Steffina franklin

INDEX 1/8

THE CURRENT GLOBAL SITUATION AND REGIONALOVERVIEWS OF VIRUSES, VECTORS, SURVEILLANCEAND SPECIFIC FEATURES

ORAL PRESENTATION

BLUETONGUE – A SOUTH AFRICAN PERSPECTIVEG.H. Gerdes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3THE CURRENT SITUATION IN MEDITERRANEAN EUROPEC. Gómez-Tejedor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4EPIDEMIOLOGICAL OBSERVATIONS ON BLUETONGUEIN SHEEP AND CATTLE IN JAPANY. Goto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5BLUETONGUE VIRUSES, VECTORS AND SURVEILLANCE INAUSTRALIA: THE CURRENT SITUATION AND UNIQUE FEATURESP.D. Kirkland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6REGIONAL OVERVIEWS OF VIRUSES, VECTORS, SURVEILLANCEAND UNIQUE FEATURES: SOUTH AMERICA I.A. Lager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7STUDIES ON BLUETONGUE DISEASE IN CHINAZ. Nianzu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8REGIONAL OVERVIEW OF VIRUSES, VECTORS, SURVEILLANCEAND UNIQUE FEATURES: NORTH AMERICAE.N. Ostlund . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9REGIONAL OVERVIEWS OF VIRUSES, VECTORS, SURVEILLANCEAND UNIQUE FEATURES: EASTERN EUROPE D.E. Panagiotatos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10OVERVIEW OF BLUETONGUE DISEASE, VIRUSES, VECTORS,SURVEILLANCE AND UNIQUE FEATURES: INDIA, THESUBCONTINENT AND ADJACENT REGIONSD. Sreenivasulu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11THE HISTORY OF BLUETONGUEAND CURRENT GLOBAL OVERVIEWT.E. Walton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

OUTBREAKS OF BLUETONGUE & OTHER ARBOVIRALDISEASES IN ISRAELH. Yadin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

POSTERS

ENTOMOLOGICAL SURVEILLANCEOF BLUE-TONGUE IN 2002 IN FRANCET. Baldet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14SEROLOGICAL AND CLINICAL EVIDENCE FOR REACTIVITY OFARBOVIRAL TERATOGENIC SIMBU SERO-GROUP INFECTIONIN ISRAEL; 2001/2002 EPISODE(S)J. Brenner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15FIRST RESULTS OF BLUETONGUE SURVEILLANCE IN SWITZERLANDA.Y. Cagienard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16EPIDEMIOLOGICAL SURVEILLANCEOF BLUETONGUE IN SICILY (SW ITALY)S. Caracappa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17SEROPREVALENCE OF BLUETONGUE VIRUS IN CATTLE, SHEEPAND GOATS OF ALBANIAM. Di Ventura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18CURRENT SITUATION OF BLUETONGUE IN TURKEYA. Ertürk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19INCIDENCE AND ISOLATION OF BLUETONGUE VIRUS (BTV)IN CATTLE OF ITUZAINGO AND SANTO TOME DEPARTMENTSOF CORRIENTES PROVINCE, ARGENTINAI.A. Lager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20THE EPIZOOTIOLOGICAL APPEARANCE OF BLUETONGUEIN THE CENTRAL BALKANSB. Djuricic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21COMPLETE SEQUENCE ANALYSIS AND COMPARISONS OF GENOMESEGMENT 2 (ENCODING OUTER CAPSID PROTEIN VP2)FROM REPRESENTATIVE ISOLATES OF THE 24 BTV SEROTYPESS. Maan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

INDEX 2/8

OVERVIEW OF BLUETONGUE IN GREECEK. Nomikou . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23MOLECULAR EPIDEMIOLOGY OF BLUETONGUE VIRUSES FROM DISEASEOUTBREAKS IN THE MEDITERRANEAN BASINS. Maan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24RESULTS OF CURRENT SURVEILLANCE ON LIKELY BLUETONGUEVIRUS VECTORS OF THE GENUS CULICOIDES IN CATALONIA (SPAIN)V. Sarto i Monteys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

EPIDEMIOLOGY AND VECTORS

ORAL PRESENTATION

MODELLING THE DISTRIBUTION OF BLUETONGUE VECTORS:THE PREDICTED IMPACT OF GLOBAL WARMINGM. Baylis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29BLUETONGUE SURVEILLANCE METHODS IN THE UNITED STATESD. Dargatz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30BTV SURVEILLANCE IN AN EMERGING AREAA. Giovannini . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31THE BIOSYSTEMATICS OF CULICOIDES VECTOR COMPLEXES –UNFINISHED BUSINESSR. Meiswinkel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32INFECTION OF THE VECTORS AND BLUETONGUEEPIDEMIOLOGY IN EUROPEP.S. Mellor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33BTV SURVEILLANCE METHODS IN AN ENDEMIC AREA:AUSTRALIAL.F. Melville . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34ENVIRONMENTAL EFFECTS ON VECTOR COMPETENCE ANDVIROGENESIS OF BLUETONGUE VIRUS IN CULICOIDES:

INTERPRETING LABORATORY DATA IN A FIELD CONTEXTB.A. Mullens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35FAO’S ROLE IN BLUETONGUE DISEASEP. Roeder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36EPIDEMIOLOGY OF BTV AND EHDV IN WILDLIFE:SURVEILLANCE METHODSD.E. Stallknecht . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37CULICOIDES GENETICS: IMPLICATIONS FOR UNDERSTANDINGTHE GLOBAL EPIDEMIOLOGY OF BLUETONGUE VIRUS INFECTIONW.J. Tabachnick . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38ORAL SUSCEPTIBILITY OF SOUTH AFRICAN CULICOIDESSPECIES TO LIVE-ATTENUATED SEROTYPE-SPECIFIC VACCINESTRAINS OF BLUETONGUE VIRUS (BTV) – PRELIMINARY DATA G.J. Venter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39POSSIBLE OVERWINTERING MECHANISM OFBLUETONGUE VIRUS IN VECTORSD.M. White . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40

POSTERS

TEMPORAL ACIVITY OF BITING MIDGES (DIPTERA:CERATOPOGONIDAE) ON CATTLE NEAR DARWIN, NORTHERNTERRITORY, AUSTRALIAG.A. Bellis ..................................................................................41FACTORS AFFECTING THE SPREAD OF CULICOIDES BREVITARSISAT THE SOUTHERN LIMITS OF ITS DISTRIBUTION IN EASTERNAUSTRALIAA.L. Bishop .................................................................................42IMPROVING LIGHT TRAP EFFICIENCY FOR CULICOIDES SPPWITH LIGHT EMITTING DIODES (LEDs)A.L. Bishop .................................................................................43BLUETONGUE SURVEILLANCE IN CAMPANIA REGION, ITALY:GIS AS A TOOL TO CREATE RISK MAPSV. Caligiuri .................................................................................44

BLUETONGUE IN ITALY – part I P. Calistri ...................................................................................45USE OF A MONTECARLO SIMULATION MODEL FOR THE RE-PLANNINGOF BLUETONGUE SURVEILLANCE IN ITALYP. Calistri ...................................................................................46TOWARDS THE IDENTIFICATION OF POTENTIAL INFECTIOUS SITESFOR BLUETONGUE IN ITALY: A SPATIAL ANALYSIS APPROACHBASED ON THE DISTRIBUTION OF C. IMICOLAA. Conte ....................................................................................47PROTECTION OF CATTLE FROM CULICOIDES SPP. IN AUSTRALIABY SHELTER AND CHEMICAL TREATMENTSW.M. Doherty .............................................................................48EPIZOOTIOLOGIC CONTROL OF BT DISEASE IN BULGARIA IN 2002G. Georgiev ................................................................................49BLUETONGUE IN ITALY – part II A. Giovannini ..............................................................................50CULICOIDES (DIPTERA: CERATOPOGONIDAE) IN ALBANIA:RESULTS OF THE 2002 ENTOMOLOGICAL SURVEY FOR BLUETONGUEM. Goffredo ................................................................................51DISTRIBUTION AND ABUNDANCE OF CULICOIDES IMICOLA,C. OBSOLETUS COMPLEX AND C. PULICARIS COMPLEX(DIPTERA: CERATOPOGONIDAE) IN ITALY M. Goffredo ................................................................................52ENTOMOLOGICAL SURVEILLANCE OF BLUETONGUE IN ITALY:METHODS OF CAPTURE, CATCH ANALYSIS AND IDENTIFICATIONOF CULICOIDES BITING MIDGESM. Goffredo ................................................................................53ENTOMOLOGICAL SURVEILLANCE FOR BLUETONGUE IN MALTA:FIRST REPORT OF CULICOIDES IMICOLA KIEFFERM. Goffredo ................................................................................54LABORATORY SURVIVAL AND BLOOD FEEDING RESPONSE OF WILD-CAUGHT CULICOIDES OBSOLETUS COMPLEX (DIPTERA:CERATOPOGONIDAE) THROUGH NATURAL AND ARTIFICIAL MEMBRANESM. Goffredo ................................................................................55SPATIAL DISTRIBUTION OF BLUETONGUE IN CATTLEIN SOUTHERN CROATIA IN THE LAST QUARTER OF 2002 A. Labrovic .................................................................................56

SEROLOGIC EVIDENCE OF BLUETONGUE AND PRELIMINARYENTOMOLOGY STUDY IN SOUTHERN CROATIAE. Listes.....................................................................................57EVIDENCE THAT BLUETONGUE VIRUS SEROTYPE 2 (BTV-2) HASPERSISTED IN THE SOUTHEASTERN UNITED STATESFOR THE PAST 20 YEARSJ.O. Mecham...............................................................................58ADULT KEY TO THE OLD WORLD IMICOLA COMPLEX(CULICOIDES; SUBGENUS AVARITIA FOX, 1955)R. Meiswinkel..............................................................................59CHRISTOPHER COLUMBUS AND CULICOIDES: WAS C. JAMAICENSISEDWARDS, 1922 INTRODUCED BY SHIP TO THE MEDITERRANEAN500 YEARS AGO AND LATER RENAMED C. PAOLAE BOORMAN, 1996?R. Meiswinkel..............................................................................60MULTIPLE VECTORS AND THEIR DIFFERING ECOLOGIES:OBSERVATIONS ON TWO BLUETONGUE (BT) AND AFRICAN HORSESICKNESS (AHS) VECTOR CULICOIDES SPECIES IN SOUTH AFRICAR. Meiswinkel..............................................................................61THE IMICOLA AND ORIENTALIS COMPLEXES OF THE SUBGENUSAVARITIA FOX, 1955: THEIR REDEFINITION BASED ON ADULTMORPHOLOGY (CULICOIDES; DIPTERA, CERATOPOGONIDAE)R. Meiswinkel..............................................................................62SEASONAL ABUNDANCE OF CULICOIDES IMICOLA ANDC. OBSOLETUS GROUP IN THE BALEARIC ISLANDS (SPAIN)M.A. Miranda ..............................................................................63SEASONAL PREVALENCE OF BITING MIDGES, CULICOIDES SP.(DIPTERA: CERATOPOGONIDAE) OF DOMESTICATEDANIMALS OF MARATHWADA REGIONB.W. Narladkar ...........................................................................64S10 SEGMENT SEQUENCE ANALYSIS OF SOME GREEK BTV STRAINSS.V. Nikolakaki ...........................................................................65PHYLOGENETIC STATUS AND GENETIC STRUCTURE OF THE ARBOVIRUSVECTOR CULICOIDES IMICOLA KIEFFER (DIPTERA:CERATOPOGONIDAE) IN THE MEDITERRANEAN BASIND.V. Nolan..................................................................................66BLUETONGUE SURVEILLANCE SYSTEM IN ITALYA. Giovannini..............................................................................67

INDEX 3/8

CULICOIDES IMICOLA IN GREECEJ. M. Patakakis............................................................................68MODELLING THE DISTRIBUTIONS OF OUTBREAKS AND CULICOIDESVECTORS IN SICILY: TOWARDS PREDICTIVE RISK MAPS FOR ITALYA. Torina....................................................................................69WHAT CLIMATIC FACTORS DETERMINE WHEN EPIZOOTICS OCCUR INTHE MEDITERRANEAN? PREDICTION OF DISEASE RISK THROUGH TIMEBY CLIMATE-DRIVEN MODELS OF THE TEMPORAL DISTRIBUTION OFOUTBREAKS IN ISRAELY. Braverman .............................................................................70EMERGENCE OF BLUETONGUE DISEASE IN THE MEDITERRANEANBASIN: MODELLING LOCATIONS AT RISK FOR POTENTIAL VECTORS(CULICOIDES SPP.) USING SATELLITE IMAGERYF. Roger.....................................................................................71FIELD DISINFESTATION TESTS AGAINST CULICOIDESIN NORTH-WEST SARDINIA G. Satta .....................................................................................72ISOLATIONS OF BLUETONGUE VIRUS (BTV) FROM FIELDPOPULATIONS OF THE OBSOLETUS COMPLEX(CULICOIDES, DIPTERA, CERATOPOGONIDAE) IN ITALYG. Savini ....................................................................................73VP-2 SEQUENCE ANALYSIS OF SOME ISOLATESOF BLUETONGUE VIRUS (BTV) RECOVERED IN THE MEDITERRANEANBASIN DURING THE 1998-2002 OUTBREAKG. Savini ....................................................................................74ASSOCIATION BETWEEN 2001-2003 BLUE TONGUE OUTBREAKSIN LAZIO AND TOSCANA (CENTRAL ITALY) AND THEDISTRIBUTIONAND ABUNDANCE OF VECTORS CULICOIDES IMICOLAAND C. OBSOLETUS G. Scavia ...................................................................................75CULICOIDES SPECIES ASSOCIATED WITH LIVESTOCKIN TAMIL NADU STATE OF INDIAK.G. Udupa.................................................................................76MOLECULAR INVESTIGATIONS OF VIRUS/VECTOR INTERACTIONSW.C. Wilson ................................................................................77

BLUETONGUE VIRUS AND BLUETONGUE DISEASE

ORAL PRESENTATION

GENETIC DIVERSIFICATION OF FIELD STRAINS OF BTVK.R. Bonneau ....................................................................81A COMPARISON OF DIFFERENT ORBIVIRUS PROTEINSTHAT COULD AFFECT VIRULENCE AND PATHOGENESISH. Huismans .....................................................................82A COMPARISON OF LABORATORY ADAPTEDAND ‘WILD’ STRAINS OF BLUETONGUE VIRUS. IS THERE ANYDIFFERENCE AND DOES IT MATTER?P.D. Kirkland .....................................................................83THE PATHOGENESIS OF BLUETONGUE VIRUS INFECTIONOF RUMINANTS AND DURATION OF VIREMIAN.J. MacLachlan .................................................................84MOLECULAR AND STRUCTURAL BIOLOGY, AND VIRUSREPLICATIONP.P.C. Mertens...................................................................85MOLECULAR EPIDEMIOLOGY OF BLUETONGUE VIRUSESIN AUSTRALIAL.I. Pritchard .....................................................................86A POTENTIAL OVERWINTERING MECHANISM FOR THE VIRUS -RECENT FINDINGSH.H. Takamatsu.................................................................87

POSTERS

GLOBAL ISOLATES OF BLUETONGUE VIRUS SEGGREGATEINTO REGION-SPECIFIC TOPOTYPES BASED ON PHYLOGENETICANALYSES OF THEIR NS3 GENESU.B.R. Balasuriya ........................................................................88

INDEX 4/8

ISOLATION AND MOLECULAR CHARACTERIZATIONOF BLUETONGUE VIRUS FROM SHEEPS.M. Byregowda ..........................................................................89CHARACTERIZATION AND SEROPREVALENCE OF BLUETONGUE VIRUSIN THE MAHARASHTRA STATE OF INDIAV.V. Deshmukh...........................................................................90REPLICATION OF EPIZOOTIC HEMORRHAGICDISEASE VIRUS IN DH 82 CELLSE.W. Howerth .............................................................................91EXCRETION OF BLUETONGUE VIRUS IN CATTLE SEMEN - A FEATURE OF LABORATORY- ADAPTED VIRUSP.D. Kirkland ..............................................................................92INVESTIGATION OF BLUE TONGUE DISEASE IN TAMIL NADU, INDIAA. Koteeswaran...........................................................................93BLUETONGUE VIRUS DOES NOT PERSISTIN NATURALLY INFECTED CATTLEL.F. Melville ................................................................................94PHYLOGENETIC ANALYSIS OF BLUETONGUE VIRUS GENOME SEGMENT6 (ENCODING VP5) FROM DIFFERENT SEROTYPESK.P. Singh ..................................................................................95POSSIBILITY OF BLUETONGUE VIRUS IN BOVINE SKIND.M. White .................................................................................96THE CHARACTERISATION AND MONITORING OF NEUTRALIZATION-RESISTANT VP2 PHENOTYPES IN BTV-1 ISOLATES FROM NORTHERNAUSTRALIA COLLECTED OVER A TWENTY YEAR PERIODJ.R. White ..................................................................................97

DIAGNOSTICS

ORAL PRESENTATION

“VIRUS ISOLATION AND IDENTIFICATION” OR “VIRUSIDENTIFICATION AND ISOLATION”?B.T. Eaton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101APPLICATION OF LABORATORY DIAGNOSTIC TESTSC. Hamblin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102BLUETONGUE DIAGNOSIS BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTIONS. Zientara. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

POSTERS

THE DIFFERENTIAL DIAGNOSIS OF BLUETONGUE VIRUS:A PCR APPROACHS. Anthony ...............................................................................104APPLICATION OF MOLECULAR BIOLOGICAL TECHNIQUES FOR RAPIDDETECTION AND DIFFERENTIATION OF BLUETONGUE VIRUSAND PALYAM ORBIVIRUSES SEROGROUP I.E. Aradaib ..............................................................................105APPLICATION OF VARIOUS DIAGNOSTIC PROCEDURES TOEPIDEMIOLOGICAL SITUATIONS ENCOUNTERED DURING ARBOVIRALINFECTIONS USING THE SIMBU SERO-GROUP AS EXAMPLEJ. Brenner ................................................................................106DETECTION OF BLUETONGUE VIRUS BY DIRECT RT-PCRAND BY NON-RADIO ACTIVE NUCLEIC ACID PROBES S.M. Byregowda ........................................................................107BLUE TONGUE: AN OVERVIEW ON RECENT TRENDS IN DIAGNOSTICSH. Dadhich ...............................................................................108

INDEX 5/8

EVALUATION OF NEW PRIMERS FOR IDENTIFICATIONOF BLUETONGUE VIRUS SEROTYPE 1 BY NESTED RT-PCRS. Dahiya .................................................................................109THE MOLECULAR DIFFERENTIATION OF FIELDAND VACCINE STRAINS OF BLUETONGUE VIRUSSEROTYPE 2 (BTV-2), USING REAL-TIME PCRBY FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET)HYBRIDISATION PROBESG. Orrù ....................................................................................110THE FIRST BLUETONGUE VIRUS ISOLATION IN YUGOSLAVIAB. Djuricic ................................................................................111BLUETONGUE LABORATORY DIAGNOSIS: A RING TEST TO EVALUATESEROLOGICAL RESULTS USING A COMPETITIVE ELISA KITR. Lelli.................................................................................................112RT-PCR BASED ASSAYS AND SEQUENCING FOR TYPINGEUROPEAN STRAINS OF BLUETONGUE VIRUS AND DIFFERENTIALDIAGNOSIS OF FIELD AND VACCINE STRAINSS. Maan ...................................................................................113COMPARATIVE EVALUATION OF SEROGROUP SPECIFIC VP3, NS1 ANDVP7 GENE BASED NESTED RT-PCR FOR THE DETECTION OFBLUETONGUE VIRUSESRamesha..................................................................................114THE FREQUENCY OF THE CROSS REACTION BETWEENTHE IBARAKI VIRUS AND BLUE TONGUE VIRUS INFECTED SERUMIN AGAR GEL IMMUNO-DIFFUSSION TESTS. Shimizu ................................................................................115BLUETONGUE IN BOSNIA: COMPARISON OF cELISAAND STANDARD AGID TESTSL. Velic.....................................................................................116FIELD-DEPLOYABLE REAL-TIME PCR DETECTION OF BLUETONGUEAND EPIZOOTIC HEMORRHAGIC DISEASE VIRAL RNAW.C. Wilson ..............................................................................117

BLUETONGUE CONTROL USING VACCINES

ORAL PRESENTATION

THE USE OF VACCINATION IN THE CONTROL OF BLUETONGUEIN SOUTHERN AFRICAB. Dungu ........................................................................121AN EXPERIENCE FROM THE MEDITERRANEAN ISLANDS INEUROPEG. Gerbier.......................................................................122BLUETONGUE VACCINATION IN EUROPE: THE ITALIANEXPERIENCEC. Patta ..........................................................................123GENETICALLY ENGINEERED STRUCTURE-BASED VACCINEP. Roy ............................................................................124

POSTERS

THE EFFECT OF VACCINATION AGAINST BLUETONGUEON MILK PRODUCTION AND QUALITY IN CATTLE VACCINATEDWITH LIVE-ATTENUATED MONOVALENT TYPE 2 VACCINEA. Giovannini ............................................................................125EFFECTS OF BLUETONGUE BIVALENT BTV2 - BTV9 VACCINEON REPRODUCTIVE PERFORMANCE IN CATTLE: A CASE STUDYIN CALABRIA REGION (ITALY)G. Lucifora................................................................................126FIELD VACCINATION WITH BIVALENT MODIFIED-LIVE VACCINEAGAINST BLUETONGUE VIRUS SEROTYPES 2 AND 9 IN CATTLE: EFFECT ON MILK PRODUCTIONF. Monaco.................................................................................127

INDEX 6/8

VACCINATION WITH MONOVALENT MODIFIED-LIVE VACCINEAGAINST BLUETONGUE VIRUS SEROTYPES 2 IN CATTLE: INNOCUITY,IMMUNGENICITY AND EFFECT ON PREGNANCYF. Monaco.................................................................................128VIROLOGICAL AND SEROLOGICAL RESPONSES IN CATTLE FOLLOWINGFIELD VACCINATION WITH BIVALENT MODIFIED-LIVE VACCINEAGAINST BLUETONGUE VIRUS SEROTYPES 2 AND 9F. Monaco.................................................................................129DEVELOPMENT AND EVALUATION OF INACTIVATED VACCINESFOR BLUETONGUE IN INDIAM.A. Ramakrishnan....................................................................130BLUE TONGUE CONTROL USING VACCINES:EMILIA ROMAGNA EXPERIENCEA. Santi....................................................................................131EFFICACY AND SAFETY STUDIES ON A LIVE AND INACTIVATEDVACCINE AGAINST BLUETONGUE VIRUS SEROTYPE 2 (BTV2) B. Di Emidio..............................................................................132FIELD VACCINATION WITH BIVALENT MODIFIED-LIVE VACCINEAGAINST BLUETONGUE VIRUS SEROTYPES 2 AND 9 IN SHEEP: EFFECT ON MILK PRODUCTIONG. Savini ..................................................................................133LIVE MODIFIED MONOVALENT VACCINE AGAINST BLUETONGUEVIRUS (BTV) SEROTYPE 2: IMMUNITY STUDIES ON COWS G. Savini ..................................................................................134NEUTRALISING ANTIBODY RESPONSE IN CATTLEAFTER VACCINATION WITH MONOVALENT LIVE-MODIFIED VACCINEAGAINST BLUETONGUE VIRUS (BTV) SEROTYPE 2 G. Savini ..................................................................................135SEROLOGICAL RESPONSES IN CATTLE AND SHEEP AFTER INFECTIONOR VACCINATION WITH BLUETONGUE VIRUSG. Savini ..................................................................................136VIROLOGICAL AND SEROLOGICAL RESPONSES IN SHEEP FOLLOWINGFIELD VACCINATION WITH BIVALENT LIVE-MODIFIED VACCINEAGAINST BLUETONGUE VIRUS SEROTYPES 2 AND 9 G. Savini ..................................................................................137

SEROLOGICAL AND PROTECTION RESULTS OBSERVEDIN A VACCINATION-CHALLENGE TEST USING LIVE AND INACTIVATEDVACCINES AGAINST BTV2 IN SHEEPS. Boutrand ..............................................................................138EFFECT OF LEVAMISOLE ADMINISTRATIONON BLUETONGUE VACCINATION IN SHEEPC. Stelletta ...............................................................................139

CONTROL AND TRADE ISSUES

ORAL PRESENTATION

BLUETONGUE VIRUS AND TRADE ISSUES -THE NORTH AMERICAN PERSPECTIVER. DeHaven .....................................................................143EU POLICY FOR THE CONTROL AND ERADICATION OFBLUETONGUEJ. Février ........................................................................144SOUTH AMERICA PERSPECTIVEI.A. Lager .......................................................................145OIE INTERNATIONAL STANDARDS FOR BLUETONGUEA. Schudel ......................................................................146

POSTERS

SUSCEPTIBILITY AND REPELLENCY OF CULEX PIPIENSAND FIELD POPULATION OF CULICOIDES IMICOLATO THE PYRETHROID LAMBDA-CYHALOTHRINY. Braverman ............................................................................147

INDEX 7/8

SANITARY MANAGEMENT OF LARGE ANIMAL POPULATION MOVEMENTIN ITALY: THE CASE OF TRANSHUMANCE AND BLUETONGUE RISKD. Nannini ................................................................................148DATA MANAGEMENT AND ANALYSIS SYSTEMSFOR BLUETONGUE VIRUS ZONING IN AUSTRALIAA.R. Cameron ...........................................................................149THE IMPACT OF CURRENT AND PROPOSED CHANGES TO OIE GENERALGUIDELINES FOR SURVEILLANCE ON BLUETONGUE SURVEILLANCEA.R. Cameron ...........................................................................150RISK ANALYSIS ON THE INTRODUCTION INTO FREE TERRITORIESOF VACCINATED ANIMALS FROM RESTRICTED ZONESA. Giovannini ............................................................................151

IMPLEMENTATION OF A NEW CONTINGENCY PLANFOR BLUE TONGUE DISEASE IN ITALYG. Filipponi ...............................................................................152INTERATED MANAGEMENT OF CULICOIDES SP(DIPTERA: CERATOPOGONIDAE) OF DOMESTICATED ANIMALS OFMARATHWADA REGIONB.W. Narladkar..........................................................................153

INDEX 8/8

AS. Anthony, 104I.E. Aradaib, 105

BU.B.R. Balasuriya, 88T. Baldet, 14M. Baylis, 29G.A. Bellis, 41A.L. Bishop, 42, 43K.R. Bonneau, 81Y. Braverman, 147J. Brenner, 15, 106S.M. Byregowda, 89, 107

CA.Y. Cagienard, 16V. Caligiuri, 44P. Calistri, 45, 46, 148A.R. Cameron, 149, 150A.Conte, 47

DH. Dadhich, 108S. Dahiya, 109D. Dargatz, 30R. DeHaven, 143P. De Santis, 110V.V. Deshmukh, 90C. Di Bella, 17C.E. Di Francesco, 151M. Di Ventura, 18B. Djuricic, 111W.M. Doherty, 48B. Dungu, 121

EB.T. Eaton, 101A. Ertürk, 19

FJ. Février, 144G. Filipponi, 152

GG. Georgiev, 49G. Gerbier, 122G.H. Gerdes, 3A. Giovannini, 31, 50, 125M. Goffredo, 51, 52, 53,

54, 55C. Gómez-Tejedor, 4Y. Goto, 5

HC. Hamblin, 102E.W. Howerth, 91H. Huismans, 82

KP.D. Kirkland, 6, 83, 92A. Koteeswaran, 93

LA. Labrovic, 56I.A. Lager, 7, 20, 145

D. Lausevic, 21R. Lelli, 112E. Listes, 57G. Lucifora, 126

MS. Maan, 22, 113N.J. MacLachlan, 84J.O. Mecham, 58R. Meiswinkel, 32, 59, 60,

61, 62P.S. Mellor, 33L.F. Melville, 34, 94P.P.C. Mertens, 85M.A. Miranda, 63B.A. Mullens, 35

NB. W. Narladkar, 64, 153Z. Nianzu, 8S.V. Nikolakaki, 65D.V. Nolan, 66K. Nomikou, 23

INDEX OF PRESENTING AUTHORS 1/2

OE.N. Ostlund, 9

PC. Paladini, 67D.E. Panagiotatos, 10J.M. Patakakis, 68C. Patta, 123L.I. Pritchard, 86B.V. Purse, 69, 70

