenesis of epididymitis caused byBrucella ovis in laboratory animals. Am J Vet Res 34:1077-85, 1973.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Alberto Cuba-Caparó, Pan American Zoonoses Center, RamosMejía, Buenos Aires Province, Argentina.


1971 - continuing.

251

EXPERIMENTAL INFECTION OF MALE GOATS WITH BRUCELLA OVIS

PROBLEM

Goats and sheep are very susceptible to Brucella melitensis, but there is noinformation on the natural occurrence of B. ovis infection in goats. It is assumedthat goats are not susceptible to this infection. The fact that goats are not raisedin the principal areas where B. ovis infection is prevalent should be taken into accountin explaining this phenomenon.

METHOD

Eighteen male goats serologically negative for B. ovis were artificially infectedwith a B. ovis strain recently isolated from a case of ram epididymitis. Lambs infectedat the same time were maintained as controls. One group of animals of both species wasinoculated by the conjunctival and another group by the prepucial route. Semen andblood samples were taken periodically to verify infection.

In a second trial all animals were inoculated by the conjunctival route and smallgroups were killed 2 months, 4 months. and six months after inoculation. Samples fromorgans were inoculated on culture media in an effort to isolate Brucella.

RESULTS TO DATE

There were 12 reactors among the 14 goats which received an inoculum of 3 x 107viable B. ovis cells by the conjunctival route and 5 serologically positive reactorsamong the 13 goats given a similar dose through the prepuce. The same inoculum stimu-lated an antibody response in all the control rams.

Similar results were obtained in a second trial.

Two months after inoculation, a strain of B. ovis was recovered from the semen,and 5 strains from the organs of male goats. Four months after inoculation all malegoats gave negative cultures from semen as well as from organs.

Some male goats developed epididymitis and orchitis but the lesions were lessapparent than those observed in rams.

SIGNIFICANCE

It is important to assess the susceptibility of male goats to B. ovis for thecontrol of the infection in goat- and sheep-raising areas.

In this study a comparison is also being made of the techniques currentlyavailable for the diagnosis of ram epididymitis.

252

PUBLICATIONS

1. García-Carrillo, C. et al. Susceptibilidad comparada de cabritos y carnerosa la infección causada por Brucella ovis: Estudios serológicos, bacterioló-gicos y patológicos. Gaceta Veterinaria 36:355-74, 1974.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Casimiro García Carrillo, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina.


1971 - continuing.

253

EVALUATION OF THE CARD TEST WITH NONDISPOSABLE MATERIALIN THE DIAGNOSIS OF BRUCELLOSIS

PROBLEM

Though this test is very reliable for the diagnosis of animal brucellosis, itsuse in Latin America is limited because the disposable material it requires is patented,costly, and has to be imported. The purpose of this study is to promote the use of thecard test as a screening tool in Latin America, by adapting the method to inexpensivenondisposable materials.

METHOD

Brucella cells are grown in a fermentation tank. The antigen is standardizedaccording to the methods described in the National Animal Disease Laboratory (NADL),U.S. Department of Agriculture (USDA), Manual 65 C. The buffered Brucella antigen test(BBA) is performed on plates, in duplicate, in order to compare the reference NADL-USDAantigen distributed with the commercial kit, and the antigen prepared by the Center.The equipment and materials used in the nondisposable technique are: the glass plate,Bang's pipette and dropper (calibrated to dispense 0.03 ml per drop) used to run thestandard plate agglutination test (Huddleson's method).

In the performance of the test, equal amounts (0.03 ml) of each serum and anti-gens are placed on the card and glass plate. The serum and antigen are carefully mixedand the pipettes containing them are tilted continuously by hand or a mechanical devicefor 4 min. The test is read immediately at the end of the 4-minute rocking period.

Each serum sample is processed by the same operator, running first the card testand immediately after the plate test, using both antigens.

RESULTS TO DATE

(1) The NADL-USDA and the Center's anti.-gens were found to be equally sensitive.

(2) The sera of 683 nonvaccinated and 486 vaccinated cattle (over 30 months old)were studied to compare the performance of the tests.

The results obtained using disposable and nondisposable materials were similar.Antigens from NADL-USDA and the Pan American Zoonoses Center were found to be of equalsensitivity.

SIGNIFICANCE

The adaptation of this simple and sensitive screening method is expected to beof great value in programs dealing with control and eradication of brucellosis inLatin America.

Reference buffered Brucella antigen and training on production of the antigenare now available through the Pan American Zoonoses Center.

254

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Raúl Casas Olascoaga, Pan American Zoonoses Center, RamosMejía, Buenos Aires Province, Argentina.


1973-1974.

255

TYPING OF BRUCELLA STRAINS ISOLATED FROM MAN AND ANIMALSIN LATIN AMERICA

PROBLEM

Few laboratories in Latin America have specialized personnel and adequate meansfor typing Brucella strains. As a result, many strains have been classified on thebasis of only one or two biochemical characteristics or epidemiologic circumstances alone.In Brazil, for example, it is claimed that Brucella melitensis has been isolated fromhuman patients, but B. melitensis in animals has not been recognized and its evidencedisclaimed (Pacheco and Thiago de Mello, 1955). A group of strains received from humanpatients in C6rdoba, Argentina, an enzootic goat brucellosis area, had been previouslylabeled as B. melitensis, and several of them turned out to be B. suis. The classifica-tion was originally made merely on the grounds that one of the patients was a shepherdand that another habitually consumed goat cheese.

The biotypes existing in Latin America and their relative frequency are not knownand it is the purpose of this study to obtain new information on the Brucella species.

METHOD

Brucella strains isolated from both humans and animals are collected from differ-ent laboratories throughout Latin America. They are subcultured and studied for disso-ciation, and colonies are selected for typing.

Typing is performed by the conventional biochemical methods and by nonspecificsera, as recommended by the WHO/FAO Expert Committee on Brucellosis. Urease activityis also studied by the method of Brauer.

All the cultures are studied for phagelysis using 1 RTD and 10,000 RTD of TbBrucella phage.

All cultures that show characteristics different from biotype 1 of each speciesare studied by the oxidative metabolic method.

RESULTS TO DATE

Of a total of 860 strains, 379 were identified as B. abortus, 309 were B. meli-tensis, and 172 B. suis. The most prevalent biotype was biotype 1 with the followingpercentages: 85.5, 97.7, and 100 for B. abortus, B. melitensis, and B. suis, respec-tively.

B. abortus was isolated in 10 of the 11 countries that cooperated in this study.Besides biotype 1, biotype 2 was found in Brazil, Colombia, and Venezuela; biotype 3in Brazil and Venezuela; biotype 4 in Argentina, Chile, Cuba, and Ecuador; biotype 6in Venezuela. The B. melitensis strains belonged to collections from Argentina, Mexico,Perú, and Chile. All B. melitensis strains were biotype 1 with 5 exceptions from Chilebelonging to biotype 2 and 2 strains from Mexico that were biotype 3.

All B. suis strains typed so far belonged to biotype 1. Nevertheless, somestrains are-pending final classification because they show characteristics not typicalof any known biotype.

256

Brucella ovis was typed from samples from Argentina, Chile, Peru, and Uruguay.

SIGNIFICANCE

The present research provides new information on the Brucella species and theirbiotypes prevailing in Latin America. These data will be useful in epidemiologic studiesand in the control of the infection.

PUBLICATIONS

1. García-Carrillo, C. et al. Tipificación de brucelas aisladas del hombre ylos animales en América Latina. Rev Latinoam Microbiol 14:117-25, 1972.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Casimiro García Carrillo, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina.

Pan American Health Organization/World Health Organization,United Nations Development Program, and the Government ofArgentina.

1966 - continuing.

257

EVALUATION OF VACCINES AGAINST SWINE BRUCELLOSIS

PROBLEM

The INTA vaccine against swine brucellosis was developed in Argentina some yearsago, but it has not found acceptance outside the country where it originated. A defini-tive evaluation of this vaccine is required and, in addition, a comparison against themost promising of the other methods that have been put forward experimentally for immu-nization against swine brucellosis would be desirable.

METHOD

In a previous experiment, the 50 percent infecting dose (ID5 0) of Brucella suischallenge strain was determined for sows.

It has been suggested that the Rev 1 B. melitensis vaccine might be suitable tovaccinate pigs against B. suis infection, and a preliminary trial to assess the durationof infection in swine was conducted.

The second phase of the evaluation involved guinea pigs and sows.

The guinea pig experiment was performed with 377 animals. Groups of guinea pigswere vaccinated using the INTA immunizing procedure (live attenuated B. abortus andkilled B. suis culture) followed by a booster of glucid-lipidic extract of B. suis, 28days later. Another group of guinea pigs received an inactivated B. abortus vaccine inan oil adjuvant and the extract of B. suis, 28 days later. A third group was vaccinatedtwice at a 4-week interval with B. suis inactivated vaccine in an oil adjuvant, and afourth group was vaccinated with a single dose of Rev 1 B. melitensis vaccine (1/10 ofthe normal dose). After 8 weeks, all the vaccinated animals and the nonvaccinated con-trols were challenged with graded doses of B. suis virulent culture.

Eight weeks after the challenge all animals were sacrificed. Blood samples forserologic tests and lymph nodes and spleen for bacteriologic examination were taken.

In the evaluation of the INTA procedure, 45 sows are vaccinated with a B. suisinactivated vaccine in oil adjuvant and 25 sows are being kept as nonvaccinated controls.Blood samples are taken periodically to study the evolution of antibodies 1h/ conventionaland supplementary tests. Mating of the experimental animals takes place at the age of 7to 8 months. The animals are challenged at 40 days of pregnancy (6-1/2 months after vac-cination). Fifteen days after farrowing all sows are sacrificed, and lymph nodes andorgans removed for bacteriologic examination.

RESULTS TO DATE

(1) Guinea pig assays

There was a marked difference in infection following challenge between each vac-cinated group when compared to the controls. All four vaccines were equally effectiveagainst the lower challenge dose of 101 organisms. The Brucella melitensis Rev 1vaccine was the only immunogen that conferred significantly higher immunity, when com-pared to the other three vaccines employing a challenge dose of 103 cells.

258

(2) Infectivity and serologic response of an atypical strain of Brucella suis

Studies on a locally isolated atypical strain of Brucella suis were carried out.The IDSO was shown to be 103.47 with a 95 percent confidence A dose of 105 cells wasnecessary to infect all the pigs studied.

On new trials the sensitivity and specificity of routine serologic tests wereinvestigated. Previous results on the poor sensitivity of the tests to detect recentlyinfected animals were confirmed. Coombs test showed high sensitivity but there weremany doubts on its specificity. The complement fixation test seemed to be the mostreliable technique to detect recently infected pigs.

(3) Protection conferred to pigs by different vaccines

The immunity conferred by INTA, Oleo-Bang INTA No. 1, Oleo-Bang INTA No. 2, Oleo-suis, and Brucella melitensis Rev 1 vaccines was studied.

The results showed no difference in infection between the vaccinated and controlgroups. Nevertheless, the rate of abortion in vaccinated groups was significantlysmaller than in the control group.

SIGNIFICANCE

There are efficient vaccines against brucellosis for cattle, sheep, and goats butnot for swine. This study aims at evaluating the immunogenic values of different anti-gens proposed for swine immunization.

PUBLICATIONS

1. García-Carrillo, C. et al. Brucelosis experimental en porcinos. 2. Eva-luación de técnicas serológicas en cerdos con infección reciente de Brucellasuis. Rev Invest Agropec INTA (Argentina) 8:99-107, 1971.

2. ----- et al. Evaluación en cobayos de vacunas contra Brucella suis. Zoonosis,in press.

3. Cedro, V. C. F. et al. Brucelosis experimental en porcinos. 1. Determina-ción de la dosis infectante. Rev Inst Agropec INTA (Argentina) 8:91-98,1971.

4. ----- et al. Determinación de la dosis infectante de una cepa atípica deBrucella suis y su evaluación de la respuesta serológica. Zoonosis, inpress.

OTHER DATA

Grantee: Dr. Casimiro García Carrillo, Pan American Zoonoses Center,Ramos Mejía, Buenos Aires Province, Argentina, and Dr. V. C. F.Cedro, Instituto Nacional de Tecnología Agropecuaria (INTA),Castelar, Buenos Aires Province, Argentina.

259


1969 - continuing.

260

Funded by:

Timetable:

STUDIES ON THE GROWTH AND DISSOCIATION OF THE REV 1 VACCINE STRAINOF BRUCELLA MELITENSIS IN CONTROLLED LIQUID CULTURE ENVIRONMENTS

PROBLEM

The Rev 1 vaccine strain of Brucella melitensis has been extensively tested asa prophylactic vaccination agent in recent years and has come to be regarded as agenerally safe and effective measure for use in the control of brucellosis in goatsand sheep. Application of this vaccine on an increasingly wider scale has underlinedthe need for improved methods of production.

Particularly important is the need to define conditions and procedures whichresult in high cell yields and which avoid development of nonsmooth variants, sincethe latter has been a longstanding and sometimes serious problem in preparing thisvaccine.

Since these defined conditions are best studied in a liquid culture system,and since liquid culture generally results in higher cell yields, it was firstnecessary to develop and refine some rather complex apparatus for the study of Rev 1in a 5-£ liquid culture "fermentation" system. A significant part of this system ofapparatus is required to satisfy safety conditions, because of the relative virulenceof Rev 1 for man. Thereafter, experiments were undertaken to determine the growthand dissociation characteristics of the Rev 1 strain under various environmentalconditions.

METHOD

Equipment: A liquid culture system safe for the study of pathogens was devised,assembled, tested, and utilized in experiments with Rev 1. The basic apparatus is a5-L fermentor with drive located in a completely closed biological safety cabinet,with access for sampling with rubber gloves. The air exhaust from the fermentor isdecontaminated in a custom designed electric air incinerator. Devices are incor-porated to sense hazardous malfunctions and sound alarms. All elements of thesystem are monitored and controlled from instrument panels located outside thecabinet. The fermentor itself is equipped for recording and control of temperatureand pH, and for recording of dissolved oxygen concentration and oxidation-reductionpotential.

Techniques: Optical density and/or viable counts are used to monitor growth.Dissociation is determined using crystal violet staining. Inocula are prepared fromfreeze-dried stocks via selection of smooth colonies and growth in liquid media inshaken flasks.

Experimental plan: Batch experiments are conducted in shake flasks and inthe fermentor to compare several media with regard to growth rates, cell yields,and dissociation tendencies. Batch fermentor experiments are conducted at severalcontrolled pH values. Continuous culture experiments are conducted to study theeffects of dissolved oxygen levels on dissociation. Finally harvest and freezedrying of suitable batches is undertaken and appropriate animal tests are conductedto demonstrate the efficacy of liquid culture vaccines.

261

RESULTS TO DATE

Early studies indicated that autoclaving Trypticase Soy Broth (TSB) in the fer-mentor resulted in severe dissociation of the culture, although this effect is not seenin shake flasks. Further investigations of this dissociation are in progress. Mediasterilized by microporous filtration proved very suitable, however.

Growth in commercial dehydrated media (TSB and Pfizer (Albimi) Brucella Broth)was fairly good,with doubling times of around 4.0 to 4.5 hours and yields of 1 or 2x 1010 viable organisms (vo) per ml. Growth in the very rich medium used by PAHO workersin Argentina was excellent with yields exceeding 1.2 x 1011 vo/ml. However, dissolvedoxygen became depleted towards the end of the growth phase, in spite of high agitationrates. An intermediate medium was devised and used in subsequent experiments, givingyields of 0.5 to 1.0 x 1011 vo/ml. Growth in several defined media was too slow forfurther practical use, with doubling times of 10 to 30 hours.

Experiments at controlled pH values resulted in the following doubling times:5.4 hr at pH 6.0, 3.3 hr at pH 7.0, 3.2 hr at pH 7.5, and 9.0 hr at pH 8.0. In addition,growth at pH 8 resulted in the complete predominance (percent) of nonsmooth cell typesin the culture. This effect is being further investigated.

SIGNIFICANCE

The maintenance of stock cultures and the production of quantities of vaccine ofRev 1 in the smooth phase is a major problem in the use of this vaccine in brucellosiscontrol. It is hoped that, by studying the environmental factors that affect the dis-sociation process in a well controlled and monitored liquid culture environment, and byrelating these observations to current agar surface culture techniques, this major pro-blems may be somewhat alleviated.

It has been estimated that the vaccine produced from two liters of rich liquidmedium is approximately equivalent to the quantity obtainable from 100 Roux bottles ofTrypticase Soy Agar. Thus, it may be hoped that liquid culture may eventually come tobe adopted as the standard method for mass production of the vaccine. If so, it willbe very important to define the necessary cultural conditions in a highly sophisticatedapparatus such as ours and then apply this knowledge to the rational design of produc-tion systems using lower levels of technology.

PUBLICATIONS

1. Williams, L. A., C. R. Wilke, and S. S. Elberg. Liquid Culture Productionof Brucella melitensis Rev 1 Vaccine. Paper accepted for presentation.Fifth International Fermentation Symposium, West Berlin, June 28-July 3,1976.

OTHER DATA

Grantee: Dr. Sanford S. Elberg, Graduate Division and School of PublicHealth, University of California, Berkeley, California, U.S.A.

262

Funded by:

Timetable:

Pan American Health Organization

1973-1975.

263

STUDY OF INACTIVATED BRUCELLA VACCINES

PROBLEM

The purpose of the project is to evaluate the immunogenic properties whichinactivated vaccines may have in the control of animal brucellosis.

Live attenuated vaccines, especially B. abortus strain 19 and B. melitensis Rev 1are generally recognized as the best immunogenic agents against bovine and caprine bru-cellosis. These vaccines have, however, some disadvantages such as persistence of agglu-tination titers. For this reason the use of young animals is recommended.

In countries where the prevalence of brucellosis is low or where the last stageof an eradication campaign has been achieved, it is advisable either to discontinue vac-cination completely or else to use vaccines that would not cause persistent agglutination.

In 1957, Renoux announced very promising results, obtained in laboratory animalsby using B. melitensis H38 vaccine inactivated with formol and suspended in an oil ad-juvant. Further work by the same author and his colleagues would indicate that vaccineH38 is more efficient than strain 19 vaccine for immunizing goats and cattle, thoughthis vaccine is also agglutinogenic. Little confirmatory information exists, however,on H38 vaccine.

There are conflicting opinions on the nonagglutinogenic 45/20 vaccine, and manyLatin American countries are requesting advice on the use of some of the inactivatedand oil-adjuvant 45/20 vaccines.

Pilet and Bonneau (1969) have developed a nonagglutinogenic vaccine by means ofsaturation with specific agglutinins of the agglutinogens of strain 19. This vaccinehas been produced on a commercial scale without sufficient testing of its immunogenicquality.

METHOD

The vaccines are tested in two phases: (a) in laboratory animals, and (b) indomestic animals. The vaccines are tried in large animals, only if they have satis-factorily passed the laboratory animal tests.

Local reactions, immediate and mediate serologic responses, and the potency ofthe vaccines are also studied.

In a first stage, PB 19 nonagglutinogenic and H38 agglutinogenic inactivatedvaccines were compared with live strain 19 and B. melitensis Rev 1 vaccines in guineapigs.

Live vaccines were inoculated in a single dose and inactivated vaccines wereadministered to two groups, one receiving a single dose, the other receiving two dosesat a 20-day interval. All vaccinated and control animals were challenged 8 weeks aftervaccination with 1,500 cells of virulent Brucella abortus strain 2308 and sacrificed 8weeks after challenge.

Animals containing any number of Brucella cells in precrural or cervical lymphnodes or spleens were considered infected.

264

A complementary study in mice was performed using PB 19 vaccine and 45/20 vaccinein an oil adjuvant. The animals were vaccinated with 0.5 ml of PB 19 and 0.25 ml of45/20. The nonvaccinated controls and the vaccinated mice were challenged 8 weeks aftervaccination using B. abortus strain 2308 at a challenge dose of 103 viable cells. Theanimals were sacrificed 8 weeks after challenge and their organs cultured.

RESULTS TO DATE

Under the conditions of the experiment, the PB 19 vaccine tested in guinea pigsoffered very poor protection with one as well as with two doses. Only three of 43 and3 of 30 animals vaccinated once and twice respectively with PB 19 vaccine yielded cul-tures negative for Brucella abortus. In the infected animals macroscopic lesions werecomparable to those observed in the control group.

The protection conferred by all other vaccines was highly significant. The per-centages of protected guinea pigs with the different vaccines were as follows: H38(1 dose): 73.2; H38 (2 doses): 92.9; strain 19: 70; and Rev 1: 86.1.

The PB 19 vaccine tested in mice afforded 80 percent and the 45/20 vaccine, 91.49percent protection. All the nonvaccinated mice were infected.

The serological responses of 3- to 6-month old female calves vaccinated with theinactivated B. melitensis H-38 showed similar antibody levels than those in calves vac-cinated with B. abortus strain 19 vaccine. Nevertheless, the persistence of reactorsamong the mice vaccinated with oily adjuvant vaccine was longer than that of mice vac-cinated with strain 19.

SIGNIFICANCE

Knowledge of the immunogenic value of different inactivated vaccines is essentialfor control purposes.

PUBLICATIONS

1. García Carrillo, C. and A. Trenchi. Estudio comparativo de la inmunidadconferida a cobayos por varios tipos de vacunas antibrucélicas. Rev Med Vet(Argentina) 55:3-11, 1974.

2. ----- and R. Casas Olascoaga. Comparaci6n de la respuesta serológica de ter-neras vacunadas a la edad de 3 a 6 meses con vacuna Brucella melitensis H-38y B. abortus cepa 19. Rev Med Vet (Argentina), in press.

OTHER DATA

Grantee: Dr. Casimiro García Carrillo, Pan American Zoonoses Center, RamosMejía, Buenos Aires Province,-Argentina.

Funded by: Pan American Health Organization/World Health Organization, UnitedNations Development Program, and the Governments of the Americas.


265

FACTORS AFFECTING THE STORAGE OF LYOPHILIZED VACCINES

PROBLEM

Strain 19 B. abortus vaccine is considered an effective immunogen to preventbovine brucellosis. Rev 1 B. melitensis vaccine is being used in control programs inseveral areas to prevent B. melitensis infections in goats. From the literature avail-able there appears to be some divergence of opinion regarding the criteria concerned inthe stability of lyophil ized vaccines when stored at different temperatures. Publishedreports state that strain 19 vaccine loses its potency a few days after it has been heldat a temperature of 370C. Nevertheless, sufficient studies have not yet been made todetermine if this loss is caused exclusively by temperature, or if other factors suchas residual humidity, integrity of vacuum, or composition of stabilizing agents play asignificant role.

METHOD

(1) Preparation of a lot of strain 19 and Rev 1 vaccines.

(2) Lyophilization using stabilizing agents of different composition.

(3) Division of the lyophilized vaccines into:

(a) Intact vacuum.

(b) Ampoules in which 8 percent of the vacuum is replaced by sterile air.

(c) Ampoules in which 80 percent of the vacuum is replaced by sterile air.

(4) Storage of ampoules from each group in incubator (370 C), room temperature(220 C), and in refrigeration (40C).

(5) Viability and dissociation tests are carried out weekly, at the beginningof this project and at several intervals.

(6) Determination of residual humidity.

RESULTS TO DATE

Testing was done on a lot of B. melitensis Rev 1 vaccine suspended in a standardstabilizing vehicle kept at different temperatures and at varying degrees of vacuum.

One hundred thirty days after starting the assays, tests showed viability lossesin all vaccines with the exception of the vaccine kept at 40C with complete vacuum.

The B. melitensis Rev 1 vaccine propagated in fermentation tank and lyophilizedin a standard stabilizing vehicle was studied at different temperatures and at varyingdegrees of vacuum.

After 2 years of storage at +40C the viable cell counb of vaccine from the vialswith intact vacuum was only marginally reduced from 2.5 x 10 to 1.5 x 109 organisms/ml,

266

whereas the viable cell counts of vials containing 8.3 percent or 80 percent of air were1.3 x 10 and 6.3 x 107 respectively.

Of the two factors studied (temperature and vacuum), increases of the temperatureof storage caused a greater reduction of viable cells than did the increase in the aircontent of the vials.

SIGNIFICANCE

The determination of optimal conditions for the production and preservation oflyophilized vaccines is of great importance for the operation of control programs every-where, especially in tropical countries.

The research is also important to improve the quality of immunogens most commonlyused in brucellosis control in Latin America.

PUBLICATIONS

1. García-Carrillo, C. Conservación de la vacuna liofilizada Brucella meli-tensis Rev 1, in preparation.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Casimiro Garcia Carrillo, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina.


1973 - continuing.

267

IMMUNITY IN GUINEA PIGS CONFERRED BY STRAIN 19 VACCINE AGAINSTDIFFERENT BIOTYPES OF B. ABORTUS PREVALENT IN LATIN AMERICA

PROBLEM

It is well recognized that strain 19 vaccine protects cattle challenged withvirulent strains of B. abortus. Taran et al have demonstrated that interspecies cross-immunity exists, and that strain 19 and Rev 1 vaccines have protective action not onlyagainst homologous infection but also against heterologous species.

Immunity produced by strain 19 challenged with different biotypes of B. abortushas not been sufficiently studied. Information about the degree of immunity producedby strain 19 vaccine against B. abortus biotypes different from biotype 1 has been re-quested by some Latin American countries.

So far, biotypes 1, 2,and 4 of B. abortus have been found to be present in LatinAmerica.

The principal objective of this program is to study the immunity conferred bystrain 19 vaccines against native strains of B. abortus, biotypes 1, 2, and 4 isolatedin Latin America.

METHOD

Latin American strains of the different biotypes with a known history were chosenand their virulence in guinea pig titrated.

Guinea pigs were vaccinated with lyophilized B. abortus strain 19 vaccine at 1/10of the cattle dose. The vaccinated guinea pigs and the control animais were challengedafter 8 weeks of vaccination with each strain previously titrated and maintained inliquid nitrogen.

Control tests of the vaccine were conducted previous to and simultaneously withthe vaccination of the guinea pigs.

In a preliminary assay 180 guinea pigs were vaccinated with 1/10 dose of B.abortus strain 19. Groups of previously vaccinated animals were challenged with 10-100infective doses of each of the biotypes. Each of the three control groups received a100-, 10-, and 1-infective challenge dose. Eight weeks after challenge, all animaiswere slaughtered, blood samples collected, and lymph nodes and spleen removed for sero-logic and bacteriologic examinations.

A second assay was performed using 468 guinea pigs; 12 groups of 18 guinea pigswere left as nonvaccinated controls. After 8 weeks the groups of vaccinated animalswere challenged with 10 , 10 , 10 , 106 virulent viable organisms of each one of thebiotypes. Nonvaccinated controls received 51, 52, 53, 54, and 55 virulent viable cellsof each one of the biotypes. After 8 weeks of challenge, all the guinea pigs weresacrificed; blood was taken for serologic tests and lymph nodes and spleen removed forbacteriologic examinations.

268

RESULTS TO DATE

Results of the first experiment were as follows:

Strain 19 vaccinated guinea pigs, challenged with 105 and 104 viable cells ofthe different biotypes, were protected against biotype 1 at 18 percent and 65 percentrespectively; against biotype 2 at 45 percent and 67 percent respectively; against bio-type 4 at 57 percent and 75-percent respectively.

The controls were exposed to doses ranging from 103 to 105 viable cells of thethree biotypes, and the infection rate varied from 95 percent to 100 percent.

In the second trial, all vaccinated guinea pigs showed a significant immunityagainst the three biotypes in comparison to the controls but no significant differencecould be demonstrated in degree of protection afforded against each biotype.

SIGNIFICANCE

It is important to learn if strain 19 vaccine, presently in use in the majorityof the Latin American countries, protects as well against the different biotypes of B.abortus as it does against biotype 1. The results in guinea pigs will indicate thesuitability of further research.

PUBLICATIONS

1. García-Carrillo, C. et al. Protección de la vacuna cepa 19 contra cepas deBrucella abortus biotipos 1, 2 y 4. Ensayos en cobayos. Rev Med Vet(Argentina), in press.

2. ----- et al. Patogenicidad para el cobayo de algunas cepas de tres biotiposde B. abortus. Rev Med Vet (Argentina), in press.

OTHER DATA

Grantee: Dr. Casimiro García Carrillo, Pan American Zoonoses Center,Ramos Majía, Buenos Aires Province, Argentina.

Funded by: Pan American Health Organization/World Health Organization,United Nations Development Program, and the Governments of theAmericas.


269

PROFICIENCY TESTING OF BRUCELLA ABORTUS STRAIN 19 VACCINE

PROBLEM

In the Americas the quality control of strain 19 vaccine is limited to the studyof absence of contaminating organisms, and of dissociation and viable cell count. Thevaccine is not routinely tested to check immunogenicity.

In the potency test recommended by the British Veterinary Codex, the vaccine doseis too high, or the challenge dose too low, to represent a critical test.

It is therefore highly desirable that a standard method be developed to test inlaboratory animals the immunogenicity of at least a proportion of the batches of thisvaccine produced commercially in Latin America.

METHOD

(1) Estimation of ID50 of different Brucella abortus strains in guinea pigs

The standard strains, 2308 and W544 were chosen. Healthy guinea pigs were ino-culated intramuscularly with graduated doses of the frozen cultures maintained in liquidnitrogen.

The strain for future work was selected according to the pathogenic characteris-tics and lesions produced.

(2) Potency test

Various trials were designed considering both alternatives:graduate challenge and graduate vaccine/constant challenge.

constant vaccine/

(3) Conventional quality testing

Samples of commercial B. abortus strain 19 vaccine from different countries wereacquired, and the samples passing the conventional quality tests were subjected to po-tency tests in guinea pigs according to the results of the potency model assays.

RESULTS TO DATE

The pathogenic activity of B. abortus 2308 and B. abortus W544 was very differentin the microscopic lesions produced in the liver, changes in spleen, and body gain.

Data collected in the experiment indicated that Brucella abortus 2308 had advan-tages over the British W544 strain for experimental work in guinea pigs.

SIGNIFICANCE

In many countries of the Region, brucellosis programs will require in the nearfuture the massive application of B. abortus strain 19 vaccine. It is important

270

that a potency test be available so as to obtain information on the immunogenicity ofthe vaccines in use.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Casimiro García Carrillo, Pan American Zoonoses Center, RamosMejía, Buenos Aires Province, Argentina.

Pan American Health Organization/World Health Organization, UnitedNations Development Program, and the Governments of the Americas.

1975 - continuing.

271

EVALUATION OF FOUR VARIANTS OF THE INDIRECT HEMAGGLUTINATION TESTFOR HUMAN HYDATIDOSIS DIAGNOSIS

PROBLEM

The indirect hemagglutination (IHA) test has been used by many scientists indifferent areas of the world for the immunodiagnosis of human hydatid disease. Antigensand red celis from different host sources, various procedures for coupling hydatid cystfluid (HCF) antigens to red cells, many modifications in the technical conditions forsensitizing cells and sera of varying reactivities have been employed in past estimatesof its sensitivity and specificity, with correspondingly varied results.

This study was designed to evaluate the comparative sensitivity and specificityof the tannic acid, glutaraldehyde, benzidine, and formol IHA test in the immunodiag-nosis of this zoonosis.

METHOD

A 10-liter pool of hydatid cyst fluid was collected from sheep livers and lungsat slaughter, and an aliquot of this characterized and standardized antigen was usedin each of the four IHA tests.

Sera from patients with confirmed hydatid disease, other parasitic diseases,

nonparasitic diseases, and healthy donors were examined by each of the four IHA tech-niques.

RESULTS TO DATE

When the same hydatid and nonhydatid sera were examined by the tannic acid,glutaraldehyde, benzidine, and formol IHA tests for hydatidosis using the same HCFpool as antigen, the number of reactors, and the degree of reactivity of the differentsera varied with each technique.

The lower rate of nonspecific reactions, and the higher sensitivity of thetannic acid IHA test based on a diagnostic titer for hydatidosis as positivity cri-terion, suggest that it is more effective than the glutaraldehyde, benzidine, andformol IHA tests for the diagnosis of hydatidosis.

SIGNIFICANCE

It is now known that the tannic acid IHA technique is the variant of choicefor the diagnosis of human hydatid disease.

The fact that the degree of reactivity of the same sera varied with each tech-nique may be related to qualitative and/or quantitative differences in the HCF anti-gens that bind to the red cells.

These findings, however, illustrate the difficulties that may be expected whenattempting to use a standard hydatid serum of known IHA titer for reference purposesin laboratories employing different variants of the IHA test for hydatidosis.

272

PUBLICATIONS

1. Varela-Díaz, V. M. et al. Evaluation of four variants of the indirecthemagglutination test for human hydatidosis. Am J Trop Med Hyg 24:304-11,1975.

OTHER DATA

Grantee: Dr. V. M. Varela-Díaz, Pan American Zoonoses Center, Ramos Mejía,Buenos Aires Province, Argentina.



273

PENETRATION OF MACROMOLECULES INTO HYDATID CYSTS

PROBLEM

The presence of species-specific antigenic determinants of host immunoglobulinsin hydatid cyst fluid obtained from different host species suggested that these mole-cules had entered the cyst from the surrounding host tissues. This conclusion wassupported by the finding of recipient IgG in hydatid fluid (HF) from cysts transplanted3 months previously into the peritoneal cavity of heterologous host species. Subse-quently, the presence of host IgG was also detected in hydatid cyst membranes and theseimmunoglobulins were shown to penetrate readily into the laminated layer of the cyst.

The present report deals with a study on the penetration of recipient IgG mole-cules into the hydatid fluid of individual cysts implanted into the peritoneal cavityof heterologous hosts.

METHOD

Competitive radioimmunoassay test was employed to measure the recipient IgG con-centration in the hydatid cyst fluid.

RESULTS TO DATE

The results showed that recipient IgG was detected in the HF of only 29.5 per-cent of mouse hydatid cysts individually implanted into the peritoneal cavity of ger-bils for variable time intervals.

SIGNIFICANCE

These results suggest that the penetration of these macromolecules into theindividual hydatid cyst may be a discontinuous or random event. This conclusion issupported both by the irregular finding of host IgG in single cysts obtained fromexperimentally infected gerbils and by the variations in recipient IgG concentrationobserved in HF obtained from mouse cysts implanted into gerbils.

This irregular penetration may be the result of a random event like the occa-sional fissuring of the cyst tegument, induced either mechanically, immunologically,or during plasma membrane turnover.

PUBLICATIONS

1. Coltorti, E. A. and V. M. Varela-Díaz. Penetration of host IgG moleculesinto hydatid cysts. Z Parasitenk, in press.

274

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. V. M. Varela-Díaz, Pan American Zoonoses Center, Ramos Mejía,Buenos Aires Province, Argentina.


1974-1975.

275

MODIFICATION OF THE IMMUNOELECTROPHORESIS TESTFOR THE DIAGNOSIS OF HUMAN HYDATIDOSIS

PROBLEM

The inimunoelectrophoresis (IEP) test based on the positivity criteria of theEchinococcus granulosus arc 5 detection is now considered the test of choice for theimmunodiagnosis of human hydatidosis, because of its specificity.

The IEP test, as originally described, involved lyophilization and reconstitutionof serum samples to one third of their initial volume, necessitating the availabilityof lyophilizing equipment in each laboratory performing the test. Such equipment, how-ever, is not within the reach of all hospital laboratories, particularly those withlimited resources or in rural areas where hydatidosis is endemic.

METHOD

In an attempt to overcome these difficulties, the diagnostic efficacy of the IEPtest for hydatidosis was examined, using unconcentrated serum, but filling the serumtrough on three different occasions during development of the slides. The results werecompared to those obtained when the test was performed with concentrated serum. Onehundred sera were examined by the IEP test under the above conditions for the immuno-diagnosis of hydatidosis.

RESULTS TO DATA

All those sera which revealed the characteristic arc 5 with the concentratedserum, also formed the diagnostic arc, when the trough had been filled 3 times withneat serum.

SIGNIFICANCE

These results show that the IEP test based on the arc 5 criterion of positivitymay now be employed using unconcentrated serum for the immunodiagnosis of hydatid dis-ease in laboratories previously unable to perform it for lack of facilities for theconcentration of sera.

PUBLICATIONS

1. Coltorti, E. A. and V. M. Varela-Díaz. Modification of the immunoelectro-phoresis test for the immunodiagnosis of hydatidosis. J Parasitol 61:155-57,

1975.

276

Dr. V. M. Varela-Díaz, Pan American Zoonoses Center, RamosMejía, Buenos Aires Province, Argentina.


1974-1975.

277

OTHER DATA

Grantee:

Funded by:

Timetable:

EVALUATION OF IMMUNODIAGNOSTIC TESTS FOR HUMAN HYDATID DISEASE

PROBLEM

The comparative sensitivity and specificity of the indirect hemagglutination(IHA), latex agglutination (LA), and immunoelectrophoresis (IEP) tests for hydatid dis-ease are not known since in previous studies, sera of varying reactivities, differenttechnical conditions and positivity criteria were employed for each of the tests.

The present study was designed to determine the relative sensitivity and speci-ficity of the IHA, LA, and IEP tests for hydatid disease by comparing the results ob-tained in the same hydatid and nonhydatid sera with a single hydatid cyst fluid (HCF)pool as the source of antigen for all tests.