RM.A. Ramakrishnan, 130Ramesha, 114P. Roeder, 36F. Roger, 71P. Roy, 124

SA.R. Samuel, 24A. Santi, 131V. Sarto i Monteys, 25

G. Satta, 72G. Savini, 73, 74, 127,

128, 129, 132, 133, 134,135, 136, 137

P. Scaramozzino, 75A. Schudel, 146C. Schumacher, 138S. Shimizu, 115K.P. Singh, 95D. Sreenivasulu, 11D.E. Stallknecht, 37C. Stelletta, 139

TW.J. Tabachnick, 38H.H. Takamatsu, 87

UK.G. Udupa, 76

VR.Velic, 116G.J. Venter, 39

WT.E. Walton, 12D.M. White, 40, 96J.R. White, 97W.C. Wilson, 77, 117

YH. Yadin, 13

ZS. Zientara, 103

INDEX OF PRESENTING AUTHORS 2/2

THE CURRENT GLOBALSITUATION

AND REGIONAL OVERVIEWSOF VIRUSES, VECTORS,

SURVEILLANCEAND SPECIFIC FEATURES

GLOBALSITUATION

EPIDEMIOLOGYAND VECTORS

BT VIRUSAND BT DISEASE DIAGNOSTICS

BT CONTROLUSING VACCINES

CONTROL ANDTRADE ISSUES

ABSTRACT BOOK

BLUETONGUE – A SOUTH AFRICAN PERSPECTIVEG.H. GerdesDepartment of Virology • Onderstepoort, 0110 • South Africa • Tel: +27 12 529 9114 • [email protected]

3

Bluetongue (BT) is a notifiable disease in South Africa in terms of the Animal DiseasesAct of 1984. Twenty-one of the 24 recognized serotypes of the virus occur here whereit has circulated in game since antiquity being by definition a disease of domestic andwild ruminants. At present serotypes 17, 20 and 21 are exotic to South Africa.Historically a French naturalist wandering through South Africa between 1781 – 84came across “Tong Sikte” and the disease was recognised in merino sheep introducedinto the Cape Colony in the late 18th C.Because a progressive Agricultural Policy was seen to be essential to the survival ofcivilization in Africa South and very little animal husbandry was possible particularlyinland, veterinary surgeons and scientists of the time worked aggressively on BTamong other African diseases and a vaccine for BT was produced through attenuationby passage through sheep using serotype 4. This monovalent vaccine was in use for40 yrs for the so-called Malarial Cattarrhal fever of sheep.BT was confined to Africa till 1943, when it spread to Cyprus, although the date is inquestion as it may have been as early as 1925. The virus was rapidly disseminatedfurther and to other continents – however limited by the distribution of its vectors.Work on the virus of BT progressed rapidly through egg propagation by yolksac andintra vascular routes to adaptation to growth in mice for the production of antigens tofinally growth in tissue culture. The plurality of the BT virus serotypes was onlyrecognized later with 12 serotypes dating to pre 1960, an additional 4 serotypes beingconfirmed between 1960-1970 and the remaining 8 serotypes recognized post 1970.Leaving history to history and looking at the present day situation – BT does not enjoy thesame attention now as then maybe because of the endemic nature of its presence in SouthAfrica. There is a “soup of viruses” out there but no great economic impact is felt becauseindigenous breeds of sheep and of course goats do not display obvious disease. When we

look for BT antibody negative sheep for whatever purpose – we need to go to a high, coldplace – and then we still have trouble particularly in sourcing negative merino sheep. Thisis not necessarily because people vaccinate regularly, but because sheep are exposed tovirus regularly.Between 1997 – 2000, we found ourselves exporting a lot of game, which naturally, ifthey were ruminants, had to be tested for BT. 14 spp out of a total of 1,200 game wereexported of which about 1/3 were seropositive. Giraffe were popular game animals butonly 23% positive and of the antelope – springbok, blesbok and gemsbok, they wereall 40% odd positive.If we summarise BT diagnostics from 1983 – 2003 we are aware of the followingstatistics. The four most prevalent serotypes were 1, 3, 4 and 2 and then the 3 leastoften seen serotypes were 10, 13, and 15. Four were not found at all during the periodunder review namely 18, 19 22 and 23. This translates roughly into groupingspreviously made of serotypes with high or low epidemic potential and those present atlow levels every season.We have already mentioned the endemic nature of the disease and in fact the virus wasisolated in all the nine Provinces of South Africa to a greater or lesser extent during theperiod under review.In conclusion, if we turn to vaccination and disease reporting, the former will be morecomprehensively discussed in a separate forum but the latter is worth a mention. BTis sporadically reported and figures are unreliable. They vary from 23 outbreaksrecorded for one year to as many as 101 recorded for yet another year.In summary, while BT may well have originated in Africa, as a disease it does notoccupy our collective consciousness to any great degree today.

The current global situation and regional overviews of viruses,vectors, surveillance and specific features

G.H. Gerdes

GLOBALSITUATION


BT VIRUSAND BT DISEASE

DIAGNOSTICS BT CONTROLUSING VACCINES



ABSTRACT BOOK

THE CURRENT SITUATION IN MEDITERRANEAN EUROPEC. Gómez-TejedorLaboratorio Central de Veterinaria • Ctra. de Algete, Km 8 • 28110 ALGETE (MADRID) • Tel: 916290300; Fax: 916290598; E-mail: [email protected]

4

BTV distribution, related to the vector distribution, has taken place untilrecently between latitudes 40º North and 35º South. Within those limits thedisease has been present in parts of America, Asia, Africa and Australia.Even though Europe has suffered several BTV epizootics, the disease hadnot been established in its territory. The situation is changing. BTV has emerged in Mediterranean Europeancountries where it had never been reported, Italy, France, as well as incountries where the virus had sporadic presence before, i.e. Spain (late50’s), Greece and Turkey (late 70’s). It is relevant to underline that someof the recently European affected areas are not in the classical latitudesfor BTV. In recent years, Europe has been affected by BTV epizootics that, evencoinciding at the same period of time, had two different origins, easternand southern boundaries of Europe.

Eastern origin: at the end of 1998, BTV was reported in the Greek Islandsand in summer 1999 in Turkey. In 2001 the disease advanced west andnorthward reaching central and northwest of mainland Greece, and non-mediterranean european countries. In this epizootics, serotypes 4, 9 and16 have been isolated.Southern origin: at the end of 1999 BTV was confirmed in Tunisia andspread to Algeria in the border area. In summer 2000, the virus reachedthe Italian island of Sardinia and spread to Sicily and Calabria (the closestItalian mainland area to Sicily). In october 2000 BTV was reported in theFrench island of Corsica and in the Spanish Balear island of Menorca fromwhere it spread to another Balear island, Mallorca. In 2001 BTV spreadSouthwest mainland Italy.In the epizootics related to the NorthAfrican origin, serotype 2 has beenisolated.

C. Gómez-Tejedor

ABSTRACT BOOK

EPIDEMIOLOGICAL OBSERVATIONSON BLUETONGUE IN SHEEP AND CATTLE IN JAPANY. Goto and O. Yamaguchi Kannondai, Tsukuba, Ibaraki, Japan • National Institute of Animal Health • Department of Infectious Diseases • E-mail: [email protected]

5

In Japan, the first described cases of Bluetongue disease were JapaneseBlack cattle in three Northern Kanto prefectures and Suffolk sheep in oneprefecture. Affected cattle and sheep show clinical signs of disease,including fibre response, and in severe cases, deglutitive difficulty,ulceration of the oral mucosa and laminitis. PCR was used to detect theBluetongue virus (BTV) in blood samples from affected cattle, and the BTviral RNA was detected in blood from affected sheep. Since then, therehave not been any reported cases of Bluetongue disease in Japan, but theresults of a national arbovirus monitoring program in cattle typicallyreveal several seroconverted cases annually. Furthermore, seven yearsafter the first cases were detected 1994, in September of 2001, there wasan outbreak of Bluetongue disease at a sheep farm in Northern Kanto, andBTV was isolated. Moreover, there have not been any reports of

Bluetongue disease in Kyushu and Okinawa, but some cattle in theseregions have been reported to have BTV antibodies, and BTV has beenisolated from some sentinel cattle blood samples and Culicoidesbrevitarsis. Therefore, while targeting the RNA segment 3 that encodesthe serogroup specific protein, RT-PCR was performed on BTV isolates andPCR products of positive blood samples from affected cattle and sheepwas produced. The results showed that the degree of homology was lowbetween the Japanese isolates and three Australian strains, and that theJapanese isolates could be roughly divided into two lineages. Thesefindings suggest that the Japanese isolates are also undergoing geneticvariation. It will therefore be important to continue conducting molecularepidemiological studies on BTV in Japan.


Y. Goto

GLOBALSITUATION





GLOBALSITUATION






ABSTRACT BOOK

BLUETONGUE VIRUSES, VECTORS AND SURVEILLANCE IN AUSTRALIA:THE CURRENT SITUATION AND UNIQUE FEATURESP.D. Kirkland Elizabeth Macarthur Agricultural Institute • PMB 8, Camden NSW, 2570, Australia • Ph: 61-2-4640 6331 Fax: 61-2-4640 6429. Email:[email protected]

6

While there are dramatic contrasts between the bluetongue (BLU) situation in Europeand Australia, there are also similarities. The purpose of this presentation is to brieflyreview the bluetongue “journey” in Australia, describing how the country hasresponded to the discovery of BLU viruses and to outline the current situation.In 1977 the Australian animal health authorities were advised that a virus isolated 2years previously had just been identified as a BLU virus. As a country with apopulation of more than 140 million sheep, Australia had exotic disease plans torespond to an incursion of BLU virus. Consequently, the livestock industries struggledwith this news, given that, although the virus did not produce disease, it halted exportof animals and animal products. During the following 15 years, 8 serotypes wereidentified. Extensive research was conducted into the distribution and pathogenicity ofBLU viruses and the distribution and competence of vectors. Research projects havefocussed on the development and evaluation of new rapid diagnostic techniques,evaluation of vaccines and studies of the basic biology of BLU virus infections in bothsheep and cattle, including teratogenicity and the excretion of virus in semen.In the last decade, research efforts have been directed at reducing the impact of BLUviruses on the export of livestock, semen and embryos. In 1993 the NationalArbovirus Monitoring Program (NAMP) was formally established as a co-operativeinitiative between the livestock industries, national and state governments. The mainemphasis of NAMP has been on the definition of the distribution of BLU viruses andtheir vectors, together with monitoring annual fluctuations of viruses and vectors.These objectives have been achieved by sampling of sentinel cattle at key locationsaround Australia and by light trap collection of insects at these sentinel sites. At themost northerly site, where BLU virus was first isolated, more intensive monitoring isconducted to accumulate BLU viruses for genetic studies. Virus isolation is also

performed at other sites where BLU seroconversions are detected. As well as type-specific serology at these locations, BLU viruses are subjected to topotyping to identifythe geographical origin of the viruses. Recently, research projects have also beendirected towards detailed studies of vector ecology, modelling of virus and vectordistribution, vector reduction strategies and improved vector collection techniques. Despite the presence of some pathogenic viruses, Australia remains free ofbluetongue disease. However, the economic impact is considerable due to disruptionto trade. Australia has a number of unique features that contrast with other countriesand continents, and influences the surveillance methods used. It is a large,geologically stable and ancient landmass. The landform is relatively flat and uniform,with each physical environment gradually merging into the next. Consequently, thereis a continuous distribution of vectors and virused through northern and easternAustralia towards a southern limit imposed by climate. Cattle are dispersedthroughout the northern and eastern regions of higher rainfall and temperature, andsheep are found in the cooler drier areas. Extensive grazing predominates and animalsare not housed. Vaccination for bluetongue has never been practiced, providing noopportunity for changed virus characteristics though genetic re-assortment betweenvaccine and wild types. Collectively these factors, when combined with virus andvector monitoring data that has been gathered over more than 25 years, have allowedthe accurate delineation of bluetongue free zones and zones of possible bluetonguetransmission in accord with OIE guidelines. Other presentations in this symposium will describe research on typing of viruses,improved vector collection techniques, data management, mapping techniques andzoning.

P.D. Kirkland

ABSTRACT BOOK

REGIONAL OVERVIEWS OF VIRUSES, VECTORS, SURVEILLANCEAND UNIQUE FEATURES: SOUTH AMERICA I.A. LagerInstituto de Virologia, INTA-Castelar, CP 1712, Hurlingham, Buenos Aires, ARGENTINA • Telephone: 54-11-4621-1447 Fax: 54-11-4621-1743 e-mail:[email protected]

7

The global Bluetongue Virus (BTV) distribution historically has beenshown to be between latitudes of approximately 40°N and 35°S (15).Eleven of the thirteen South America (SA) countries are in that area.Only Chile and Argentina have part of their territory south of theparallel 35º. However Argentina found , by a serological survey, thatthe infected region of its territory, only involved Misiones Provinceand two Departments of Corrientes Province (parallel 29 south).The first published report of BTV infection in SA was done by Brazil in1979. They detected serological evidence of infection in livestock in SaoPaulo and Rio de Janeiro States. Since then several serological surveyshave determined that the infection is widespread in SA. The susceptible hosts that had antibodies included sheep, bovine, goatsand water buffaloes with a big range of seroprevalence percentages.Other species have been analysed for the presence of antibodies aspossible hosts or reservoirs. In Argentina, llamas, guanacos, vicuñas andPampean deer were negative but Peru found that the alpaca can beinfected.Only four outbreaks of bluetongue disease have been reported so far inSA and they all occurred in the last three years, in Párana State, Brazil.The first was in June 2001, with 9 cases that affected seep and goats. Thesecond was in February 2002 in goats and the last two in March 2002affecting sheep and goats.

The first BTV isolation from SA naturally infected animals was BTVserotype 4 from zebu cattle that were imported from Brazil to the USA.Brazil and Argentina are the only SA countries where BTV has beenisolated. The Brazilian isolation was made in Panaftosa from blood andtissue samples of clinically affected sheep and goats of the June 2001outbreak, confirmed by RT-PCR and typed by VN as type 12. In ArgentinaBTV was isolated at INTA-Castelar from blood of sentinel bovines withoutclinical signs and the serotyping was performed by the IAH, Pirbright, UKas type 4.By serology, the serotypes detected in SA are : 4, 6, 14, 17 and 19 inBrazil; 12, 14 and 17 in Colombia; 14 and 17 in Guyana and 6, 14 and 17in Suriname. These results should be considered as indicative because ofthe cross reactions among virus serotypes. Culicoides insignis is the predominant vector detected in the area.However, as BTV was isolated from C. pusillus in Central America and theCaribbean and this species is also present in SA, it is possible that C.pusillus could be also a BTV vector in the area. The virus has not yet beenisolated from the vector in the region.The pattern of BTV transmission in SA, seems to be very similar to otherendemic areas. The highest incidence of infection occurs when the climaticconditions favour the breeding of competent vectors i.e. in the latesummer and autumn (February to May).


I.A. Lager

GLOBALSITUATION





GLOBALSITUATION






ABSTRACT BOOK

STUDIES ON BLUETONGUE DISEASE IN CHINAZ. Nianzu, L. Zhihua, Z. Fuqiang, Z. Jianbo and L. HuachunLaboratory of Tropical and Subtropical Animal Virology, Ministry of Agriculture, Jindian, Kunming 650224, PR China • E-mail: [email protected]

8

Bluetongue is an important infectious, noncontagious, insect-borne viraldisease of ruminant and belonged to list A disease according to OIEanimal health regulation. Since first discovery and diagnosis of thisdisease in Shizong county of Yunnan Province in 1979, we havedeveloped the systematic studies of epidemiology, experimentalepidemiology, aetiology, pathology, molecular characteristics of viralnucleic acid, diagnostic techniques, virus identification (grouping andtyping) methods, vaccines and immunization mechanism of bluetonguein China. Viruses have been isolated from seven provinces and theirmajor serotypes have been identified. Epidemiology surveys have beencarried out in sixteen provinces and a distinct law of bluetongueepidemiology has been found in China. Sentinel herds have beenestablished to monitor bluetongue regularly from 1995 to 1998.Experimental epidemiology study revealed the laws of regionaldistribution of serotypes and dynamic spread of bluetongue in differentialhabitat of distinct nature condition, ecological environment and climatein China. Researches on aetiology and pathology have beenaccomplished. The technical system of diagnosis for bluetongue havebeen established, included agar gel immunodiffusion (AGID), the indirect

ELISA, the competitive ELISA (cELISA), the virus neutralisation (VN) test(for bluetongue antibodies detection), and the serum neutralisation (SN),the immunofluorescence (IF) and immunoperoxidase (IP) methods, theantigen capture ELISA (AC-ELISA), the virus inoculation methods, theimproved procedure for virus isolation, nucleic acid electrophoresisanalysis, nucleic acid probe technique, polymerase chain reactionmethod( for bluetongue antigen or viral nucleic acid detection andidentification). Attenuated vaccine and killed vaccine of BTV serotypes 1and 16 have been developed and new immunisation mechanism has beenfound. The L3, S7, S10 and partial of the L2 gene segment of Chineseprototype strains of BTV serotypes 1, 2, 3, 4, 12, 15 and 16 have beensequenced and compared to the same strains of prototype and fieldstrains of BTV from the US and Australia. Phylogenetic analyses indicatethe genetic relations of these viruses and the correspondence relationsbetween viral nucleic acid sequences to serotypes or geographic origin.“The National Standard Technical Regulations of Bluetongue Diagnosis,PR China” has also been enacted. The remarkable social benefits andeconomic benefits have been obtained since application anddissemination of these achievements in China.

Z. Nianzu

ABSTRACT BOOK

REGIONAL OVERVIEW OF VIRUSES, VECTORS, SURVEILLANCEAND UNIQUE FEATURES: NORTH AMERICAE.N. Ostlund, K.M. Moser, D.J. Johnson, J.E. Pearson and B.J. Schmitt Diagnostic Virology Laboratory, National Veterinary Services Laboratories. P.O. Box 844 Ames, IA 50010 USA. Tel: 515- 663-7551; Fax: 515-663-7348;e-mail: [email protected]

9

Bluetongue virus (BTV) distribution in North America is limited by therange of the vector, Culicoides spp. With the exception of sporadicoccurrences in the Okanagan Valley in British Columbia, Canada isconsidered free of BTV. Regional differences exist in the United States.The northeastern U.S. states are free of bluetongue. BTV activity occursmost of the year in the southern region of the United States and inMexico. In more temperate regions of the United States, evidence of BTVinfection is seasonal. Peak transmission in the temperate zones occurs inlate summer and early autumn. In the United States, the seroprevalence of BTV antibodies in slaughtercattle from selected northeastern and northern states is examinedregularly. From 1992 through 2002, eight serosurveys were conducted.Samples collected from cattle slaughtered in early winter were examinedfor BTV antibodies. Approximately 600 samples from each of 10geographic regions were tested in each survey. Results of testing have

verified negligible bluetongue antibody prevalence in cattle from thenortheastern and northern states. Antibody against BTV is often detectedin cattle originating from states bordering the BTV free regions of theUnited States, but there is year to year variability in BTV antibody statusof these states. Bluetongue disease in sheep or cattle is infrequently reported in theUnited States. In addition to diagnostic testing, examination for evidenceof BTV infection is often required to certify U.S. animals or animalproducts for international export. BTV serotypes previously identified inNorth America are serotypes 2, 10, 11, 13, and 17. Isolates of BTVserotypes 10, 11, 13 and 17 are encountered nearly every year, althoughthe total number of isolates per year is small. Isolation of BTV serotype 2in the United States is quite rare. However, a BTV serotype 2 isolate wasobtained in 1999 from sheep with clinical disease residing in Florida. Allother strains of BTV are exotic to the United States.


E.N. Ostlund

GLOBALSITUATION





GLOBALSITUATION






ABSTRACT BOOK

REGIONAL OVERVIEWS OF VIRUSES, VECTORS, SURVEILLANCEAND UNIQUE FEATURES: EASTERN EUROPE D.E. PanagiotatosMinistry of Agriculture, Athens Center of Veterinary Institutes, Department of Epidemiology and Bio-statistics. 25 Neapoleos Str., 153 10 Ag.Paraskevi. Athens,GREECE. E-mail: [email protected]

10

After an interminable period of historical freedom, presumed freedom or– at worst – minor, sporadic and geographically confined incidents of sero-conversion, massive and multiple incursions of blue tongue (BT) wererecorded in South - Eastern Europe starting in 1998 and spilling overduring subsequent years.Between autumn of 1998 and summer of 2003, successive waves of BTepidemics swept through the Balkans giving rise to a number of clinicaloutbreaks, causing severe direct losses and involving several countries,namely Greece, Bulgaria, Serbia and Montenegro, the Former YugoslavRepublic of Macedonia, Bosnia-Herzegovina and Albania (Source: OIE)and, probably, Turkey.Affected countries employed different control, safeguard, preventionand epidemio-surveillance measures against BT and, with theexception of Greece and Bulgaria, comprehensive and reliableepidemiological data are by and large lucking.However, through an analysis and extrapolation of the epidemiologicalprofiles and patterns observed in Greece and Bulgaria – and assuming

that the evolution of BT in these two countries reflects closely thesituation of BT in the Balkans – certain general comments can be madeand some questions of outstanding epidemiological significance emergewhich shed new light on our conventional perceptions and clearly call fora new risk assessment and prevention / control strategy against BT.Such outstanding questions arising form the study of BT in the Balkansare, in particular:• The geographical occurrence and abundance of efficient vector(s),• The potential involvement of other, more common and wide-spread,

vectors,• The occurrence and distribution of BTV sero-types, in particular those

perceived as “exotic”,• The possibility and the mechanisms of “overwintering” of BTV,• The appropriate prevention, control and safeguard measures.This presentation reviews the history of BT in the Balkans in the period1998 - 2003, highlights certain key epidemiological points and identifiesareas for needed future research.

D.E. Panagiotatos

ABSTRACT BOOK

OVERVIEW OF BLUETONGUE DISEASE, VIRUSES, VECTORS, SURVEILLANCEAND UNIQUE FEATURES: INDIA, THE SUBCONTINENT AND ADJACENT REGIONSD. Sreenivasulu, M.V. Subba Rao, Y. Narasimha Reddy and G.P. Gard Associate Professor, Department of Microbiology, College of Veterinary Science, TIRUPATI-517502, Andhra Pradesh, INDIATelephone No: (0) 091- 877- 2249155, 2248068; Res No: 091-877- 2253994; Fax: 091-877-2249563 e-mail address: [email protected]

11

Bluetongue is endemic in India causing clinical disease in native,crossbred and exotic breeds of sheep. Bluetongue antibody is common incattle, buffaloes and goats but overt disease has not been reported. Exoticbreeds of sheep and their crosses are more susceptible to disease thannative breeds. Overall, morbidity, mortality and case fatality rates of9.3%, 2.7% and 28.8% respectively have been reported in rural flocks;rates significantly higher than in organised farms. Bluetongue disease in Andhra Pradesh native sheep has been investigatedsince 1983. The annual case fatality rate ranges from 2.4% to 38.1%. Theincidence of disease is cyclical with most outbreaks occurring between Juneand September during the South West monsoons when livestock-bitingmidges greatly increase. However, studies to identify the virus vector

species have not yet been undertaken.Bluetongue viruses have been isolated from native sheep, and sentinelherds have been used to demonstrate virus activity in several parts ofIndia. A total of 21 bluetongue virus serotypes have now beenreported in the country. Major impediments to disease control includethe presence of multiple virus serotypes, the broad vertebrate hostrange and a lack of detailed knowledge of vectors. Inactivatedvaccines prepared from local isolates are currently under field trials.Bluetongue virus occurs in regions adjacent to India. An antibodyprevalence of 48.4% has been reported from Pakistan with serotypes3, 9, 15, 16 and 18 present. Bluetongue antibody, but not disease, hasbeen reported in Bangladesh and Sri Lanka.


D. Sreenivasulu

GLOBALSITUATION





GLOBALSITUATION






ABSTRACT BOOK

THE HISTORY OF BLUETONGUE AND CURRENT GLOBAL OVERVIEWT.E. Walton USDA, APHIS, VS, CEAH; 2150 Centre Avenue; National Resources Research Center, Building B; Fort Collins, CO 80526-8117; USA. Tel.: 970-494-7201; fax: 970-472-2668; [email protected]

12

Bluetongue first was reported more than 125 years ago with the introduction ofEuropean breeds of sheep into southern Africa. Sheep experienced a severefebrile disease with high morbidity and high mortality. The viral etiology of thedisease was demonstrated in 1906. Bluetongue viruses have been identified inmany tropical and temperate areas of the World since that time from a latitudeof 40Þ North to 35Þ South. However, bluetongue, the disease, is a phenomenonof ruminants in the temperate zones. There is little, if any, clinical disease inthe tropical and subtropical areas of the World, except when non-nativeruminants are introduced into a virus-endemic area. At least 25 serotypes ofbluetongue virus have been described internationally. While the viruses areclassified antigenically and taxonomically as bluetongue viruses, each serotypeis unique and may not cause bluetongue, the disease. The bluetongue virusesare transmitted among ruminants by competent vector species of the genusCulicoides, which are known as biting gnats or midges. With the evolution ofimproved virologic and serologic techniques, bluetongue viruses werediscovered outside of Africa. With such discoveries of bluetongue viruses andthe extension of clinical bluetongue into temperate areas previously consideredto be free of bluetongue, it was presumed that bluetongue viruses wereemerging from Africa. More recently, it has been shown that bluetongue virusserotypes exist with vector species of Culicoides in predictable, but finite,geographic and ecologic cycles or ecosystems around the world. Despite thealmost certain movement of bluetongue viruses, livestock, and Culicoides

species between these ecosystems, there has been little evidence thatbluetongue virus serotypes have been moved between and persist in theseecosystems. Rather, periodic cyclic extensions and remissions of these virus-vector ecosystems permit the viruses and the disease to move into and recedefrom adjacent non-endemic areas in a pattern characteristic of many otherknown arthropod-borne viruses (arboviruses).There are Culicoides species on every continent except Antarctica. However, allCulicoides are not proven vector species. Like Koch’s Postulates which must besatisfied before a microorganism can be considered as the cause of a disease,there are similar requirements that must be satisfied to consider ahematophagous insect as a vector of a pathogen.Publications during the period from 1970 through mid-1980 suggested thatpersistent viremia; concurrent circulating virus and antibody; activation ofviremia by feeding of non-infected Culicoides sonorensis, the known US vectorof bluetongue viruses; virus excretion in semen; and reproductive anomalies inin uterine-infected fetuses occurred in bluetongue virus-infected cattle.Subsequent studies in the same laboratory and similar studies byinternationally recognized scientists at the same and other laboratories wereunable to reproduce or confirm the hypotheses. No subsequent naturalexperiences and research have supported the conclusions. The hypotheseshave not been validated and the conclusions of the research are no longeraccepted by the scientific community.