METHOD

The tannic acid, IHA, LA, and IEP tests were performed under the conditionsdescribed in the Scientific Monograph No. 7 of the Pan American Zoonoses Center.

Sera from patients with confirmed hydatid disease, other parasitic diseases,nonparasitic diseases, and healthy donors, were employed in this study. The same HCFpool antigen characterized by IEP was employed in all tests.

RESULTS TO DATE

The results showed a comparable sensitivity for the LA and IEP tests, whichalso correlated better than that of the IEP and IHA tests (the latter based on a posi-tivity criterion which eliminates cross reactions). The LA test showed a very lowrate of nonspecific reactions.

These findings suggest that the LA test is the screening technique of choicefor field and seroepidemiologic studies of hydatid disease.

SIGNIFICANCE

Because the results of the IHA and IEP tests do not correlate as well as thoseof the LA and IEP tests, a higher proportion of arc 5-positive hydatidosis cases couldescape detection if sera were screened with the IHA rather than with the LA tests.Furthermore, the simultaneous use of both LA and IHA tests as screening techniqueswould not seem to increase significantly the detection rate of arc 5-positive sera.

These considerations suggest that the LA test is the screening technique ofchoice for field or seroepidemiologic studies of hydatidosis to select sera that canbe immunologically confirmed as positive for hydatidosis by IEP tests based on thedetection of the diagnostic arc 5.

278

PUBLICATIONS

1. Varela-Díaz, V. M. et al. Evaluation of three immunodiagnostic testsfor human hydatid disease. Am J Trop Med Hyg 24:312-19, 1975.

OTHER DATA



1974-1975.

279

Grantee:

Funded by:

Timetable:

EVALUATION OF THE CROSSED-OVER ELECTROPHORESIS TESTFOR THE IMMUNODIAGNOSIS OF HUMAN HYDATIDOSIS

PROBLEM

The crossed-over electrophoresis (COE) test has received in recent years in-creasing attention for the immunodiagnosis of human hydatid disease, but its relativemerits and limitations are not well known. Available data on its sensitivity areconfused and the rate of nonspecific reactions with sera from persons with other para-sitic infections has not been reported.

METHOD

The diagnostic sensitivity and specificity of the COE test were compared withthose obtained by the immunoelectrophoresis (IEP) test in the same group of sera usingthe same lot of antigen. The COE was evaluated with sera from hydatidosis patients,healthy donors, and persons with other parasitic and nonparasitic diseases.

RESULTS TO DATE

The COE test was found to be inadequate for the immunodiagnosis of human hydati-dosis because of the high rate of positive results obtained in nonhydatid sera. It wasnot possible to differentiate hydatid from nonhydatid sera by the COE test. The IEPtest based on the detection of the E. granulosus arc 5 as criterion of positivity, wasfound to be the test of choice for this purpose.

SIGNIFICANCE

The present results indicate that the COE test is not adequate for the diagnosisof human hydatidosis.

PUBLICATIONS

1. López-Lemes, M. H. and V. M. Varela-Diaz. Evaluation of the crossed-overelectrophoresis test for the immunodiagnosis of human hydatid disease.Trop Geograph Med, in press.

OTHER DATA

Grantee: Dr. Victor M. Varela-Díaz, Pan American Zoonoses Center, RamosMejía, Buenos Aires Province, Argentina.

280


1974-1975.

281

Funded by:

Timetable:

EVALUATION OF A SEROLOGIC SCREENING APPROACH FOR DETECTINGHYDATIDOSIS CASES IN FIELD SITUATIONS

PROBLEM

A serological approach for the detection of hydatid cyst carriers in a populationby the immunoelectrophoresis (IEP) test has been proposed since no false positive re-actors have been encountered to date when the criterion of test positivity lies in theappearance of the Echinococcus granulosus arc 5. Considering that the time involved inthe performance of the test may render it impractical for the examination of large num-bers of sera, the possibility of conducting a preliminary screening with the latexagglutination (LA) or the indirect hemagglutination (IHA) test to select arc 5-positivesera was suggested on the basis of data obtained with confirmed hydatid and nonhydatidsera.

This study was designed to evaluate the use of the LA and tannic acid IHA testsas screening techniques for the detection of arc 5-positive sera in a serologic survey.

METHOD

For this purpose, 662 sera collected in hydatidosis endemic areas of the Provinceof Neuquén, Argentina, were each examined by these immunodiagnostic techniques.

RESULTS TO DATA

The results showed an excellent correlation between the LA and IEP tests, a find-ing in agreement with previous observations on clinically-confirmed cases.

The results demonstrated that the LA test is the screening technique of choicefor use in examining large numbers of sera for the detection of arc 5-positive sera.

The IHA test based on the minimum nonspecificity-positivity criterion would alsoseem adequate as a screening technique but the superiority of the LA test for thesepurposes was evident.

SIGNIFICANCE

From the viewpoint of the detection of the maximum possible number of hydatidcyst carriers who could be immunologically confirmed by the IEP test and subsequentlyprovided with medical attention before the onset of complications, the LA test was themost effective screening technique. This observation is in agreement with the goodcorrelation between LA and IEP test results observed in sera from clinical hydatidosiscases.

282

PUBLICATIONS

1. Varela-Díaz, V. M. et al. Evaluation of immunodiagnostic techniques forthe detection of human hydatid cyst carriers in field studies. Submittedfor publication.

OTHER DATA

Grantee:

Funded by:

Timetable:



1974-1975.

283

EVALUATION OF DOUBLE DIFFUSION TESTS FOR THEIMMUNODIAGNOSIS OF HUMAN HYDATIDOSIS

PROBLEM

Previous studies have shown that the latex agglutination (LA) test was the screen-ing technique of choice for human hydatidosis. The present study was undertaken to de-termine if the double diffusion in gel (DDG) test was an equally effective screeningtechnique for these purposes.

METHOD

The comparative sensitivity and specificity of the LA, DDG, and immunoelectro-phoresis (IEP) tests for hydatid disease were evaluated in the same group of sera fromconfirmed hydatidosis patients, healthy donors, and persons with other parasitic andnonparasitic diseases.

RESULTS TO DATE

The results showed that DDG was not useful as a screening technique for thediagnosis of human hydatidosis because of its lack of specificity.

SIGNIFICANCE

These findings show that the LA test, because of its simplicity, the excellentcorrelation of its results with those of the IEP test, its good sensitivity and supe-rior specificity, is preferable to the DDG test as a screening technique for the de-tection of arc 5-positive sera when examining large numbers of sera in field studies.

PUBLICATIONS

1. Guisantes, J. A. and V. M. Varela-Díaz. Las pruebas de aglutinación dellátex y doble difusión en gel en el inmunodiagnóstico de la hidatidosishumana. Rev Chil Parasitol 30:1975.

OTHER DATA

Grantee:

Funded by:

Timetable:



1974-1975.

284

EPIDEMIOLOGIC STUDIES ON HYDATID DISEASE IN BOLIVIA, ECUADOR, AND GUATEMALA

PROBLEM

Previous studies indicated that in Latin America, Argentina, Brazil (Rio Grandedo Sul), Chile, Peru, and Uruguay were highly endemic areas for hydatid disease. Coun-tries of Central America were generally considered free from infection, and other coun-tries were said to have only sporadic cases of hydatid disease.

No human cases were reported from Bolivia and Guatemala and dogs also appearedfree of infection. Hydatid cysts were, however, observed in bovines, sheep, goats, andpigs. Human cases of hydatidosis were reported from Ecuador but there was no detailedinformation about the prevalence of infection in animals in that country. Thus, it wasdeemed worthwhile to study in detail the prevalence of hydatid disease in Bolivia,Ecuador, and Guatemala. Epidemiologic, social, and ecological factors influencing thespread or limit of hydatid disease were also taken into consideration.

METHOD

Contact was made with national authorities and detailed information obtainedthrough questionnaires sent to appropriate departments. Field visits were made tocountries about which no information was available or about which additional informa-tion was required to perform necessary surveys. In other cases retrospective hospitalsurveys or diagnostic surveys of dogs or livestock were carried out in collaborationwith national authorities.

RESULTS TO DATE

Results of a 1974 survey of the state of hydatidosis in Latin America showedthat major problem areas include Argentina, Chile, southern Brazil, the mountainousportion of Peru and Uruguay, where, aside from the magnitude of the problems in humans,large economic losses are suffered because of the existence of the disease in live-stock.

SIGNIFICANCE

A realistic assessment of the present situation is essential for effectivefuture evaluation of control efforts against hydatidosis. This research should pro-vide much of the data needed.

PUBLICATIONS

1. CEPANZO. Hidatidosis. Boletín Informativo, vol. 11, November 1975.

285

I

Drs. Amar S. Thakur and Héctor L6pez Adaros, Pan AmericanZoonoses Center, Ramos Mejía, Buenos Aires Province, Argentina.


1976-1977.

286

OTHER DATA

Grantee:

Funded by:

Timetable:

EPIDEMIOLOGIC STUDIES OF HYDATID DISEASE IN ARGENTINA

PROBLEM

The paucity of information on the incidence, prevalence, and economic and publichealth significance of hydatid disease in the Americas is a barrier to the effectiveevaluation of control measures. Investigative efforts need to be initiated in each ofthe severely affected countries of Latin America, and prophylactic steps taken to pre-vent introduction of the disease into those areas now free of infection. Hydatiddisease incidence is reportedly high in the province of Entre Ríos (Argentina). Epi-demiological studies have been previously carried out in the Province of Río Negro(Patagonia), and similar ones, in the province of Entre Ríos, would permit the deter-mination of the prevalence of hydatid disease in entirely different climate and soilconditions (Mesopotamia),as well as of the livestock industry pattern. The purpose ofthis research is to conduct an intensive survey of two or three departments of thisprovince to study the actual prevalence of the infection.

METHOD

The annual incidence of new surgical cases of hydatid disease and mortality wereobtained by a review of hospital records and of clinics performing major surgery.

The number of intrathoracic cysts uncovered during screening for tuberculosis,and of hydatid cysts found at autopsy was also recorded.

Similarly, infection in dogs was identified by arecoline dosing and by necropsyof dogs killed by rabies control authorities.

The prevalence of infection in animals was recorded from slaughterhouse and"frigoríficos" data.

RESULTS TO DATE

Data collected during this year showed that in the province of Entre Ríos,Argentina, there were 36 cases (27 new and 9 old cases) of hydatid cyst in humans.The prevalence of infection in animals was reported to be 17.9 percent in bovines,11.3 percent in sheep, and 9.6 percent in pigs.

SIGNIFICANCE

A realistic assessment of hydatid disease in different ecologic conditions inan endemic area is essential for effective future evaluation of control efforts againsthydatidosis. The research underway should provide much of the data needed.

PUBLICATIONS

1. CEPANZO. Hydatidosis. Boletín Informativo. Vol. II, November 1975.

287

I

6. Thakur, A. S. and H. López Adaros. La distribución geográfica y algunosaspectos epidemiológicos de la hidatidosis en las Américas en el año 1974,in preparation.

OTHER DATA

Grantee:

Funded by:

Timetable:

Drs. Amar S. Thakur and Héctor López Adaros, Pan American ZoonosesCenter, Ramos Mejía, Buenos Aires Province, Argentina.


1975 - continuing.

288

ESTIMATION OF ECONOMIC LOSS IN SHEEP DUE TO HYDATID DISEASE IN URUGUAY

PROBLEM

There is no report in the scientific literature, which convincingly demonstratesthe presence or absence of deleterious effect of hydatidosis on growth or wool produc-tion of sheep. Investigators attempting to do so have been hampered by an insufficientnumber of animals (Popov, 1964) or other technical problems (Moya and Santamarina, 1963).Echinococcosis in sheep is often manifested by the presence of large number of cyststhat nearly replace the parenchymatous tissue of the lung and liver; there is usuallyno obvious functional disability. Economic losses attributed to hydatid cyst infection,therefore, are usually measured in terms of the value of infected organs that are re-jected for consumption at slaughter. Such losses are not always felt by the farmerwho raised the animal because he often sells his animal alive. Determination of reducedmeat or wool production in sheep because of hydatid disease, would help estimate eco-nomic losses correctly, and would stimulate farmers into doing something to prevent thedisease in his farm.

METHOD

In an isolated península of Rio Negro, Uruguay, 400 lambs (200 males and 200females) are being maintained for this study. Two hundred lambs (100 male and 100female) will be infected with E. granulosus eggs. Infection will be given 3 times ata 6-month interval and each lamb will be infected with 1,000 eggs each time. Two hun-dred lambs (100 male and 100 female) will be kept as control. All the animals will beweighed periodically and differences in weight gain, quantity and quality of wool pro-duced, and reproductive capabilities determined.

RESULTS TO DATE

In October 1975, 24 dogs were infected with 0.5 cc each of freshly packed scolicesof E. granulosus. These dogs were sacrificed 11 weeks later and parasite eggs collectedto infect experimentally the study lambs.

SIGNIFICANCE

Knowledge of the economic losses caused by hydatidosis in sheep would stimulatefarmers into doing something to prevent the disease. This information would also beuseful to animal health officials in their effort to convince governments that organizedcontrol program against this disease should be initiated.

PUBLICATIONS

None thus far.

289

Dr. Amar S. Thakur, Pan American Zoonoses Center, Ramos Mejía,Buenos Aires Province, Argentina.

Pan American Health Organization/World Health Organization, UnitedNations Development Program, Government of Argentina, and ComisiónHonoraria de Lucha contra la Hidatidosis, Uruguay.

1975 - 1978.

290

OTHER DATA

Grantee:

Funded by:

Timetable:

STRUCTURE AND FUNCTION OF PILOT HYDATID CONTROL PROGRAMSIN ARGENTINA, URUGUAY, PERU, CHILE, AND BRAZIL

PROBLEM

The structure and functions of hydatid control projects play an important rolein the efficient output and flow of information influencing decision-making processes.Control programs exist in the Neuquén Province, Argentina, the Department of Flores,Uruguay, and Tupac Amaru, Peru, and a variety of methods are now being utilized inpilot control efforts.

At present, there is a great need to evaluate the problems encountered by thevarious control programs already existing in Latin American countries and to determinetheir effectiveness in controlling the parasite population. The cost-benefit of theprogram should also be studied with a view to introducing an effective drug within theframework of ongoing control efforts.

METHOD

The structure and function of the control program in relation to the progressmade was studied. The existing parasite population was compared to the initial oneand analyzed. Difficulties encountered during the implementation of the control pro-gram were studied. Local conditions influencing the control program were also takeninto consideration.

RESULTS TO DATE

Visits were made to pilot control program areas and information collected.analysis of the prevailing conditions will be made.

An

SIGNIFICANCE

Knowledge of the structure and function of pilot control programs in relationto parasite-population and cost-benefit studies is important to the introduction of anew and effective drug like EMBAY 8440 for the control of hydatidosis.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Drs. Amar S. Thakur and José Germán Rodríguez Torres, PanAmerican Zoonoses Center, Ramos Mejía, Buenos Aires Province,Argentina.

291


1976 - 1977.

292

Funded by:

Timetable:

DRUGS FOR THE TREATMENT OF CANINE ECHINOCOCCOSIS

PROBLEM

At present arecoline hydrobromide is the only drug used for the elimination ofEchinococcus granulosus. This taeniafuge is, however, of erratic performance. It sel-dom removes all the parasites with a single dose; sometimes nine repeated doses may berequired to clear up the infection. Another drug used for the treatment of E. granu-losus is bunamidine hydrochloride. The dose required to eliminate all worms has beenestimated to be greater than 500 mg/kg. It may, however, be lethal to some dogs at1/10 that rate. In addition, at the latter dose, three treatment may be required beforea dog is considered free from tapeworms.

Development of a drug more effective than arecoline hydrobromide and bunamidinehydrochloride is an active area of hydatid disease investigation. A truly effectiveand one-hit compound may be of particular value in heavily infected areas.

In New Zealand and in India, Embay 8440 (Bayer Laboratory) has been shown tohave 100 percent efficacy against E. granulosus. It would be worthwhile to examine theefficacy of Embay 8440 against E. granulosus under Latin American conditions, so as torecommend it in hydatid control programs, if found effective.

METHOD

Dogs, obtained from reputable sources, are vaccinated against distemper and ra-bies and dosed immediately twice with bunamidine hydrochloride. They are subsequentlychallenged with 0.5 cc of fresh packed protoscolices of Echinococcus granulosus. Theappropriate treatment schedule with Embay 8440 is commenced 28 days postinfection (withor without food) and the drug is administered at the doses of 1.25 mg/kg, 2.5 mg/kg,5 mg/kg, and 10 mg/kg body weight. Autopsy is carried out 35 days postinfection andresults are compared with those of controls.

RESULTS TO DATE

Various drugs have previously been screened against Echinococcus granulosus.The drug fospirate (Dow Chemical Co., Michigao), was found active against immatureE. granulosus, although it did not eliminate all strobilae from all dogs at any ofthe treatment schedules tested.

SIGNIFICANCE

A truly effective drug would be of tremendous value in heavily infected areasof Latin American countries, where an intensive educational campaign failed to produceexpected results because of existing socioeconomic conditions.

293

PUBLICATIONS

1. Schantz, P. M. and U. Prezioso. The efficiency of divided doses of fospirateagainst immature Echinococcus granulosus in dogs, in preparation.

OTHER DATA

Grantee:

Funded by:

Timetable:


Bayer Laboratory, Pan American Health Organization, World HealthOrganization, United Nations Development Program, and the Govern-ments of the Americas.

1976.

294

FINE STRUCTURE OF THE GERMINAL MEMBRANEOF ECHINOCOCCUS GRANULOSUS CYSTS

PROBLEM

The information available on the fine structure of the hydatid cyst is centeredaround the ultrastructure of the laminated layer, brood capsules, and protoscoleces.

The present study deals with the fine structure of the germinal layer of Echino-coccus granulosus, pointing out certain morphologic details not considered in previousstudies.

METHOD

Conventional procedures in electron microscopic studies were employed.

RESULTS TO DATE

The germinal layer tegument is a syncytial band formed by the distal cytoplasmof the tegumental cells. The perinuclear cytoplasm of these cells is connected withthat of adjacent cells also forming a syncytium. Narrow and deep plasma membrane in-vagination present in tegumental cells do not interrupt their cytoplasmatic continuity.

Muscular, glycogen, lysosomal, flame, and duct cells and several subcellularstructures were observed in the germinal membrane.

SIGNIFICANCE

The present observations suggest that the tegumental plasma cells membranes areresponsible for the regulation of the movement of molecules between the hydatid cystfluid and the laminated layer.

PUBLICATIONS

1. Lascano, E. F. et al. Fine structure of the germinal membrane of Echino-coccus granulosus cysts. J Parasitol 61:1975.

OTHER DATA

Grantee: Dr. Victor M. Varela-Díaz, Pan American Zoonoses Center, RamosMejía, Buenos Aires Province, Argentina.

295


1974-1975.

296

Funded by:

Timetable:

PRACTICAL METHODS OF KILLING SCOLICES OF ECHINOCOCCUS GRANULOSUSTO MAKE VISCERA SAFE FOR DOG FEEDING

PROBLEM

The best approach to hydatid disease control is to cut the life cycle of theparasite by not feeding infected viscera to dogs. In Tasmania infected viscera iscondemned, but in New Zealand, Argentina, and Uruguay, infected viscera may be fed todogs if it is boiled for 40 minutes. During this process, all the scolices of E.granulosus are killed and the infected viscera is rendered safe for dog-feeding. How-ever, poor economic conditions and lack of time make it difficult to practice thismethod very efficiently, which results into insufficient cooking of the viscera. Inhighly endemic areas of Latin America, discarding infected viscera represents a tre-mendous loss of animal protein. Development of a suitable method of killing E. granu-losus scolices to make viscera safe for dog-feeding would, therefore, be of great helpto these countries, so that a huge quantity of infected viscera, which would go as awaste, may be utilized properly for feeding dogs and other animals.

METHOD

Infected viscera are collected from slaughterhouses and "frigoríficos" andtreated with different concentrations of salt and other cheap chemicals that are safefor consumption for different periods of time. Scolex survival is determined by flamecell activity of the scolex and by experimental infection of rodents.

RESULTS TO DATE

None thus far.

SIGNIFICANCE

Chemical treatment of infected viscera would make them safe for dog-feedingand would prevent, huge waste of animal protein.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. Amar S. Thakur, Pan American Zoonoses Center, Ramos Mejía,Buenos Aires Province, Argentina.

297


1976-1977.

298

Funded by:

Timetable:

INDUCTION OF IMMUNITY AGAINST LARVAL E. GRANULOSUSUSING X-IRRADIATED EGGS

PROBLEM

Production of artificial immunity in intermediate hosts may help decrease theprevalence of infection in these animals and the availability of infected viscera as dogfood. The development of a practical method of vaccination of sheep in the field wouldbe of great help to any control program. A vaccine has been developed by the irradia-tion of larvae of a nematode parasite, and various workers (Gemmell, 1964, 1967; Rickardand Bell, 1971), have induced variable degrees of immunity against several taeniid sp.by inoculation of antigenic materials obtained from the parasites. Williams and Colli(1972) have shown that when eggs of E. granulosus were irradiated and inoculated to mice,there was a reduction in the number of animals becoming infected and the number of cystsformed. It is possible that embryos attenuated by irradiation may produce sufficientimmunogenic stimulus to immunize hosts against further challenge. This possibilityshould be examined towards the development of a vaccine against E. granulosus.

METHOD

Suitable experimental animals are infected orally with eggs exposed to severalradiation doses (10 kr, 20 kr, 30 kr, and 40 kr). After varying periods, they arechallenged, as are control animals, with nontreated eggs in order to determine whetherresistance to the subsequent challenge has been acquired.

Initially, susceptible laboratory animals (mice and jird) are used. If thesetrials are successful, they will be repeated in sheep under both laboratory and fieldconditions. Simultaneously immunologic response of animals to the immunizing proce-dures and egg challenges will be monitored by immunodiagnostic and histologic procedures.

RESULTS TO DATE

None thus far.

SIGNIFICANCE

It is possible that embryos attenuated by irradiation may produce sufficientimmunogenic stimulus to immunize hosts against further challenge. Exploitation of thispossibility may lead to production of a vaccine against E. granulosus.

PUBLICATIONS

None thus far.

299


Pan American Health Organization/World Health Organization,United Nations Development Program, and the Government ofArgentina.

1976-1977.

300

OTHER DATA

Grantee:

Funded by:

Timetable:

EFFECT OF BUNAMIDINE HYDROCHLORIDE AND EMBAY 8440 ON EGGS(FREE OR WITHIN PROGLOTTIDS) OF ECHINOCOCCUS GRANULOSUS

PROBLEM

A large number of chemical compounds have been tested as potential ovicidal agentsfor taeniid eggs, but no practical ovicide has been found. A chemical agent that wouldkill E. granulosus ova exposed to reasonable concentrations for short periods of timewould be of considerable use to veterinary officials concerned with the disinfection ofclinic sites contaminated during mass anthelmintic treatment of dogs. It might alsolessen the risk of infection among those engaged in research on echinococcosis and, ifsufficiently nontoxic to dogs or man, might be of further use as a dip for dogs follow-ing anthelmintic treatment.

Williams et al (1973) found that bunamidine hydrochloride affected the morpho-logical appearance of taeniid embryos and that incubation of E. granulosus eggs in thisdrug completely eliminated the infectivity of eggs. However, the effectiveness of drugagainst eggs within the proglottids or otherwise protected by organic material needsto be investigated. As such, it would be of much use to study the effect of bunamidinehydrochloride and EMBAY 8440 (a drug proved to be 100 percent effective against E. gra-nulosus in New Zealand and India) on the eggs of this parasite, either free or containedwithin the proglottids.

METHOD

Adults of E. granulosus were obtained through necropsy of experimentally infecteddogs. Gravid proglottids were collected and washed several times with sterile 0.85 per-cent normal saline (4 C) and stored in refrigerator at 40C in normal saline containingantibiotics. Eggs were obtained by macerating gravid proglottids, which were washedin saline and kept at 4 C for further use. Gravid proglottids as well as eggs wereincubated at 37 C in various dilutions of drugs. Control proglottids and eggs wereincubated in distilled water. Eggs were freed from incubated proglottids. The eggswere inoculated intraperitoneally to mice, which were killed after 3 months to recordthe development of cysts.

RESULTS TO DATE

The anthelmintic drug bunamidine hydrochloride had a marked effect on the mor-phological appearance of taeniid embryos and incubation of E. granulosus in bunamidinehydrochloride completely eliminated the infectivity of eggs.

SIGNIFICANCE

There now exists no known chemical compound which, at practical concentrations,effectively destroys E. granulosus eggs and thus permits the sterilization of laboratorysurfaces and instruments. The findings of the ovicidal properties of bunamidine hydro-chloride and EMBAY 8440 may have important consequences in the treatment of infecteddogs.

301

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:



1976-1977.

302

SURVIVAL AND VIABILITY CRITERIA OF HYDATID CYSTS MAINTAiNED IN VITRO

PROBLEM

No information is available on the maintenance of fully formed Echinococcusgranulosus cysts in vitro.

The conditions required for maintenance of these metacestodes in synthetic mediashould be of interest for studies on the biochemistry and physiology of the organismand possibly for work on the action of chemotherapeutic agents or immunologic effectorson cyst viability.

The criterion for hydatid cyst viability has traditionally been the microscopicdetection of motility in the flame cells of protoscolices or germinal membrane. Thiscriterion is, however, limited in that flame cell motility may mean that only thesecells are viable and may not necessarily reflect cyst germinal membrane integrity.

METHOD

The present work was designed to study the maintenance of hydatid cysts in vitrousing synthetic media and to evaluate the macroscopic appearance of these cysts follow-ing implantation into the peritoneal cavity of gerbils as a criterion of cyst viability.

RESULTS TO DATE

Hydatid cysts showed no alterations in their appearance, shape, or elasticityafter 10 days of in-vitro maintenance in 199, BHK-21, or phosphate buffered saline(PBS) medium. After their implantation into gerbils these cysts were found to retaintheir original aspect, turgor, shape, and elasticity.

Cysts showed no changes in morphology and appearance after 10 days in the samemedium or PBS to which a metabolic poison (sodium azide) or an antimetabolite (5-fluorouracil) had been added. However, after their implantation into gerbils theywere found to be dead, as indicated by their flattened appearance, loss in fluid con-tent, and torn germinal membrane.

These observations suggest that the macroscopic appearance of hydatid cysts aftertheir implantation into the gerbil peritoneal cavity provides an adequate method forthe assessment of cyst viability.

SIGNIFICANCE

The present experimental model permits the detection in vivo of irreversibledamage to a cyst by a substance or procedure to which it has been exposed in vitro.This model may be employed in studies on the effect of chemotherapeutic agents ormediators of the immune response on hydatid cysts. The present findings -re also ofvalue for studies on the biochemistry and physiology of hydatid cysts.

303

PUBLICATIONS

1. Coltorti, E. A. and V. M. Varela-Díaz. Survival and viability criteriaof hydatid cysts maintained in vitro. J Parasitol 61:974-76, 1975.

OTHER DATA

Grantee:

Funded by:

Timetable:



1974-1975.

304

SURVEYS OF CYSTICERCOSIS AND FASCIOLIASIS IN MAN AND ANIMALS IN THE AMERICAS

PROBLEM

The VII Interamerican Meeting of Ministers of Health in Trinidad and Tobago,recommended in 1974 that careful surveys be undertaken of the public health problemsof cysticercosis and fascioliasis in man and animals, which are responsible for greateconomic losses throughout Latin America.

METHOD

Data published in the literature and official government bulletins about cysti-cercosis and fascioliasis in man and animals in the Americas are collected and analyzed.

Questionnaires are designed and sent to national authorities to get informationon these parasitic zoonoses.

Visits are made to endemic areas to investigate further, in collaboration withnational authorities, the prevalence of the infection and to study epidemiologic andsocioeconomical factors responsible for parasite transfer, in order to design ultimatelya control program.

RESULTS TO DATE

Literature on these parasitic zoonoses has been partially collected.

SIGNIFICANCE

A realistic assessment of the present situation of cysticercosis and fascioliasisin the Americas is essential to plan effective future control programs.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:



1975-1978.

305

ISOLATION OF SEROTYPE FORT-BRAGG IN BARBADOSAND ITS ASSOCIATION TO LEPTOSPIROSIS OF CATTLE AND MAN

PROBLEM

Leptospirosis is considered the most important zoonosis in Barbados. The latestavailable epidemiologic survey covering a 5-year period shows that 146 human cases ofleptospirosis were reported, 30 percent of which terminated in death. Today still, theserotype causing human death has not been determined. Serologic studies in cattlethroughout the island carried out during 1971 and 1972 have shown a high percentage ofreactors to leptospiral antigens of the Autumnalis group. As a result of these find-ings, wild animals were trapped with the intent to isolate Leptospira. One rat (Rattusrattus) yielded an Autumnalis. serotype that proved to be serologically homologous toserotype fort-bragg. The purpose of this study was to correlate the finding of thisserotype from a rat to the problem of leptospirosis in cattle and man.

METHOD

Absorption studies on selected bovine sera were conduc-ted using the rat-isolatedstrain and closely related Autumnalis serotypes. Serologic diagnostic tests and serumabsorption studies on human sera received from Barbados were also carried out.

RESULTS TO DATE

The fort-bragg rat isolate, when used in absorption tests with selected bovinesera known to contain agglutinins to Autumnalis serotypes, removed all agglutinins toabsorbing culture, serotype fort-bragg, and cross-reactivity to the other serotypes ofthe Autumnalis group. These findings show to a high degree of probability that theserotype fort-bragg is involved in cattle infections.

About 189 human sera were received and tested for leptospiral antibody againsta battery of 17 Leptospira screening antigens. Thirty-four of the sera were fromfebrile hospital patients, 100 from cane field workers, 42 from cattle workers, and13 from abattoir employees. Aside from one serum sample from an abattoir worker, whichwas positive to serotype canicola at a serum dilution of 1:400, 49 (29.8 percent) ofthe sera were positive to serotype fort-bragg of the Autumnalis group with titers ashigh as 1:6,400. Four of the higher titered positive sera were used for absorptiontests with the reference fort-bragg strain and with the rat isolated fort-bragg cul-ture. On retesting the absorbed sera, all agglutinins to the reference fort-braggstrain and the fort-bragg rat isolate were removed. These findings also indicate toa high degree of probability that serotype fort-bragg is the cause of human leptospi-rosis in Barbados. No significant difference was noted in reactor rates between thegroups of sera (cane field, cattle, and abattoir workers or febrile patients), whichmay reflect an abundant rat population on the island.

SIGNIFICANCE

Serotype fort-bragg of the Autumnalis group has never been reported outside ofthe United States and has never been isolated from an animal host. The isolation of

306

the Leptospira serotype causing human death and bovine infections in association with

the identification of a host-carrier for this serotype to transmit disease, are extremelyimportant for the prevention and control of leptospirosis in Barbados.

PUBLICATIONS

1. Myers, D. M. and C. J. Jones. Leptospira fort-bragg isolated from a rat inBarbados. Bull PAHO 2:208-11, 1975.

OTHER DATA

Mr. Donald M. Myers, Pan American Zoonoses Center, Ramos Mejia,Buenos Aires Province, Argentina.


1974-1976.

307

Grantee:

Funded by:

Timetable:

EQUINE LEPTOSPIROSIS: SEROLOGIC STUDIES AND ISOLATIONS OF SEROTYPEHARDJO AND LEPTOSPIRA BIFLEXA STRAINS FROM HORSES OF ARGENTINA

PROBLEM

Equine leptospirosis is a disease of considerable importance to countries wherehorses are still used as a major source of labor and transportation. Comparativelylittle has been reported in the literature on leptospiral isolation and clearly proveddisease. Available worldwide data show that only 11 leptospires from natural equineinfections have been isolated and identified. Evidence of infection in most cases hasgenerally depended upon serologic tests and the demonstration of clinical signs ofocular involvement. Numerous recorded serologic studies show a diversity of multipleLeptospira interrogans serotype agglutinins occurring in horses. The actual signifi-cance of these positive serologic findings has not been clearly understood because con-

current attempts at cultural isolation for leptospires were for the most part unsuccess-ful.

The present investigations have the following purposes: (1) serologic determina-tion of the prevalence of leptospirosis in a random equine population of Argentina;(2) isolation and identification of leptospires from renal tissue of horses collectedin an abattoir; (3) determination of the significance of the multiple cross-reactivityin agglutination tests to pathogenic serotypes which is frequently encountered in horsesera.

METHOD

The program involves the collection of sera for serologic testing against abattery of leptospiral antigens from a random equine population. Simultaneously, en-tire kidneys of horses collected in groups in an abattoir are cultured in an attemptto isolate leptospires. Isolates are identified by cross-agglutination-absorptionprocedures and tested for virulence by laboratory animal inoculations. The isolatesare also used in absorption studies on selected horse sera to determine their relation-ship to the multiple agglutination reactions of antigenically unrelated Leptospirainterrogans serotype agglutinins. In addition, a suitable animal model will be inocu-lated with an isolate to demonstrate localization of leptospires in the kidney thatproduce the antigenic stimulus required to explain the significance of persistent, mul-tiple leptospiral serum titers observed in horses.

RESULTS TO DATE

Three pathogenic strains of Leptospira interrogans and 12 Leptospira biflexastrains were isolated from 72 apparently normal horse kidneys collected in an abattoirin Argentina. Cross-agglutination reaction patterns of the pathogens showed that theywere antigenically identical and members of the Hebdomadis group. When one of thestrains was compared to Hebdomadis serotypes in reciprocal agglutination-absorptiontests it was found to be serologically homologous to serotype hardjo. This is thefirst known report of an isolation of this serotype from horses.

Serological tests were carried out on randomly collected abattoir sera from 245

horses to determine the prevalence of equine leptospiral agglutinins. Significantantibody titers (1:100 or greater) were found in 74.6 percent of the sera. Predominant

308

reactions occurred with the antigens pomona, Hebdomadis group (hardjo, wolffi, sejroehebdomadis), pyrogenes, tarassovi, and canicola. Agglutination tests also carried outemploying antigen prepared from one of the saprophytic biflexa isolates showed seroposi-tive reactions in 99.1 percent of the equine sera with agglutinating titers ranging from1:100 to 1:3,200. Selected horse sera absorbed with this saprophytic strain removed theagglutinins to Leptospira interrogans serotypes. The results obtained so far demonstratethe probability that Leptospira biflexa strains can act as an antigenic stimulus, andmay account for the majority of persistent multiple cross-reaction patterns in equinesera against pathogenic Leptospira interrogans serotypes.

SIGNIFICANCE

The research provided new and important information on equine leptospirosis re-sulting in a better understanding of the disease in this animal. Three strains of sero-type hardjo were isolated from kidney tissue of apparently normal horses. This is thefirst known isolation of this serotype from horses. Horses, because of their closeecologic relationship to cattle and men, may play an important role in the epidemiologyof leptospirosis. The findings obtained with biflexa isolates is expected to proveuseful in demonstrating a continuing source of leptospires in the kidney to provide anantigenic stimulus. Such a stimulus would account for the persistent leptospiral serumtiters and may elucidate the pathogenesis of ocular reactions commonly associated withleptospiral infection in horses.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Mr. Donald M. Myers, Pan American Zoonoses Center, Ramos Mejía,Buenos Aires Province, Argentina.



309

HISTOLOGIC AND HEMATOLOGIC STUDIES IN APPARENTLY NORMALLABORATORY AND WILD ANIMAL SPECIES

PROBLEM

The use of new animal species as experimental models requires previous knowledgeof their normal histology and hematology. These values serve as reference for altera-tions that may occur in the course of an experiment.

This project involves the study of the above constants in gerbils (Merionesunguiculatus) and two species of Argentinian armadillos: Dasypus hybridus (mulita)and Chaetophractus villosus (peludo).

METHOD

Standard hematologic and histologic techniques are used: fixation in neutralformalin, imbedding in paraffin, hematoxylin-eosin and Giemsa staining. Electronmicroscopy techniques are used for "mulitas" and gerbils: fixation in glutaraldehydeand osmium tetroxide, imbedding in epon 812, sections in ultramicrotome Porter BlumMT1, and observation with a Siemens 101 electron microscope.

Studies are also made of the leukocyte ultrastructure of peripheral blood in"mulitas" and of the epididymis and testicle ultrastructure in gerbils.

RESULTS TO DATE

(1) Twenty-six seven-banded armadillos (Dasypus hybridus), 10 males and 16 fe-males, were used, from which blood was obtained by cardiac puncture at time of capture.The following hematologic determinations were performed by standard methods: number oferythrocytes and leukocytes per mm3; erythrosedimentation; hematocrit; percentage andabsolute count of leukocytes; mean globular hemoglobin and concentration of globularhemoglobin; study of the ultrastructure of neutrophile leukocytes and lymphocytes; anddetermination of rectal temperature and of the sexual chromatin of leukocytes.

(2) The study of hematologic constants in the gerbil has been completed. Onehundred gerbils of both sexes were used and the following determinations were performedby standard hematologic techniques: erythrocyte and leukocyte count, erythrosedimen-tation, hematocrit, and percentage and absolute count of leukocytes.

(3) The histologic study of Dasypus hybridus and Chaetophractus villosus isunder way. An atlas of the normal histology of these species is being completed.