T.E. Walton

ABSTRACT BOOK

OUTBREAKS OF BLUETONGUE & OTHER ARBOVIRAL DISEASES IN ISRAELH. Yadin and M. van HamKimron Veterinary Institute, Israel • E-mail: [email protected]

13

Israel is a small country of 20.500 square km, with a livestock population of 275.600bovines and 618.200 small ruminants. Israel is situated at the junction of Asia, Africaand Europe, within the global band of arbovirus activity. Since 1950 four arboviraldiseases have been recorded and identified in Israeli livestock: bluetongue (BT) insmall ruminants and cattle, bovine ephemeral fever (BEF) in cattle, West Nile virus(WNV) in geese and horses, and Akabane in cattle and small ruminants. All fourdiseases appear in springtime, with the majority of outbreaks occurring at the end ofsummer. This observation was confirmed by an annual serosurvey, conductedbetween 1981 and 1995 at the beginning and end of summer, in sentinel calves in11 dairy operations. In 2002, following a wave of congenital blindness andhydrocephaly in calves, a serosurvey for BT antibodies in cattle revealed that 79% ofthe herds were seropositive while in sheep only 30% were positive. What role BTVhas in causing congenital malformations in dairy cattle is still not clear. Of the fourarboviral diseases, BT is the only one that appears regularly in Israel; since 1976,318 outbreaks were recorded in sheep herds, only 4 years (1980-1981, 1999-2000)were BT free. Viewing the annual number of BT infections it can be observed thatevery few years there is a peak in the number of outbreaks. Five serotypes - 2, 4, 6,10, and 16 - have been implicated in BT outbreaks in Israel; serotypes 4, 6, and 16are the most common. BT vaccination is voluntary. The annual number of appliedvaccine doses decreased from 77.000 in 1989 to 9.000 last years.BEF was first reported in 1930; thereafter the disease appeared at intervals of severalyears in 1936, 1939, 1951, 1990 and 1999 – 2001. Considerable study was devotedto the last outbreaks. In 1990(?) BEF first occurred along the Jordan Rift Valley, thenspread west and south along the coastline. In 1999 a new incursion of BEF affected170 herds with sever economic losses. The following year (2000) the disease occurred

in 75 dairy operations and in 2001 only a few were affected. The economic damageto the affected dairy operations was morbidity of 3% in calves and 31% in theproductive cows with a milk loss of 350 liter per sick cow. The average mortality was0.4% for calves and 3.6% for dairy cows. Control by vaccination is still underdiscussion.Akabane disease first appeared in the small ruminant and dairy cattle industry inIsrael in 1969/70. The first outbreak was impressive: 3000 calves, 700 lambs, and600 kids were affected. In following years sporadic cases occurred. Seromonitoring was performed 1988 – 1994 in young sentinel calves once in springand the second in autumn, showed that 80 – 90% of calves negative in March/Aprilseroconverted by November. In the last 2 years there has been an increase incongenital blindness and hydranencephaly in calves and lambs. In one case Akabaneviral genome was detected by real time PCR.The first outbreaks of WNF were reported in Israel 1950 – 1954 with several hundredhuman cases. The second and the third outbreaks were described in 1957 and 1980,in which mostly young soldiers were involved. The latest outbreak started in spring1997 and lasted till 2001. In this epizootic commercial goose flocks were involved. Outof 50 goose flocks 8 to 15 were affected, 8 flocks with 9269 geese were destroyed in2000. In the same year 417 human cases of WNF were diagnosed, 326 hospitalizedwith mortality of 8.4%. During this time 3 horses died and virus was detected andisolated. Serosurveillance for WNV showed 40% seroprevalence in soldiers, while inhorses it varied between 0 – 85% depending on geographic location. The monthlydistribution of WNV is similar to the other arboviruses with peaks of disease inSeptember. The control of WNF is based on eradication and control of dump waterreservoirs repellents, and vaccination of commercial goose flocks.


H. Yadin

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The current global situation and regional overviews of viruses,vectors, surveillance and specific features A 1

ABSTRACT BOOK

ENTOMOLOGICAL SURVEILLANCE OF BLUE-TONGUE IN 2002 IN FRANCET. Baldet(1), J.C. Delecolle(2), B. Mathieu(3) and S. De La Rocque(1)

(1) CIRAD-EMVT, TA 30 E, Campus International de Baillarguet, 34 398 Montpellier (France). Phone: 33 4 67 59 38 68 / Fax: 33 4 67 59 37 99 / E-mail:[email protected](2) Université Louis Pasteur de Strasbourg, Strasbourg (France)(3) EID- Méditerranée, Montpellier (France)

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Bluetongue (BT) is an arboviral disease that has emerged in the north ofthe Mediterranean basin since 2000. The main vector, C. imicola Kieffer1913 was detected for the first time in Corsica (France) in October 2000and then important outbreaks due to BT serotype 2 appeared in autumn2000 and 2001. In parallel to the vaccination campaigns conducted between 2001 and2003, an entomological surveillance network was established in 2002: (i)in Corsica, to study the population dynamics of C. imicola and otherpotential vector species associated with livestock (C. obsoletus, C.scoticus, C. newsteadi, C. pulicaris); (ii) on the coastal mainland inFrance, to survey the introduction of C. imicola and study the populationdynamics of others Culicoides species. For this purpose, we selected 12 representative sites in Corsica (farmsaffected by BTV 2 outbreaks in 2000 and/or 2001) and 19 sites at risk onthe coastal mainland at intervals of 50 km. One-night-catching on siteusing UV-light traps with a suction fan was performed each 3 weeks inCorsica and monthly on the mainland. In Corsica, from February to December 2002, 84 790 Culicoides belongingto at least 50 species were collected in a total of 180 nights of catching. C.imicola accounted for 18.3% of the specimens collected with a maximumand an average catch size of 4 814 and 86 respectively. The main vectorwas predominant in 2 sites located at the extreme south of the island.

Adult densities reached a peak in September-October at the end of thesummer. The presence of C. imicola at all the sites, and during 8 months(from May to December) confirms that the main vector of BT over-winteredand is now well established in Corsica. The other more abundant andwidespread species were C. newsteadi (36.8%, 4 018, 174), C. scoticus(18%, 4 016, 85), C. obsoletus (9.2%, 1 912, 43), C. circumscriptus(4.2%, 507, 20) and C. pulicaris (3.8%, 626, 18). The 43 others speciesaccounted for less than 2% of the total. On the mainland, from April to November 2002, 16 197 Culicoidesbelonging to 44 species were collected in a total of 109 nights of catching.No specimens of C. imicola were found. The more abundant andwidespread species were C. newsteadi (73.5%, 3 655, 109), C. obsoletus(8%, 201, 12), C. scoticus (5.4%, 417, 8), C. circumscriptus (3.2%, 177,5) and C. griseidorsum (2.7%, 337, 4). The 39 others species accountedfor less than 2% of the total. The geographical and seasonal distribution of C. imicola and others speciesof interest were discussed in relation with their bio-ecology andenvironmental factors, mainly climatic ones specific to each area. Thesedatasets appear essential to contribute to a better comprehension of BTepidemiology, and to build up and validate predictive models based onremote sensing in order to identify areas at risk of BT.


ABSTRACT BOOK

SEROLOGICAL AND CLINICAL EVIDENCE FOR REACTIVITY OF ARBOVIRAL TERATOGENICSIMBU SERO-GROUP INFECTION IN ISRAEL; 2001/2002 EPISODE(S)J. Brenner, T. Tsuda, H. Yadin, D. Chai, Y. Stram and T. Kato Kimron Veterinary Institute, 50250 Bet Dagan, Israel, Tel 972-3-9681688; Fax- 972-3-9681788; Email [email protected]

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Within the last 35 years two major outbreaks of Akabane Virus (AKAV)infections were recorded in Israel, 1969/70 and 2002/3, respectively.An outbreak of congenital malformations in ruminant newbornscharacterized by the appearance of an arthrogryposis andhydranencephaly syndrome (AHS) first appeared in Israel in 1969. Basedon serological, epidemiological, clinical, pathological, histopathological,and serological data, AHS has shown to be strongly connected withreactivity to AKAV, which is a member of the Bunyaviridae, Simbu sero-group. Since 1969/70, malformations have been seen sporadically in Israel butall attempts failed to isolate a teratogenic arbovirus that belonging to theSimbu sero-group. To date the evidence for the presence of AKAV, andpossibly Aino (AIV) viruses is based only on the results of serologicalsurveys. A serological survey by the serum neutralizing test (SNT) performed byKalmar et al., has confirmed that the episode of 1969/70 was stronglyconnected with AKAV infection and that between 1970 and 1973/4, noserological evidence of the presence of AKAV could be established. In mid-February 2002 the first cases of “blind newborn calves” appearedin the Northern Valleys of Israel. This particular outbreak, allowed us to

investigate the possible introduction of an agent that was suspected tohave (probably) teratogenic properties. In an attempt to identify the causative agent of this epidemic ofcongenital abnormalities, eighty-one serum samples were taken fromcattle raised in different geographical zones, of which 54 samples werefrom dams that had delivered affected calves or of calves born withmalformations and 27 from non-affected (control) zones. SNT was carried out by micro-titer methods using AKAV (strain OBE-1)and AIV (strain JaNAr 28) viruses respectively. The results are as follows: antibody to AKAV virus was detected at muchhigher rate (47/54, 87%) in the sera taken from cattle raised in the affectedzones than in unaffected zones (1/27, 3.7% only). The percentage ofpositively reacting sera to AIV was relatively low in both affected and non-affected farms. This reactivity was found in about 30% (8/27) of the seraoriginating from the unaffected zones in 2001. This probable reintroduction of AKAV into our region with the evidence of AIVpresence and the epidemics of bluetongue in the south of Europe and theeast Mediterranean basin, together with the potential alert due to West Nilevirus in Israel and in Italy, are evidence of the potential spread of arbovirusesinto uninfected areas.

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ABSTRACT BOOK

FIRST RESULTS OF BLUETONGUE SURVEILLANCE IN SWITZERLANDA.Y. Cagienard, F. dall’Acqua, B.Thür, C. Griot and K.D.C. StärkSwiss Federal Veterinary Office, Schwarzenburgstrasse 161, PO Box CH-3003, Bern - Switzerland • Ph +41 31 323 23 94; Fax +41 31 323 95 43; E-mail:[email protected]

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Recently reported outbreaks in several Mediterranean countries documentthat BT has moved in Europe north as far as latitude 45°north. With globalwarming and climate as a major determinant of insect distribution and vectorcompetence, BT virus positive C. imicola could establish itself in Switzerland.Using occurrence modelling, Rawdon (2002) identified Switzerland as anarea at risk for occurrence of C. imicola. So far, the presence of C. imicola inSwitzerland is unknown. Additionally, Swiss livestock populations have as yetnever been tested for BTV-antibodies. The objective of this first survey wasi) to establish the immune status of cattle regarding BTV, and ii) to obtainbaseline data on vector populations, predominantly Culicoides spp. and theirdistribution within Swiss areas at risk.A serological survey was conducted using a two-step sample calculationon Swiss cattle farms. The sample size was calculated using OIErecommendations, so that a minimal prevalence of 2% could be detectedwith a probability of 95% (Anonym, 2002). Switzerland was divided into46 equal quadrates. Taking into account that quadrates have a differentacreage because of lakes and mountains, a proportion of km2

pasture/quadrate was calculated for each quadrate (range from 6 km2 to1600 km2). The number of farms to be sampled within each quadrate wascalculated proportional to its pasture size (range from 2 to 148 farms per

quadrate). Out of 2384 randomly selected Swiss cattle farms, sampled forroutine disease surveillance purposes in 2003, a total number of 660farms were randomly selected. On each farm, 5 blood samples weretaken. A total number of 3300 blood samples will be analysed using acommercial BT ELISA kit (Biological Diagnostic Supplies Limited, BDSL). The entomological survey of vectors was limited to high-risk areas in theSouth of Switzerland. In three cantons (Grisons, Ticino, Valais) adjacentto Italy, black-light traps (Onderstepoort Veterinary Institute) forCulicoides spp. were set up. Trapping sites were selected by severalcriteria, such as farm type (cattle), altitude, average temperature,average rainfall and humidity. Traps will be positioned 5 times at differenttrapping sites in each of the three Cantons in July and in September2003. The traps were geo-referenced by GPS (Global PositioningSystem). At each trapping site, the same trapping protocol was followed.In addition to collection site parameters, an interview based on aquestionnaire was performed in order to obtain information on livestockmanagement data. The samples will be analysed to identify theabundance of the Culicoides family, specifically C. imicola and C.obsoletus.Results of both surveys will be presented.

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ABSTRACT BOOK

EPIDEMIOLOGICAL SURVEILLANCE OF BLUETONGUE IN SICILY (SW ITALY)S. Caracappa(1), M. Bagnato(2), P. Sghembri(2), A. Guercio(3), F. Prato(4), G. Tumino(5), A. Migliazzo(6), F. Geraci(6), S. Vullo(6), S.Agnello(6) and C. Di Bella(6)

(1) Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri” – Direzione Sanitaria (2) Assessorato Regionale alla Salute – Ispettorato Regionale Veterinario (3) Istituto ZooprofilatticoSperimentale della Sicilia “A. Mirri”- Area di Diagnostica Virologica (4) Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”- U.O. Sistema Informativo e Statistica (5) IstitutoZooprofilattico Sperimentale della Sicilia “A. Mirri” – Area Ragusa (6) Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri” – Area Sorveglianza Epidemiologica

Istituto Zooprofilattico Sperimentale in Sicily “A. Mirri”, Via G. Marinuzzi, 3. 90129 Palermo. Phone (39) 916565309 – Fax (39)916565294; e-mail: [email protected]

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Starting from 13 th October 2000, which is when the first outbreak ofbluetongue in Sicily was recorded, the development of the disease isdescribed in this paper, and the results of the Epidemiological SurveillanceProgramme are also illustrated, relating to sentinel animals distributedover the whole region.In Sicily the incidence of the disease is relatively low compared to otherareas in the Mediterranean basin. 75 outbreaks of the disease wererecorded in the first 3 epidemics (October 2000 – May 2003). An overallmorbidity of 4.58% and mortality of 2.09% were recorded and casefatality rate of 45.74%. Province of Catania seems to have been hit the

worst; the incidence rate in August 2002 was at 0.8%. The monthlyincidence rate was calculated for sentinel animals. There is estimated tobe 3,654 of these animals, divided into 63 areas. It is important tounderline that in the period being considered, an average of 2,382animals were examined. During the surveillance period, which went fromSeptember 2001 to May 2003, the incidence peaked in Septeber 2002, at5.91% ± 0.979. The cumulative incidence rate for the period fromSeptember 2001 to August 2002, and September 2002 to March 2003was recorded at 4.53% ± 0.76 e 20.03% ± 1.85. The circulation of serumtypes 2, 4, 9 and 16 is also described, as seen by the sentinel animals.

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ABSTRACT BOOK

SEROPREVALENCE OF BLUETONGUE VIRUS IN CATTLE, SHEEP AND GOATS OF ALBANIAM. Di Ventura(1), M.Tittarelli(1), G. Semproni(1), B. Bonfini(1), G. Savini(1) and A. Lika(2)

(1) Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo, Italy(2) Institute of Veterinary Research “Bilal Golemi”, Department of Virology, Tirana, Albania

Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo, Italy. Tel.: +39 0861 332235, Fax: +39 0861 332251, e-mail: [email protected]

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Following the diffusion of Bluetongue virus (BTV) in the Mediterraneanbasin, numerous Countries involved in the epidemic have organisedentomological and serological surveillance plans to define the infectiousarea and better control the infection. No data on the presence anddiffusion of BTV in Albania were available. One of the target of the“National survey on Foot-and-Mouth Disease and Bluetongue”, a joinproject realized between the Albanian Veterinary Services, the VeterinaryResearch Institute of Tirana and the Istituto Zooprofilattico Sperimentaledell’Abruzzo e del Molise “G. Caporale”, was to survey a population ofsusceptible domestic ruminants for the presence of BTV antibodies.Between October and November 2002 serum samples from 857 cattle and870 sheep and goats were collected by the Albanian Veterinary Services.

Animals were from 15 Albanian districts, some bordering Yugoslavia,Macedonia and Greece, others facing the Adriatic sea. At the AlbanianVeterinary Research Institute the samples were tested for the presence ofBTV antibodies using a c-ELISA (Bluetongue Antibody Test Kit – IZSAM-Teramo) and in Italy serum-neutralisation have been used to confirm theELISA positive results. The overall prevalence was 18,9% in cattle and4,4% in sheep and goats. Positive animals were found in all examineddistricts. The Tirana district was the region with the highest prevalenceresulting 61% of cattle and 20% of sheep and goats positive to c-ELISA.The SN confirmed the c-ELISA results revealing the presence of antibodiesagainst BTV serotype 9.

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ABSTRACT BOOK

CURRENT SITUATION OF BLUETONGUE IN TURKEYA. Ertürk, Ö. Kabakli, S.G. Çizmeci and F.M. Barut Ministry of Agriculture and Rural Affairs, Etlik Central Veterinary Control and Research Institute, Viral Diagnosis Laboratory, 06020 Etlik/Ankara/TürkiyePhone Number: 00 90 312 326 00 90 / 144 Fax Number: 00 90 312 321 17 55 E-mail Address: [email protected], [email protected]

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The first reported bluetongue outbreak was in 1977 in Ayd¦n province, inthe west of the country. Disease spread between 1977 and 1979 andbecome endemic in the provinces bordering the Aegean andMediterranean Sea. Casual agent originating from sheep and calf sampleswas isolated and identified as BTV type 4. Epidemiological investigationsshowed that not only sheep but also goats and cattle were involved inthese outbreaks. The vector was Culicoides imicola.The disease was halted in sheep successfully by vigorous control measures(quarantine, animal movement control, disinfection, insecticide treatment

and vaccination campaigns) in sheep in western provinces with attenuatedBTV type 4 vaccines, which produced in Etlik Central Veterinary Control andResearch Institute. Unexpected outbreaks of bluetongue have beenoccurred in Edirne province in northwest of Thrace in 20 July 1999 andspread to adjacent villages. Serotypes of the disease have been reportedin 2000 from Pirbright Laboratory as BT serotypes 9 and 16. No cases wereobserved in 2001. The last case has been in August 2000.The diagnosis of bluetongue was based on clinical findings, serologyand virus isolation.

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ABSTRACT BOOK

INCIDENCE AND ISOLATION OF BLUETONGUE VIRUS (BTV) IN CATTLE OF ITUZAINGOAND SANTO TOME DEPARTMENTS OF CORRIENTES PROVINCE, ARGENTINAI.A Lager, S. Duffy, J. Miquet, A. Vagnozzi,C. Gorchs, G. Draghi, B. Cetrá, C. Soni, C. Hamblin, S. Maan, A. Samuel, P. Mertens,M. Ronderos and V. RamirezInstituto de Virologia,INTA-Castelar, CP 1712, Hurlingham, Buenos Aires, ARGENTINA. Telephone: 54-11-4621-1447; Fax: 54-11-4621-1743; e-mail:[email protected]

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The objective was to study the pattern of transmission of BTV in cattle andisolate the virus. Sentinel herds were monitored serologically during two periods. Firstly, fromJune 1999 to August 2000 and secondly from September 2000 to April 2001.Herds were located in Santo Tome (H1 and H2) and Ituzaingo (H3) areas whereBTV activity was known to occur. In the first period, 30 female 6-9 months oldcattle seronegative for BTV (screened by a BTV-cELISA kit*) were selected ateach herd (H1, H2, H3). Blood samples were collected monthly with sodiumcitrate, except in January-99 and July-00. In the second period, 40 females ofsame age as before were selected at each herd (H1 and H2). Samples werecollected on September-00, December-00, March-01 and April-01. Light trapswere located close to sentinel herds to collect potential vectors of BTV at H1. From June-99 to August-00, the cumulative incidence (CI) of BTV infection were0% (0/34); 35% (11/31) and 25% (8/32) in H1, H2 and H3, respectively. Cattlebecame seropositive from October to May in H2 and from September to May inH3. The highest monthly CI was recorded in March-April in H2 and February-March in H3. No seroconversion was detected later than May. From September-00 to April-01, the CI of BTV infection were 10% (5/50), and 97% (39/40) inH1 and H2, respectively. Cattle became infected from December to April in H1and from September to March in H2. Red blood cells from those animals that showed seroconvertion were processedfor virus isolation by inoculation into embryonated chicken eggs and cell cultures(OIE,2000) .Four samples showed CPE in BHK cells and were positive by direct

and indirect inmmunofluoresce with BTV specific reagents (Brewer ,1994). Thesesamples were examined by electro microscopy and showed virus particles withBTV morphological characteristics. Blood samples and tissue culture supernatantswere positive by the RT-PCR with primers corresponding to the segment 3 of theBTV genome (Shad,1997;OIE,2000). Micro-serum neutralization assays and RT-PCR with primers corresponding to BTV genome segment 2, followed by sequenceanalysis of the amplified cDNA segment and comparison to reference BTV strains,were used to identify the four samples as serotype 4.The average of the monthly maximum temperature was always above 19ºC.However, the average monthly median temperature was below 19ºC and theaverage monthly minimum temperature was below 15ºC from May-00 toAugust-00. There was no viral activity detected at that time. Culicoides insignis was identified as the predominant species of potentialvectors (99%) trapped near sentinel herds (Wirth,1988).Discussion: Though clinical disease has never been reported in Argentina, viralactivity was detected and the virus isolated in sentinel herds. This first isolationof BTV in Argentina corresponds to serotype 4 .There was a marked variabilityin the CI of BTV infection among herds and between years. Absence of BTVactivity from May to September suggests that low temperatures result in low ornil vector activity. Culicoides insignis seems to be the most likely vector of BTVin this area (Mellor,2000).

*Veterinary Diagnostic Technology, Inc.

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THE EPIZOOTIOLOGICAL APPEARANCE OF BLUETONGUE IN THE CENTRAL BALKANSB. Djuricic, D. Nedic, D. Lausevic and M. Pavlovic Katedra za zarazne bolesti, Fakultet veterinarske medicine Beograd, Bulevar JNA 18. Phone: ++381 11 685 080; E-mail: [email protected]

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Bluetongue is an acute viral disease of sheep, goats and cattle, to which deerare also susceptible. It appears as enzootic, in the form of natural foci, and istransmitted by haematophagous insects (ticks and mosquitos of theCulicoides genus). Its symptoms are fever, catarral-necrotic changes of thenasal and oral cavity mucous, the tongue, digestive organs, toe crowns, aswell as degenerative changes in skeletal muscles. There can appear face ortongue swelling, especially in cattle, as well as abortions and still births ofdegenerated youngs.The worldwide incidence of the disease is connected with the habitus of thecarriers. With the increased warming up of the north hemisphere, there hasbeen a spreading and moving of the carriers (from 40° to 43°). In theMediterranean region the disease has, in the last 5 years, been registered inAlgiers, France, Greece, Italy, Spain, Tunisia and Turkey. In the countries ofthe Balkan peninsula the disease has been present in Bulgaria (since 1999),Macedonia and Serbia and Montenegro, Croatia, Bosnia and Herzegovina, andAlbania. Bluetongue was first carried into the epizootiologial regions of theBalkans from Turkey in 1999. The disease was transmitted by the infectedmosquitos in the border regions with Turkey. In Serbia and Montenegro thedisease was first registered in July 2001, in Zubin Potok region (Kosovo), anduring August, in neighbouring regions- Novi Pazar, Tutin, Rozaje, Leposavic,etc. The disease was simoultaneously registered in East Serbia, in the borderregions with Bulgaria (citiy regions of Bosilegrad, Pirot, Knjazevac). Based onthe epiyootiological appearance of the disease and the clinical symptoms, it

can be concludede that the disease was in East Serbia present also in 2001,but low lethality and mild clinical signs caused it to remain unregistered.During 2002. wider serological investigations on animals belonging toYugoslav regions,have been carried out, and the spreading of the disease wasregistered in the North of the country. The first serological cases of thedisease were at that time also registered in the East parts of Republika Srpska(BiH). Seen from the epizootiological point of view,it can be said that thenorthernmost point of disease origin in Serbia is on the River Sava. Diagnosticresearches currently performed in Serbia and Montenegro, as well as inRepublika Srpska, and which are in accordance with the prescribed MeasuresProgramme for 2003, will show the real epizootiological picture of the diseasein the central Balkans. It is necessary to investigate this territory seen fromthe carriers of the Culicoides genus point of view, which is not yet being done,mostly due to financial problems. Having in mind the epizootiological appearance of Bluetongue in the Balkans,a conclusion can be drawn that no matter how good the results in the study ofthe occurrence and spreading of the disease, it still remains a majorinternational problem, which demands constant attention of the nationalveterinary services, as well as of the OIE, working together on the preventionof the spreading of the disease, and not only in the countries where thisdisease is present.It is clear that further investigations demand a team workingof all the regional services, as well as showing the need for deciding on a globalproject for studying the character of the disease in Southern Europe.

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COMPLETE SEQUENCE ANALYSIS AND COMPARISONS OF GENOME SEGMENT 2 (ENCODINGOUTER CAPSID PROTEIN VP2) FROM REPRESENTATIVE ISOLATES OF THE 24 BTV SEROTYPESS. Maan, N. Maan, R. O’Hara, A.R. Samuel, A. Meyer, S. Rao and P.P.C. Mertens Department of Molecular Biology, Institute for Animal Health, Pirbright laboratory, Ash Road, Pirbright, Surrey GU24 ONF. Phone +44(0) 1483 232441; e-mail:[email protected]

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Bluetongue is an economically important disease of sheep (classified within a'list A' by the OIE), caused by bluetongue virus (BTV), which can also infectcattle, deer and goats. Bluetongue virus is the prototype species of the genusOrbivirus within the family Reoviridae. BTV is transmitted by certain species ofbiting midges (Culicoides sp.) and has a global distribution between latitudes50° North and 30° South that depends primarily on the distribution and activityof these vector insects. The BTV genome is composed of ten separate segments of linear double-stranded RNA that are packaged within the central space of the icosahedralvirus particle (approximately 80nm in diameter). The virion is composed ofthree concentric protein shells or capsid layers (sub-core, core and outer-capsid). The outermost capsid layer contains two major structural proteins, VP2and VP5, which are encoded by genome segments 2 and 6 respectively. Theseproteins elicit a neutralising and protective antibody response in infectedmammalian hosts and are the most variable of the virion components (possiblyas a result of antibody selective pressure). The specificity of interactionsbetween the outer capsid proteins (particularly VP2) and neutralisingantibodies, determines virus serotype. Incomplete sequence data was previously available for genome segment 2 from14 of the 24 BTV serotypes, including full-length sequences for only ninedifferent serotypes (see http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/btv_sequences.htm). We have now completed an analysis of the full-length genome segment 2 sequence from representative isolates of each of the

remaining uncharacterised, or partially characterised serotypes, as well assegment 2 from multiple isolates of some BTV serotypes. Phylogenetic treeswere drawn to show relationships between isolates of the same and differentserotypes (see: http://www.iah.bbsrc.ac.uk/dsRNA_ virus_proteins/orbivirus-phylogenetic-trees.htm). Some BTV types were shown to be genetically moreclosely related than others. For example there is a closer relationship betweenthe North American serotypes (10, 11, 17 and 20) and BTV-4, which is foundin Europe and Africa but has recently also been identified in South America (seeabstract by Lager et al). Isolates of the same serotype were more also closely related than isolates ofdifferent serotypes, suggesting that it will be possible to identify the serotypeof any new BTV isolate by analysis of the sequence of genome segment 2,without the need for standardised and therefore very expensive serologicalreagents. This will facilitate the more rapid and appropriate selection of vaccinestrains in the face of BTV outbreaks. Control measures that may becomeincreasingly important as climate change continues to affect the epidemiologyand spread of the disease. These sequence data are also being used to develop molecular techniques (RT-PCR, serotype or strain specific primers and sequencing) for diagnostic testingand the identification of BTV serotype (see abstracts by Maan et al).Phylogenetic data for segment 2, in conjunction with studies of the othergenome segments of specific virus isolates, will also help us to identify andunderstand the role of BTV genome segment re-assortment in the field.

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OVERVIEW OF BLUETONGUE IN GREECEK. Nomikou, O. Mangana-Vougiouka and D. Panagiotatos Ministry of Agriculture, Institute of Infectious and Parasitic Diseases, Virus Laboratory. National Reference Laboratory for Bluetongue, Epizootic Hemorrhagic Diseaseand African Horse Sickness. 25, Neapoleos Str., Aghia Paraskevi, 153 10, Athens, GREECE. Tel: +30 210 6011499; Fax: +30 210 6011499; E-mail:[email protected]

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Three major epidemics of Bluetongue ( BT ) occurred in Greece , in 1979which was due to serotype BTV 4, in 1998-1999 mainly to serotypes BTV 9,BTV 4 and less to serotype BTV 16 and in 2001 mainly to serotype BTV 1.The first incursion of BT in Greece occurred in the Autumn of 1979 on theisland of Lesvos, which is situated in the eastern part of the Aegean Sea,6-12 miles from the Asian coast. About 500 sheep were clinicallyaffected,mostly at the eastern regions of the island. Serotype BTV 4 wasisolated and the responsible vector present was Culicoides imicola.Regionalization of the area was applied and an eradication program wasinitiated. All the approximately 5 000 cattle of the island were serologicallytested each year. Between 1980 and 1986 aproximately 560 seropositivecattle were slaughtered. Surveillance and control measures were applied alsoin the islands of Eastern Aegean in the years 1980 – 1991. The successful application of surveillance and control measures resultedin official lifting of EU restrictions in 1991.Between the years 1991-1998 the surveillance measures continued in theislands of Eastern Aegean and no clinical disease was reported.In Autumn and Winter of 1998 clinical outbreaks of BT were reported in 3Eastern Aegean islands (Rhodos, Kos, Leros). Serotype BTV 9 wasisolated, while C. imicola was involved.In Summer and Autumn 1999,11 out of 51 perfectures of NorthEastern,

Eastern and mainland Greece had experienced clinical outbreaks while 5perfectures had serological evidence of BTV. Serotypes BTV 9 , BTV 4 andBTV 16 were isolated while C. imicola and C. obsoletus were present.In Autumn of 2000, serotype BTV 4 was isolated from clinical casesreported only in 1 perfecture.In Summer – Winter of 2001, 11 out of 51 perfectures of Western (W),Northwestern (NW), Eastern (E) (island of Lesvos) and central Greecewere clinically affected. Serotype BTV 1 was isolated from cases reportedin the W., NW., and E. prefectures while serotypes BTV4, BTV9 wereisolated from NW. and central Greece. Again C. imicola and C. obsoletuswere involved.Safeguard measures in domestic trade of live animals, control ofCulicoides spp., control and epidemio-surveillance measures were appliedfrom 1998 until today.The sero-monitoring of sentinel bovines in 18 perfectures out of 51 in2002 resulted in no seroconversions. Sero surveillance of sentinel bovines for virus activity is working in 18perfectures of Eastern, North, Western and central Greece for 2003.Results of nation-wide sero-prevalence indicate that, despite repeatedwaves of the disease, a large proportion of susceptible animals remainnaïve and conversely could sustain a new clinical epidemic.

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MOLECULAR EPIDEMIOLOGY OF BLUETONGUE VIRUSES FROM DISEASE OUTBREAKSIN THE MEDITERRANEAN BASINS. Maan, A.R. Samuel, S. Rao and P.P.C. Mertens Department of Molecular Biology, Institute for Animal Health, Pirbright laboratory, Ash Road, Pirbright, Surrey GU24 ONF. Phone +44(0) 1483 232441 e-mail:[email protected]

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Since 1998 five bluetongue virus (BTV) serotypes (1, 2, 4, 9 and 16) havecaused outbreaks of disease in across much of Southern Europe. Thedistribution of previous disease outbreaks in the region (both bluetongueand African horse sickness) had mirrored the distribution and abundanceof the major vector species (Culicoides imicola). However, bluetongue hasnow spread northwards into new areas of Europe (Bulgaria, Serbia andCroatia) that are beyond the range of C. imicola, suggesting theinvolvement of novel insect vector species (see Abstract by P. Mellor).Indeed the virus has recently been isolated in several areas, from both C.obsoletus and C. pulicaris (species that are abundant across much ofnorthern Europe). These observations, together the effects of globalclimate change on the distribution and activity of vector species, and theexistence of an effective overwintering mechanism for BTV (see abstractby Takamatsu et al; Takamatsu et al 2003), suggest that the virus maycontinue its spread northwards. New areas of Europe must therefore beconsidered to be 'at-risk' from the disease. BTV serotype is primarily controlled by the variable outer coat proteinVP2, encoded by segment 2 of the virus genome. Phylogenetic analysesof segment 2 shows that recent Mediterranean isolates of BTV-2 have asimilar genetic lineage to those from sub Saharan Africa and NorthAmerica but are distinct from Asian strains. In contrast the isolates ofBTV-9, which caused outbreaks in the Eastern Mediterranean, are related

to a genetic lineage from Asia (phylogenetic trees are available athttp://www. iah.bbsrc.ac.uk/dsRNA_virus_prote ins/orb iv i rus-phylogenetic-trees.htm). The strain of BTV-1 that caused diseaseoutbreaks in Greece during 2001 is most closely related to BTV isolatesoriginating from India, also suggesting virus movement from East toWest. BTV-4 isolates from outbreaks in the Mediterranean basin show thatGreek and Turkish isolates are similar to each other, but that they aredifferent from the Turkish type 4 vaccine strain. The results of the BTV genome segment 2 sequencing studies that will bepresented, are also being used to establish a database for molecularepidemiological studies (see: http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/orbivirus-accession-numbers.htm, see also abstracts by S. Maanet al). This will provide a resource to support and improve BTV serotypeidentification methods by using sequence comparisons (via the Web),rather than the conventional serological techniques that requirestandardised (and very expensive) serological reagents. More rapid andaccurate BTV strain and serotype identification will also provide valuableinformation concerning the route and direction of virus spread that cannotbe obtained from existing serological typing assays. This information islikely to be of considerable importance if we are to understand andcombat disease transmission that occurs during the increasingly complexmovement of animals in the livestock trade.

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ABSTRACT BOOK

RESULTS OF CURRENT SURVEILLANCE ON LIKELY BLUETONGUE VIRUS VECTORSOF THE GENUS CULICOIDES IN CATALONIA (SPAIN)V. Sarto i Monteys, C. Aranda, R. Escosa, N. Pagès and D. Ventura Departament d'Agricultura, Ramaderia i Pesca Fundació CReSA/Entomologia; Universitat Autònoma de Barcelona; Campus de Bellaterra, edifici V 08193; Bellaterra(Barcelona) Spain. Phone: +34 93 581 1420; Fax: +34 93 581 3142; E-mail: [email protected]

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Midges of the genus Culicoides (Diptera: Ceratopogonidae) were trapped weekly in eightsites located along the Catalonian Mediterranean coast (Spain). This trapping programmewas carried out within the framework of an investigation, entrusted by the CatalonianDepartment of Agriculture, Livestock and Fisheries, into the occurrence/absence ofpossible vectors of bluetongue virus (BTV) in Catalonia. The surveillance was triggeredas a consequence of the year 2000 BT outbreak and the discovery of C. imicola in theBalearic islands.Because of several difficulties encountered while selecting the farms (all stocked with eithersheep or goats or both) the first trapping date varied considerably among the farms. Thefollowing table summarizes this:

Trapping sites Peralada Vilanova de Dosrius Bellaterra S. Just Vallirana Vinallop La Galera(from north to south) la Muga Desvern

First trapping date 31.7.03 31.7.03 1.1.03 1.1.03 21.5.03 14.5.03 21.5.03 21.5.03

The knowledge of the Culicoides fauna in Catalonia was extremely poor. In fact, the onlypublished reference quoting species from this region was that of Strobl (1906) for C.pulicaris and C. obsoletus, both found in Malgrat, a coastal town in the province ofBarcelona. Later, due to the 1990 AHS epizootic that affected southern Spain, the SpanishVeterinary Services set 70 collecting traps in 17 Spanish provinces. The traps were locatedmostly in southern Spanish localities but three were in coastal Catalonia: Castellód’Empúries (Girona), 18m asl, Viladecans (Barcelona), 17m asl and Amposta (Tarragona),8m asl. A total of 699 Culicoides specimens were collected in those three traps during 1990and 1048 in 1991, comprising nine species, being C. obsoletus and C. pulicaris the only likelybluetongue virus vectors found (the main vector, C. imicola, was not found). Very recently,Sarto i Monteys & Saiz-Ardanaz (2003) reported the presence of nine Culicoides species,

trapped from May 2001 through December 2002, in Dosrius and Bellaterra, in the provinceof Barcelona; those included C. scoticus and C. imicola as new to Catalonia. Concerning C.imicola, only a single female was collected on 8 August 2002 at Dosrius; it was heavily gravidand might have been wind blown from the Balearic Islands of Majorca or Menorca, where alarge population existed and a BT epizootic occurred in 2000. It is known that adultCulicoides can be carried on the wind for long distances, as far as 700 km (e.g. Sellers,1992). The west coasts of these two islands are only 209 km (Menorca) and 200 km(Majorca) away from the Barcelona coast in mainland Spain. This significant finding of themain vector of BTV and AHSV in Europe was the first ever in Catalonia and stood as thenorthernmost point in the Iberian Peninsula (parallel 41º 35’ N). Previously, in continentalSpain, the northernmost point known for imicola was that of Talavera de la Reina (Toledo),at 40º00’N (Rawlings et al., 1997; Ortega et al., 1998). At present (2003), the result of this ongoing surveillance has shown that C. imicola seemsto be quite spread along all coastal Catalonia, as several specimens have been found in alltrapping sites. Specimens began appearing at the end of August, after the first heavy rainsin Catalonia, and were also collected through September. Catches were not very high(maximum, so far, 46 specimens per trap), however its continuous presence is significant,specially keeping in mind that only one single specimen had been detected in 2002.This current situation in Catalonia seems to agree with that foreseen by Wittmann et al.(2001), who had developed a logistic regression model to identify locations were C. imicolacould become established in European areas. The model indicated that under currentconditions, the distribution of C. imicola in Spain, Greece and Italy could extend and othereastern Mediterranean countries could be invaded.The presence in Catalonia of C. pulicaris, C. obsoletus, C. scoticus and, specially, C. imicola,all likely bluetongue virus vectors, is a worrying fact that must be addressed properly byregional Animal Health authorities, since the risk of a bluetongue outbreak in Catalonia andneighbouring regions cannot be discarded.

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ABSTRACT BOOK

MODELLING THE DISTRIBUTION OF BLUETONGUE VECTORS:THE PREDICTED IMPACT OF GLOBAL WARMINGM. Baylis and B. Purse Institute for Animal Health, Ash Road, Pirbright, Surrey GU24 0NF, UK. Tel +44 1483 232441. Fax +44 1483 232448. E-mail: [email protected]

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The 1987-1991 outbreak of African horse sickness in Spain, Portugal andMorocco, and the 1998-2003 outbreak of bluetongue (BT) across much ofthe Mediterranean basin, have stimulated attempts to develop models ofthe distribution of the Culicoides vectors in terms of climate data andsatellite imagery. The purposes of the modelling are (1) to improve our

understanding of the biotic and abiotic determinants of the distribution ofthe vectors; (2) to identify the limits to the distribution permitted bysuitable climate, and thereby to define new areas at risk and areas thatmight remain disease-free; and (3) to investigate how the distributionmight change under scenarios of future global warming.

Epidemiology and vectors

M. Baylis

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Epidemiology and vectorsABSTRACT BOOK

BLUETONGUE SURVEILLANCE METHODS IN THE UNITED STATESD. Dargatz(1), G. Akin(2), A. Green(1), M. Herrero(1), S. Holland(3), A. Kane1, D. Knowles(4), T. McElwain(5), K. Moser(6), E. Ostlund(6), M. Parker(1), E.Schmidtmann(7), A. Seitzinger(1), L. Schuler(8), G. Stevens(2), L. Tesar(9), L. White(10), L. Williams(11), N. Wineland(1) and T.E. Walton(1)

(1) USDA:APHIS:VS Centers for Epidemiology and Animal Health. E-mail: [email protected] (2) USDA:APHIS:VS Nebraska Area Office, (3) State ofSouth Dakota, (4) USDA:ARS Pullman, WA, (5) Washington Animal Disease Diagnostic Laboratory, Pullman, WA, (6) USDA:APHIS:VS National Veterinary ServicesLaboratories, (7) USDA:ARS Arthropod-borne Animal Diseases Research Laboratory, Laramie, WY, (8) State of North Dakota, (9) USDA:APHIS:VS South Dakota AreaOffice, (10) USDA:APHIS:VS North Dakota Area Office, (11) State of Nebraska

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Bluetongue viruses (BTV) have been and continue to be a significant impediment tointernational livestock trade. Various activities have been undertaken over the years todevelop information on the risk associated with movement of animals from certaingeographic areas to other areas thought to be free of the disease. The United Stateshas used a variety of methods to monitor the prevalence and distribution of BTVinfection in various parts of the country. Historically the U.S. has conductedsurveillance on alternate years by testing serum collected from adult animals atslaughter. This program has focused on the northeast segment of the country wherethe vector for the U.S. serotypes of BTV has been absent. More recently a BluetongueSurveillance Pilot Project (BSPP) was designed and implemented to evaluate a sentinelherd surveillance system for BTV and to evaluate the ecology of BTV and the vector ofBTV in the north-central part of the U.S.Cattle herds in North Dakota, South Dakota, and Nebraska were selected to participatein the study. Blood samples were collected from cows in each of the herds before andafter a vector season. Blood samples were tested for antibody to BTV using acommercial competitive ELISA (cELISA) test. When only a single animal on anoperation had a positive cELISA test, that sample was tested by virus neutralization(VN) to five serotypes (2, 10, 11, 13, and 17) of BTV. Operations were consideredpositive if they had two or more cELISA positive samples or a single cELISA positivesample that was positive on the VN. Insect traps were set on approximately half of theoperations to determine presence of Culicoides sonorensis, the primary North Americanvector of BTV. Data also were collected on operation management, proximity to vectorhabitats, and animal characteristics.Overall, 149 operations initially were enrolled in the study, mostly (93%) beef cattleoperations. Of the 144 operations where blood samples were collected prior to the

vector season, 7,226 samples were tested. Of these, 1,018 samples (14.1%) werepositive by the cELISA test and 54 (37.5%) operations were considered positive (2 ormore cELISA positives or 1 cELISA positive also positive by VN). Fewer operations werepositive in the northern reaches of the study area than in southern latitudes. Of the128 operations with blood samples collected after the vector season, 49 (38.3%) wereclassified as positive. Again, a similar geographic pattern of positives was seen asbefore the vector season. For the second sampling, 975 (17.3%) of the 5,627 animalstested were positive by the cELISA.Insect (vector) trapping was conducted on 68 operations. Of these operations, 31(45.6%) had catch samples with C. sonorensis. The spatial distribution of C.sonorensis-positive catch samples was similar to that of the positive blood samples asfewer samples were positive in the more northern and eastern parts of the study area.The results of the BSPP suggest the following:1. The prevalence of BTV infection in the more northern latitudes is very low or zero.2. The distribution of C. sonorensis is consistent with previously drawn maps.3. The sentinel herd system is useful to explore the spatial distribution of disease agents

and vectors and to generate hypotheses regarding the ecology of animal diseases.Plans for the future include further descriptive analysis of the data and epidemiologicmodeling to evaluate risk factors for BTV serostatus. In addition, the data will beanalyzed for geo-spatial factors (weather and topographic) related to vectordistribution.This project was an extensive effort made possible by the cooperation and resourcesfrom many state and Federal agencies and groups. The collaboration of these groups,the individuals within them, and the participating operation owners is gratefullyacknowledged.

D. Dargatz

ABSTRACT BOOK

BTV SURVEILLANCE IN AN EMERGING AREAA. Giovannini, P. Calistri, A. Conte, L. Savini, D. Nannini, C. Patta, U. Santucci and V. Caporale Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo, Italy. e-mail: [email protected]

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DIAGNOSTICSBT CONTROL

USING VACCINESCONTROL ANDTRADE ISSUES

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The penetration of bluetongue in densely animal populated areas with intensiveanimal trade and movement may cause a derangement of the national trade andhusbandry structure. This occurred in Italy after the detection of bluetongue inAugust 2000. In such conditions, surveillance can be the tool to delimitate asprecisely as possible the areas where virus circulation occurs and, therefore, needingmovement restrictions. Furthermore, surveillance data can provide usefulinformation to assess the risks originating from animal movement and trade.A structured surveillance system for bluetongue was implemented in Italy sinceAugust 2001. It was based on the periodical testing of unvaccinated sentinel cattleuniformly scattered throughout the Italian territory according to a grid of 400 km2

square cells. The initial number of sentinel sites and sentinel animals and the widthof the restricted area originated by a positive serological result in a sentinel animalwhere based on conservative criteria, e.g. animal movements were restricted in abuffer of 20 km radius around any positive serological result. The area of this bufferis about 1257 km2, equivalent to the area of three grid cells, irrespective ofserological results in the adjacent cells to the seroconversion. After the start ofvaccination campaigns, the sentinel surveillance system was also the only way toevaluate the effectiveness of campaigns and to estimate the incidence of infection inthe non immunized strata of ruminant populations.Data collected during a two years running of the system allowed to assess the riskposed by the adoption of less conservative criteria for the delimitation of infectedareas and by the progressive relax of movement restrictions in vaccinatedpopulations. Concerning the delimitation of restricted areas, a new approach wastested and validated in the field, based on a bayesian analysis of the positive and

negative results obtained by the testing of geographically referenced sentinelanimals. Concerning the risks related to animal movement, the surveillance dataallowed to perform risk assessments concerning the movement of slaughter andbreeding animals from vaccinated infected and surrounding areas to free areas.These risk assessments led to a consequent modification of the relevant legislation.Finally, a Montecarlo simulation model has been developed in order to simulatedifferent sentinel system scenarios and to decrease the total number of sentinelanimals and sites required by the surveillance system.The sentinel surveillance system was complemented by an entomologicalsurveillance system based on the use of a number of permanent blacklight traps runweekly all year round and a number of blacklight traps moved through the grid cellsduring the summer and fall each year. The aim of the entomological surveillance wasto define the maximum areal of vectors (traps operated during summer and fall) andtheir population dynamics (permanent traps). Furthermore, the permanent trapsystem can provide an early warning on the start of a new epidemic peak.The data of the entomological surveillance system were also analyzed to generateprobability maps of the presence of the main bluetongue vector (Culicoides imicola)and to define the geographical risk for bluetongue on a national basis, and toforecast the geographical distribution and the short-term diffusion of Culicoidesimicola in Sardinia, using spatio-temporal data. The detection since 2001 ofbluetongue outbreaks in absence of C. imicola and the recent identification of thebluetongue virus in Culicoides obsoletus midges, led to the development ofentomological research encompassing also other possible vector species (namely C.obsoletus and C. pulicaris).


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A. Giovannini

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THE BIOSYSTEMATICS OF CULICOIDES VECTOR COMPLEXES –UNFINISHED BUSINESSR. Meiswinkel, L. Gomulski, J.C. Delecolle, M. Goffredo and G. Gasperi Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Campo Boario, 64100, Teramo, Italy. Tel: 0039+0861-3321 (ext. 324); Fax: 0039+0861-332251; e-mail: [email protected]

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Morphology: “…the most interesting department of natural history, [which] may besaid to be its very soul”. Charles Darwin, 1859.The explosive outbreaks of bluetongue (BT) and African horse sickness (AHS) thatcyclically decimate livestock in the Mediterranean Basin are fuelled by the classicalAfro-Asiatic insect vector Culicoides (Avaritia) imicola Kieffer, 1913. In recent seasons(1998-2002) this link between C. imicola and the re-emergence of BT has beenreaffirmed following the large outbreaks to affect Italy. However, for the first time, thespread of this disease shifted marginally northwards and eastwards, and in addition,appeared (and persisted) in areas that are apparently devoid of C. imicola. This pointsclearly to the involvement of novel Culicoides vectors. Prime candidates are species inthe Obsoletus and Pulicaris Complexes, which (within the genus Culicoides) belong inthe subgenera Avaritia Fox, 1955 and Culicoides Latreille, 1809, respectively. Togetherthese two complexes comprise 20 species or more, many of which occur in abundancethroughout the Palaearctic Region, and attack both man and his livestock. Currentuncertainty as to precisely which species play a role in virus transmission is rooted ina lack of taxonomic clarity at the species level, and higher.Using both morphological and molecular data the systematics of these two subgenera(comprising the Imicola, Obsoletus and Pulicaris Complexes) are reviewed. Currentevidence indicates the Imicola Complex (subgenus Avaritia) in Italy (and throughoutthe Mediterranean Basin) to be represented by C. imicola only. However, at least sixmore species of Avaritia occur in Italy, but only three (C. obsoletus, C. scoticus and C.montanus) belong in the Obsoletus Complex. The remaining taxa, though previouslyreferred to this complex, and encompassing C. chiopterus (Meigen), 1830 and C.dewulfi Goetghebuer, 1936, should be placed elsewhere in Avaritia. Present usage ofthe subgenus Culicoides is also too broad as the handful of species related to C.pulicaris sensu stricto are usually ‘lumped’ with those related to C. grisescens Edwards,

1939 and C. fagineus Edwards, 1939. More correctly the latter two represent separatecomplexes (or subgenera). Of these, the Grisescens clade (= Cockerellii group in NorthAmerica) has already been raised (justifiably) to the level of subgenus, C. grisescenssensu stricto being the type species of Silvicola Mirzaeva & Isaev, 1990. The data alsoindicate that both C. remmi Damian-Georgescu, 1972 (subgenus Silvicola) and C.lupicaris Downes & Kettle, 1952 (subgenus Culicoides) should be resurrected fromsynonymy.The very recent discovery of large and extensive populations of C. imicola in Italyillustrates perfectly the paucity of our knowledge as regards the distribution of vectorCulicoides in the Mediterranean Basin. Ironically nearly 250 years have passed sinceLinneaus described C. pulicaris in 1758, and yet no monograph has appeared on the200+ species of Culicoides believed to occur across the Palaearctic Region. Althoughsome excellent regional studies exist, these have appeared in a number of languages,and vary greatly in style. Such regional “individuality” introduces a subjective elementinto species determinations, and errors follow. Harmonisation (and objectivity) will bestbe achieved using an integrated morphological and molecular approach, andconsidering the complexity of the task, should be undertaken using large collaborativeresearch networks. Simultaneously, the redefinition of species (and species complexes)should be linked to ecological studies on the life-habits of both the adult and immaturestages. This refined knowledge is essential if the distributions of known and potentialvectors are to be modelled accurately across the Palaearctic Region. Indeed it wouldnot be unwise to extend these investigations to cover the entire Holarctic as some ofthe 20 or more species of the Obsoletus and Pulicaris Complexes, and of the subgenusSilvicola found in North America, could also display increased vectorial competenceunder the influence of global warming.

R. Meiswinkel

ABSTRACT BOOK

INFECTION OF THE VECTORS AND BLUETONGUE EPIDEMIOLOGY IN EUROPEP.S. Mellor Institute for Animal Health, Pirbright Laboratory, Ash Rd., Pirbright, Woking, Surrey, GU24 0NF, UK • Tel: 00 44 1483 232 441; Fax: 00 44 1483 232 448; e-mail:[email protected]

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This paper describes some of the factors controlling the infection andtransmission of bluetongue virus (BTV) by vector species of Culicoides. Italso outlines certain important features of the recent bluetongue epizooticin the Mediterranean Basin, concentrating on those aspects involvingvector transmission and overwintering of the virus. The regions affected

by the outbreaks and the BTV serotypes involved are set out, thedistribution of the major vector, C. imicola is described and the impact ofnovel vector species of Culicoides and a possible overwinteringmechanism for the virus in Europe, are discussed.


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P.S. Mellor

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BTV SURVEILLANCE METHODS IN AN ENDEMIC AREA: AUSTRALIAL.F. MelvilleDepartment of Business, Industry and Resource Development • GPO Box 3000 • Darwin NT 0801 Australia • Ph: 61 8 8999 2251; Fax: 61 8 8999 2024; Email:[email protected]

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Surveillance for bluetongue viruses has been carried out at a number ofsites in the Northern Territory since 1980. The number of sites andintensity of monitoring has varied during this period.Since the commencement of the National Arbovirus Monitoring Program(NAMP) in 1993 between six and fourteen sentinel herds have beenmonitored each year. Each herd consists of 10 – 20 young bovines.At all sites, monthly serology has been conducted for the bluetongue,epizootic haemorrhagic disease and Palyam group viruses, Akabane virusand bovine ephemeral fever virus. Trapping for Culicoides sp. has alsobeen carried out at these sites on a regular basis. In 2002 and 2003sentinel herd monitoring has been supplemented with an annual

bluetongue survey of 18 – 24 month old cattle on selected properties. Thisis designed to enable the bluetongue free and surveillance zones to bemore accurately defined under the OIE guidelines.Weekly virus isolation has been performed on samples from the sentinelherd at the site of greatest known arboviral activity. This has enabled theisolation of all eight serotypes of bluetongue virus found in Australia. Nonew serotypes have been isolated since 1986, however genetic analysisof isolates obtained since 1991 has shown incursions of bluetongueviruses of Southeast Asian origin in 1992, 1994 and 1995. Marked annualvariation is seen in the number and identity of viruses isolated.

L.F. Melville

ABSTRACT BOOK

ENVIRONMENTAL EFFECTS ON VECTOR COMPETENCE AND VIROGENESIS OFBLUETONGUE VIRUS IN CULICOIDES: INTERPRETING LABORATORY DATA IN A FIELD CONTEXTB.A. Mullens (1), A.C. Gerry (2), T.J. Lysyk (3) and E.T. Schmidtmann (4)

(1) Department of Entomology, University of California, Riverside, CA 92521 (USA). Telephone (909) 787-5800, FAX (909) 787-3086, email [email protected](2) Department of Entomology, University of California, Riverside, CA 92521 (USA) (3) Agriculture and Agri-Food Canada, P.O. Box 3000, Lethbridge, Alberta T1J4B1 (Canada), (4) USDA-ARS, Arthropod-Borne Animal Diseases Research Laboratory, P.O. Box 3965, University Station, Laramie, WY 82071 (USA)

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35

Environmental factors can have profound effects on vectorial capacity ofa vector population for a disease agent. Environment governs dynamicsand intensity of vertebrate-vector contact in both time and space (e.g.seasonal vector population densities, biting rates, and feedingfrequencies). Temperature has a dramatic and consistent influence,altering vector developmental rates and life history parameters, andprobably modifying vector competence of the invertebrate host to somedegree (ability of a vector to be infected by and transmit a pathogen). Temperature is best known to control rates of virogenesis and thus timeto transmission for bluetongue viruses in Culicoides. Past research onenvironmental modification of vector competence and virogenesis in thissystem will be reviewed. Such work to date has been done in thelaboratory, and generally at constant temperatures. While laboratorystudies are indispensable, it is important to recognize the limitations oftranslating those data directly to more complicated and variable field

settings. We will delineate field biology studies that are both helpful andnecessary in applying laboratory-derived rate information. Studies shouldevolve towards iteratively moving from field to laboratory, as we attemptto understand complex epidemiological patterns. To this end, simulation models can be extremely helpful in identifying andhopefully predicting geographic and seasonal trends in virus occurrence.We will present field and laboratory data from the C. sonorensis-bluetongue system in North America, incorporating them into preliminarymodel estimates of virus prevalence and geographic occurrence along alatitudinal gradient. Geographic information systems technology is likelyalso to be helpful in understanding and predicting vector and virusoccurrence on a broader scale, especially in temperate latitude situationsthat typify sporadic or emerging transmission zones and thus are areas ofparticular concern for animal shipment.


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B.A. Mullens

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FAO’S ROLE IN BLUETONGUE DISEASEP. RoederAnimal Health Officer for Infectious Disease Emergencies. Empres Animal Health Service, Animal Production and Health Division - FAO. E-mail: [email protected]

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Recent years have witnessed a dramatic northwards extension of thebluetongue virus-infected and disease-affected area within theMediterranean basin and the Balkans. Serious losses have beenexperienced for the first time in some areas never before known to havebeen infected. Associated with this are also changes in vector distributionand possible changes in vector competence. Similarly in East Asia changesare occurring in virus and vector distribution which might be matched bynortherly extension of bluetongue virus infection in Central Asia andsoutherly extension in South America. These events are not fully definednor are the determinants of their occurrence understood. Whether or notthe events witnessed are due to climate change associated with globalwarming needs to be determined. The UN Food and Agriculture

Organization is concerned to ensure that bluetongue virus evolution ismonitored on a global scale to ensure that all countries, but especiallydeveloping countries and those in transition, can be prepared in advancefor future problems, if necessary. Concern over bluetongue virus does notstop there for there are several other vector-borne viruses and theirassociated diseases which could threaten livestock agriculture invulnerable areas for which bluetongue could serve as a useful model. FAOhas a responsibility to assist member countries to establishepidemiological systems and specific investigational expertise andcapacity to fill what could otherwise be lacunae in monitoring networks.Tactical and strategic responses to virus spread also merit unitedinternational action by FAO, OIE and partners in animal disease control.