SIGNIFICANCE

The determination of hematologic values and the knowledge of the normal histol-ogy in apparently normal animals is basic, as a term of reference for future studies inwhich the animal species will be used as experimental models.

310

PUBLICATIONS

1. Cuba-Capar6, A. Some hematological studies in the 7-banded armadillo(Dasypus hybridus). Submitted for publication.

2. -----. Atlas of normal histology of the 7-banded armadillo (Dasypushybridus), in press.

3. Gonzáles, M. I. and R. Báttolla. Determinación de algunos valoreshematológicos normales del merión (Meriones unguiculatus). Rev Med Vet(Buenos Aires) 54:3-8, 1973.

OTHER DATA

Grantee:

Funded by:

Timetable:



1973-1976.

311

THE ARMADILLO AS A POSSIBLE RESERVOIR HOST FOR LEPTOSPIRALSEROTYPE HARDJO INFECTION IN CATTLE

PROBLEM

In recent years, as a result of improved culture methodology, Leptospira inter-rogans serotype hardio has been recognized as the most prevalent serotype in cattle,causing abortions, still-births, weak calves, and reduced milk production. In Argentina,earlier studies carried out on armadillos (Chaetrophractus villosus) using inadequateculture methods for the isolation of serotype hardjo, re sulted in 24 leptospiral isola-tions in 438 animais examined. All of the isolates proved to be Bataviae group sero-types. Serological examination of these animals showed a high number of reactors tothe Hebdomadis group antigens, mainly serotype hardjo, in which cross-agglutinins toBataviae serotypes do not occur. All of the armadillos were captured on farms wherecattle are raised.

The aim of the present investigation is to continue attempts at cultural isola-tion of leptospiras, including serotype hardjo, using improved culture methods. Posi-tive isolations of serotype hardjo are expected to show a relationship to bovineinfection and may identify the armadillo as an abundant wildlife animal reservoir hostfor this serotype.

METHOD

Armadillos are being captured in groups of 10 on cattie raising farms inArgentina and transported live to the laboratory for processing. Blood is collectedby cardiac puncture and the sera examined by the microscopic-agglutination test usinga battery of 15 live screening antigens. On autopsy, the kidneys of each animal areaseptically removed and used for cultural isolation attempts and comparative histo-pathologic studies.

Improved cultural methods include the use of the modified bovine albumin-polysorbate 80 culture medium containing sodium pyruvate. For contamination control,one set of the culture tubes were inoculated with the suspensions of renal tissueafter having been exposed to the combined action of 25 pg/ml each of neomycinand furazolidone for 1 hour. The other set of culture tubes were used without anti-microbial agents. Leptospiral isolates are being identified on the basis of cross-agglutination reaction patterns against hyperimmune antisera representative of the 17serogroups of pathogenic leptospiras and serogroups Semaranga and Andamana. Definitiveclassification of the cultures is being done employing cross-agglutination-absorptionprocedures.

RESULTS TO DATE

A total of 15 (16.8 percent) Leptospira isolates were obtained from the kidneysof 89 armadillos. Three of the strains proved to be members of the serogroup Canicola,9 of the Bataviae group, 2 strains appeared to be L. biflexa species, and 1 isolatebelonged to the Hebdomadis group. All cultures are being identified by serotype.

312

Serologic examination of these 89 animals showed that 16 (17.9 percent) hadagglutinin titers of 1:100 or greater. The predominant reactions occurred with theantigens of the Hebdomadis group (hardjo, wolffi, serroe, hebdomadis), and serotypesbataviae and canicola.

Histopathologic studies of the kidneys of 13 of the armadillos revealed evidenceof renal lesions in 11 of the animals. The most significant lesions in the order oftheir intensity and frequency were: chronic interstitial nephritis; thickening of theBowman's capsule; congestion of the glomerular tufts with hyaline deposits; and necroticdegeneration of the renal tubules, which were distended by hyaline casts.

SIGNIFICANCE

The armadillo has been shown to be a potential wild-animal reservoir for thetransmission of leptospires of the serogroups Bataviae, Canicola, and Hebdomadis.Should this Hebdomadis isolate prove to be serotype hardjo, it will be the first timethat this serotype has been isolated from the armadillo.

PUBLICATIONS

None thus far

OTHER DATA

Grantee: Mr. Donald M. Myers, Pan American Zoonoses Center, Ramos Mejía,Buenos Aires Province, Argentina.



313

INFLUENCE OF LEPTOSPIRAL GROWTH MEDIA ON MICROAGGLUTINATIONSERUM TITERS IN THE DETECTION OF LEPTOSPIRAL AGGLUTININS

PROBLEM

The microscopic agglutination test is the serologic procedure most often usedfor the diagnosis of leptospirosis in man or animal. The test is considered highlysensitive and specific. The antigens used in the test are diverse Leptospira sero-types that are maintained by continual subculture into one of various leptospiralmedia. The selection of the medium may vary among laboratories. Many laboratoriesare converting to the exclusive use of the new bovine albumin-polysorbate 80 mediumwith its modifications. The main reasons for this change are easy preparation of themedium and elimination of the need for a continual source of pooled rabbit sera. Dis-crepancies in results occasionally encountered between diagnostic laboratories withdivided serum samples may in part reflect the medium used in preparing antigens.Notably lacking in the literature are reports dealing with the effect of growth mediaor their components on the sensitivity and specificity of leptospiral antigens.

The purposes of the present investigation were: (1) to compare the results oftiters with antisera of reference leptospiral strains against their homologous strainsas antigens after growth in the various media; and (2) to reexamine bovine field serawhich contained a high percentage of antibody to serotype autumnalis, and correlatetiters against serotype autumnalis antigens prepared from cultures grown in Stuart,Fletcher, Korthof, and bovine albumin media.

METHOD

Leptospiral strains used as antigen were initially grown and maintained in semi-solid Fletcher and bovine albumin media. Transfers were then made from each intoStuart, Korthof, and bovine albumin liquid media. Cultures were adapted to each mediumby three successive weekly transfers. Antigens were 4, 7, and 14 days old when usedin the microscopic agglutination tests with hyperimmune antisera. Cell density and theperformance of the test followed the recommendations for the microscopic-agglutinationtest. All of randomly selected bovine sera were divided, coded, and tested by two in-dividuals who performed daily the microscopic-agglutination test. Results with anti-gens grown in each of the various liquid media were evaluated by statistical analysis.

RESULTS TO DATE

Comparison of results of four Leptospira serotypes showed that antigens preparedfrom leptospiral cultures growing in either Stuart or Korthof media were equally sen-sitive when used in the microscopic-agglutination test for the detection of antibody.When employing the albumin-based medium for the same purpose, a significantly lowerantigen sensitivity (P < 0.001) was evident using twofold serum dilutions of both hyper-immune antisera and bovine field sera. The specificity of the antigen/antibody reactionwith the antigens prepared from leptospires grown in each medium was 100 percent. Thecriterion of positivity indicating present or past infection was a serum agglutinintiter of 1:100 or greater. Bovine sera examined with antigens prepared in either Stuartor Korthof media showed no statistical difference in antigen sensitivity with values of92.1 percent and 94.4 percent, respéctively, using the 95 percent confidence intervalmeasurement. A significantly lower value (39.3 percent) was obtained with the albumin-

base medium used to prepare antigen and to test the same sera. Antigenic sensitivityof this latter culture was restored after 3 passages into media containing rabbit serumenrichment to 92.1 percent.

SIGNIFICANCE

These findings demonstrate the importance of taking into consideration the cul-ture medium used for antigen production in the diagnostic microscopic-agglutinationtest. Since leptospires are antigenically stable organisms and paradoxical or anoma-lous serologic reactions are probably due to the medium used, serological titers ofclinical specimens are open to question, unless the culture medium is taken into con-sideration. Consequently, an urgent need exists to upgrade procedures for the diagno-sis of leptospirosis to bring uniformity in laboratory methodology.

PUBLICATIONS

1. Myers, D. M. Effect of culture medium on the agglutinability of lepto-spires by the microscopic-agglutination test. Prepared and submittedfor publication.

OTHER DATA

Grantee:

Funded by:

Timetable:

Mr. Donald M. Myers, Pan American Zoonoses Center, RamosMejia, Buenos Aires Province, Argentina.


1974-1976.

315

DETERMINATION OF THE EFFICACY OF THE OUTER ENVELOPE OF LEPTOSPIRAAS A POTENTIAL VACCINE AND/OR DIAGNOSTIC REAGENT

PROBLEM

Leptospirosis exists as a zoonotic problem in the Western Hemisphere, with agreater intensity recently focused in the Caribbean countries specifically. Sero-epidemiologic surveys indicate an average reactor rate of 20 percent in humans. Also,vaccination programs for domestic animals do not exist in these countries.

The purpose of this research was to develop, from strains of Leptospira isolatedfrom Barbados, outer envelope preparations that experimentally would be effective as avaccine and/or diagnostic agent.

METHOD

The program involved the experimental development and evaluation of reagents(outer envelope preparations and heat-killed whole cell bacterins) of avirulent(L. canicola Hond Utrecht IV and pomona S-91) and corresponding virulent (L. canicolamoulton and pomona HCE) leptospiral strains as possibly effective vaccines. Theexperimental animals used were 3- to 4-week old pure-bred weanling hamsters. Heter-ologous and homologous immunization trials were done at 4 dosage levels of antigenusing 25 animals per dosage level. With proper controls, all animais were challengedwith the corresponding highly virulent leptospiral strain (LD10 0 = 2 cells for canicolamoulton; LDl 00 = 20 celis for pomona HCE) in hamster and subsequently checked for deathand kidney infection over a 21-day period.

RESULTS TO DATE

Heat-killed whole cell bacterins and outer envelope preparations generally provedto be potent immunogens. However, it is clear that variations in potency exist whencomparing bacterins or preparations from homologous virulent and avirulent strains ofserotypes pomona and canicola.

All antigen preparations protected against death and renal infection withexceptions; renal infection was observed at the lowest dosage level of those preparedfrom avirulent organisms. It is possible that loss of virulence may be accompaniedby a corresponding reduction in antigenicity. In these exceptions, heat-killed wholecell bacterins were more effective in preventing renal infection than outer envelopepreparations.

SIGNIFICANCE

The development of a vaccine that is simple and economic to prepare and hasproved to be experimentally effective is the necessary first step from which to studylarger animals and humans in high-risk groups. The fact that heated whole cellbacterins are easily prepared with a minimum laboratory requirement allows worldwideuse relative to the unique serotype-geographical characteristics of strains causing

316

leptospirosis. That such a preparation protects against kidney infection and organismalresidence eliminates a significant stage (shedder) in the transmission of the organismwhich should markedly decrease the incidence of this disease in a vaccination/eradicationcampaign.

PUBLICATIONS

1. Zeigler, J. A. et al. Immunization trials of heat-killed whole cell andouter envelope leptospiral antigens in hamsters. Submitted for publicationto Bull PAHO, 1976.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Ronald H. Jones, Medical Research Institute, FloridaInstitute of Technology, Melbourne, Florida, U.S.A.


1974-1975.

317

EFFECTS OF TUBERCULIN TEST SENSITIVITY BY TUBERCULIN POTENCY, TEST SITE, AND TESTINTERPRETATION SCHEME IN ARGENTINE CATTLE INFECTED WITH MYCOBACTERIUM BOVIS

PROBLEM

The proper selection of a tuberculin test system for cattle in a national programagainst bovine tuberculosis requires that estimates of tuberculin test sensitivity andspecificity be made in the environment in which the program will operate.

It has been shown that tuberculin test responses vary in size with tuberculinsof different types, potencies, and production strains of Mycobacteria, and accordingto the test site used. Classification errors about the tuberculosis status of animalsvary with the criteria used to interpret test results.

A number of tuberculin test systems employing different tuberculins, test sites,and interpretation schemes have been used with excellent results in various countries.However, data on the percentage of errors of these various test systems are not readilyavailable, nor has the accuracy of the test systems been thoroughly studied in Argentinecattle.

The objective of this study is to demonstrate that variations of tuberculin testsystems and tuberculin potency affect the tuberculin test sensitivity.

METHOD

A lot of 113 beef cattle with a high risk of bovine tuberculosis were tuberculintested both with the comparative tuberculin test (using avian and human PPD in the neckarea) and the simple caudal fold test (using human PPD alone in the caudal fold) em-ploying PPDs produced by a commercial laboratory (PL) and PPDs produced at the PanAmerican Zoonoses Center (CPZ). Each animal also received one injection of CPZ bovinePPD in the neck.

All test results were measured and recorded in millimeters of increase of skinthickness. The animals were then submitted to a routine post-mortem examination at alocal slaughterhouse. Samples of granulomatous lesions and/or normal lymph node tis-sues were collected for laboratory examination. The various test results were thencompared for all animals from which M. bovis was recovered.

RESULTS TO DATE

Mycobacterium bovis was recovered from 50 of the 113 cattle. The results ofcomparisons of the various tests follow tendencies shown in previous work in other coun-tries. The number of positive tuberculin responses was greater in more potent PPDs andin the neck site. Bovine PPD gave larger responses than did human PPD and the overalltuberculin test sensitivity varied from a low of 44 percent to a high of 88 percentdepending on the test system chosen.

318

SIGNIFICANCE

The results of this trial demonstrate the importance of the choice of tuberculintype, tuberculin dose, test site, and interpretation on overall tuberculin test sen-sitivity. The importance of quality control of tuberculin potency is also clearlyshown. It is suggested that the factors studied could have a dramatic effect on thecost of a bovine tuberculosis control program.

The need for further work on the caudal fold test at different dose levels issuggested as well as the need for parallel studies on the effects of tuberculin testspecificity in Argentina.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. J. D. Roswurm, Pan American Zoonoses Center, Ramos Mejia,Buenos Aires Province, Argentina.

Pan American Health Organization and the Instituto Nacionalde Tecnología Agropecuaria.

1975-1976.

319

TRANSPORT MEDIA FOR BOVINE TUBERCULOUS LYMPH NODES

PROBLEM

Selection of an appropriate transport medium for specimens collected at post-mortem examination is of great importance in programs for the control of bovine tuber-culosis, since specimens must be sent to the laboratory, often from the place where theywere taken. Such is the case, for example, of tuberculin-positive or negative animals,presenting doubtful lesions at post-mortem examination.

The purpose of this study is to determine which antiseptic should be used for theshipment of lymph nodes to the laboratory.

Results of this study could also be applied to the shipment of human samples(sputum, biopsies, and others).

METHOD

A comparative study was conducted, using three different antiseptics: 1 percentboric acid, 1 percent cetylpyridinium bromide, and saturated solution of sodium borate.

Respiratory tract lymph nodes taken in slaughterhouses were divided into foursimilar pieces: one was placed in a flask without antiseptic, and the other three, inflasks containing the above mentioned solutions. Samples were kept at room temperatureand, at 5 and 10 days, were decontaminated with 4 percent sodium hydroxide. Each sam-ple was inoculated into two tubes of Stonebrink and 2 of Lowenstein-Jensen media. Thesecultures were incubated at 370 C and checked every 7 days for 60 days after inoculation.

Positive cultures and contamination findings were recorded.

The mycobacteria isolated were typed taking into consideration the followingfactors: growth rate, the most suitable medium, aspect of colonies, photochromogenicity,nitrate reduction, and niacin tests. In some instances additional tests were applied.

RESULTS TO DATE

Of the 100 lymph nodes processed, 91 gave a positive culture for M. bovis. Theremaining 9 were negative and thus were not included in this comparison.

Eighty-one percent of the cultures made on the first day were positive; 17.6 per-cent of them were negative, and 1.1 percent of them contaminated.

In the cultures made on the fifth day, the degree of positivity from samples keptwithout antiseptic fell to 14.3 percent, whereas contamination levels rose to 47.2 per-cent. Depending on the antiseptic used (boric acid, sodium borate, or cetylpyridiniumbromide), the above two percentages were: 72.5 and 4.4; 76.9 and 2.2; 73.6 and 0.0,respectively.

In the cultures made on the 10th day, the degree of positivity from samplesmaintained without antiseptic dropped to 9.9 percent and contamination level reached42.9 percent. With the addition of boric acid, these two values were 61.5 percent

320

and 6.6 percent, respectively; with sodium borate, they were 63.7 percent and 4.4 per-cent; and with cetylpyridinium bromide, they were 64.8 percent and 2.2 percent.

Results showed a great similarity in the efficacy of the three antiseptics. Thelowest number of positive cultures, and the greatest number of contaminated cultureswere found when using boric acid, whereas the lowest rate of contamination was obtainedwith cetylpyridinium bromide.

SIGNIFICANCE

From the results obtained and taking into account the cost of drugs, the use ofsodium borate (saturated solution) is recommended as the preservative of choice to sendbovine lymph specimens to tuberculosis bacteriology laboratories.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Isabel N. de Kantor, Pan American Zoonoses Center, RamosMejia, Buenos Aires Province, Argentina.


1974-1976.

321

ISOLATION OF MYCOBACTERIA FROM TWO SPECIES OF ARMADILLOS:DASYPUS HYBRIDUS (MULITA) AND CHAETOPHRACTUS VILLOSUS (PELUDO)

PROBLEM

The choice, by Kirchheimer and Storrs, of the armadillo (Dasypus novemcinctus)as a model for the study of leprosy, and their successful essays in this field, haveincreased the interest of researchers in other types of armadillos, for use in the re-search of leprosy and other mycobacterial infections.

Two species of armadillos: Dasypus hybridus and Chaetophractus villosus, areabundant in the pampa region of Argentina.

The purpose of this study is to establish whether these two species, in theirnatural habitat, are carriers of mycobacteria and, if such is the case, to determinewhich types of mycobacteria may be isolated from them.

METHOD

Different lymph nodes were aseptically removed from armadillos during necropsyand processed for the culture of mycobacteria. A general method for the treatment ofsamples was followed: suspension of the mixture in a mortar with sterile sand andwater, followed by decontamination of the suspension with 4 percent NaHO. The sampleswere then inoculated into two tubes of Lowenstein-Jensen and two of Stonebrink media.The following tests were performed on growth rate, growth temperature, niacin pro-duction, nitrate reduction, catalase and peroxidase activity at 250C and 700C, pigmen-tation in dark, pigment photoactivation, Tween hydrolysis, growth in presence of 5 per-cent NaCl, iron uptake and, in special cases, inoculation into guinea pigs and chickens.

RESULTS TO DATE

The following mycobacteria were isolated from 164 lymph nodes and samples oflung tissues of normal peludos:

Scotochromogenic slow growers: two strainsM. avium-intracellulare complex: four strainsM. terrae-triviale complex: two strainsM. fortuitum: two strainsM. fortuitum-chelonei: six strainsM. chelonei: three strainsM. phlei: one strain

The strains considered M. fortuitum-chelonei were positive to the nitrate re-duction test and negative to the iron uptake test.

Only six nodes from "mulitas" were processed and no mycobacteria were isolated.

322

SIGNIFICANCE

Results to date show that a wide variety of mycobacteria can be isolated fromlymph nodes of apparently normal Chaetophractus villosus.

This finding should be taken into account when armadillos are used for experimen-tal mycobacterial infections.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Isabel N. de Kantor, Pan American Zoonoses Center, RamosMejía, Buenos Aires Province, Argentina.


1975-1976.

323

DEVELOPMENT OF MICROBIOLOGICAL CRITERIA FOR THECONTROL OF CHEESE IN ARGENTINA

PROBLEM

Microbiological standards for the control of cheese in Argentina are currentlybeing reviewed by a special commission in charge of updating Argentine food legislation.The commission lacks sufficient information on the microbial contamination of productsderived from the use of standardized sampling and examination techniques, a situationalso observed in many countries of the Region.

In view of the botulism outbreak, which caused several deaths and which was asso-ciated with the ingestion of cheese spread, health authorities decided to increase con-trol over cheese products. This task was hindered by the lack of standardized analyticaltechniques and adequate microbiological criteria to interpret results.

The purposes of this project are: (1) to study the structure of cheese productionand marketing in Argentina; (2) to determine the degree of microbial contamination incheese manufactured in Argentina, relating findings to the sanitary practices applied invarious establishments; (3) to obtain figures for the establishment of microbiologicalcriteria to be enforced in the future; (4) to select and standardize sampling and exami-nation techniques in order to recommend their adoption by official control laboratories;(5) to propose sanitary measures to be taken by processing plants and distributors;and (6) to train technical personnel from the Centro de Investigaciones Tecnológicasde la Industria Láctea (CITIL) in microbiological analytic techniques used in the con-trol of cheese.

METHOD

Processing plants and distributors are classified into two groups, one observingexisting regulations and applying adequate sanitary measures in handling of cheese, andthe other, using inadequate sanitary practices.

Random cheese samples from several establishments are examined weekly.

In all samples, counts of total coliforms, fecal coliforms, coagulase positiveStaphylococcus aureus, mould and yeast, and of clostridium perfringens, are taken, andthe presence of salmonellae investigated.

In order to have a guide to follow for the classification of products on thebasis of their microbiological composition, some interim criteria are suggested. De-pending on the results obtained when the study is completed, the criteria most suitablefor domestic production will be recommended to the Food Legislation Commission.

The style to be used will be the one recommended by the International Commissionon Microbiological Specifications for Foods (ICMSF).

RESULTS TO DATE

To date 117 lots of soft cheese ("quartirolo" type) from 6 establishments havebeen examined.

324

The interpretation of the results in accordance with the criteria referred toabove, showed that counts of total coliforms were satisfactory in 2.5 percent and notacceptable in 63.2 percent of the lots. For fecal coliforms, 25.6 percent of the sam-ples were satisfactory and 41.9 percent acceptable; Staphylococcus aureus coagulasepositive counts were satisfactory in 70 percent of the samples and acceptable in 17.5percent. The differences between the percentages cited and 100 percent correspondedto the percentage of condemned lots.

No salmonellae could be isolated from any of the samples and no sample was con-demned because of excessive fungi, yeasts, or Clostridium perfringens.

According to these results, two cheese manufacturers could be classed as "good",two as "fair", and two as "deficient".

Some samples of semi-hard cheese ("pategras" type) were also examined.

SIGNIFICANCE

Results obtained in the examination of "quarti¡rolo" type cheese served as a basisfor recommendations made to authorities on microbiological criteria to be adopted in thecontrol of this type of products. The figures obtained reflect the actual situation ofthe industry and the technological developments and hygienic status of the country.

The legislation to be drawn from these results will provide an appropriate ex-perimental base line, will promote better control, and will stimulate progress in thecheese industry.

Advice on the analytical methodology to be used can also be given.

PUBLICATIONS

1. Quevedo, F. and S. Tesone. Desarrollo de criterios microbiológicos parael control de quesos. 1. Quesos de pasta blanda tipo quartirolo, inpreparation.

OTHER DATA

Grantee: Dr. Fernando Quevedo, Pan American Zoonoses Center, Ramos Mejía,Buenos Aires Province, Argentina.



325

NEW METHOD FOR MICROBIOLOGICAL SAMPLINGOF ANIMAL CARCASSES AND SURFACES

PROBLEM

Official control laboratories and those belonging to the meat industry mustcertify the sanitary condition of animal carcasses and of the facilities in meat pro-cessing plants. One of their main concerns is to search for pathogenic organisms,especially salmonellae, in the products and places they control.

Yet these controls present some problems that make it necessary to use very sim-ple, economical, and quick sampling techniques. The diverse nature of the materialsto be tested, for example, metallic or wooden surfaces, concrete walls and floors, thevarious utensils, the state of the hands and clothing of personnel, substances of ani-mal origin, and others, is an important factor to keep in mind. Speed is a must inorder not to interfere with production. Every effort is made to avoid resistance andill feelings towards laboratory staff on the part of both labor and management groups.

Another aspect to be considered during sampling is the inadvisability of usingglass instruments such as tubes because they may break, cause accidents, and contaminatethe meat with glass fragments. Similarly and for obvious reasons, nondestructive sam-pling techniques should be preferred.

Several techniques obviate some of these difficulties, but they are not altogethersatisfactory. Therefore, the search for a more adequate technique must be pursued.

The purpose of this study is to develop a simple, quick, and economical samplingtechnique to search for pathogenic organisms in carcasses and abattoir surfaces.

METHOD

The material required for sampling consists of an unused plastic bag and an or-dinary polyurethane sponge that can be easily sterilized by autoclaving. Using thebag pulled inside out like a glove, the sponge, previously moistened with a peptonesaline solution is applied to the surface to be studied. The same is then kept in thebag, which is labeled for identification. Once in the laboratory, lactose broth isadded to the bag, which is securely fastened and incubated for 18-24 hours. Then sal-monellae (or other organisms) are examined in that broth by the usual laboratory methods.

The presence of salmonellae in equine and porcine carcasses is investigated em-ploying the technique described above.

RESULTS TO DATE

Through this new technique, 116 (80 percent) of 145 samples studied were foundto be contaminated with salmonellae.

The technique proved effective for studies on hospital infection and contamina-tion of hospital kitchens. Salmonellae sp., enteropathogenic E. coli, Pseudomonas sp.,and Staphylococcus aureus were isolated from places where the classical swab had notyielded comparable results.

326

SIGNIFICANCE

This technique offers very important advantages, such as application to differentsurfaces and low cost. It is more economical, faster, and simpler than the methodscurrently used for the same purpose. It does away with glassware material in meat andother food-processing areas.

The technique is very sensitive and can also be used in the hygienic control ofother products and premises, and may be employed in the collection of samples for coli-form or total bacterial counts.

PUBLICATIONS

1. Quevedo, F. et al. Control microbiológico de superficies con esponjas depoliuretano. Rev Latin Microb, submitted for publication.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Fernando Quevedo, Pan American Zoonoses Center, Ramos Mejía,Buenos Aires Province, Argentina.


1974 - 1975.

327

SALMONELLAE AND FECAL COLIFORMS AS POLLUTION INDICESIN SUPERFICIAL WATERS OF THE GREATER BUENOS AIRES AREA

PROBLEM

The participation of superficial waters in environmental pollution and their roleas collectors of enteropathogenic organisms are being regarded with increasing interest.Indeed, superficial waters constitute an important link in the cycle that permits organ-isms to recontaminate animals, food, and humans.

In the course of the investigation currently being conducted at the Pan AmericanZoonoses Center on the presence of salmonellae in animals, food, and food handlers, agreat variety of serotypes have been isolated with clear predominance of some. What isnot well known, however, is the routes followed by salmonellae in contaminating animalsand through them foodstuffs and humans.

Another problem concerns the best temperature at which enrichment media shouldbe incubated, a point not yet settied. Temperatures of 41°C and 430C are usually re-commended for the recovery of salmonellae in superficial waters, but further tests mustbe carried out in order to determine the most appropriate.

The degree of fecal pollution in the superficial waters of the Greater BuenosAires area is not known either; the determination of the most probable number (MPN) offecal coliforms will permit the obtainment of the required information.

The objectives of this program are to study the degree of fecal contamination inthe superficial waters of the Greater Buenos Aires area; to find out the extent of thecontamination with salmonellae in those waters and the distribution of serotypes; todetermine the behavior of the serotypes found in the presence of antibiotics; and tocompare temperatures of 410C and 430C in the incubation of enrichment media for sal-monellae.

METHOD

River, brook, and stream waters in the greater Buenos Aires area are studied,and representative points, accessible for sampling chosen.

For the study of salmonellae, water sampling is done, using Moore gauze swabs,placed at selected spots. As swabs are picked up, a water sample is taken for fecalcoliform analysis.

A comparison is made of two incubation temperatures, 410C and 430C, for enrich-ment media, in an electronically regulated water bath. In addition to the biochemicalstudy of the salmonella strains isolated, serological assays and execution of anti-biograms are performed.

The most probable number of fecal coliforms are determined by culturing theseries and dilutions considered necessary in each case. The incubation is at 430C,with the usual confirmations.

The pH is determined for each sample.

328

RESULTS TO DATE

A total of 89 samples have been analyzed but the presence of salmonellae couldonly be confirmed in 79 of them because of the collecting swab losses. At two placeswater samples for coliform analysis could not be taken. Salmonellae were isolated from54 samples, which constitutes a high degree of positivity, and they were isolated in allthe areas studied.

Fecal coliforms were present in all but one of the samples studied and in severalof them in large numbers.

No direct relationship was found between the MPN of fecal coliforms and the pre-sence of salmonellae, and there was no marked difference between the results obtainedin the isolation of salmonellae at 410C and 430C.

Serotypes isolated to date are anatum, bredeney, meleagridis, derby, give, new-port, haelsinborg, infantis, tennessee, typhimurium, montevideo, livingstone, oranien-burg, agona, glostrup, carrau, panama, poona, minnesota, thompson, and eimsbuettel.Anatum was the most commonly encountered serotype. Many of these strains show resist-ance to antibiotics such as tetracycline and ampicillin.

SIGNIFICANCE

Results obtained to date confirm the important role played by superficial watersin environmental pollution. The part they play as natural collectors of salmonellae--and possibly other enteropathogenic organisms--should induce health authorities to takemore decisive corrective action.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. Fernando Quevedo, Pan American Zoonoses Center, Ramos Mejía,Buenos Aires Province, Argentina.



329

PATHOLOGY OF LABORATORY ANIMALS

PROBLEM

The main purpose of this study is the health surveillance of laboratory animaisthat are used by the different units of the Pan American Zoonoses Center.

To achieve the aforementioned objective, two classes of animais are being studied:(1) apparently healthy animals, and (2) sick animals.

METHOD

(1) Apparently healthy animals: the health of these animals is controlledthrough necropsies on breeders discarded because of their advanced age.

At necropsy, material is collected for microbiologic and histopathologic examina-tions of organs showing macroscopic lesions.

(2) Sick animais: If an illness occurs in an animal colony, necropsy is per-formed and samples are taken for the microbiologic and histopathologic examination oforgans involved.

RESULTS TO DATE

(1) Apparently healthy animals: Focal hepatic necrosis was found at necropsyof guinea pigs discarded because of their advanced age. According to the informationavailable, this type of necrosis has never been described in guinea pigs. This con-dition is widely spread in the colony and affects mostly older animals, although ithas also been observed in younger, and even suckling animals.

Focal hepatic necrosis progresses asymptomatically, without clinical signs, inspite of the intensity and the severity of the hepatic lesions observed in some cases.

(2) Sick animals: An illness, reported in guinea pigs maintained under over-crowding conditions, and characterized by uncertain, nonspecific symptomatology (ano-rexia, adynamia, and dyspnea), has been studied. The general condition of the animaiswas rapidly impaired and they died in a few days. Bacteriologic and histologic studieshave shown unspecific changes.

It is concluded that the process studied is not specific but is caused by over-crowding and poor hygiene, and by temperature and feeding deficiencies.

SIGNIFICANCE

It is hardly necessary to emphasize the importance of monitoring the health oflaboratory animals to be used in scientific experiments. The early diagnosis of dis-eases that may affect a laboratory animal colony is of great importance to prevent itsfurther spread.

330

PUBLICATIONS

1. Battolla, L. E. and J. E. Roncarrolli. Estudio sobre una mortandad encobayos en un criadero destinado a proveer animales de experimentación.Rev Med Vet (Buenos Aires) 55:99-107, 1974.

2. Cuba-Caparó, A. et al. Focal hepatic necrosis in clinically normal guineapigs. Submitted for publication.

OTHER DATA

Grantee:

Funded by:

Timetable:


Pan American Health Organization/World Health Organization,United Nations Development Program, and Governments of theAmericas.

1973 - continuing.

331

INTER-AMERICAN INVESTIGATION OF MORTALITY IN CHILDHOOD

PROBLEM

The overall aim of the Inter-American Investigation of Mortality in Childhood wasto develop death rates as accurate and comparable as possible in selected communities,taking into account the nutritional, sociological, and environmental factors responsiblefor excessive mortality. The data provided by the study were used to compare underlyingand associated causes of death, investigate relationships with infectious diseases,nutritional deficiency states, and sociological conditions, and to recommend actionsindicated by the findings.

METHOD

The deaths of 35,095 infants and children under 5 years of age in 15 widely sepa-rated areas of the Americas were investigated through interviews in the home, at thehospital or clinic, and with the physician to obtain as complete a record as possibleof the fatal illness, including results of laboratory and other examinations and autopsy.To permit a full evaluation of the factors resulting in the death of these infants andyoung children, tabulations on the pregnancy history, the status of the infant at birth,breast feeding, growth and development, and social and environmental conditions havebeen included.

In order to compare the data on the children who died with similar data for thegeneral population, approximately 20,000 living children under 5 years of age have beenstudied through household surveys in which probability sampling techniques have beenused.

RESULTS TO DATE

A comprehensive report on the investigation was published in English and Spanishin 1973 under the titles of Patterns of Mortality in Childhood and Características dela Mortalidad en la Niñez, PAHO Scientific Publication No. 262. This geographic study,and the previous one on urban mortality, PAHO Scientific Publication No. 151, 1967,in several areas of the Continent, point to distinctly different and intriguing pat-terns. They show the great value of comparing mortality in widely separated areas,thereby leading to a better understanding of health problems.

Besides these two books, the pamphlets, Excerpts and Datos Seleccionados werealso published in 1973 for use in teaching and at meetings.

Nutritional deficiency was the most serious health problem uncovered in theInvestigation. When found in conjunction with low birthweight, it endangered thesurvival of infants, young children, and probably of future mothers, and hamperedtheir growth and development. The distribution of neonatal deaths by infant weightat birth suggested excessive frequencies of low birthweight infants in Latin Americanprojects. Further study of hospital births showed that the proportion of live births,with birthweights of 2,500 g or less, is much higher in Latin America than in theUnited States and Europe. In addition, the number of babies weighing at birth 2,501-3,000 g was relatively high, and of those 3,501 9 and over, low. Infants with birth-weights of 3,501-4,000 g have the lowest mortality in New York City (Kessner, D. M.

332

et al. Infant Death: An Analysis by Maternal Risk and Health Care. Contrast in HealthStatus, Vol. 1. Institute of Medicine of the National Academy of Sciences, 1973). Be-cause of the synergistic relationships of immaturity, nutritional deficiency, and in-fectious diseases, these babies with low and deficient weights are at great risk ofdeath and if they survive, their growth and development may be hampered. The resultsof the Investigation clearly point to the need for programs directed to health of womenin the reproductive period, especially during pregnancy. Infants of healthy mothershave better chances to be born with normal weights and defensive mechanisms than do ba-bies of unhealthy mothers.

Of the 35,095 deaths studied in the Investigation, 2,107 were not registered andwere discovered through searches by the staff of the projects. In the four cities,Santiago, San Salvador, La Paz, and Cartagena, over 30 percent of neonatal deaths inhospitals were not registered. During the Investigation, it was found that 56.1 percentof the neonatal deaths in large maternity sections of six hospitals were not includedin official statistics records. Discovery of such unregistered deaths, many of whichwere of low birthweight babies, resulted in uncovering the serious problem of immaturity.Based on findings of the Investigation, recommendations for the improvement of registra-tion of births and deaths were made in a paper prepared for use at the United NationsExpert Committee Meeting for the Improvement of Demographic Statistics. Hospitalsshould have the responsibility for preparing and registering certificates of births anddeaths occurring in their institutions.

The Investigation uncovered excessive frequencies of deaths from congenital ner-vous system anomalies in three projects. Further exploration in the El Salvador andSherbrooke projects has revealed relatively high frequencies of anencephalus in fetaldeaths. Thus research into causation of congenital anomalies of the nervous system intwo Latin American countries, El Salvador and Mexico, and in the Quebec Province ofCanada is indicated.

Study of biologic and socioeconomic factors revealed that lack of breast feedingwas involved in excessive infant mortality from diarrheal disease and nutritional defi-ciency. The mother's level of education was shown to be a valuable indicator of socio-economic status. A negative correlation was established between postneonatal deathrates and the percentages of homes with piped water among families of infants dying inthe postneonatal period. Coordinated, community-centered research was clearly demon-strated to be a sound method of uncovering health problems and providing data neededfor the establishment of effective programs.

New analyses have revealed the three important determinants of infant mortality:birthweight, birth order, and maternal age group.

Because of the value of data on the three determinants for health planning,procedures for collection and analysis of the outcome of pregnancies were developedin 1973 in Recife and were followed in three large maternity hospitals in 1974. Theoutcome of 24,598 pregnancies in Brazil was followed. The results indicate serioushealth problems. In the hospital with the most complete data, half of the live bornhad low (16.8 percent) and deficient (33.1 percent) birthweights. Of the young mothersunder 20 years, 22.6 percent had low and 37.0 percent deficient birthweights. Thedeath rate in the first day of life of 19.1 per 1,000 live births was excessive.

Since official data in Latin America are not available for study of problemsconnected with the outcome of pregnancies, the utilization of hospital birthweightsand mortality on the first day of life was recommended for knowledge of those seriousproblems and as the basis for a vital statistics system in which such data could beobtained.

Fourteen additional papers and reports utilizing new information as a resultof the Investigation have already been published, and two papers from the Recife projectin Brazil have been submitted for publication in Bol Of Sanit Panam in 1976.

333

SIGNIFICANCE

The importance of this research program has been recognized widely in the Con-tinent for orientation of actions in maternal and child health. In December 1971, theMinisters of Health of the Andean Region stated the need for strengthening community-centered research in their countries in order to measure and uncover the real magnitudeof basic health problems and also to find the best methods for the efficient use ofresources for solution of the problem disclosed. They pointed out the value of joiningtheir efforts for coordinated research such as has been done in the Inter-AmericanInvestigation of Mortality in Childhood.