P. Roeder

ABSTRACT BOOK

EPIDEMIOLOGY OF BTV AND EHDV IN WILDLIFE: SURVEILLANCE METHODSD.E. Stallknecht and E.W. Howerth Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, The University of Georgia, Athens, GA, USA 30605. Telephone: (706) 542-1741;FAX (706) 542 5865; e-mail: [email protected]

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The bluetongue viruses (BTV) and epizootic hemorrhagic disease viruses(EHDV) infect ruminant species of wildlife as well as domestic animals.Clinical disease in wildlife, however, is common only in North Americawhere mortality has been documented in white-tailed deer (Odocoileusvirginianus), mule deer (O. hemionus), pronghorn (Antilocapraamericana), and bighorn sheep (Ovis canadensis). To date, mortality hasbeen associated with BTV-10, -11, -13, and -17 and EHDV-1 and –2infections. With the exception of BTV-2, this represents all of the NorthAmerican BTV and EHDV serotypes. Surveillance methods directed at wildlife in the United States have reliedon standard serological and virus detection methods, and both passiveand active surveillance strategies have been effectively employed.Because these viruses potentially impact wildlife populations, considerablesurveillance has been done. This is especially true for white-tailed deer.Work related to BTV and EHDV in this species has not only led to a betterunderstanding of potential disease impacts but also has served to betterdefine the epidemiology of these viruses within the United States. Long-term surveillance of BTV and EHDV in white-tailed deer populations has

demonstrated geographical related patterns ranging from areas ofenzootic stability, characterized by high rates of annual infection with littleor no clinical disease, to areas of epizootic disease, characterized byinfrequent infection and high mortality rates. This surveillance also hasdemonstrated cyclic patterns of disease that are most likely driven byherd immunity. Wildlife-based surveillance for BTV and EHDV is effective in the UnitedStates for three reasons. First, the presence of disease and mortality hasresulted in interest from wildlife management organizations resulting inboth funding and cooperation in obtaining research and diagnosticsamples and mortality reports on a national level. Secondly, theavailability of samples from hunter-killed deer provides for very cost andtime efficient sampling of these populations. Finally, the broad distributionof these species, especially white-tailed deer and mule deer, providesnational coverage for such surveillance activities. Wildlife surveillance forBTV and EHDV benefits and adds value to these wildlife resources andmay provide unique opportunities to better understand the epidemiologyof these diseases.


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D.E. Stallknecht

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CULICOIDES GENETICS: IMPLICATIONS FOR UNDERSTANDINGTHE GLOBAL EPIDEMIOLOGY OF BLUETONGUE VIRUS INFECTION W.J. Tabachnick Florida Medical Entomology Laboratory, Department of Entomology and Nematology, University of Florida – IFAS, 200 9 th St., SE, Vero Beach, FL 32962, Tel: 772-778-7200, FAX: 772-778-7205, E-mail: [email protected]

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It is well established that the distribution of the bluetongue virusesthroughout the world is dependant on the presence of suitable species ofCulicoides able to become infected and transmit the viruses to susceptibleanimal hosts. Only a few different species of Culicoides have beenimplicated in bluetongue virus transmission. Each broad geographicregion of the world has a different Culicoides species as the primarybluetongue virus vector: Culicoides sonorensis in North America,Culicoides insignis in Central and South America, Culicoides imicola inAfrica, the Middle East, Asia, and Culicoides brevitarsis in Australia are theprinciple bluetongue vectors in these regions.The genetic and environmental differences between these species thatcontrol the traits enabling them to transmit the bluetongue viruses have

yet to be explored. The relationship between the different species and thedifferent bluetongue virus serotypes in a region is not understood. Finally,it is clear that even within specific vector species there is extensivevariation in the ability of individual insects and populations of insects totransmit the bluetongue viruses.Understanding the genetic and environmental controlling factors thatcontribute to the vector competence of Culicoides vector species of thebluetongue viruses is essential in order to assess the risk to livestock frombluetongue disease in a specific geographic region. These issues will bediscussed using research on the genetics of Culicoides sonorensis and theCulicoides variipennis complex to illustrate the complexity and importanceof this information to understand bluetongue virus epidemiology.

W.J. Tabachnick

ABSTRACT BOOK

ORAL SUSCEPTIBILITY OF SOUTH AFRICAN CULICOIDES SPECIESTO LIVE-ATTENUATED SEROTYPE-SPECIFIC VACCINE STRAINS OF BLUETONGUE VIRUS(BTV) – PRELIMINARY DATA G.J. Venter, G.H. Gerdes, P.S. Mellor and J.T. Paweska Department of Entomology, Onderstepoort Veterinary Institute. E-mail: [email protected]

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Preliminary results on susceptibility of South African Culicoides species tooral infection with the tissue culture-attenuated vaccine strains ofbluetongue virus (BTV) currently in use are reported. Field collectedCulicoides were fed on sheep blood-virus mixtures each containing one ofthe four serotype-specific vaccine strains of BTV (serotypes 1, 4, 9, 16) anda South African field isolate of BTV-1. Titres of vaccine strains in bloodmeals ranged from 5.1 to 6.1 log10TCID50/ml; the titre of the non-attenuatedfield isolate of BTV-1 was 7.1 log10 TCID50/ml. Vaccine strains recovery ratesin Culicoides assayed immediately after feeding were below 10%. Thisdemonstrates that the concentrations of vaccine viruses in blood mealswere too low to ensure that all individuals ingested detectable amounts ofvirus. Consequently it appears that the majority of individuals assayed werenot effectively exposed to a challenge virus. Thus, the oral susceptibility ofCulicoides to infection with BTV vaccine strains might be higher than thosedetermined in this study.Of a total of 10.506 Culicoides fed 6.540 survived a 10 day extrinsicincubation period at 23.5°C. Due to bacterial and fungal contamination,results could be obtained for only 6.004 midges assayed. Despite relativelow virus titres in the blood meals, the two known South African BTV

vectors, C. imicola and C. bolitinos, become infected with at least one ofthe four of BTV vaccine strains. Of a total of 124 Culicoides that testedpositive for the virus, 65 individuals become infected after feeding onblood containing vaccine strains, and the remaining 59 following feedingon blood containing field isolate of BTV-1. For the vaccine strains theinfection prevalence ranged from 11.0 % in C. bolitinos fed on a bloodcontaining 6.1log10/ml of BTV-1 to 0.3% in C. imicola fed on a bloodcontaining 5.3 log10/ml of BTV-4. The infection rate for C. imicola and C.bolitinos fed on field isolate of BTV-1 was 9.5% and 36.0%, respectively.In Culicoides infected with the vaccine strains of BTV their titres rangedfrom 0.65 to 4.4 log10TCID50/midge. Although in most infected midges thereplication level of vaccine strains was below the postulated threshold fora systemic infection with an orbivirus (>2.5 log10TCID50/midge), someindividuals replicated vaccine strains to high titres.The high level of viral replication in some individual midges suggests thatthe BTV vaccine strains can be potentially transmitted from vaccinated tonon-vaccinated animals. However, practical significance of our findingsrequires further assessment.


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G.J. Venter

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POSSIBLE OVERWINTERING MECHANISM OF BLUETONGUE VIRUS IN VECTORSD.M. White (1), W.C. Wilson (1), C.D. Blair (2) and B.J. Beaty (2)

(1) USDA, ARS, Arthropod-borne Animal Diseases Research Laboratory, PO Box 3965, Laramie, WY 82072, USA. voice: 307-766-3601; fax: 307-766-3500; e-mail:[email protected] (2) Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Colorado State University

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The overwintering mechanism of bluetongue virus (BTV) has eludedresearchers for many years. While overwintering in the vertebrate hosthas been the main hypothesis, it has been shown that severalarboviruses overwinter in their invertebrate vectors. OverwinteringCulicoides sonorensis larvae were collected from long-term study sites innorthern Colorado and examined for the presence of BTV nucleic acid byRT-nested PCR. Sequences from the S7 segment of BTV RNA weredetected in 17 of 56 (30%) pools comprised of larvae and pupaecollected in 1998, and in 32 of 319 (10%) pools comprised of adultsreared from larvae collected in 1996. BTV was not isolated from the adultpools. Additionally, cell lines derived from culicoid embryos collected atthe same site, or derived from material collected during a BTV outbreak,

were positive for BTV nucleic acid. Interestingly, in contrast to the S7segment, the L2 RNA segment could only rarely be detected in any of thefield-collected larvae, and was not detected in the culicoid cell lines.These data suggest that BTV may not require abundant expression of theouter coat genes to persist in the insect vector. This could also explainthe low rate of isolation of virus from insects. If the vertebrate cellreceptor ligand VP2 (which is encoded by L2) is expressed at very lowlevels in the insect, traditional vertebrate cell-based isolation methodswould be inefficient until the virus had amplified itself sufficiently toexpress all virus genes required for vertebrate cell infection. Furtherresearch in this area will define and characterize the role of the vector inthe overwintering of BTV, and will help in focusing control efforts.

D.M. White


TEMPORAL ACIVITY OF BITING MIDGES (DIPTERA: CERATOPOGONIDAE) ON CATTLENEAR DARWIN, NORTHERN TERRITORY, AUSTRALIAG.A. Bellis, L. Melville and N. Hunt Northern Australia Quarantine Strategy, GPO Box 3000 Darwin, NT Australia 0801. Ph: +61 8 8999 2345; Fax +61 8 8999 2312; Email: [email protected]

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The activity of 9 species of biting midges on cattle was recorded in the lateafternoon, evening and early morning at a site near Darwin, NT. Somespecies, C. actoni and Lasiohelia sp., were mostly active during daylighthours while others, C. peregrinus, C. bundyensis and C. brevipalpis, werealmost exclusively nocturnal. C. brevitarsis was crepuscular but activity

peaked prior to sunset which is slightly earlier than that observed at othersites. The remaining species, C. fulvus, C. marksi and C. oxystoma did nothave a definite peak period of activity but were found on cattle before andafter both sunset and dawn.

B 1

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B 2ABSTRACT BOOK

FACTORS AFFECTING THE SPREAD OF CULICOIDES BREVITARSIS AT THE SOUTHERNLIMITS OF ITS DISTRIBUTION IN EASTERN AUSTRALIAA.L. Bishop, L.J. Spohr and I.M. BarchiaNSW Agriculture, Locked Bag 26, Gosford NSW 2250, Australia. Tel: +61-2-4348-1928; Fax: +61-2-4348-1910; Email: [email protected]

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The southern limits of the bluetongue vector Culicoides brevitarsis Kieffer(Diptera: Ceratopogonidae) in eastern Australia are found in New SouthWales (NSW). The vector is endemic on the northern/mid-northerncoastal plain. It moves south in summer, but normally retains a coastaldistribution. These movements depend on temperature, wind speed, winddirection, and the distance to be travelled from the endemic area. Thevector moves at different rates in different areas and it was thought thatthe rate of dispersal was related to geographical features, such as largeurban areas to the south and the altitude/escarpment of the GreatDividing Range to the west. The Range would act as a physical barrierslowing movements to the western slopes and plains where there are highconcentrations of susceptible sheep or cattle.

An eight-year study has been made of C. brevitarsis movements from theendemic coastal plains, and up coastal valleys of mid-northern NSW to thetop of the Range. Models were developed to show that dispersal can occuron more than one occasion, and can be explained by the distance to betravelled, and the altitude of sites of destination. Altitude was found todelay movement by about 5 weeks for the first and 11 weeks for thesecond time that the vector reached the top of the Range atapproximately 1000 m of altitude. Southern and western movements cannow be predicted from a series of models. Periods of possible activity atsites of destination, and the probability of survival through winter at eachnew site, can be estimated from temperature data. Activity, subsequentmovements, and survival at the top of the Range are extremely limited.

Epidemiology and vectors B 3ABSTRACT BOOK

IMPROVING LIGHT TRAP EFFICIENCY FOR CULICOIDES SPPWITH LIGHT EMITTING DIODES (LEDs)A.L. Bishop, R. Worrall, L.J. Spohr, H.J. McKenzie and I.M. Barchia NSW Agriculture, Locked Bag 26, Gosford NSW 2250, Australia. Tel: +61-2-4348-1928; Fax: +61-2-4348-1910; Email: [email protected]

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Light traps with incandescent globes are used to monitor the presence andabundance of Culicoides spp. responsible for the transmission ofbluetongue and other arboviruses to livestock in Australia. Modificationsto the traps have been made recently with the addition of stainless steelfittings and substantial changes to the circuitry. The result is traps thatare lightweight yet robust enough to enable their ease of use in harshenvironments of the Australian outback. These traps have allowedefficient monitoring in remote areas for many years. However, recentadvances in the development of light emitting diodes (LEDs) haveproduced LEDs that are more energy efficient and can produce a greaterphoton flux than incandescent globes, making them even more suitablefor battery operation. The LEDs can also provide closely defined outputsover narrow or wide spectral ranges which can be comparedexperimentally. Responses of eight Culicoides species to red, yellow, green and blue LEDs

were assessed. Three species were attracted to blue light, and responsesto blue and green light could not be separated for four other species. C.brevitarsis, the main vector of bluetongue in cattle in Australia, respondedsignificantly to green light. There was a significant increase in thefrequency of successful collections of C. brevitarsis in an environmentwhere it can be difficult to detect its presence. Further, the numberscollected were significantly higher than with incandescent globes. Catchesof C. brevitarsis were also related to the intensity of green LEDs whichwere always more effective than the incandescent globes at intensitiesbetween 46% and 142% of the incandescent intensity. Further field evaluations in areas with a wide range of vector densities andenvironmental conditions are now planned to confirm that these modifiedlight traps can be used routinely to increase the sensitivity and efficiencyof vector monitoring.

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BLUETONGUE SURVEILLANCE IN CAMPANIA REGION, ITALY:GIS AS A TOOL TO CREATE RISK MAPSV. CaligiuriOsservatorio Epidemiologico Veterinario Regione Campania,Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute, 2 Portici 80055 – Napoli, Italia. Tel.081 7865270; fax 081 7865267; e-mail: [email protected]

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Aim of this project is the implementation of a Geographic InformationSistem (GIS) in order to study areas of the Campania Region that arepotentially at risk for Blue Tongue (BT). The work has been divided inthree steps.As a first step we studied the habitat of Culicoides imicola with regard toenvironmental features (ground and climate) and presence of susceptibleanimals. Firstly satellite imaging of the Region, has been used to identifyareas (grasses, woods, water fields) with increasing risk based on theprobability of presence of culicoides and susceptible animals. Taking intoaccount slope, sun exposure and altimetry, various maps have beenobtained by the Regional DEM (digital earth model).As a second step we performed a meteorological study. Temperatures,rainfall and humidity reported in the last three years were linked to theclimate requirements for the survival of Culicoides spp. Data collected

from the weather stations have been entered in ArcGis and interpolatedto obtain continuous values for every pixel of the raster image.Therefore maps accounting for different territorial, geographical andenvironmental patterns have been created. Four zones have been defined:marshy/cold, slightly marshy/cold, marshy/warm and slightlymarshy/warm. Then data from culicoides-traps have been checked andrelated to the population density of sheep, goats, bovines and buffalos, inorder to obtain a definitive risk map.As a final step a validation of the risk maps was carried out. The mapswere compared to the results of BT surveillance data of 2002 i.e. monthlymean number of Culicoides captured and BT outbreaks. Outbreaks datawere used in terms of presence/absence in each area, whereas data ofcaptures were divided in two periods and the monthly mean number ofcaptures for each period and each trap was counted.

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BLUETONGUE IN ITALY – part I P. Calistri, A. Giovannini, A. Conte, D. Nannini, U. Santucci, C. Patta, S. Rolesu and V. Caporale Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo – Italy. Phone: +39 0861-332232; Fax: +39 0861-332251; email: [email protected]

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Bluetongue in the Mediterranean was first recorded in Cyprus and Israelin 1943. Some authors claim that it has been present in theMediterranean since at least 1924. In Turkey, Syria, Israel and Egypt,bluetongue is considered endemic and outbreaks involving serotypes 2, 4,6, 9, 10 and 16 are periodically reported. In the years 1956-1960, anepidemic involving BTV serotype 10 occurred in Spain and Portugal, whereit caused the loss of about 179.000 sheep. In Greece, epidemics havebeen intermittently reported since 1979, principally on the islands facingthe Turkish coast, namely Lesbos and the Dodecanese islands of Léros,Kos and repeatedly Rhodes.Since 1999, bluetongue has been detected also in continental Greece, andin the south-east of Bulgaria adjoining both Turkey and Greece. SinceAugust/September 2000 the infection has spread progressively throughthe Balkans [affecting Bosnia, Croatia, Kosovo, Albania, Macedonia(FYROM) and Serbia and Montenegro]. The serotypes that circulate in theBalkans were BTV 4, -9 and –16, of which BTV 9 in the whole region, theothers only in Greece. In December 1999, bluetongue (serotype 2) wasrecorded in Tunisia and, the following summer, also in Algeria. In August 2000, bluetongue disease, involving serotype 2, was reportedfor the first time in Italy, in the Balearic Islands (Spain), and in Corsica

(France). By the fall of 2000 a second serotype (BTV-9) had beendiscovered to occur in southern Italy. This incursion of virus into theNorth-Western Mediterranean region resulted in the largest epidemic ofbluetongue ever to affect Europe. The features of this epidemic differsignificantly from those observed in previous outbreaks of bluetongue.The main differences are: 1) its penetration further northwards(reaching the 44th latitude in both Italy and the Balkans); 2) itspersistence across four years in a some areas (in southern Italy and inthe Balkans) brings with it the serious risk of endemisation across awide geographical area, and 3) its successful invasion of areasseparated from previously infected ones by wide expanses of sea(Sardinia, Sicily, Calabria, and the Balearic islands).The present paper describes the pattern of the spread of infection acrossItaly before the vaccination of susceptible sheep and cattle populationswas introduced, and the seasonality observed. Hypotheses are presentedon the possible role of certain ecological conditions (climate, soil andvectors) on the incidence of the disease and the overwintering ofinfection. Some hypotheses are also advanced as to the possible originsand modes of introduction of bluetongue virus into Italy.

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USE OF A MONTECARLO SIMULATION MODEL FOR THE RE-PLANNINGOF BLUETONGUE SURVEILLANCE IN ITALYP. Calistri, A. Giovannini, A. Conte and V. Caporale Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo (Italy). Phone: +39 0861-332232; Fax: +39 0861-332251; e-mail: [email protected]

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Since August 2000 Italy has lost almost 500,000 sheep in the largestincursion of bluetongue (BT) yet to affect Europe. The Italian BTsurveillance system is composed by a serological and an entomologicalprograms. The main objective of the serological program is an earlydetection of BT virus (BTV) circulation in the protection and surveillancezones (and throughout the remaining of Italy) by periodical testing ofmore than 30,000 sentinel cattle distributed across Italy.The sentinel network is in force since October 2001. During this period, itdemonstrated to be accurate and timely in detecting viral circulation in theterritories. However, the periodical testing of more than 30,000 sentinelanimals across the whole Italy during year 2002 required a significanteffort by veterinary services and a costly engagement by farmers.

Therefore, a Montecarlo simulation model has been developed in order tosimulate different sentinel system scenarios, comparing each other theexpected results. The model has been validated using data derived fromthe serological surveillance activities in Sardinia during October 2001 –December 2002.The model permitted to identify a combination of numbers of sentinelherds and sentinel animals within herds able to give the same confidencefor the overall system to detect infection, if present, with a lesser burdenfor both veterinary services and farmers. In fact, the comparison amongdifferent simulation scenarios demonstrated the equivalence between thesentinel network in force at that time, and a sentinel system based on lessanimals equally distributed on the territory.

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TOWARDS THE IDENTIFICATION OF POTENTIAL INFECTIOUS SITESFOR BLUETONGUE IN ITALY: A SPATIAL ANALYSIS APPROACHBASED ON THE DISTRIBUTION OF C. IMICOLAA.Conte, C. Ippoliti, P. Calistri, S. Pelini, L. Savini, R. Salini, M. Goffredo and R. Meiswinkel Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo, Italy – tel. +39 0861 332336; fax +39 0861 332251;email: [email protected]

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Geographic Information Systems (GIS) have become an important tool togain a clearer understanding of information relating to the Earth and itsenvironment and for the compilation and analyses of all available geo-spatial data. Whatever the application, all the geographically relatedinformation available can be inputted and prepared in a GIS, so that userscan combine and display that information which is of interest toinvestigate specific problems. GIS and remote sensing technologies arebeing used increasingly in epidemiology, especially in the study of spatialand temporal patterns of infectious diseases. A great deal of research onthe distribution of bluetongue (BT) and its principal vector C. imicola inthe Mediterranean basin has recently been spawned following theincursions of the disease into southern Europe between 1998 - 2002.Special attention has been focused on modelling the diffusion of C. imicolaand of the effect of climatic and geographic factors on its presence. TheNational Reference Center for Veterinary Epidemiology (Teramo – Italy)developed a grid-based GIS to identify potential infectious sites forbluetongue in Italy through the analysis of areas most suitable for thepersistence of C. imicola. In particular, a Spatial Process Model (SPM) hasbeen created to identify those areas into which bluetongue virus (BTV) ismost likely to be spread by the vector C. imicola. The following geographicand climatic grids and datasets have been taken into consideration toexplain the spatial interactions: Digitalized Elevation Model, landuse,remote sensing images describing the Normalized Difference Vegetation

Index (NDVI), aridity index, soil type, temperature, and animal populationdensity. The model has been developed through four steps: 1) datasetinput; 2) manipulation of datasets to create new information; 3)reclassification of each dataset to a common scale, and 4) the weightingand combination of datasets for the identification of suitable locations.Since NDVI and temperature are seasonally dependent, different periodsof the year have been selected to represent possible scenarios. Newdatasets have been created through spatial analyses and are included inthe model: 1) a grid with animal population density information; 2) aslope grid derived from the elevation model, and 3) surface grids fortemperatures calculated through geostatistical interpolation methods(ordinary kriging). The classes of each dataset have been ranked givinghigher values to those in which C. imicola has been found most frequentlyduring the widespread entomological surveillance program. No weightshave been applied when obtaining the final risk maps. The comparisonbetween the geographic model’s results and those of actual BT outbreakswas then made using data collected over the past two years.The BToutbreaks spread also into areas where C. imicola was not found, andwhere geographic and climatic conditions appear not favourable for thepresence of this vector. The correctness of a spatial model should beverified through sampling of the potential sites in the field, and in theprocess perhaps also identify previously unconsidered variables that mayhelp sustain the insect vector locally.

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PROTECTION OF CATTLE FROM CULICOIDES SPP. IN AUSTRALIA BY SHELTER ANDCHEMICAL TREATMENTSW.M. Doherty, A.L. Bishop, L.F. Melville, S.J. Johnson, G.A. Bellis and N.T. Hunt Queensland Department of Primary Industries, Oonoonba Veterinary Laboratory, Box 1085/Abbott St Townsville Q 4810 Australia. Ph Int: + 61 7 47222609; FaxInt: + 61 7 47784307; e-mail: [email protected]

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Australia exports live cattle to a number of other countries. Exports toarbovirus-sensitive markets could be enhanced if cattle could be safelyexported from ports in northern Australia. However this would requirecattle to be transported to the ports by truck or train through areas wherethere is risk of infection with arboviruses transmitted by Culicoides spp.C. brevitarsis, the most widespread of the Australian vector species isconsidered exophagic. The relevant habits of other vector species havenot been studied. Improvised covers on livestock transport compartmentsmay reduce feeding on cattle by exophagic Culicoides. Some chemicaltreatments for livestock ectoparasites have been shown to inhibit feedingby various Culicoides. Trials were conducted in 3 separate regions ofAustralia to investigate the potential for additional chemical treatmentsand improvised shelters to reduce feeding by Culicoides on cattle andminimise the risk of arbovirus transmission.In New South Wales, fewer C. brevitarsis were collected in vacuumsamples taken from cattle held in pens covered by a tarpaulin than fromsamples from similar cattle in uncovered pens. However in a subsequenttrial which used light traps over penned cattle to collect the Culicoides,this cover failed to reduce the numbers of C. brevitarsis. Pens with wallsbut no roof also failed to reduce C. brevitarsis numbers but pens with

walls and roof yielded fewer C. brevitarsis in this latter trial. In Northern Territory, the numbers of C. brevitarsis, C. actoni and C.fulvus in vacuum collections from cattle held in pens with tarpaulincovers were no lower than in similar collections from uncovered pensalthough the covers reduced the numbers of some other Culicoidesspecies. In a later trial, light traps under similar covers but in theabsence of cattle generally collected fewer of a variety of species thansimilar traps in the open.In Queensland, simulated transport compartments comprising slattedwalls around penned cattle were tested with and without tarpaulin covers.The slatted walls alone reduced the numbers of C. wadai in vacuumcollections from the cattle but not the numbers of C. brevitarsis. Slattedwalls and cover reduced the numbers of C. brevitarsis and reduced thenumbers of C. wadai more than walls alone. The chemical treatments“Flyaway” (a blend of repellents) and fenvalerate both reduced thenumbers of C. brevitarsis and C. wadai in vacuum collections from pennedcattle up to 50hours post-treatment. Improvised shelters and chemical treatments can reduce but not yetentirely eliminate the risk of Culicoides spp. transmitting viruses to cattlein Australia.

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EPIZOOTIOLOGIC CONTROL OF BT DISEASE IN BULGARIA IN 2002G. Georgiev, N. Nedelchev, Y. Ivanov and E. VelevaNational Diagnostic and Research Veterinary Medical Institute – Sofia, Bulgaria; E-mail: [email protected]

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According to the National Surveillance Program for the control of BTDisease in 2002 serum surveillance were performed in 22 sentinel serum-negative animal herds located in Western Bulgaria in a distance of 40 km.outside the settlements affected during the epizooty in 2001. Another 42sentinel villages (herds) located in South Bulgaria in a 10 km border-stripzone in Bourgas, Yambol, Haskovo, Kardjali, Smolyan and BlagoevgradDistricts have been established. The implementation of the 2002 programwas started on 15th of April and continued till 15 th of November 2002.Evidences for presence of active BTV infection on the whole territory ofBulgaria have not been found on the basis of the complex viral,serological, epidemiological and clinic-pathological investigations in aperiod before 26th of August 2002. In the period before 26-th of August2002 was no trans-border penetration of BTV in Bulgaria by persistentlyinfected animals or by infected Culicoides sp. as well. More than 7200serum samples were tested with negative resultat.On 26th of August 2002 BTV serum positive sentinel animals were detectedwhen the regular samples from animals located closely to the SouthBulgarian border have been investigated. Consequently serum positive bycELISA animals were found in more than 20 villages inside these two

districts, but clinical symptoms of the disease have not been occurred.A Culicoides trapping program were performed in 23 of out 58 quadrants50 x 50 km each on the whole territory of the country and total 92culicoides catches were done. In a three year Culicoides surveillance inBulgaria no C. imicola have been detected. Dominating Culicoides specieswere C. obsoletus, C. pulicaris and C. punctatus. In Culicoides catchesfrom South-East part of Bulgaria trapped in August 2001 C. puncticolis forthe first time have been recorded.The most spread Culicoides species trapped in these regions were C.obsoletus followed by C. pulicaris and C. punctatus.We performed phenology investigations in 2 villages – Vacsevo andBersin, Kiustendil district affected with BTV in a last Bluetongue epizootyin 2001. We started to trap the Culicoides from 1st of March 2002 till 15 th

November 2002. The active Culicoides seasonal dynamics in 2002 started in a third decadeof April. Roughly, it is possible to estimate two or three picks - during thesecond half of May, in August and probably in beginning of October of theiractivity. Their seasonal activity calm down in the second half of November.

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BLUETONGUE IN ITALY – part II A. Giovannini, P. Calistri, D. Nannini, C. Paladini, U. Santucci, C. Patta and V. Caporale Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo Italy. E-mail: [email protected]

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In summer 2000, Bluetongue infection entered Italy and caused a wideepidemic involving in 3 years a total of 10 southern and central Italianregions. From the date of the first case (18th August 2000) to 14th May2001, when the minimum in the epidemic curve was observed, a total of310.234 animals in 6.869 flocks of 3 regions were involved. During theperiod from 15th May 2001 to 14th April 2002, when a second epidemicwave swept through central and southern Italy, a total of 323.635 animalsin 6.807 flocks of 7 regions were involved. During years 2000 and 2001virtually no susceptible ruminant was vaccinated, despite the ItalianMinistry of Health ordered on 11th May 2001 the vaccination of susceptibleanimals of all domestic ruminant species (i.e. sheep, goats, cattle andwater buffaloes) in infected and surrounding areas. The vaccinationstrategy stemmed from a risk assessment demonstrating the possibility toprevent both most direct economic losses and to significantly reduce alsovirus circulation through.The vaccination of the target populations started in January 2002.Nevertheless, in July 2002 when the new epidemic peak started, different

proportions of vaccinated populations were achieved in the variousregions. The different levels of vaccination had clear consequences on thedisease and infection spread. The relationships between vaccination coverage of the target populationsand (1) animal losses due to the disease, or (2) virus circulation asdetected by the sentinel surveillance system, are analyzed. The effectiveness of the vaccination campaign to decrease the restrictedareas and consequently indirect losses is analyzed and discussed.Furthermore, at the end of 2002 a new risk assessment led to (1) theauthorization of movement of vaccinated slaughter animals from infectedareas where at least 80% of susceptible population was vaccinated and(2) a new approach to define the areas under movement restrictions.Following the 2nd vaccination campaign (January-May 2003) a further riskassessment and the results of vaccination trials performed in controlledand in field conditions open the possibility to devise a procedure for thetrade of breeding animals.