The success of the Investigation requires application of the recommendations forreduction of mortality and improvement of the health of future generations. To stimu-late action in each project, tabulations were reproduced and discussed with the localstaff in the projects. This material serves for analysis prepared locally and forteaching. A new project has been developed for full utilization of the results inmaternal and child health programs and for teaching students of public health, medicineand midwifery. This regional project should be influential throughout Latin America.

The World Health Organization is conducting studies of perinatal, infant, andchildhood mortality in areas of Afghanistan, Algeria, Sierra Leone, Sudan, and Trinidadand Tobago, using procedures adapted from the ones followed in the Investigation. Thusthe continental study is being utilized to reveal problems in other areas of the world.As stated by the director of one of the divisions of WHO, the Investigation will be abasic reference for studies of child mortality for many years to come.

Interests and actions in implementing recommendations of the Investigation aregreat in Brazil. In Sao Paulo, the Secretary of Health has developed a supplementalfood program for pregnant women and nursing mothers and their babies. A quota of 400calories per day will be provided for women attending prenatal clinics. A program ofsupplemental feeding will be undertaken in Recife and a research project for study ofhuman reproduction before and after the introduction of supplemental feeding is alreadyunderway, in which weight gain in pregnancy and the results of pregnancy are beingobtained. A film is already being used to encourage breast feeding. Because of themany new programs and projects in Brazil, a volume of nine papers in Portuguese is beingplanned for release in 1976.

In addition to these specific actions in Brazil, the results of the Investigationhave been discussed in several conferences, seminars, and meetings and are affectingchanges and reorientation of maternal and child health programs to the improvement ofthe condition of the child at birth. Emphasis is on healthy newborn babies with pre-vention of low and deficient birthweights and nutritional deficiency in contrast totreatment.

PUBLICATIONS

1. Aceves-Sainos, D. Investigación Interamericana de Mortalidad en la Niñez.Planificación de los trabajos en el área de Monterrey, N.L., México.Salud Pública de México 11:471, 1969.

2. -----. Investigación Interamericana de Mortalidad en la Niñez: resultadosseleccionados del proyecto realizado en la área de Monterrey, N.L., México.Bol Of Sanit Panam 79:485-507, 1975.

3. Laurenti, R. A Investigaçao Interamericana de Mortalidade na Infancia enSao Paulo, Brasil. Rev Saude Pub (Sao Paulo) 3:225-29, 1969.

334

4. -----. Alguns aspectos particulares referentes aos resultados a investigaçaoInteramericana de Mortalidade na Infancia, na área de projeto de Sao Paulo,Brasil. Bol Of Sanit Panam 79:1-14, 1975.

5. ----- et al. Sub-registro de nascimento de criancas falecidas com menos deum ano de idade. Rev Saude Pub (Sao Paulo) 5:237-42, 1971.

6. Legarreta, A. et al. Omissions in the registration of deaths in maternityhospitals in Santiago, Chile. Bull PAHO 7:35-40, 1973.

7. -----. Omisión del registro de defunciones de niños ocurridas en maternidad,Santiago, Chile. Bol Of Sanit Panam 75:308-14, 1973.

8. Mortality from nutritional deficiency. Editorial. J Trop Pediat 20:1-2, 1975.

9. Pan American Health Organization. Mortality in Childhood. PAHO Gazette19:2-15, 1973.

10. Puffer, R. R. Initial phases of the Inter-American Investigation of Mor-tality in Childhood. Bol Of Sanit Panam 65:114-26, 1968; English edition,65:35-46, 1968; and 66:341-43, 1969.

11. ----- and C. V. Serrano. Inter-American Investigation of Childhood Mortality.Washington, D.C. Technical Front Section of War on Hunger. U.S. Agency forInternational Development, Vol. 3, No. 11, November-December 1969.

12. ----- and -----. Patterns of Mortality in Childhood. PAHO ScientificPublication No. 262, 1973.

13. ----- and -----. Características de la Mortalidad en la Niñez. OPS Publica-ción Científica No. 262, 1973.

14. ----- and -----. The role of nutritional deficiency in mortality. Bull PAHO(English edition) 7:1-25, 1973.

15. ----- and -----. La deficiencia nutricional y la mortalidad en la niñez.Bol Of Sanit Panam 75:1-30, 1973.

16. ----- and -----. Datos básicos esenciales sobre nacimientos y defuncionespara la planificación de salud y las estadísticas demográficas. Bol Of SanitPanam 76:187-208, 1974.

17. ----- and -----. Interrelación de varios factores que intervienen en lamortalidad infantil. Bol Of Sanit Panam 77:509-33, 1974.

18. ----- and -----. Birthweight, Maternal Age, and Birth Order: Three Im-portant Determinants in Infant Mortality. PAHO Scientific PublicationNo. 294, 1975.

19. ----- and -----. El peso al nacer, la edad materna y el orden de nacimiento:Tres importantes determinantes de la mortalidad infantil. OPS PublicaciónCientífica No. 294, 1975.

20. ----- and -----. Challenging problems of Inter-American Investigation ofMortality in Childhood. Courrier 25:357-65, 1975.

21. ----- and -----. Teenage pregnancies: High risk for infants. DraperWorld Population Fund Report 1:16-17, 1975.

335

22. ----- et al. The Inter-American Investigation of Mortality in Childhood.Assignment Children. United Nations Children's Fund 15:72-90, 1971.

23. Serrano, C. V. and R. R. Puffer. Utilization of hospital birthweights andmortality as indicators of health problems in infancy. Bull PAHO 8:325-46,1974.

24. ----- and -----. Datos del peso al nacer y de la mortalidad en hospitalesutilizados como indicadores de los problemas de salud en la infancia. Bol OfSanit Panam 78:93-119, 1975.

25. Teruel, J. R. el al. Investigación Interamericana de Mortalidad en la Niñez:Peso al nacer en la región de Ribeirao Preto, Sao Paulo, Brasil. Bol OfSanit Panam 79:139-45, 1975.

26. Wallace, H. M. et al. Patterns of infant and early childhood mortality inthe California project of a collaborative inter-American study. Bull PAHO9:32-38, 1975.

27. -----. Características de la mortalidad en la infancia y la niñez tempranaen el proyecto de California de un estudio interamericano cooperativo. BolOf Sanit Panam 79:15-23, 1975.

OTHER DATA

Grantee: Dr. Ruth R. Puffer, Consultant to the Pan American HealthOrganization, Washington, D.C., U.S.A.

Funded by: U.S. Agency for International Development (in part).


336

RURAL SANITATION

PROBLEM

The purpose of this project is to collect information on some of the problemsthat affect rural areas, specifically water supply, disposal of excreta, and generalsanitation. The study will be restricted to the Río María Linda Basin, but the use ofsewage for irrigation in Chichicastenango will also be investigated.

METHOD

A bibliographic research will be made to study the environmental informationavailable on the Río María Linda Basin and the practices of using sewage for irrigationin Chichicastenango. Sampling station sites will be selected and samples taken of thesewage used to irrigate the apple orchards in Chichicastenango. Samples will also betaken for analysis in the Lake of Atlitán in the vicinity of San Lucas Tolimán. Sur-veys will be made and data collected on the water supply systems in existence and onthe situation of disposal of excreta within the area.

RESULTS TO DATE

None thus far.

SIGNIFICANCE

The project will shed light on the sanitary conditions of a rural area, theRío María Linda Basin, in Guatemala.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Eng. Francisco Campos, Escuela Regional de Ingeniería Sanitariapara Centro América y Panamá, Universidad de San Carlos, Guate-mala City, Guatemala.

Funded by:

Timetable:

Pan American Health Organization, Swiss Government.

1974-1977.

337

CHEMICAL AND BACTERIOLOGIC QUALITY OF THE WATER OF LAKE PEÑUELAS

PROBLEM

The Lake Peñuelas is one of the sources of water for the city of Valparaíso. Itis a natural lake. A dam has been built to increase its capacity. The possibility ofdiverting water from the Mapocho River into this lake has made it necessary to studythe parameters of quality of the water of the lake.

METHOD

Sampling points were selected, and a sampling program developed considering dirfferent seasons of the year. Physical, chemical, and bacteriologic analyses of these sam-ples were conducted.

RESULTS TO DATE

Results showed that (a) the salt content of the water was low; (b) alkalinityincreased with depth; (c) algae influenced oxygen balance and there was less dissolvedoxygen by night than by day; (d) the total coliform content of the water was low; and(e) fecal contamination existed.

It was concluded that the Lake Peñuelas water had not reached a degree of markedpollution.

SIGNIFICANCE

The study provides accurate information about the state of pollution of theLake Peñuelas. It could therefore be used as base for any ulterior study or eventualwater pollution control program.

PUBLICATIONS

1. Castillo, J. and A. M. Sancha. Estudio de la calidad química y bacterioló-gica del Lago Peñuelas. Santiago, 1975.

2. ----- and -----. Aprovechamiento de los recursos hídricos de la cuenca delLago Peñuelas. Santiago, 1975.

OTHER DATA

Grantee: Eng. Jorge Castillo, Facultad de Ciencias Físicas y Matemáticas,Sección de Ingeniería Sanitaria, Universidad de Chile, Santiago,Chile.

338

Funded by:

Timetable:


1974-1975.

339

SEWAGE TREATMENT WITH TOWER-LIKE FILTERS

PROBLEM

The project aims to study the performance of a tower-like trickling filter, treat-ing sewage from a well-defined section of Guatemala City. The sewage to be treated canbe considered as domestic sewage. Tower-like trickling filters are innovative approachesto the process of treatment known as biologic percolation. The structures to build willserve also for teaching purposes.

METHOD

It is planned to construct a primary settling tank, the tower-like filter, anImhoff tank, and drying beds. Once the plant is built, it will be connected to thesewage outlet and operated and maintained by the University. Flow rates will be ad-justed at will through by-passes. Efficiency studies will be carried out involving aseries of physical and chemical determinations, among them the biochemical oxygen de-mand (BOD), the dissolved oxygen (DO), total solids, settleable solids, bacterial andcoliform content. Students will participate in the research activities.

RESULTS TO DATE

None thus far.

SIGNIFICANCE

The project will try to establish the efficiency of a tower=like trickling filterunder different loads and volumes of domestic sewage coming from a definite section ofGuatemala City. This is not a laboratory study. This is field research operation.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Eng. Arturo Pazos, Escuela Regional de Ingeniería Sanitariapara Centro América y Panamá, Universidad de San Carlos,Guatemala City, Guatemala.

Pan American Health Organization, and the Swiss Government.

1974-1976.

340

SEDIMENTATION TANKS

PROBLEM

No definite conclusions have been reached concerning the advantages and disad-vantages of vertical-flow settling tanks as opposed to horizontal-flow installations.A number of factors affect the performance and efficiency of each type.

At the Vizcachas water treatment plant in Santiago, Chile, there are circularsettling tanks, originally of horizontal-flow design, which have been modified tofunction through combined vertical and horizontal flow. The purpose of the present re-search is to gain a better understanding of this particular type of tank and to comparethe performance of horizontal- and vertical-flow installations.

METHOD

A compilation of existing literature on the subject was prepared.

Two different types of pilot settling tanks were designed and built, and theefficiency of both units was measured. They were made to work under different condi-tions but under the same hydraulic head and with water of the same quality.

RESULTS TO DATE

Pilot plants were designed and built. Conditioning of the settling tanks at theVizcachas plant was completed. Tests were run on speeds varying from 15 cm/min to 60cm/min on horizontal- and vertical-flow basis. Tests were also run at Las Vizcachitasplant and at Lo Gallo plant. Retention periods of 2 to 4 hours were considered. Nosignificant differences were found in the performances of the vertical-flow and thehorizontal-flow (circular) types. Studies were carried out to design, build and oper-ate a model of a rectangular horizontal-flow settling tank at Las Vizcachitas watertreatment plant.

SIGNIFICANCE

The results derived from the research on vertical-flow and horizontal-flowsettling tanks can be incorporated in the design of future circular settling tanks.

PUBLICATIONS

1. Madariaga, M. and J. P. Schifini. Análisis y estudios de procesos de sedi-mentación y de floculación mecánica. Santiago, 1973.

2. Schifini, J. P. Memorial acerca de investigación sobre sedimentadores.Santiago, 1970.

341

3. Universidad de Chile, Facultad de Ciencias Físicas y Matemáticas, Departamentode Obras Civiles, Sección de Ingeniería Sanitaria. Estudios sobre sedimenta-dores. Informe de avance. Santiago, December 1970.

4. Vidal, J. Sedimentadores: construcción y puesta en funcionamiento de unmodelo rectangular de flujo horizontal. Santiago, 1974.

OTHER DATA

Eng. Juan Pablo Schifini, Facultad de Ciencias Físicas y Matemá-ticas, Universidad de Chile, Santiago, Chile.


1970-1975.

342

Grantee:

Funded by:

Timetable:

Ja

STABILIZATION PONDS

PROBLEM

Stabilization ponds, because of their relatively low cost, are being increasinglyrecognized as one of the most suitable approaches to sewage treatment in the developingcountries. There has been no previous experience in Chile with this process. The pur-pose of the research is: (a) to study the efficiency of stabilization ponds under theclimatic conditions prevailing in the city of Melipilla, Chile; (b) to compare perform-ances with conventional sewage treatment systems; and (c) to determine the load capacityof the lagoons.

METHOD

The project, originally planned for 4 years, (1970-1973), has been extended atthe request of the national agency responsible for the sewage programs in Chile. Thelatter agency participated in the project through the building of the lagoons and theconventional sewage treatment plant at Melipilla, and by involving university authori-ties.

The project started with the building of one stabilization pond by the Ministryof Public Works in the city of Melipilla, by the side of a conventional sewage treat-ment plant being built by same Ministry. The project is now in its fourth stage, andis responsible for the creation by the end of 1975, of three operating stabilizationponds.

The existing literature on stabilization ponds was reviewed. Meteorologic datawere collected. Different loads and flow rates were applied to the lagoons, and thebiochemical oxygen demand (BOD), the dissolved oxygen (DO), and total solids were mea-sured and recorded.

RESULTS TO DATE

Marked reductions in the BOD of the influent resulted when sewage passed throughthe facultative stabilization ponds (from 32 percent to 68 percent overall BOD reduc-tion). Algae developed well. Records of sunshine hours and of water and air tempera-tures were registered. Correlation of these data with BOD reduction and DO content areunder way.

Three lagoons were prepared to operate in "series". In winter time, the lessfavorable season for effective lagoon operation, the three lagoons in series reducedthe BOD by 84.3 percent. Reduction of fecal coliform was greater than when the lagoonsoperated in other arrangements. The three lagoons in series significantly reduced thealgae content in the effluent. After 4 years of operation of the lagoon system, thelagoons were found to have polluted the ground water table. This pollution disappearedhowever, 40 to 50 m away from the lagoons.

Through radioactive tracers used in the lagoons, the real retention period (asmeasured in the lowest outlet) was estimated to be 50 percent greater than the designretention period. In the middle outlet, the real retention period appeared to be verysimilar to that of the design.

343

In view of the results obtained with the three lagoons in series, loading designparameters for this type of arrangement and for temperatures of or above 130C, are sug-gested as follows: (a) Primary Lagoon--from 700 to 1200 Kg BOD/Ha-day; (b) SecondaryLagoon--350 to 600 Kg BOD/Ha-day; and (c) Tertiary Lagoon--100 to 150 Kg BOD/Ha-day.

SIGNIFICANCE

Results will provide realistic information about the feasibility of using thistreatment process in Chile.

PUBLICATIONS

Eng. Juan Pablo Schifini, Facultad de Ciencias Físicas yMatemáticas, Universidad de Chile, Santiago, Chile.


1970 - continuing.

344

Grantee:

Funded by:

Timetable:

STREAM POLLUTION IN THE WATERSHED OF THE LAKE AMATITLANAND IN THE VILLALOBOS RIVER

PROBLEM

Lake Amatitlán is one of the most important water resource bodies in Guatemala.It is only about 25 miles from the capital of the country and is one of the most attrac-tive spots for tourism in Guatemala. Considerable investments have been made in thebuilding of residences, resort houses, and club facilities. The Amatitlán lake and theVillalobos river are suffering now from increasing pollution, the extent of which hasnot yet been determined. This project aims to develop a system of sampling stations,to collect and analyze regularly samples taken from these two bodies of water, to mea-

sure up the degree of pollution and to identify the sources of the main causes of pol-lution.

METHOD

To achieve the goals of the study, the project's steps are:

(1) To limit the extension in geography and distances of the area to be studied.

(2) To determine the sources of pollution: residential, agricultural, indus-trial, or others.

(3) To build a number of sampling stations.

(4) To determine the physical parameters in the region.

(5) To sample and analyze periodically to establish the extent and degree ofpollution.

(6) To study the eutrophication of the lake Amatitlán.

(7) To establish the present and future restrictions in the uses of water fromthe lake AmatitlIn and from the Villalobos river according to the degree of pollution.

(8) To suggest steps to be taken for water pollution control in these two bodiesof water.

RESULTS TO DATE

The building of seven sampling stations has been already completed. A systematicsampling schedule has been established and the logistics for the periodic analysis havebeen put into effect. Data collected are being processed, classified, grouped, corre-lated, and studied.

345

SIGNIFICANCE

,The project will be a contribution to establishing the scientific and technicalbackground to sustain the regulatory official decisions in a water pollution controlprogram.

PUBLICATIONS

None thus far.

OTHER DATA

Dr. Carlos E. Muñoz Palacios, Escuela Regional de IngenieríaSanitary para Centro América y Panamá, Universidad de San Carlos,Guatemala City, Guatemala.

Pan American Health Organization and the Swiss Government.

1972-1976.

346

Grantee:

Funded by:

Timetable:

TOXIC PARAMETERS IN THE WATER SUPPLY SYSTEMSOF THE MAJOR CITIES IN CHILE

PROBLEM

In the increasingly industrial development of a country, use is constantly madeof elements that may form toxic compounds when in contact with water, compounds thatmay affect human health. The purpose of this project is to search for the toxic ele-ments that may be appearing in water supply systems.

The parameters considered in the research project include, among others, arsenic,chromium, lead, copper, nitrogen, mercury, radioactive compounds, pesticides, and non-biodegradable detergents.

METHOD

A number of cities of above 10,000 people will be selected. During the first 2years the study will be restricted to 30 cities. Two sampling points will be estab-lished in each water supply system: the first will be at the effluent of the watertreatment plant, the second in the downtown area. Three samples should be taken everyweek and immediately sent to the laboratory. Sampling will be conducted during twodifferent seasons of the year, summer and winter. Analytic techniques will follow thestandard methods recommended by the American Water Works As-sociation, 13th ed., 1971.For radioactive materials, the methods used by the U.S. Department of Health, Education,and Welfare will be used.

RESULTS TO DATE

None thus far.

SIGNIFICANCE

The study will bring a valuable contribution to the improvement of the qualityof the drinking water. Findings from the research will be submitted immediately toappropriate authorities so that corrective measures may take place.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Eng. Francisco Unda Opazo, School of Public Health, Universityof Chile, Santiago, Chile.

347

Funded by:

Timetable:


1974-1978.

348

AIR POLLUTION IN GUATEMALA CITY

PROBLEM

The project aims to determine what are the major air pollutants of GuatemalaCity, and to obtain data on the concentration, origin, and sources of these pollutants,and their possible relationship to other atmospheric conditions. The project also per-mits graduate students at the University to familiarize themselves with research method-ology and praxis.

METHOD

Methodology involves: (a) determination of concentration of carbon monoxide(CO) in major traffic-congested areas of the city of Guatemala; (b) study of the possi-ble relationship between this pollutant and traffic and atmospheric conditions pre-vailing at the time of sampling; and (c) evaluation of the research process requiredfor the study of CO to see how many and which other pollutants could be incorporatedin the research.

RESULTS TO DATE

None thus far.

SIGNIFICANCE

This is a much needed research as Guatemala is a fast growing city and the cen-ter of all industrial activities in the country. The project is also used in theteaching of research processes for university students.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Eng. Rodolfo González Morasso, Escuela Regional de IngenieríaSanitaria para Centro América y Panamá, Universidad de SanCarios, Guatemala City, Guatemala.

Pan American Health Organization, Swiss Government.

1974-1977.

349

MATHEMATICAL MODELS FOR SELF-PURIFICATION CONSTANTS IN SURFACE WATERS

PROBLEM

The project aims to the formulation of mathematical models so that the impactthat pollutants may cause in various bodies of water may be followed and even predicted.These models could be used in the studies that are being contemplated for water usagesby the national planning agencies.

METHOD

A revision of existing self-purification theories and modeling techniques willbe undertaken and the bodies of water to be studied selected. Sampling points will bechosen in each of the streams selected and systematic sampling programmed and conducted.Physical, chemical, and bacteriological analyses will be made. Adjustments in the math-ematical models will be made to take into consideration the factual results of analyses.

RESULTS TO DATE

The compilation of literature for the revision of theories and modeling tech-niques dealing with the process of self-purification of streams has been completed.

SIGNIFICANCE

By offering scientifically supported bases for decisions at various levels, theproject will help in defining priorities to develop better use of the water resourcesof the country.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Eng. José M. Gundelach, Facultad de Ciencias Físicas y Matemá-ticas, Secci6n de Ingeniería Sanitaria, Universidad de Chile,Santiago, Chile.


1974-1978.

350

NATIONAL SYSTEM FOR THE MAINTENANCE OF HEALTH CARE FACILITIES

PROBLEM

The maintenance of health care facilities in Venezuela was generally bad becauseof the lack of a national maintenance system. The problem was so urgent as to requireemergency action. The immediate objectives of this program are: (1) to reduce thebacklog of essential maintenance and repairs of health care facilities; (2) to increasethe normal life of facilities and equipment; and (3) to establish procedures for main-tenance and engineering. The long-range objectives are: (1) to develop human resourcesfor maintenance and engineering; (2) to realize economic resources and financing; (3) todevelop a national maintenance system; and (4) to improve utilization of maintenanceresources.

METHOD

Development and implementation of national maintenance and engineering servicesfor health care facilities are carried out at four levels of operation--central, re-gional, subregional, and local--by clearly identifying the functional relationship ofthe various levels of the system. Organizational structures are developed and intro-duced at each level within the framework of the administrative reform. A managementcomponent is introduced at all levels, at the same time as education, training, spe-cial studies, technical assistance, and maintenance manuals.

RESULTS TO DATE

The objectives of this program are being carried out effectively. All necessarytechnical manuals have been written or are in preparation. Programs have been devel-oped for the training of personnel. Interministerial technical committees have beenestablished, and through them, technical recommendations and modifications can be in-corporated during the course of design and construction. Comprehensive technicalcourses are being instituted in electrical systems, hospital maintenance and engineer-ing, refrigeration and air conditioning, boiler operation and maintenance, emergencypower plants, environmental sanitation, sterilization equipment, and laundry super-vision.

Policy-makers have recognized the need for a national maintenance system, andhave increased annual funds from Bs. 6,000,000 in 1971 to Bs. 55,500,000 for 1976.

Implementation of the total system is under way.

SIGNIFICANCE

These special studies, which are also utilized in other Latin American coun-tries, have provided the basis for the establishment of an effective national main-tenance system for health care facilities and for the publication of technical manualsand procedures on the subject. The program, the first of its kind in Latin America,has generated a great awareness by policy-makers in many other countries, of the needfor preventive and curative maintenance of health care facilities.

351

Formalized technical training is now instituted in Venezuela and other countriesare utilizing this capability.

PUBLICATIONS

One hundred sixty-three manuals and monographs have been published by the staffof the Center for Hospital Engineering and Maintenance on the maintenance and repairof health care facilities.

OTHER DATE

Mr. John E. Donahue (Project Coordinator), Department of MedicalCare, Division of Health Services, Pan American Health Organiza-tion, Washington, D.C., U.S.A., and Mr. John Z. Anders (ProjectManager), Division of Engineering, Ministry of Health,Caracas, Venezuela.

United Nations Development Program.

1972-1977.

352

Grantee:

Funded 'by:

Timetable:

INFORMATION SYSTEM FOR THE CONTROL OF SUPPORTING SERVICESIN HOSPITAL WARDS

PROBLEM

In 1969 the Pan American Health Organization/World Health Organization, aidedby grants from the W. K. Kellogg Foundation, developed a Progressive Patient CareProject in six university hospitals in Latin America, with a view to strengthening thecoordination of their supporting services. To this effect, an information system wasprepared to monitor the effectiveness of these services and was used as a diagnosticinstrument in one of the six hospitals under study.

The specific objectives of this research are: .(a) identification of quantifiablecriteria; (b) development of a data collecting procedure; (c) establishment of a dataprocessing method; (d) elaboration of a chart in which nonconforming signals stimulatethe initiation of corrective measures in the supporting services; and (e) monitoringof supporting services in hospital units in which the concept of unit administrationis being applied.

METHOD

Quantifiable criteria for the evaluation of supporting services in hospitalwards were determined by means of an information matrix representing the network ofsupporting services. On its ordinate, 31 characteristics, such as ipatient information,maintenance, supplies, and others, were grouped into functional subsystems. Theabscissa bore information on 20 different sites within the wards.

Data were gathered through random sample procedures using 80-item (YES/NO)questionnaires filled 3 times a day for 10 days. These data were recorded on dailymatrices through which mean values and ranges could be established.

Statistical control charts, prepared from values given in the above matrices,display the daily mean values and signal-ed deviations from standard behavior in thesupporting services. These signais then stimulated the administrator's decision-makingat three levels of control.

RESULTS TO DATE

The methods described above were applied to the surgery and internal medicinewards of a university hospital. The results obtained during the 10-day experimentalperiod were entered on various matrices in daily averages and ranges. A comparativestatistical analysis of supporting service indices by functional subsystems was madebetween the two wards and the degree of control of the system for each ward wasdetermined, using the control charts described under METHOD.

SIGNIFICANCE

This systems control approach (management by exception) provides a usefulinstrument for the identification and quantification of the various problems in the

353

area of nonsupportive services, leading to an accurate analysis of the results and toa decision for corrective measures.

Such an approach has already been used in a hospital management training programas a tool for the diagnosis of services and for developing the decision-making traitsrequired for the solution of problems.

PUBLICATIONS

1. Peña, J. and J. Ortiz. Sistema de Información Dinamico de Evaluación delApoyo a Unidades de Pacientes. PAHO Document RD 13/16, 1976.

OTHER DATA

Grantee: Mr. Jorge Ortiz, Department of Research Development andCoordination, Division of Human Resources and Research,and Dr. Jorge Peña, Department of Medical Care, Divisionof Health Services, Pan American Health Organization,Washington, D.C., in collaboration with Erika Goldstuker,Docente de Registros Hospitalarios y Estatistica del Centrode Educación de Administración de Salud, Bogotá, Colombia.



354

PAHO REGIONAL LIBRARY OF MEDICINE AND THE HEALTH SCIENCES

PROBLEM

A survey of library resources in Latin America conducted by the Pan AmericanHealth Organization in 1965 revealed serious deficiencies in the supply of informationto the biomedical community and an urgent desire on the part of most of the institutionsconcerned to resolve the problem.

METHOD

The PAHO Regional Library of Medicine and the Health Sciences (RLM) was estab-lished in 1967 to contribute to the strengthening and improvement of the teaching,research, and practice of medicine and allied professions in Latin America. Servicesfor users,started in 1969 in Brazil, were later extended to other Latin American coun-tries. The Library carries out regular functions designed to accomplish the followingaims:

-To provide workers in the health sciences with better access to a more com-prehensive collection of publications in these sciences, and, by so doing, to makeLatin America self-sufficient in library resources and services in the biomedicalfield;

-To use modern methods of data processing and communication, including theMEDLINE system, to make these materials more accessible, and to disseminate biomedicalinformation to the health sciences community;

-To build up a pilot biomedical audiovisual unit and to establish a dissemina-tion system for audiovisual materials;

-To serve as a demonstration and training center to foster and encourage thedevelopment of medical libraries in Latin America:

-To encourage cooperative library programs among medical libraries in LatinAmerica; and

-To build up its own facilities through the development of journals and mono-graph collections, as well as selected secondary reference materials.

RESULTS TO DATE

A library building provided by the Escola Paulista de Medicina has been adaptedfor RLM's use, and equipment is being obtained as needed. Since 1971 the building hasbeen expanded from 1,950 m2 to 3,340m2 to satisfy RLM's growth in services and to pro-vide space for audiovisual activities.

The systematic acquisitions and maintenance of bibliographic materials, serialpublications, monographs, and documents is being continued. A collection of periodi-cals from 1960 onward of 5,089 biomedical journal titles--1,706 of which are obtained

355

by subscription, 271 by exchange, and 1,051, by gifts from the editors--is well on itsway to being completed.

The staff is trained for international library service. Its role in the improve-ment of local services including audiovisuals is also being developed.

A Scientific Advisory Committee on the Library has held eight annual meetingsso far.

Up to 1975, RLM processed 253,058 interlibrary loan requests from 100 librariesand expanded its services to most Latin American and Caribbean countries, includingGuyana, Trinidad and Tobago, and Surinam. RLM satisfied 74 percent of the requestsfrom its own resources. Other libraries in Sao Paulo participating in the programfilled 24 percent of them, and the U.S. National Library of Medicine (NLM) in Bethesda,Maryland, answered the remaining requests. Up to 1975, 7,449 requests for specialbibliographies were received: 2,521 of these were satisfied by manual searches at RLM;147 were sent to NLM for retrieval by the MEDLARS computer-based system; and 4,781 werefulfilled by the MEDLINE system operating in Sao Paulo.

Twelve library subcenters have been established in Brazil, in Belém, Belo Hori-zonte, Brastlia, Curitíba, Florianopolis, Fortaleza, Manaus, Niteroi, Porto Alegre,Recife, Rio de Janeiro, and Salvador, and nine specialized subsystems have been createdfor dentistry, nursing, pharmacy, basic sciences, biomedical sciences, gastroenterology,zoology, genetics, and veterinary medicine. RLM pooled the resources of these libra-ries and coordinated their operations throughout Brazil. It then initiated agreementswith national centers elsewhere in Latin America to establish a Pan American networkfor biomedical communications. Gift and exchange activities were conducted with 210libraries in Brazil and 195 similar institutions in other Latin American countries.A total of 288,289 duplicate journal volumes or issues were distributed since 1969.

In 1970, a Selective Dissemination of Information Prog.ram (SDI) was begun forworkers in hospital administration, medical education, health and population dynamics,maternal and child health, nuclear medicine, preventive and social medicine, and para-sitology and tropical medicine. Ulp to the present time, 204,625 articles were deliveredto 767 participants.

An SDI program on cancer was started in 1975 with the financial support of theBrazilian Ministry of Health. Through this program 17 biomedical libraries of Brazilwill have in their holdings additional cancer journals.

MEDLINE service was initiated on a pilot basis in March 1974. By arrangementwith NLM, which provides the software, and the State of Sao Paulo's Atomic Energy Insti-

tute, which leases computer time, MEDLINE is routinely performing 2 hours of search perweek. A three-phase MEDLINE deployment program has been initiated under grants from

the United Nations Development Program and the Government of Brazil. Since September1975, MEDLINE searches are provided in S5o Paulo at RLM, Brastlia, Rio de Janeiro, andRecife. The system will be extended later to other areas of Brazil.

Hardware and software have been acquired for RLM's audiovisual center, and ar-rangements have been made for coordination with the Latin American Center of EducationalTechnology for Health (CLATES).

Advanced courses have been offered to medical librarians throughout Latin America.In 1975, 25 librarians obtained PAHO fellowships to attend the courses and 22 assistantlibrarians received in-service training at RLM. Several seminars for guidance and ori-entation of medical educators and librarians were also offered.

356

SIGNIFICANCE

By providing specialists in the health sciences with increased access toscientific literature, the PAHO Regional library of Medicine and the Health Sciencesis contributing to the acceleration and improvement of biomedical education, research,and practice in Latin America.

Satisfactory and prompt service and improved bibliographic resources are gen-erating an increase in requests for scientific literature. Biomedical librariansthroughout Brazil are cooperating with RLM by producing collective catalogs and im-proving the interlibrary loan services among local medical libraries.

Cooperative efforts have been made to improve scientific information in mostLatin American countries. An inter-American network of biomedical communications isbeing developed and studies are being made of existing facilities and needs. Theinstallation of the MEDLINE data-base in Sao Paulo has been the first step towardestablishing this network in Brazil and other countries of Latin America. RLM'saudiovisual activities are also bringing a sizable contribution to medical education.

PUBLICATIONS

1. Bibliographic Reference Lists: 10 issues.

2. Catalog of Latin American Biomedical Journals existing at RLM.

3. Catalog of Recent Acquisitions: 4 issues.

4. Information Bulletin: 7 volumes.

5. List of Joint Duplicates for Exchange.

6. Neghme, A. Operations of the Biblioteca Regional de Medicina (BIREME).Bull Med Lib Assoc 63:173-79, 1975.

7. -----. Problems of Health Sciences Communications in Latin America.In: Kargel Basel, Communication of Scientific Information, 1975,pp. 69-82.

OTHER DATA

Grantee: Dr. M. Martins da Silva (Project Coordinator), Pan AmericanHealth Organization, Washington, D.C., and Dr. Amador Neghme,Director, PAHO Regional Library of Medicine and the HealthSciences, Sao Paulo, Brazil.

Funded by: Commonwealth Fund; W. K. Kellogg Foundation; United NationsDevelopment Program; Brazil's Ministries of Health and ofEducation and Culture; Secretariat of Health of the State ofSao Paulo; U.S. National Library of Medicine; Pan AmericanHealth Organization; and Escola Paulista de Medicina.


357

STRENGTHENING THE BRAZILIAN BIOMEDICAL INFORMATION NETWORK

PROBLEM

The high rate of change of biomedical science, the diversity of biomedicalpublications, and the generally very limited store of such publications in LatinAmerican biomedical libraries combine to make access to specific, up-to-date informa-tion particularly tedious and slow in many Latin American locations.

MEDLINE is a powerful system for interactive search and retrieval of biblio-graphic citations from the world's biomedical journals. Access is on-line from anylocation equipped with a data terminal and telephone connection to a central computer.

The purpose of this program is to establish MEDLINE in Brazil as an independentteleprocessing network reaching the country's 10 largest cities from Belém to PortoAlegre.

The software is provided on a continuing basis by the U.S. National Library ofMedicine, through an arrangement with PAHO.

The 10-city MEDLINE/Brazil network centered at PAHO's Regional Library of Medi-cine and the Health Sciences (RLM) in Sao Paulo, is planned as a basic infrastructurecapable of further growth by addition of terminals, further geographic spread, andassimilation of complementary data bases.

A supplemental objective of this program is to create an audiovisual center atRLM and an audiovisual acquisition and dissemination net generally paralleling MEDLIUIE.

METHOD

The program covers: (1) the establishment of a data transmission network con-sisting of dedicated and leased voice channels and, eventually, dial-up voice links;(2) selection, procurement, and installation of data terminals; (3) establishment,management, financial structures, and operating procedures for the teleprocessing net-work; (4) negotiation of participatory agreements with institutional members of thenetwork; (5) training courses and maintenance arranagements; and (6) recommendationsfor continuing evaluations.

Development of the MEDLINE/Brazil system is proceeding in three steps:

(1) System planning and equipment acquisition (1974).

(2) System start-up, and deployment (1975).

(3) Training of operating personnel and system consolidation (1976).

Deployment is planned in two phases:

I. Experimental service to Brasilia, Rio de Janeiro, Recife, and Sao Paulo.

II. Extension and consolidation of service to the 10-city network.

358

RESULTS TO DATE

Phase I deployment was completed in September 1975 and was followed by 3 monthsof network teleprocessing tests and experimental service and by the training of thefirst group of MEDLINE librarians. Terminal output during Phase I averaged 8.5 biblio-graphies per terminal hour.

The planning for the second phase of deployment has been completed and installa-tion finished in March 1976.

Terms of agreement for MEDLINE service have been delivered to the participatinginstitutions. Ratifications are expected soon.

Updating of the data base with tapes from the U.S. National Library of Medicineis being completed. As of February 1976, the system has been kept current and updatedmonthly.

It is too early to evaluate user satisfaction, but the demand for bibliographiesthrough searches by MEDLINE/Brazil has been generally higher than expected.

SIGNIFICANCE

The MEDLINE/Brazil system is routinely providing on-line, selective, up-to-datebibliographic reference information on biomedical subjects to researchers, practitioners,and professors in Brasília, Rio de Janeiro, Recife, and Sao Paulo. In February andMarch 1976, the service will be extended to include the 10 major cities of Brazil.

The use of MEDLINE/Brazil has greatly reduced the time to obtain a bibliographyespecially in the more remote cities. From Recife for example, bibliographic servicetime from RLM has been cut from 2 to 4 weeks to 3 days or less. Reduction in the man-hours required to compile a bibliography manually is even more dramatic, from 15 hoursto 5 or 7 minutes with MEDLINE.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. John D. Wilkes, Project Manager, PAHO Regional Library ofMedicine and the Health Sciences, Sao Paulo, Brazil.

Funded by: United Nations Development Program.


359

U A~-lY"1·~%~5~~~~0, ~S

irProyes^ - IRIS PAHO Home

Documents

Transcript of irProyes^ - IRIS PAHO Home