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CULICOIDES (DIPTERA: CERATOPOGONIDAE) IN ALBANIA:RESULTS OF THE 2002 ENTOMOLOGICAL SURVEY FOR BLUETONGUEM. Goffredo, J.C. Delecolle, G. Semproni and A. LikaIstituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” , Campo Boario, 64100 Teramo, Italy. Tel.: +39 (0)861 332285, Fax: +39 (0)861332251, e-mail: [email protected]

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A survey for Culicoides Latreille, 1809, was made in Albania in 2002 toestablish whether Culicoides imicola Kieffer, 1913, the main vector ofbluetongue virus (BTV) in the Mediterranean Basin, or any othersuspected vector species, was present. The collections and the analyseswere performed according to the protocols of the National ReferenceCentre for Exotic Diseases (CESME), Italy. A total of 43 catches weremade in October and November in 15 districts: Bulqizë, Devoll, Diber,Durrës, Fier, Gjirokastër, Kolonjë, Korçë, Librazhd, Mat, Permet, Pogradec,Shkodër, Tirana, Tropojë. With the only exception of Kolonje, the number of midges/catch wasalways less than 400. In Kolonje district, where three catches wereperformed, the number of midges/catch ranged between 2,620 and

20,884 and 99% of the midges belonged to the Obsoletus complex.Culicoides imicola was never captured during the survey. The species of Culicoides identified in the collections were: C. alazanicusDzhafarov, 1961, C. cataneii Clastrier, 1957, C. circumscriptus Kieffer,1918, C. festivipennis Kieffer, 1914, C. gejgelensis Dzhafarov, 1964, C.kibunensis Tokunaga, 1937, C. maritimus Kieffer, 1924, C. newsteadiAusten, 1921, C. nubeculosus Meigen, 1830, C. obsoletus Meigen, 1818,C. odiatus Austen, 1921, C. pulicaris Linnaeus, 1758, C. punctatusMeigen, 1804, C. puncticollis Becker, 1903, C. riethi Kieffer, 1914, C.saevus Kieffer, 1922, C. scoticus Downes & Kettle, 1952, C. sejfadineiDzhafarov, 1958, C. submaritimus Dzhafarov, 1962, C. univittatusVimmer, 1932.

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DISTRIBUTION AND ABUNDANCE OF CULICOIDES IMICOLA, C. OBSOLETUS COMPLEXAND C. PULICARIS COMPLEX (DIPTERA: CERATOPOGONIDAE) IN ITALY M. Goffredo, A. Conte and R. Meiswinkel Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” , Campo Boario, 64100 Teramo, Italy. Tel.: +39 (0)861 332285, Fax: +39 (0)861332251, e-mail: [email protected]

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During the years 2000-2002 approximately 16,000 light-trap collectionswere made for Culicoides throughout Italy, and a detailed distribution mapfor C. imicola Kieffer, 1913 compiled. This principal vector of bluetonguevirus (BTV) was captured in 11 regions (Abruzzo, Basilicata, Calabria,Campania, Lazio, Liguria, Molise, Puglia, Sardinia, Sicily and Tuscany), andup to latitude 44° 30' (in the province of Genova, Liguria). However, in someareas where clinical BTV occurred amongst sheep and fatalities resulted, nospecimens of C. imicola could be captured; in such instances bluetongue

virus was successfully isolated from biting midges of the Obsoletus complex.It thus became paramount to map also the distribution of this complexacross Italy, and also that of the Pulicaris complex (which has also beenpreviously implicated in the transmission of orbiviruses to livestock).Accordingly we here report on, and map the distribution and abundanceof the Obsoletus and Pulicaris Complexes in Italy, as determined fromapproximately 3,000 light-trap collections analysed. We compare theseresults to those obtained for C. imicola.

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ENTOMOLOGICAL SURVEILLANCE OF BLUETONGUE IN ITALY: METHODS OF CAPTURE,CATCH ANALYSIS AND IDENTIFICATION OF CULICOIDES BITING MIDGESM. Goffredo and R. Meiswinkel Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” , Campo Boario, 64100 Teramo, Italy. Tel.: +39 (0)861 332285, Fax: +39 (0)861332251, e-mail: [email protected]

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To elucidate the epidemiology of vector-borne diseases that can affectlivestock in the Mediterranean Basin and elsewhere, it is essential to obtaina fairly intimate understanding of the life-cycle and habits of the vectorinsect involved. The most important first step is to be able to identify thevector accurately; unfortunately the identification of insects is a “yes/no”and not a “maybe” activity. Only accurate identifications provide the firmfoundation for subsequent studies (all of which depend upon informationthat is reliable). These studies include the following: 1. Unravelling thebiology of the vector (with special emphasis on defining its larval habitat);2. Mapping its geographic distribution (the rock upon which predictive riskmaps are built); 3. Establishing its periods of peak flight and feedingactivity (both daily and seasonal); 4. Assessing its abundance levelsaround livestock; 5. Knowing its specific (or catholic) host preferences andbiting rates, and 6. Clarifying in the laboratory its capacity for transmittingsuch orbiviral livestock diseases as bluetongue (BT) in sheep, goats andcattle, and African horse sickness (AHS). Another purpose of such investigations is to feed an epidemiological

surveillance system. As this depends most heavily upon the collection ofspecimens in the field it becomes necessary to establish (once again indetail) what kinds of information are required, and how they are to becollected. This requires, in turn, that the method (and instrument) ofcapture be standardised, so that all data are as complete as possible, arecomparable, and are informative at many levels. Within the surveillance system for bluetongue in Italy, the National Centrefor Exotic Diseases (CESME) is leading an intensive and countrywidesurvey for Culicoides (Diptera: Ceratopogonidae), using standardisedmethods and protocols developed in collaboration with the OnderstepoortVeterinary Institute, South Africa. These have now also been implementedoutside of Italy in Malta, Croatia, Albania and Romania. This systemincludes the field protocols developed for the collection of Culicoides, andthe laboratory protocols developed around the analysis and computer-based recording of all field data. Finally, we provide an “easy-key” for therapid identification of C. imicola, and for species that belong to either theObsoletus or to the Pulicaris complex.

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ENTOMOLOGICAL SURVEILLANCE FOR BLUETONGUE IN MALTA:FIRST REPORT OF CULICOIDES IMICOLA KIEFFERM. Goffredo, M. Buttigieg, R. Meiswinkel, J.C. Delecolle and S. Chircop Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” , Campo Boario, 64100 Teramo, Italy. Tel.: +39 (0)861 332285, Fax: +39 (0)861332251, e-mail: [email protected]

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A survey for Culicoides Latreille, 1809, was made on Malta in 2002 toestablish whether Culicoides imicola Kieffer, 1913, the main vector ofbluetongue virus (BTV) in the Mediterranean Basin, or any othersuspected vector species, was present. The collections and the analyseswere performed according to the protocols of the National ReferenceCentre for Exotic Diseases (CESME), Italy. A total of 84 catches weremade between May to October at six permanent sites: Mellieha, Rabat,San Gwann, Zejtun (Malta island), Gharb and Sannat (Gozo island). Thetraps were placed near cattle (in 4 farms), cattle and sheep (1 farm,Rabat) and at sheep and goats (1 farm, Mellieha).More than 7,000 Culicoides were collected and the highest number of

midges/catch was 1,726. Culicoides imicola was identified to occur onMalta for the first time in October 2002 and was found at four sites (SanGwann, Sannat, Gharb and Mellieha) but at very low abundance levels(less than1%). Culicoides paolae Boorman, 1996 was the most widespread and abundantspecies (more than 80% of total Culicoides). Other species of Culicoides identified in the collections were: C.submaritimus Dzhafarov, 1962, C. cataneii Clastrier, 1957 or C.gejgelensis Dzhafarov, 1964, C. circumscriptus Kieffer, 1918, C.maritimus Kieffer, 1924, C. kurensis Dzhafarov, 1960, C. newsteadiAusten, 1921, C. schultzei complex, and C. obsoletus complex.

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LABORATORY SURVIVAL AND BLOOD FEEDING RESPONSE OF WILD-CAUGHTCULICOIDES OBSOLETUS COMPLEX (DIPTERA: CERATOPOGONIDAE) THROUGH NATURALAND ARTIFICIAL MEMBRANESM. Goffredo, G. Romeo, F. Monaco, A. Di Gennaro and G. Savini Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” , Campo Boario, 64100 Teramo, Italy. Tel.: +39 (0)861 332285, Fax: +39 (0)861332251, e-mail: [email protected]

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The Obsoletus complex included a group of Culicoides species belonging to thesubgenus Avaritia (Diptera: Ceratopogonidae). Many evidences indicated species ofthe Obsoletus complex as suspected vectors of bluetongue virus (BTV). They havebeen found associated with BT outbreaks in areas where the main vector, C.imicola, was absent. They have also been shown capable of sustaining BTVreplication when inoculated intrathoracically and, more importantly, serotypes 4, 2and 9 of BTV were isolated from parous individuals caught during clinicaloutbreaks. Being abundant and wide distributed they might play an important rolein spreading BTV in Europe.A crucial step to assess the vector competence of a Culicoides species is to isolatethe virus 10 days after artificial feeding. According to the literature, up to dateall the attempts to feed Culicoides obsoletus under laboratory conditions havebeen unsuccessful. The aim of this study was to set up methods for improvingthe insect survival rates and feeding under laboratory conditions. In late summer2002, alive wild-caught midges of the Obsoletus complex were collected usingblacklight traps placed nearby a horse stable in Teramo (Abruzzo, Italy). In thesame site, 2 years of daily catching showed already that the Obsoletus complexrepresented 90-95% of the total Culicoides and that the males present in thecatches belonged to at least two species of the group, Culicoides obsoletus s.s.and C. scoticus. For the survival study under laboratory conditions, 1.500 Obsoletus complex midgeshave been kept at 17-25 °C and provided only with sugar solution. Of these, 150(10%) survived for at least 40 days and 3 midges were still alive after 92 days. Thisis up to date the longest life span recorded for Culicoides. Additionally, 10 midgeswere able to survive 10 days at 4°C.

For the feeding trials a total of 40 blood meals (9.440 midges, 200-300 midges foreach meal) have been performed and 27 were successful (67.5%). Sheepdefibrinated blood was used. During the meal it was kept at 37°C and stirred by amagnet. To infect the insects, field strain BTV2 isolated from a spleen of sheepduring the 2000 Italian outbreak was added to the blood meal. Two different viralsolutions titering 106 TCID50/ml and 107 TCID50/ml were prepared. Similar feedingrates (U di Mann-Whitney=129.5 p>0,05) were obtained when natural (1 daychicken skin) and artificial (stretched parafilm) membranes were employed. In the 27 successful blood meals the feeding rate ranged from 0.3 to 16.7%, witha total of 592 engorged midges out of 7.140. Uninfected blood (12.68%;341/2.690) resulted significantly (U di Mann-Whitney=88.5 p<0,05) moreappetising than the infected meal (5.64%; 251/4.450) and midges of theObsoletus complex preferred (U di Mann-Whitney=48 p<0,05) to feed with bloodcontaining BTV2 at lower titer. After the infected blood meal, the engorged midges have been incubated at 23-25 °C and fed with sugar solution. Dead insects have been daily collected andanalysed for virus evidence. Of the 251 engorged midges, 54 (21.5%) died in thefeeding chambers or during the sorting on the chill table, 136 within the first 10days and 61 survived longer. BTV was isolated only from those which died justafter feeding (52.6%; 10/19) or 24 hours later (47.8%; 11/23). Considering thenumber of midges tested after 10 days of incubation, the prevalence which couldbe detected in this study (95% probability) would have been higher than 4,74%.These preliminary results appeared very promising as this is the first time thatmidges of C. obsoletus complex have been successfully fed under laboratoryconditions.

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SPATIAL DISTRIBUTION OF BLUETONGUE IN CATTLE IN SOUTHERN CROATIAIN THE LAST QUARTER OF 2002 A. Labrovic, Z. Poljak, S. Separovic, B. Jukic, D. Lukman, E. Listes and S. Bosnic Ministry of Agriculture and Forestry – Veterinary Administration, Ulica grada Vukovara 78, 1000 Zagreb, Croatia. Tel/fax: 00385-1-6106670/6109207; e-mail:[email protected]

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After the first outbreak of the bluetongue disease in domestic ruminantsin southern Croatia, in the late 2001, there was an interest to learn moreabout the spread as well as the distribution of the disease amongdomestic cattle population in the districts Dubrovacko-neretvanska (DN)and Splitsko-dalmatinska (SD).A sentinel pilot scheme was employed from September to December2002. The use of sentinel cattle in a protection zone (which was definedas a part of country having a radius of at least 100 km around the infectedholding and consisted of district DN and southern part of district SD) wasaimed to detect a seroconversion in cattle at the prevalence of 5%. Forthat purpose 60 cattle in 10 villages (6 cattle per village) were sampledapproximately every 15 days.Sentinel cattle in the surveillance zone (which was defined as a radius ofabout 100 km from the protection zone and consisted of northern part ofSD district) was aimed for an early detection of seroconversion in cattleat the prevalence of 2%. For this purpose 150 cattle in 15 villages (10

cattle per village) participated in the sentinel scheme. In this zone cattlewere tested approximately every 30 days. The criteria for selection of a place to post a sentinels were: a density ofruminant population, vicinity of places for which the local veterinaryservice knows or suspects that are suitable for development of a vectorsas well as readiness of the owner to participate in the sentinel scheme.The criteria for inclusion of cattle into a sentinel pilot study were: cattleolder than 6 months that are serologically negative.Serum samples were screened with Commpetitive Enzime LinkedImmunosorbent Assay (C-ELISA, VMRD Inc, USA). The risk factorsassociated with BT occurrences were collected through the questionnaire.The map indicating the spatial distribution of sentinel herds in protectionand surveillance zones will be produced. The results of 2002 sentinelcattle scheme in Croatia as well as spatial distribution of cattle foundBTV seroconverted during the 2002 sentinel program, will be presentedon the poster.

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SEROLOGIC EVIDENCE OF BLUETONGUE AND PRELIMINARYENTOMOLOGY STUDY IN SOUTHERN CROATIAE. Listes, S. Bosnic, M. Benic, M. Lojkic, Z. Cac, Z. Cvetnic, J. Madic, S. Separovic, A. Labrovic, G. Savini and M. Goffredo Veterinary Institute Split, Polji_ka cesta 33, HR-21000 Split, Croatia. Phone/fax: 00385 21 370755, e-mail: [email protected]

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Bluetongue is a viral noncontagious infectious disease of domestic andwild ruminants, transmitted by biting midges of the genus Culicoides(Diptera: Ceratopogonidae). The disease occurs in Africa, South and NorthAmerica, Asia, Australia, and occasionally in Europe. During the 1998 –2002 period, the disease spread across the Mediterranean, occurring incountries where it had never been recorded before. In November 2001, clinical signs of the disease were observed in thesheep from the Dubrovnik – Neretva County, especially in the Konavleregion near the border to Montenegro and Bosnia and Herzegovina. InDecember 2001, the disease was serologically verified at the CroatianVeterinary Institute by the method of competitive ELISA (cELISA, VMRDInc., USA).Results of serologic testing of blood samples of ruminants from theDubrovnik – Neretva County, and preliminary studies of the Culicoidesmidges in the area are presented. A total of 3318 sera of ruminants from53 herds were examined. Blood sampling was performed from the end of

January 2002 till June 2002. Because of the small number of animals perherd in the area, all ruminants from a particular village were considereda herd on result analysis. The area was divided into three parts: Konavle,where blood sampling was performed in the nearly complete ruminantpopulation; Dubrovnik area; and Dubrovnik littoral, the latter two in thenorth, where blood sampling was done by random selection.A total of 357 bovine sera, 178 (49.9 %) of them positive, 1268 ovine sera(174 or 13.7% positive), and 1693 caprine sera (270 or 16% postive)were tested. Antibodies to bluetongue virus serotype 9 were detected in212 positive sera by serum neutralization test. A preliminary light-trap survey for midges of the genus Culicoides wascarried out in September 2002 in the Dubrovnik – Neretva County.Fourteen light-trap collections from 7 different locations were examined.A total of 4872 Culicoides spp. were collected, 4492 (92%) of thembelonging to the species C. obsoletus.The mean air temperature at the time of the study was 20°C.

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EVIDENCE THAT BLUETONGUE VIRUS SEROTYPE 2 (BTV-2) HAS PERSISTEDIN THE SOUTHEASTERN UNITED STATES FOR THE PAST 20 YEARSJ.O. MechamUSDA, ARS Arthropod-Borne Animal Diseases Research Laboratory, P.O. Box 3965 Laramie, WY 82071-3965 USA. Tel: +1-307-766-3620; Fax: +1-307-766-3500;e-mail: [email protected]

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Bluetongue virus serotype 2 (BTV-2) is the most recent bluetongueserotype to be introduced into the U.S. It was first isolated from sentinelcattle and Culicoides insignis at Ona, Florida in 1982. The initial isolatesdemonstrated two RNA electropherotypes and were designated Ona-A andOna-B. The electropherotype of the Ona-A variant was identical to that ofthe South African BTV-2 prototype. It was isolated in September andOctober of 1982 and then disappeared. The Ona-B variant was isolated inlate October and November of 1982 at the same site and at additionalsites in Florida the following year. Isolations of Ona-B were subsequentlymade in 1984 and 1985 from cattle in Alabama. No isolations of either

variant were reported after 1987, and it was widely assumed that BTV-2had disappeared from the southeastern United States. However, BTV-2was recently isolated from sheep in Florida, suggesting that this serotypehad been reintroduced or had persisted since its first appearance in 1982.To investigate this question, selected genome segments of this recentisolate were sequenced and the sequences compared to those of theprototype isolates and recent Florida isolates of other BTV serotypes.Phylogenetic analysis suggests that reassortment may have generatedthe Ona-B variant; and that this virus has persisted in the southeasternUnited States for the past 20 years.

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ADULT KEY TO THE OLD WORLD IMICOLA COMPLEX(CULICOIDES; SUBGENUS AVARITIA FOX, 1955)R. Meiswinkel (1)(2)

(1)Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Campo Boario, 64100, Teramo, Italy; e-mail: [email protected];(2) Research affiliate: Onderstepoort Veterinary Institute, Onderstepoort, 0110, South Africa

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A key is provided to the adults of both sexes of the nine described species of the Old WorldImicola Complex (the section for identifying the females is given below). A photograph of thefemale wing of each species is provided also. The known geographic distribution of each speciesis mapped (according to published records), and is accompanied by brief notes on its generalbiology and vectorial capacity. A shortcoming of the key is that it assumes a working knowledge of the character states thathelp place a species within the Imicola Complex. (A definition of the complex is to be foundelsewhere in this volume.) Until the six or more species complexes comprising the subgenusAvaritia are clearly defined, it will remain problematical to utilise a key as precisely aimedas that presented below for the Imicola Complex (and which therefore does not deal withthe 30 or more other species of the subgenus Avaritia known to occur across the Old World).

Key to slide-mounted females of nine world species of the Imicola Complex

1. Third palpal segment inflated; long, blunt-tipped sensilla trichodea on flagellomeresIII-X also inflated; sensilla coeloconica usually on flagellomeres III, XI-XV . . . . . . . .2Third palpal segment not inflated but is either short and roundish,or moderately long and slender; blunt-tipped trichodea slender;coeloconica usually on flagellomeres III, XII-XV . . . . . . . . . . . . . . . . . . . . . . . . . .3

2. Antennal ratio (AR) 0.98-1.13, mean 1.06; the dark wing spot straddling the radial cellsis joined to the dark spot at the base of M1; legs predominantly brown . .C. tuttifruttiAntennal ratio (AR) 1.13-1.24, mean 1.19; dark wing spot straddlingradial cells clearly separated from the dark spot at the base of M1;legs predominantly pale . . . . . . . . . . . . . . . . . . . . . . . . . .C. pseudopallidipennis

3. Anal angle of wing with an obvious, though variable-sized, dark smudge . . . . . . . . .4Anal angle pale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

4. Scutum brown bearing two yellow admedian vittae; halter knobs brown;eyes sparsely pubescent; wing dark . . . . . . . . . . . . . . . . . . . . . . . . . . .C. miomboScutum entirely brown; halter knobs pale; eyes bare; wing pale . . . . . . . .C. kwagga

5. Scutellum with two median long bristles; flagellomeres VI and VIIIwith four sensilla chaetica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C. loxodontisScutellum with one median bristle; flagellomeres VI and VIIIwith three sensilla chaetica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

6. Wing vein M2 with a broad, well-defined, preapical excision; proximalmargin of distal pale spot in R5 moderately to noticeably pointed;third palpal segment moderately long and slender . . . . . . . . . . . . . . . . . . . . . . . .7Vein M2 without preapical excision; proximal margin of distal pale spotin R5 straight or gently rounded; third palpal segment shorter, almost round . . . . . .8

7. Proboscis/head (P/H) ratio low (0.66-0.73); restricted to south-eastAsian islands east of the Wallace line . . . . . . . . . . . . . . . . . . . . . . . .C. nudipalpisProboscis/head (P/H) ratio higher (0.82-1.02); widespread in Africa,southern Europe, and the Near and Middle East, and the mainland Far East(including southern China, Thailand, Vietnam and Laos) . . . . . . . . . . . . . .C. imicola

8. Antennal flagellomeres VI-IX shorter (approximately 1.4x as long as wide);restricted to the Oriental, Eastern Palaearctic and Australasian Regions .C. brevitarsisAntennal flagellomeres VI-IX longer (1.4-1.7x as long as wide);restricted to Afrotropical Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C. bolitinos

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CHRISTOPHER COLUMBUS AND CULICOIDES: WAS C. JAMAICENSIS EDWARDS, 1922INTRODUCED BY SHIP TO THE MEDITERRANEAN 500 YEARS AGO AND LATER RENAMEDC. PAOLAE BOORMAN, 1996?R. Meiswinkel, K. Labuschagne and M. Goffredo Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100, Teramo, Italy. Tel: 0039-0861-332324; Fax: 0039-0861-332251,e-mail: [email protected]

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In 1994 an apparently new species of Culicoides was captured at a horse stable inPellaro, southern Italy. It was subsequently described and named C. paolae Boorman,1996 (after its discoverer Paola Scaramozzino). Based upon resemblances in the wingpattern C. paolae was first thought to belong in the Old World Schultzei Complex(=subgenus Remmia Glukhova, 1977), but closer scrutiny confirmed it to differ inmany other taxonomic features. For example, C. paolae bears multiple sensillacoeloconica on all antennal flagellomeres 3-15 whereas in the Schultzei Complex farfewer sensillae occur (and usually only on flagellomeres 3, 8-10). Despite thisdifference, and others, the superficial resemblance between C. paolae and theSchultzei Complex was emphasised during its description, and led to it being labelled(perhaps unjustifiably) a potential vector of livestock orbiviruses (as Epizootichaemorrhagic Disease of Deer Virus (EHDV) had previously, in the Sudan, been isolatedfrom C. kingi Austen, 1912, a species of the Schultzei complex). Following the incursion of bluetongue virus (BTV) into Italy in August 2000 a nationalsurvey was implemented and Culicoides collected countrywide. Onderstepoortblacklight traps were deployed throughout the islands of Sardinia and Sicily and onthe southern peninsular mainland of Italy, and soon revealed C. paolae to bewidespread, and sometimes locally abundant (on occasion being captured in 100sbut never in 1000s). In 2001 it was noticed adventitiously that the wing of C. paolae strongly resembled thatof C. jamaicensis Edwards, 1922, but this resemblance was initially ascribed tocongruence as many species of world Culicoides share similar wing patterns. Also, thefact that C. jamaicensis was a Central American (New World) species seemed to weightoo heavily against its dispersal into the Mediterranean Basin (Old World) across sucha wide expanse of ocean. However, doubts persisted, firstly because the publisheddescriptions of the two species showed them to be inseparable in both sexes, and,

secondly, because C. paolae seemed not to be taxonomically related to any other OldWorld species. These two facts heighten the likelihood of C. paolae being alien to theMediterranean, and so indicate that it had somehow, in the past, been introduced intothe Basin.A number of New World Culicoides are associated with the indigenous cacti of Centraland South America; these include C. jamaicensis and C. loughnani Edwards, 1922.Unexpectedly, the latter species was 30 or more years ago discovered to occur inAustralia. Likely it had arrived there after boatloads of parasite-laden rotting cactistems had been introduced to Australia in the 1920’s as part of a biological controleffort against the spread of jointed cactus. C. loughnani was subsequently found tobreed in rotting cacti stems in Australia (just as the closely related C. jamaicensis hasbeen found to do in Mexico). Therefore it must now be considered whether thewidespread occurrence of the alien Fico d’India cactus (Opuntia ficus-indica) in theMediterranean, and reputedly introduced by Christopher Columbus from the New Worldsome 500 years ago, is not the original link that first aided the dispersal of C.jamaicensis into the region, and which today also sustains it locally (but under the newname of C. paolae). However, we have twice failed to rear C. paolae from rottingOpuntia fruits (and joints), and so the apparent correlation between its occurrence andthat of the cactus still remains to be proven. Also, in pursuing further the possibility ofC. paolae being a synonym of C. jamaicensis it would be nous to compare molecularlyMediterranean populations of C. paolae against Central American ones of C.jamaicensis. A final observation: The fact that C. paolae has a markedly inflated thirdpalpal segment, and bears multiple sensilla coeloconica on all antennal flagellomeres,would indicate it to feed preferentially on birds, and not on mammals. If so C. paolaemay have originally been feeding on birds roosting around the Pellaro stable, and notnecessarily on the horses inside.

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MULTIPLE VECTORS AND THEIR DIFFERING ECOLOGIES: OBSERVATIONS ON TWOBLUETONGUE (BT) AND AFRICAN HORSE SICKNESS (AHS) VECTOR CULICOIDES SPECIESIN SOUTH AFRICAR. Meiswinkel, K. Labuschagne, M. Baylis and P.S. MellorIstituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100, Teramo, Italy. Tel: 0039-0861-332324; Fax: 0039-0861-332251,e-mail: [email protected]

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In all affected regions it is emerging that Culicoides-borne orbiviral diseases of livestock are beingtransmitted by more than one vector species. This multiplicity complicates the epidemiology ofthese diseases; another consequence is that it invalidates (partially) “single-vector” risk models.In South Africa the two most important vectors of the orbiviruses of bluetongue (BT) and Africanhorse sickness (AHS) are C. imicola Kieffer, 1913, and C. bolitinos Meiswinkel, 1989. Differencesin their relative abundances, and seasonal and geographic prevalences, were determined using anetwork of 40 blacklight traps operated weekly (from September 1996 to August 1998) acrossSouth Africa. It emerged that:1. In a total of 3.346 light-trap collections made, and yielding >3 million Culicoides representing

86 species, a mere 10 species made up 90% of all Culicoides captured. These included C.imicola and C. bolitinos, which were the 1st- and the 5th-most abundant species respectively,and together comprised >50% of all biting midges collected. They were also the most prevalentspecies being found respectively at 39 and 38 of the 40 sites sampled;

2. Culicoides imicola was most abundant in the warmer northern and eastern areas of SouthAfrica; these areas are considered enzootic for the orbiviral diseases listed above, and may actas source points for their southward spread. However, C. imicola was found abundantly also inthe southern half of the country where devastating outbreaks of AHS have occurred in the past.C. bolitinos was almost as widespread as C. imicola, but was an order of magnitude lessabundant. It predominated in cooler, higher-lying areas, which mostly abut those areas in whichC. imicola predominates; this “allopatry” effectively increases the total area at risk to orbivirustransmission in South Africa;

3. It is a common belief that areas experiencing the coldest winters (including snow) are also thosewith the smallest populations of C. imicola. This is generally the case but, unexpectedly, C.imicola was found to be even rarer (or entirely absent) at three sites having a mild, frost-freeclimate. This demonstrates that, in certain instances, climate is secondary to determining theabundance and distribution of C. imicola. These sites occurred along the coastline, and impliesthat windiness and sandy soils suppress the development of large populations of C. imicola.However, the dominance of the second vector, C. bolitinos, at one of these sites would seem tomitigate against wind being an important inhibitor of Culicoides activity. This suggests that

sandy soils, in draining too rapidly, disrupt larval development in C. imicola. In the case of C.bolitinos soil-type does not seem to act as a barrier. Due to its predilection for cattle dung as alarval habitat it is able to penetrate into sandy areas, and also into steeply sloped, mountainousterrain (where C. imicola is unable to persist);

4. The geographic distribution of these two vectors was found to be stable, and so can beharnessed as a predictive tool in risk analyses. However, there are important caveats: Forexample, C. imicola is the only species able to develop extraordinarily large populations duringthe episodic rains that affect South Africa every 10-15 years, and correlates perfectly with theold adage that “…horse sickness appears as a plague following heavy rains...”. Not only mustsuch cyclical phenomena be factored into predictive risk maps, but implies also that theconstant irrigation of grazing pastures (coupled to the maintenance of fenced livestock) mustartificially inflate local foci of C. imicola. As for C. bolitinos cattle husbandry may induce similarmodifications in its “natural” distribution across South Africa;

5. It was discovered that vector-free areas do exist in South Africa. One of these sites, which hasconsistently escaped the ravages of AHS over a century or more, was monitored weekly for twoyears. Not a single specimen of C. imicola was captured, and demonstrates again that thisvector is not able to penetrate all landscape types (even in the long term). If these C. imicola-free zones were to be characterised precisely, and their distribution mapped across the OldWorld range of C. imicola, vector-free enclaves could be identified. Whilst these would be ofinestimable value for the quarantining and export of livestock the absence of other vectors mustbe established.

The presence of the insect vector principally determines whether a given area is at risk to diseaseincursion and maintenance. The ecologies of the two most important vectors of BT and AHS inSouth Africa, in differing markedly, not only increases the total area at risk, but complicates alsothe development of rational livestock movement and disease control strategies. Similarlyannectant patterns of vector prevalence occur in other regions of the world. This warns us thatvector surveillance studies should attempt to map the distribution of ALL species of Culicoides,especially in those instances where the disease transmission potentials of “suspected” vectors stillremain unknown.

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THE IMICOLA AND ORIENTALIS COMPLEXES OF THE SUBGENUS AVARITIA FOX, 1955:THEIR REDEFINITION BASED ON ADULT MORPHOLOGY (CULICOIDES; DIPTERA,CERATOPOGONIDAE)R. Meiswinkel (1)(2)

(1)Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Campo Boario, 64100, Teramo, Italy; e-mail: [email protected]; (2) Research affiliate:Onderstepoort Veterinary Institute, Onderstepoort, 0110, South Africa

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Thirty-six subgenera comprise the biting midge genus Culicoides Latreille,1809. One of these, the relatively small subgenus Avaritia Fox, 1955, isimportant as it contains nearly half of the 16 species of world Culicoides knownto act as vectors of orbiviral diseases, and which can induce high, and usuallyswift, mortality rates in livestock pantropically. The subgenus Avaritia isdistributed globally and is at present subdivided into seven species groups andfour subgroups. Although most of these groups follow along natural lines theyremain inadequately defined and include less than half of the 70 describedspecies of world Avaritia. These 11 informal categories are here reduced to sixspecies complexes, and to one subgroup. The principal focus of this study is toredefine two of these complexes, namely the Old World Imicola and OrientalisComplexes, their separation based upon 18 differential character states foundin the adult stage of both sexes. Evolutionary radiation within the Imicola Complex (previously referred to as thePallidipennis or the Imicola group) appears to have been centred in the lower-rainfall woodland, grassland, and semi-desert regions of the AfrotropicalRegion, where 10 of the 12 known species occur, and where some have co-evolved in exclusive association with large herbivores (the African elephant, theblack and the white rhinoceros, the plains zebra, the blue wildebeest, and theAfrican buffalo). The remaining two species of the Imicola Complex arerestricted to the Indo-Australasian Region. Only one species, C. imicola Kieffer,1917, is common to both Africa and Asia (including the Mediterranean Basin).It is one of the most widespread of world Culicoides, and a formidable vector of

the animal viral diseases of bluetongue (BT) in sheep, cattle and goats, and ofAfrican horse sickness (AHS) and equine encephalosis (EE) in equids. In the Orientalis Complex adaptive radiation has been more tropic-centred;viewed broadly, this complex occurs in allopatry to the Imicola Complex as thebulk of the described species (eight) are restricted to the higher-rainfall, moreequable temperature regimes of the Oriental-Australasian Region. Only threespecies occur in tropical Africa, these placed previously in the Trifasciellussubgroup (but here transferred to the Orientalis Complex). Compared to theImicola Complex comparatively little is known about the biology of specieswithin the Orientalis Complex, especially as regards their vectorial role; onlyone species, C. fulvus Sen & Das Gupta, 1959, has been shown capable ofreplicating bluetongue virus (BTV) in the laboratory. Of the 19 Afro-Asiaticspecies previously assigned to the Orientalis Complex eight only are retained:four are moved to the Imicola Complex, whilst the remaining seven areadjudged to belong to still unknown species complexes. At the species level thetaxonomy of the Orientalis Complex appears in disarray believed due to thedescriptive format currently employed world-wide in the genus Culicoides beingtoo superficial and stylised. It is argued that if our understanding of the systematics of the subgenus Avaritiais to reach fruition new and seldom-used character states should be employed;these are presented and their taxonomic value discussed. It remains axiomaticthat correct identifications at the species level are essential to advanceknowledge on all fronts ranging from basic biology through to vector capacity.

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SEASONAL ABUNDANCE OF CULICOIDES IMICOLA AND C. OBSOLETUS GROUPIN THE BALEARIC ISLANDS (SPAIN)M.A. Miranda, C. Rincón and D. BorràsDepartment of Biology. University of the Balearic Islands. Cra. Valldemossa km 7.5. CP: 07122. Palma of Majorca. Spain. Phone: int+34971173351. Fax:int+34971173184. E- mail: [email protected]

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During September and October 2000, an important outbreak ofbluetongue was declared in the Balearic Islands (Spain). In thefollowing years 2001 and 2002, an intensive survey of the majorvectors in the Mediterranean basin, Culicoides imicola and C. obsoletus,was carried out in cattle farms located in Majorca and in Minorcaislands, where the disease was detected during 2000. Adult Culicoideswere obtained once a week from June 2001 to December 2002 usingCDC light traps.

The results obtained from 121 samples reported that both C. imicola andC. obsoletus group populations are well established in Majorca andMinorca, as well as other species of genus Culicoides. Furthermore, bothspecies showed a different seasonal abundance pattern, thus C. obsoletusgroup adult population peaked at July, meanwhile C. imicola peaked atOctober. This findings indicate that probably the recent outbreak of BT inthe Balearic Islands was associated with C. imicola as the major vector.

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SEASONAL PREVALENCE OF BITING MIDGES, CULICOIDES SP. (DIPTERA:CERATOPOGONIDAE) OF DOMESTICATED ANIMALS OF MARATHWADA REGIONB. W. Narladkar, P. D. Deshpande and P.R. Shivpuje Assistant professor of parasitology, College of Veterinary and animal sciences, Maharashtra Animal and Fishery Sciences University PARBHANI – 431 402Maharashtra State (INDIA). E-mail: [email protected]. Telephone: 0091–2452–229890 (R); Fax: 0094–2452–226188

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Consecutive studies for two years on Culicoides insects from 18 sites ofMarathwada region revealed the prevalence of Culicoides peregrinus(Kieffer, 1910), C. schultzei (Enderlein, 1908) C. actoni (Smith, 1929)species. Composition of these three midge species corresponditing to55.91, 41.81 and 2.28 percent among 9526 of cattle; 53.42, 42.66 and3.92 per cent among 6013 of bulfaloes; 51.74, 46.78 and 1.48 among4322 of sheep and 52.85, 43.35 and 1.88 per cent among 666 of goats,respectively. Host wise preference was in the order of Cattle, buffaloes,Sheep and goats.

Meteorological factors such as maximum temperature, more number ofbright sunshine hrs, low rainfall and high wind velocity deterred; whileminimum temperature, high relative humidity and rainfall favouredpositively the build up of Culicoides population significantly. Build up ofCulicoides population started from June and reached to peak duringOctober month.The seasonal composition of Culicoides in the year 2001 was 78.51, 16.91and 4.58 per cent whereas in the year 2002 it was 79.01, 17.87 and 3.06per cent in the monsoon, winter and summer seasons respectively.

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B 25Epidemiology and vectorsABSTRACT BOOK

S10 SEGMENT SEQUENCE ANALYSIS OF SOME GREEK BTV STRAINSS.V. Nikolakaki, K. Nomikou, O. Mangana-Vougiouka, M. Papanastassopoulou, M. Koumbati and O. Papadopoulos Laboratory of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine. Aristotle University of Thessaloniki, 54 124 Greece. Phone: +30 2310 999924,Fax: +30 2310 999959. E-mail: [email protected]

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This study provides preliminary data on the sequencing analysis of theNS3/NS3A gene of some Greek BTV isolates from the 1979-2001Bluetongue epizootics.The sequences of the S10 gene segment of 8 Greek BTV field strains ofserotypes BTV1, BTV 4, BTV 9 and BTV 16, isolated from sheep, weredetermined to define the molecular epidemiology of BTV infection inGreece. Phylogenetic analysis of the S10 gene segregated the Greek BTvirusesinto two monophyletic groups. In the first one were included all Greek BTVstrains of serotype 4 regardless of the area and year of isolation. Highpercentage of homology (98-100%) is observed among different strains

in this group, which in turn resembled strains from Tunisia and Corsica.The strains of serotypes BTV1, BTV9, and BTV16 segregated into a secondmonophyletic group, showing high percentage homology (97-99%).Phylogenetically, the latter group is closely related to the Chinese,Australian and South African BTV strains. From these preliminary results it may be concluded that two differentgroups of BTV strains seemed to coexist in Greece during the epizooticsof 1979-2001. Whether the deduced amino acid sequences of NS3/NS3Aproteins coded by this gene also maintain the same pattern is beingdiscussed.

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PHYLOGENETIC STATUS AND GENETIC STRUCTURE OF THE ARBOVIRUS VECTORCULICOIDES IMICOLA KIEFFER (DIPTERA: CERATOPOGONIDAE) IN THE EDITERRANEAN BASIND.V. Nolan, J.F. Dallas, M. Patakakis, Y. Braverman, A. Torina, A. Salvador Martinez, Y. Lhor, A. Ozkul, J-C. De Le Colle, S.De La Rocque, M. Capela, I. Peña, M. Baylis, P.S. Mellor and A.J. Mordue (Luntz) School of Biological Sciences (Zoology), University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, Scotland, UK. Tel: +44 (0) 1224 272868, Fax: +44 (0)1224 272396, E-mail: [email protected]

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The midge Culicoides imicola Kieffer (Diptera: Ceratopogonidae) is themost important Old World vector of African horse sickness (AHS) andbluetongue. Recent outbreaks of bluetongue have affected many parts ofsouthern and central Europe. Although bluetongue epidemiology isstrongly linked to the phylogenetic status and genetic structure of itsNorth American vector, studies on these aspects of C. imicola in theMediterranean basin have only recently been initiated. Building on thiswork the present study has analysed sequences of the mitochondrialcytochrome oxidase subunit I gene (COI) to determine the phylogeneticrelationship of C. imicola from Morocco, Portugal, Spain, Corsica, Sicily,Greece, Turkey and Israel with five species of the Culicoides imicolaspecies complex from southern Africa, and begun to characterise thespatial scale of genetic subdivision within C. imicola. The 133 C. imicolaanalysed represented 18 COI haplotypes. These haplotypes formed one

well-supported clade in a neighbour-joining tree providing strongevidence for the presence of only C. imicola in the Mediterranean basin.When these C. imicola samples were analysed, genetic subdivision withinthe Mediterranean basin was evident. We identified one clustercorresponding to the samples from Morocco, Portugal, Spain and Corsicain the western Mediterranean, and two clusters corresponding to thesamples from Greece, Turkey and Israel in the eastern Mediterranean.Interestingly, Sicily appears to be a focal point for incursions of C. imicolafrom both the western and eastern Mediterranean basin. These resultssuggest that female-mediated gene flow in C. imicola outside of thecentral region of the Mediterranean basin is either limited or non-existent,and imply that vector-mediated bluetongue outbreaks in those regions ofthe Mediterranean basin will be independent of each other.

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BLUETONGUE SURVEILLANCE SYSTEM IN ITALYA. Giovannini, C. Paladini, P. Calistri, A. Conte, P. Colangeli, U. Santucci, D. Nannini and V. Caporale Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo (Italy). Phone: +39 0861-3321; Fax: +39 0861-332251;e-mail: [email protected]

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The Authors analyze the bluetongue (BT) surveillance systemimplemented in Italy and describe the internet-based BT informationsystem for the collection and diffusion of data and information regardingthe health status of animal populations all over the Italian territory. Thesurveillance system and the internet-based information system aredesigned to collect and spread all information needed to support thedecision making process and to manage control activities. These systemsare also the kernel of the Italian BT early warning system.Furthermore, the analysis of information generated by the BT surveillance

system permitted to (a) increase the knowledge on BT epidemiology andon the dynamics and distribution of its vectors, (b) demarcate the infectedareas, (c) draw a risk map in relation to vector presence and (d) quantifythe risk due to animal movement. The results of these analyses ofinformation and risk assessments were also used to make proposals to theEU Commission and possibly contributed to scientifically support a greaterflexibility in the planning of control measures that now characterizes theEU legislation on BT.

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CULICOIDES IMICOLA IN GREECEJ.M. PatakakisK.K.I.A., Parasitology Department, 25 Neapoleos str., Agia Paraskevi, ATHENS – 153 10. Tel: + 210 60 80 838; Fax: + 210 60 80 838. E-mail: [email protected]

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C. imicola was present in Lesvos island during the 1979 Bluetongueoutbreak. Since then, many hundrends of insect catches were examinedfrom various parts of the country (mainland and islands ).We found thatalthough C. imicola was never seen in mainland Greece 12 years ago, it

is now present in many areas of northern, central and southern Greece.During the 1998-1999 bluetongue outbreak, C. imicola was present inlarge numbers in the infected areas.

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MODELLING THE DISTRIBUTIONS OF OUTBREAKS AND CULICOIDES VECTORS IN SICILY:TOWARDS PREDICTIVE RISK MAPS FOR ITALYA. Torina(1), S. Caracappa(1), A.M.F. Marino(1), A.J. Tatem(4), D.J. Rogers(4), P.S. Mellor(3), M. Baylis(2) and B.V. Purse(3)

(1) Istituto Zooprofilattico Sperimentale della Sicilia, “A. Mirri”, Palermo, Italy (2) Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN, UK (3) Institute forAnimal Health, Ash Road, Pirbright, Surrey, GU24 0NF, UK (4) TALA Research Group, Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK.

Institute for Animal Health, Ash Road, Pirbright, Surrey, GU24 0NF, UK. [email protected], Tel: 01483 232441 ext 1021, Fax: 01483 232448

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Sicily was one of the first regions in Italy to be affected by outbreaks ofbluetongue (BT) (Calistri, et al., 2003). The first outbreak was detectedon 13th October 2000 and outbreaks have occurred in late summer andautumn in every subsequent year (mostly involving serotype 2 with someserotype 9). In collaboration with C.E.S.M.E, a vector and serologicalsurveillance programme across the entire landmass of Sicily wasestablished in 2000. This programme involved regular monitoring ofsentinel animals and the operation of an extensive light-trappingprogramme.In this poster, we present surveillance data from 1137 summer and autumncatches (2000-2002) in 268 sites. We map the spatial distribution of notonly of Culicoides imicola, the main European vector, but also otherpotential vector species such as C. pulicaris and C. obsoletus Bluetonguevirus has been isolated from wild-caught individuals of C. pulicaris in Sicily(Caracappa et al. 2003) and from those of C. obsoletus in other Italianregions (Savini et al., 2003). We quantitatively compare their distributionswith that of the virus from sentinel surveilllance data to investigate the

relative role of these vectors in BT transmission in Italy. Studies elsewhere have shown that maximum catches in summer andautumn are consistently related to the annual abundance of Culicoidesacross sites (Baylis et al., 1997) and thus provide a measure of howfavourable a particular site is for a species. We modelled the observedmaximum catches of C. obsoletus, C. pulicaris, C. newsteadi, C.circumscriptus and C. imicola, (divided into zero and non-zero catches forpresence-absence modelling, and, for C. imicola, also into threeabundance) in relation to remotely-sensed environmental variables usingdiscriminant analysis. These remotely-sensed variables were available, ata 1km grid square resolution, in a continuous layer across theMediterranean and provide information on microclimatic variables such astemperature, moisture availability, soil surface water content, variablesthat affect Culicoides growth and survival and, in turn, abundance. Themodels are then used to compare the climatic requirements betweenspecies and to predict the presence and abundance of different Culicoidesvectors in mainland Italy at a 1km grid square resolution.

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WHAT CLIMATIC FACTORS DETERMINE WHEN EPIZOOTICS OCCUR IN THE MEDITERRANEAN? PREDICTION OF DISEASERISK THROUGH TIME BY CLIMATE-DRIVEN MODELS OF THE TEMPORAL DISTRIBUTION OF OUTBREAKS IN ISRAELY. Braverman(1), A.J. Tatem(4), D.J. Rogers(4), M. Baylis(3), P.S. Mellor(3), B.V. Purse(3)

(1) Kimron Veterinary Institute, PO Box 12, 50250, Bet Dagan, Israel. (2) Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN, UK (3) Institute for AnimalHealth, Ash Road, Pirbright, Surrey, GU24 0NF, UK (4) TALA Research Group, Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK.

Institute for Animal Health, Ash Road, Pirbright, Surrey, GU24 0NF, UK. [email protected], Tel: 01483 232441 ext 1021, Fax: 01483 232448

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Identification of the climatic factors that favour the occurrence ofepizootics of Culicoides-borne viral disease in particular years may allowcontrol measures and surveillance to be implemented earlier and moreefficiently. To investigate such factors, we can analyse the relationshipsbetween time series of vector, virus and host information for a particularset of points in time. Although Israel has remained relatively unaffectedby the recent Mediterranean epizootic (with single cases of bluetongue(BT) occurring only in 1998), periodic epizootics occurred therethroughout the last century, with high numbers of outbreaks (< 30) in1975, 1987, 1988, 1994 (Braverman et al., 2001). Previous studies havesuggested high BT incidence in Israel is preceded by warm, possibly wetwinters (Braverman et al., 2001). However these studies were based onweather station data for a limited number of sites, for limited portion ofthe time series, and multicollinearity between climate variables was notaccounted for. Where continuous time series of climate data are available,in the Republic of South Africa, the timing of epidemics could be linked toan interannual climate cycle, the El Nino Southern Oscillation, through itseffect on the annual rainfall pattern (Baylis et al., 1999).

The Israel Ministry of Agriculture has accumulated 30 years of data on themonthly incidence of bluetongue and for many of these years, monthlylight-trap catches of Culicoides have also been obtained from Bet Dagan,a site in the central coastal plain. In addition, we have obtained monthlyclimatic variables from remotely-sensed AVHRR data, available at a 8kmgrid square resolution, from 1982 to 2000 – variables such as theNormalised Difference Vegetation Index (NDVI) which has be shown to bea significant determinant of the spatial distributions of many vectorsincluding that of the main Buropean BT virus vector Culicoides imicola(Baylis et al., 1998; Baylis et al., 1999). Here we employ Time Series and regression analyses to determinewhether there is significant periodicity in outbreaks between 1968 and2002 at a country level and a regional level. We analyse whether thisperiodicity is determined by particular climatic, vector and livestockfactors. The resulting models are used to predict the risk of diseasethrough time elsewhere in the Mediterranean, within the range of C.imicola.

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EMERGENCE OF BLUETONGUE DISEASE IN THE MEDITERRANEAN BASIN: MODELLINGLOCATIONS AT RISK FOR POTENTIAL VECTORS (CULICOIDES SPP.) USING SATELLITEIMAGERYF. Roger, A. Tatem, S. De La Rocque, P. Hendrikx, M. Baylis, J.C. Delecolle and D. RogersCentre de coopération internationale en recherche agronomique pour le développement (CIRAD), Département Vétérinaire. TA 30/G, Campus International deBaillarguet, 34398 Montpellier Cedex 5, France. Tel.: +33 4 67 59 37 06; Fax: +33 4 67 59 37 98; Email address: [email protected].

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Following an epizootic of bluetongue on the Island of Corsica (France) in2000, C. imicola and 4 other potential vectors (C. newsteadi, C. obsoletus,C. scoticus and C. pulicaris) were trapped on 41 sites. Models weredeveloped for predicting the abundance of these species. Discriminantanalysis, using Mahalanobis distance, was applied to identify the bestmodel - for three levels of abundance of C. imicola, C. newsteadi, C.obsoletus and C. pulicaris, and for the presence/absence of C. scoticus -from 40 Fourier-processed remotely-sensed variables and a digitalelevation variable. The best model correctly predicted the abundance levels with moderate togood agreement (Kappa values from 0.57 to 0.78 according to Culicoidesspp.). The model was used to generate a map of predicted C. imicolaabundance in the Mediterranean area. It predicted high levels ofabundance in many areas recently affected by bluetongue, including theBalearics, Sardinia, Sicily, Lazio and Puglia in Italy, eastern Greece,western Turkey, Tunisia and northern Algeria. The model suggests thatseveral areas in southern mainland France and in various areas aroundMediterranean Sea may be at risk of bluetongue in the future. Usinganother source of data (Portugal trapping results) a model joining

Corsican and Portuguese data also suggests that C. obsoletus could be apotential vector of BTV in the Balkan area. A preliminary validation wasconducted using independent datasets based on trapping in progress(2002) of Culicoides spp. in Corsica and the South of France. Predictionswere satisfactory for the occurrence of Culicoides spp., especially C.imicola in Corsica and C. newsteadi in the South of France. Correlationwas investigated with prediction of C. imicola and outbreaks at communelevel in Corsica. The averaged values of the prediction (using predictedpixel values) showed a statistically significant difference between affectedand non-affected communes (Mann-Whitney U test, p-value<0.001).Moreover, a receiver-operating characteristic analysis showed that thetest has a substantial level of accuracy (area under the curve=0.73). Onepotential cause for the expansion of bluetongue in the Mediterraneanarea, and in particular in Corsica, is the climate change (global warming). These predictive results underlined the necessity to strengthen thesurveillance – entomological monitoring C. imicola but also the otherpotential vectors as well as clinical and serological surveillance - in Francebut also in European countries under a framework of cooperation thatshould be developed.

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FIELD DISINFESTATION TESTS AGAINST CULICOIDES IN NORTH-WEST SARDINIA G. Satta, M. Goffredo, S. Sanna, L. Vento, G.P. Cubeddu and E. MascherpaIstituto Zooprofilattico Sperimentale della Sardegna G. Pegreffi, Via Duca degli Abruzzi, 8 07100 Sassari Tel: +39 (0)79 2892300 e-mail: [email protected]

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Bluetongue disease first affected Sardinia in August 2000, spreadingrapidly across the island with more than 6,000 outbreaks, and causingheavy economical damages. Culicoides imicola Kieffer (Diptera:Ceratopogonidae) was identified as the main vector of the disease and itresulted to be the most abundant Culicoides species on Sardinia. In 2002 season a field trial was carried out with the aim to evaluate theefficacy of an insecticide spraying treatment on local Culicoides populationin the north-west Sardinia.A pyrethrum synthetic derivative named "Microchip" (produced by ICF)was used in two farms where active outbreaks were going on and a thirdsimilar farm was used as control. The same treatment was repeated after15 days.

Two Blacklight traps for Culicoides were placed in each farm and operatedevery two days, for two weeks before and after the disinfestations. The collections and the analysis of the catches were performed accordingto the protocols of the Italian National Reference Centre for ExoticDiseases. For each collection the number of total insect, total Culicoidesand of C. imicola was determined.Comparing to the control farm, a low decrease for few days of thecollected Culicoides in the treated farms was registered.In this paper we report and discuss the results of the trials, and alsoevaluate the decrease of Culicoides population considering the changes ofweather conditions (temperature, humidity, wind).

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ISOLATIONS OF BLUETONGUE VIRUS (BTV) FROM FIELD POPULATIONS OF THEOBSOLETUS COMPLEX (CULICOIDES, DIPTERA, CERATOPOGONIDAE) IN ITALYG. Savini, M. Goffredo, F. Monaco, A. Di Gennaro, P. De Santis, R. Meiswinkel and V. Caporale Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo (Italy). Phone: +39 0861-332219; Fax: +39 0861-332251; e-mail: [email protected]

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In August 2000 bluetongue (BT) appeared for the first time in Italy andspread across the islands of Sardinia, to a lesser extent, Sicily, and intothe southerly regions of the mainland. Since then many thousands of fociof BT disease have been reported. Culicoides imicola appeared to be themost likely vector involved as it occurred widely and abundantly, with anoverall distribution matching almost perfectly that of the disease. BT insubsequent seasons, however, began to appear in areas of southern Italywhere Culicoides imicola was either scarce (usually < 10 specimens/lighttrap collection) or absent. In these areas extensive BT outbreaks with highmortality rates were observed. Between July and September 2002, onthree affected sheep farms in the communities of San Gregorio Magno(Salerno, Campania), Laviano (Salerno, Campania) and Carpino (Foggia,Puglia) the presence of serotypes 2 and 9 of the bluetongue virus (BTV)was confirmed with high mortality. A Culicoides survey on three affected

farms resulted in the capture of approximately 10.000 specimens,representing fifteen species. Not a single specimen of the classical Afro-Asiatic bluetongue vector, C. imicola Kieffer, was found, while species ofthe Obsoletus Complex dominated the light-trap collections; the specieswere C. obsoletus (Meigen), C. scoticus Downes & Kettle and C. dewulfiGoetghebuer, and together comprised 90% of all Culicoides captured.Fifty eight pools of the Obsoletus Complex (excluding C. dewulfi), eachnumbering 100 individuals/pool, and containing only parous and gravidfemales, were assayed for virus. BTV-2 was isolated from two pools (S.Gregorio and Carpino) and BTV-9 from one (Laviano). These resultsindicate clearly that a species other than C. imicola is involved in thecurrent re-emergence of BT in the Mediterranean Basin, but whether thisis either C. obsoletus sensu stricto, or C. scoticus, or both, has yet to beestablished.

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VP-2 SEQUENCE ANALYSIS OF SOME ISOLATES OF BLUETONGUE VIRUS (BTV)RECOVERED IN THE MEDITERRANEAN BASIN DURING THE 1998-2002 OUTBREAKG. Savini, A.C. Potgieter, F. Monaco, O. Mangana, K. Nomikou, H. Yaidin and V. Caporale Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo (Italy). Phone: +39 0861-332219; Fax: +39 0861-332251; e-mail: [email protected]

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Since 1998 five different serotypes of BTV, BTV-1 BTV-2, BTV-4, BTV-9 eBTV-16, have been reported in Countries facing the Mediterranean basin.Preliminary data on the sequencing analysis of the VP2-genes of BTVisolates recovered during the 1998-2002 epizootic of BT in Italy, Greeceand Israel are presented. The VP2-genes of the Italian BTV-2 and BTV-9,Greek BTV-4 and BTV-9, Israeli BTV-4 and BTV-16 and South African BTV-2, BTV-4, BTV-9 and BTV-16, together with those of their correspondingSouth African serotype reference and vaccine strains, were cloned and thesequences of their terminal ends determined. These sequences as well asthose of all the BTV VP2-sequences currently available on GENBANK wereused to compile a phylogenetic tree in order to determine probablegeographic origin of BTV incursions in Europe. Italian isolates included inthis study were from different regions, species and years (2000-2002).The results showed that the sequencing data analysis of terminal ends ofVP2-genes of BTV can be used for topotyping. According to thephylogenetic analysis, the Italian BTV-2 and BTV-9 isolates were stabledespite species, region of origin and year of isolation. They were identical

to a BTV-2 isolate from Corsica. There was 97% identity between theItalian and Corsican BTV-2 isolates and the BTV-2 vaccine and referenceisolates. Italian BTV-9 isolates were also identical with Greek BTV-9isolates (99%). Surprisingly they showed only 67% identity to thereference BTV-9 isolate from South Africa. Conversely BTV-9 field isolatesfrom Australia and Europe had 89% identity on the nucleic acid level.Greek and Israeli BTV-4 isolates were almost identical (98% identity) andthey shared 90% homology with the BTV-4 South African reference andvaccine strains. Israeli BTV-16 and South African BTV-16 reference strainswere also similar. From these results it may be concluded that Italian and Corsican BTV-2,Israeli and Greek BTV-4, South African and Israeli BTV-16 had a commonorigin. The Greek BTV-9 isolate had more than 99% identity with theisolates from Italy showing that these isolates had a common origin. TheEuropean BTV-9 isolates grouped as "eastern isolates" grouping with theAustralian isolates rather than the South-African reference strains.

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ASSOCIATION BETWEEN 2001-2003 BLUE TONGUE OUTBREAKS IN LAZIO AND TOSCANA(CENTRAL ITALY) AND THE DISTRIBUTION AND ABUNDANCE OF VECTORS CULICOIDESIMICOLA AND C. OBSOLETUSG. Scavia, G.L. Autorino, C. De Liberato, F. Farina, G. Ferrari, M. Guidoni, A. Magliano, M. Miceli, F. Scholl, M.T. Scicluna and P. ScaramozzinoIstituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Via Appia Nuova 1411, 00178 Roma, Italia. Tel.+390679099424; Fax.+390679340724; E-mail:[email protected]

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Bluetongue first appeared in Italy in summer 2000 (Sicilia and Sardegna). In July 2001 serological,entomological and clinical surveillance were commenced in Lazio and Toscana regions (central Italy)in the framework of a BT National Surveillance Program. After the first epidemic, central Italyexperienced BTV circulation for three consecutive years in the period 2001-2003. Culicoides imicolaKieffer, 1913 is the only known certain BTV vector in the Mediterranean region, and its presence inSardegna, Sicilia and mainland Italy was confirmed during the entomological surveillance program.Nevertheless in Lazio and Toscana, C. imicola was absent in many sites interested by virus circulationand, where present, its population resulted usually very scarce; on the other hand Culicoides spp.resulted almost ubiquitous, active throughout the year, with some catches exceeding 30000specimens per night. C. obsoletus (Meigen, 1818) is a species already known to be capable insustaining BTV replication and also strongly suspected, on the basis of epidemiological evidences, tobe vector of BT virus in eastern Europe, with particular reference to the 1999 BT outbreak in Bulgaria.Actually, C. obsoletus is known to be a group of at least 4 species (C. obsoletus s.s., C. chiopterus,C. dewulfi and C. scoticus), all present in Italy and, when referring to C. obsoletus, the C. obsoletusgroup must be intended. Serological surveillance based on regular sampling (every 15 or 30 days depending on the period ofthe year and on the epidemiological status of the area) of susceptible livestock (58 bovine wherepossible, or ovine sentinels) in each 20X20km cell, was performed according to the BT NationalSurveillance Program. Evidence of BTV circulation was assessed when in a herd, symptoms of BTwere present and subsequently serologically confirmed or when serological diagnosis was performedin a previously seronegative animal (seroconversion). Once clinical BT, seroconversion or BTserological diagnosis in a never previously tested herd was confirmed, each susceptible animal inthe herd was tested twice at a 15 days interval. Following the first clinical outbreaks in 2001 clinicaland serological surveillance was implemented in the two regions, particularly in provinces underrestriction by systematic clinical examination of the entire ovine population. Entomologicalsurveillance was based on trappings performed weekly in sites defined according to geographicalcriteria and on extra catches, at least two, performed in sites where virus circulation was detectedor suspected. On these basis, a total of 2517 catches were performed from July 1st 2001 up to May31st 2003. Studies on the presence and abundance of C. obsoletus were performed on a subset of1783 out of the 2517 total catches, in 400 different trapping sites, distributed on the whole territory

of the two regions. For a comparable estimate of C. obsoletus and C. imicola population sizes in thedifferent sites, maximum catches were considered more appropriate than mean ones, as Culicoidesactivity may vary considerably according to weather conditions. For epidemiological purposes alldata were grouped and analysed according to geographical units (twenty eight cells each 1600 km2)as defined in the entomological national plan. Two cells, never interested by BTV circulation, werenot considered in the present study because of scarceness of entomological surveillance. Thepresence and the maximum catch for each species were compared between BTV-present and BTV-absent cells. All the catches were classified into 3 equally sized classes using log-transformed valuesof the abundance for C. imicola and C. obsoletus. Comparison between seasonality of viruscirculation and activity of C. imicola and C. obsoletus was performed, in sites were the trappingactivity resulted more continuous.One hundred and fifty-eight BT clinical outbreaks were diagnosed in Toscana in 2001, 62 and 16 werereported in Lazio respectively in 2001 and 2002 (Toscana never experienced clinical outbreaks during2002). Nevertheless virus circulation (seroconvertions) was confirmed in both regions respectively in34, 84 and 32 herds in the three years, until June 2003. A total of 580507 specimens of Culicoidesspp. were caught in the 1783 catches. C. obsoletus resulted by far the most abundant, making upthe 83.3% of all individuals, whereas C. imicola constituted only the 1.8% of the total catch. Themaximum catch of C. obsoletus in a sample reached 34000 specimens, whereas C. imicola neverexceeded 1500. C. obsoletus was present in every cell except one in the highest class of abundance,whereas C. imicola was found in 18 out of the 26 cells. In 4 out of the 20 (20%) BTV present cell C.imicola was not found; 45% of BTV present cells were in high abundance C. imicola class. Aboutseasonality, during 2002-2003 seroconversions in sentinel bovines were recorded all year round, evenin winter months, (December-March). In this period C. imicola was not active, whereas activity of C.obsoletus was detected, even if at a low level, along the whole period. These data allow to confirm the essential role of C. imicola in BTV circulation. Nevertheless theoccurrence of outbreaks and seroconversions in areas and during periods of the year with absence ofC. imicola activity, and the extremely abundant and constant presence of adult active C. obsoletus inquite all the cells, suggest an active role of this species in the epidemics considered in the study.Effectively in October 2002 BTV serotype 2 was isolated from a pool of C. obsoletus, caught at a farmsituated in southern Lazio, where clinical BT cases among sheep were occurring.

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CULICOIDES SPECIES ASSOCIATED WITH LIVESTOCK IN TAMIL NADU STATE OF INDIAK.G. Udupa, J. Ramkrishna, S. Nedunchelliyan and R. MeiswinkelDept. Of Veterinary Epidemiology and Preventive Medicine, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University Vepery, Chennai - 600 007Assistant Professor, Dept. of Veterinary Epidemiology and Preventive Medicine, Veterinary College, Bidar, Karnataka State, India. 585401, E-mail:[email protected]

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Study on Culicoides species (Diptera: Ceratopogonidae) associated withlivestock was carried out during July to September 2000 (monsoonseason), December 2000 to February 2001 (post monsoon season) andMay and June 2001 (summer season) using 220 volts, 8 Watts down-draught light trap at various livestock farms located at differentagroclimatic zones of Tamil Nadu state of India.A total of 26 species of Culicoides were identified during the study.Culicoides imicola and Culicoides oxystoma were found to be higher inabundance in all the farms where collections were made during themonsoon period, followed by Culicoides peregrinus, Culicoides orientalisand Culicoides similis. In most of the farms abandance of Culicoidesoxystoma was found to be high over other species. Abundnace of

Culicoides was high in post monsoon season than during monsoon inmajority of the farms. Culicoides imicola and C. oxystoma wereprevalent in all the 12 farms followed by C. peregrinus. The upsurge ofC. imicola was noticed during post monsoon season in all the farmswhile the abundance of C. oxystoma was low during this season. Duringsummer season when Culicoides midges were collexted from 4 livestockfarms, the abundance of C. imicola was found to be reducedsubstintially. The species diversity was found to be high during monsoonseason over post monsoon and summer seasons. Thus the studyindicated that C. imicola, C. oxystoma and C. peregrinus are widelydistributed in the state and its implication as probable vectors ofbluetongue has been discussed.

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MOLECULAR INVESTIGATIONS OF VIRUS/VECTOR INTERACTIONSW.C. Wilson and C.L. CampbellArthropod-Borne Animal Diseases Research Laboratory, USDA, ARS P.O. Box 3965, University Station Laramie, WY 82071-3965. Ph: (307)766-3622; Fx: (307)766-3500; Em: [email protected]

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In order to design predictors and control strategies for insect-transmitted virus (arbovirus) disease cycles, we must betterunderstand virus/insect molecular interactions. We have beeninvestigating the molecular evolution of Bluetongue viruses (BTV) andthe closely related epizootic hemorrhagic disease viruses (EHDV) fromnumerous geographic regions. Some genes display geographic types(topotypes) and may reflect a relationship between the viruses and theinsect vector populations present in these regions. To address thishypothesis, we have initiated genetic characterization studies of theprimary US vector species, Culicoides sonorensis. Using subtractivelibraries and reverse Northern blot analysis, we have identified 50+cDNAs that appear to be more abundant in midge midguts or heads at

various times during a orbivirus (EHDV) infection (Campbell and Wilson2002). We also have identified the BTV protein that interacts with a C.sonorensis putative receptor protein (Xu et al., 1997). Phylogeneticanalysis of the S7 gene that encodes the attachment protein did notclearly display topotypes (Wilson et al., 2000). More recently, we havedeveloped C. sonorensis tissue-specific cDNA libraries and havegenerated over 700 EST sequences. This developing information will aidour understanding of the genetic factors that may constitute barriers orenhancers of arbovirus infection. Once these factors are identified,these hypotheses will be tested using population genetics orcomparative genomics and may provide a scientific basis for assessingthe risk of importing exotic virus strains or insect species.

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BLUETONGUE VIRUSAND BLUETONGUE DISEASE

ABSTRACT BOOK

GENETIC DIVERSIFICATION OF FIELD STRAINS OF BTVK.R. Bonneau and N.J. MacLachlan 2165 Carlmont Dr. #302, Belmont, CA 94002 • Telephone # (415) 476-8363 (day); (650) 595-8572 (evening); (415) 476-8365 (fax); e-mail: [email protected]

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Genetic heterogeneity of field strains of bluetongue virus (BTV) occurs asa consequence of both genetic drift and shift, the latter as a consequenceof reassortment of individual viral genes. Furthermore, Australian workersfirst proposed the term of virus “topotypes” for the region-specificgrouping of BTV strains that they observed after sequence analysis oftheir L3 genes. Thus, we compared the S10 and portions of the L2 genesof Chinese and US strains of BTV. Phylogenetic analysis of the S10 genesegregated the Chinese viruses into a monophyletic group distinct fromthe US viruses, whereas analysis of the L2 gene segregated strains of BTVaccording only to serotype, regardless of geographic origin. These studiesshowed not only that BTV genes evolve independently of one another, butalso confirmed that BTV strains from distinct geographic locations can beclassified as topotypes based on the sequence of a conserved gene thatassigns a virus isolate to a specific geographic region regardless ofserotype. In subsequent studies to further characterize the geneticdiversity of field strains of BTV that co-circulate at a single site, the S10gene of field strains of BTV contained within Culicoides sonorensis (C.

sonorensis) collected from a dairy in southern California was sequenced.Phylogenetic analysis established that the S10 gene of BTV in C.sonorensis collected from the site existed as a heterogeneous but relatedpopulation, likely arising from genetic drift. Thus, we hypothesized thatviral genes undergo genetic drift during alternating passage of BTV in itsruminant and insect hosts. To test this hypothesis, variation in theconsensus sequence and quasispecies heterogeneity of the L2 and S10genes of BTV was determined during alternating infection of C.sonorensis, a sheep, and a calf. This study demonstrated that individualBTV gene segments evolve independently of one another by genetic driftin a host-specific fashion, generating quasispecies populations in theruminant and insect hosts. A unique viral variant randomly ingested by C.sonorensis that fed on the viraemic sheep resulted in fixation of a novelgenotype, thereby demonstrating founder effect. Therefore, genetic driftcoupled with founder effect offers a model for the diversification of BTVgene segments at a single site, and can be extrapolated to explaindiversification of BTV into distinct topotypes worldwide.

Bluetongue virus and Bluetongue disease

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K.R. Bonneau

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Bluetongue virus and Bluetongue diseaseABSTRACT BOOK

A COMPARISON OF DIFFERENT ORBIVIRUS PROTEINSTHAT COULD AFFECT VIRULENCE AND PATHOGENESISH. Huismans(1)(2), V. van Staden(1), W.C. Fick(1) , M. van Niekerk(1), T.L. Meiring(1) and L. Texeira(1) Department of Genetics, University of Pretoria, Pretoria, South Africa, 0002(2) [email protected]

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The factors that determine the viral virulence characteristics ofbluetongue virus (BTV), African horse sickness virus (AHSV) and otherorbiviruses are not well known. The viral proteins that are expected tobe involved include the outer capsid proteins of the virus, VP2 and VP5.These proteins affect cellular tropism and the attachment of virusparticles to target cells. It is also likely that a protein such as non-structural protein NS3 that is associated with the release of virusparticles from a target host or vector cell, is important in determiningvirulence. With respect to the pathogenesis of bluetongue, Africanhorse sickness and other oribivirus associated diseases the sameproteins may be involved. However, of particular importance may bethose proteins, such as NS3 and VP5 that have a cytotoxic effect. In thispaper we have made a comparison of a number of the commonstructural features amongst the VP2, VP5 and NS3 amino acidsequences of a large number of different orbiviruses. Although there islittle sequence homology amongst the different cognate proteins of thedifferent serogroups, a number of highly conserved features are

observed. Based on the hypothesis that virus-specified cytotoxicproteins may play a role in pathogenesis we have focused in particularon common features in VP5 and NS3 sequences that could affect apossible cytotoxic effect. In the case of NS3 we have also compared thesequence of regions on the protein that are associated with attachmentof NS3 to cellular or viral proteins. These regions may affect thetransport of virus particles from a cell. A unique feature of AHSV NS3proteins is that it is highly variable amongst the different AHSVserotypes. This is in contrast to the NS3 of BTV and equine encephalosisvirus (EEV) which appears to be much more conserved. In order tocompare the cytotoxic effect of NS3 of different orbiviruses, we haveexpressed the NS3 proteins of BTV, AHSV and EEV in both eukaryoticand bacterial cells. A large range of NS3 deletion mutants were preparedand compared with respect to their cytotoxic effect on bacterial cells. Asmall region on the NS3 protein of different orbiviruses, that appears toplay an important role in membrane damage, was identified. Truncationof the flanking regions of this domain enhances the cytotoxicity.

H. Huismans

ABSTRACT BOOK

A COMPARISON OF LABORATORY ADAPTED AND ‘WILD’ STRAINSOF BLUETONGUE VIRUS. IS THERE ANY DIFFERENCE AND DOES IT MATTER?P.D. Kirkland and R.A. Hawkes Elizabeth Macarthur Agricultural Institute, PMB 8, Camden NSW, 2570, Australia. Ph: 61-2-4640 6331; Fax: 61-2-4640 6429. Email:[email protected]

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The biological characteristics of bluetongue (BLU) viruses are extremely complex. TheBLU virus serogroup is relatively large and, although all of the viruses, by definition,share certain features, there is also considerable diversity. Shared antigeniccharacteristics group these viruses, but the assignment of a virus to a serotype alsoimplies that there are differences between members of the group. Interestingly, theelements that define serotype are not directly linked to those that influence perhapsthe most important elements – the determinants of pathogenicity and virulence. Withina serotype, there can be virus strains that are highly pathogenic and others that, atbest, cause very mild disease. For example, strains of serotype 1 in South Africa orChina have caused large disease outbreaks while Australian viruses that are membersof the same serotype are non-pathogenic.In respect of virulence, it has been known for many years that it is possible to reduceor even eliminate the ability of a virus to cause disease. Such adaptation, mostcommonly the result of passage of a virus in cell culture, has been the basis for thedevelopment of live vaccines. At a molecular level, the basis for attenuation is stillpoorly understood and the outcome of attempts to modify a virus cannot be easilycontrolled. Adaptation of a virus to cell culture induces desirable changes that result inattenuation. These vaccines have been beneficial and, in many countries, havesignificantly reduced the impact of BLU infection. Are there any adverse outcomes resulting from modification of BLU viruses inlaboratory systems? Unfortunately, the passage of BLU viruses in cell culture caninduce undesirable properties. Some of these changes appear to occur after relativelylimited manipulation in cell cultures. One of the most prominent features of laboratoryadapted virus (for example, some attenuated or modified live vaccine viruses) is theability of the virus to cross the placenta, causing abortion and foetal abnormalities. Theteratogenic effects of modified live vaccines for BLU are well recognised and vaccination

of pregnant ewes is contraindicated. In contrast, there is no conclusive evidence offoetal infection following natural exposure of sheep, cattle or goats to ‘wild-type’ strainsof BLU virus. Sometimes abortion has occurred in sheep after infection with pathogenicstrains of BLU, but this has been considered to be secondary to the febrile illnessaffecting the ewe. In countries where live vaccines are not used, there is no evidenceof virus crossing the placenta. For example, in Australia, in some years up to 0.5 millioncattle may be infected with a strain of BLU virus, without adverse sequelae. Another of the well-known properties of BLU virus that has had a profound impact ontrade between countries is the excretion of virus in semen. Studies undertaken toinvestigate this phenomenon have collectively shown that old bulls infected with cellculture adapted virus may excrete virus in semen during and soon after the period ofviraemia. A parallel study with experimental infections of old bulls with ‘wild-type’viruses failed to demonstrate virus excretion in semen. Virus transmission to ‘novel’species such as canids, together with foetal infection, has also been associated withexposure to cell culture adapted virus.Are there any other implications arising from these undesirable properties of laboratoryadapted virus? For simplicity and convenience, it has been usual practice to undertakestudies of BLU with viruses that have been passaged in laboratory systems, mainly cellcultures. As there appear to be profound differences between cell culture passagedvirus and true ‘wild’ strains, any in vivo studies of the basic biology of BLU viruses mustinclude a parallel study of animals infected with virus that has not been adapted tolaboratory systems. There should also be strict limitations placed on the repeatedpassage of virus between animals. Failure to observe these precautions could produceresults that may not be indicative of natural BLU virus infection and incorrectconclusions may be drawn.


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P.D. Kirkland

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THE PATHOGENESIS OF BLUETONGUE VIRUS INFECTIONOF RUMINANTS AND DURATION OF VIREMIAN.J. MacLachlan Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA; phone USA 530 754 8125;Fax 530 752 3349; e-mail [email protected]

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Bluetongue virus (BTV) infection of sheep sometimes results in disease,particularly in incursional regions of the world where infection is notendemic. In marked contrast, BTV infection of cattle typically is inapparent.Recent studies suggest that inherent, species-specific differences in theresponse of microvascular endothelial cells may explain the very differentconsequences of BTV infection of cattle and sheep. Despite this remarkabledichotomy in clinical outcome, the pathogenesis of BTV infection of cattleand sheep is similar. Initial replication of BTV occurs in the regional lymphnode after virus is deposited in the skin by the bite of an infected vectorCulicoides insect. Virus then is disseminated to secondary sites ofreplication, especially lymph nodes, spleen and lungs. A prolonged cell-associated viremia then ensues, during which BTV principally is associatedwith erythrocytes in which the virus cannot replicate. This novel interactionof BTV with the cell membrane of ruminant erythrocytes facilitates bothprolonged infection of the ruminant host as well as infection of thehematophagous Culicoides vector. Viremia in BTV-infected sheep and cattletypically persists anywhere between 2 and 9 weeks, depending on virusstrain. BTV nucleic acid can be detected for considerably longer in the bloodof infected ruminants using the sensitive nested PCR assay, up toapproximately 8 months. However, exhaustive studies have shown thatbloods that are positive by PCR and negative by virus isolation rarely are

infectious to either susceptible ruminants or vector Culicoides insects.Although most virus is associated with erythrocytes during viremia, BTV istransiently associated during the initial phases of viremia with all blood celltypes, including mononuclear cells. Interestingly, in vitro studiesrepeatedly have shown that BTV replicates in both macrophages andreplicating T lymphocytes that are stimulated with interleukin-2 (IL-2) andphytomitogens, although complete cytopathic effect (CPE) often does notdevelop leading to a persistent “treadmill” infection of these cultures; BTVreplicates best and causes most extensive CPE in cultured bovinemonocytes and blast-transformed CD4+ T cells, and less so in CD8+ andgamma-delta T cells. The real significance of this in vitro phenomenon,however, remains uncertain given that attempts to isolate BTV from IL-2/phytomitogen stimulated lymphocytes from the blood and tissues ofcattle and sheep that previously were infected with BTV failed todemonstrate long-term persistent infection of these animals. Similarly,despite the appearance of insect vectors in the spring, BTV-infection ofruminants annually occurs only relatively late in the vector season in BTV-endemic areas such as Northern California where BTV infection is highlyseasonal. In summary, BTV-infected ruminants have a prolonged infectionbut there is no convincing experimental or epidemiologic data to supportthe existence of any true persistent BTV carrier state in ruminants.

N.J. MacLachlan

ABSTRACT BOOK

MOLECULAR AND STRUCTURAL BIOLOGY, AND VIRUS REPLICATIONP.P.C. Mertens Institute of Animal Health. Orbivirus Group, Ash Road, Pirbright, Woking, Surrey, UK • GU24 ONF Phone: + 44 (0) 1483 23 1017. E-mail:[email protected]

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Bluetongue virus (BTV) particles can initiate infection by binding to host cells via componentsof the outer capsid (proteins VP2 and VP5). The BTV outer capsid structure has previously beendetermined by cryo-electron microscopy and is composed of 180 copies of the Mr 111 x 103,‘sail-shaped’ VP2 protein, arranged as 'triskellion’ structures, together with 360 copies of aninter-dispersed and underlying VP5 protein (Mr 59 x 103), which may also be arranged as 120trimers. These are the most variable of the bluetongue virus (BTV) proteins and by controllingthe specificity of reactions with neutralising antibodies, they determine the identities of the 24BTV serotypes. Complete sequence analyses of genome segments 2 and 6 (which code for VP2and VP5 respectively) of each serotype, have shown that their variation correlates with BTVserotype. Consequently RT-PCR and / or sequencing can be used both for serotypedetermination and molecular epidemiology (see: poster abstracts by Maan et al, & Singh et al).Infecting virus particles are taken up via an endosomal route. The reduction of pH within earlyendosomes is thought to be essential for removal of outer capsid components from the viruscore, which is then released into the host cell cytoplasm. Individual expression of VP5 causescell fusion (P. Roy; personal communication) and it is consequently thought to play a role inthe release mechanism. However, the BTV core particle contains no VP5 but is also infectiousin its own right for both Culicoides cell systems and some mammalian cells. Antibodies to the outer core protein VP7(T13), will bind to and neutralise core particles, butnot intact virus, suggesting that VP7 (which is not exposed on the intact virion surface) canmediate both cell attachment and penetration. The BTV core can bind to the cell surface viathe interaction of VP7 with Glycosaminoglycans, although other receptors may be required forinitiation of infection. Core infectivity is not affected by ammonium chloride or concanamycinA, which suppress the normal reduction in endosomal pH (presumably because there are noouter capsid components to be removed). This indicates that VP7(T13) alone can also mediaterelease of the core particle from the endosome to the cell cytoplasm. The core particle represents a biochemically active compartment. It contains the 10 dsRNAgenome segments, as well as enzymes needed to synthesise cap and methylate ssRNA copies

of each segment. These mRNAs are released into the cell cytoplasm, where they are translatedinto viral proteins, initiating the molecular events of virus replication and morphogenesis. Thestructure of the BTV core, as well as binding sites for enzyme substrates, have previously beendetermined by x-ray crystallography. The size and overall morphology of the virion appears tobe controlled by the initial assembly of the innermost capsid or 'sub-core' layer, which iscomposed of 120 copies of the highly conserved protein, VP3(T2). Indeed, when expressed byitself VP3(T2) can self assemble to form a subcore like structure (P. Roy personalcommunication). The subcores of some but not all orbivirus species are also relatively stableand can be purified [e.g. Equine encephalosis virus (EEV)].The manner in which the genome segments (together with the three viral enzymes [VP1(Pol),VP4(CaP) and VP6(Hel)] are selected for packaging within the subcore is still unresolved andis one of the challenges that faces molecular virology. Based on the molecular interactionsbetween VP3(T2) and VP7(T13), the outer core layer is thought to be assembled on thecompleted subcore shell, by sequential addition of VP7 trimers, starting from the 3 fold axis ofsymmetry and progressing towards the vertices of the icosahedral particle at the 5 fold axesof symmetry. BTV sub-core and core particles are assembled within large granular matrices, orviral inclusion bodies (VIB), within the cytoplasm of infected cells. The outer capsid proteinsappear to be added to the progeny core surface as it is released from the VIB surface. Progeny BTV particles are released from the infected mammalian cell either by budding, or bydirect cell membrane penetration, which appears to damage the cell, culminating in cell lysis.Previous expression studies have demonstrated that viral protein NS3 (encoded by genomesegment 10) can mediate cell release in insect cells. Since BTV infection does not cause cell lysisoccurs in Culicoides cells, NS3 may be necessary, both for cell exit and for virus spread from theinitial site of infection in the insect mid gut to the insect's salivary glands. It may thereforecontrol the insect's ability to transmit the virus. NS3 is a highly variable protein in both BTV andsome other orbiviruses [e.g. African horse sickness virus (AHSV)], suggesting that variation inNS3 may correlate with variations in insect vector populations and / or species.


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P.P.C. Mertens

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MOLECULAR EPIDEMIOLOGY OF BLUETONGUE VIRUSES IN AUSTRALIAL.I. Pritchard, P.W. Daniels, L.F. Melville, P.D. Kirkland, S. Johnson, R. Lunt and B.T. EatonAustralian Animal Health Laboratory. CSIRO, 5 Portarlington Road, Geelong, Australia 3220. Tel: 61 3 52275468. Fax: 61 3 275555. E-mail: [email protected]

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Bluetongue viruses (BTV) are a distinct serogroup within the Orbivirusgenus in the Reoviridae family. They contain 10 segments of doublestranded RNA that undergo genetic mutation and reassortment tocreate new progeny viruses. Since the discovery that bluetongueviruses were present in Australia, in 1977, the genetic identity of BTVisolates has been analyzed using nucleotide sequences of RNAsegment 3, which codes for the conserved core protein VP 3.Previously, we have analyzed the genetic variability amongbluetongue viruses from different regions and shown they can beclassified into regional groupings or ‘topotypes’.Work done under Australia’s National Arbovirus Monitoring Program(NAMP) for bluetongue viruses used genetic analysis to identifyseveral novel RNA segment 3 clades or genotypes in South East Asia

and tracked their distribution. Recent incursions of Southeast Asiangenotypes into northern Australia were thought to have occurred in1992, 1994 and 1995. Genetic analyses showed both apparentgeographic restriction on movement of these genotypes withinAustralia and reassortment of viral genes segments betweenSoutheast Asian and Australian isolates. A lack of correlation betweentheir electrophoretic RNA profiles and different phylogenetic trees forparticular gene segments suggests that reassortment has occurred innature, and may be an important mechanism for genetic variation. Asa consequence of differences in RNA profiles, further genetic analysiswas been done on gene segments 2, 9 and 10. Specifically the role ofgene segment 10 has been hypothesized to be involved in both vectorcompetence and geographic restriction of bluetongue viruses.

L.I. Pritchard

ABSTRACT BOOK

A POTENTIAL OVERWINTERING MECHANISMFOR THE VIRUS - RECENT FINDINGSH.H. Takamatsu, P.S. Mellor and P.P.C. Merten Institute for Animal Health, Pirbright Laboratory, Pirbright, Woking, Surrey GU24 0NF, UK. • Telephone: +44(0)1483-232441(ext 1089), Fax: +44(0)1483-232448,e-mail: [email protected]

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Bluetongue virus (BTV) is transmitted between its mammalian hosts almostexclusively via bites from the adults of certain species of Culicoides biting midges.Thus theoretically, spread of BTV into the more northerly areas of Europe shouldbe terminated by the harsh winters when adult midges disappear for extendedperiods of time. However, it has been shown that BTV can survive in suchlocations with no detectable cases of viraemia, overt disease or sero-conversionin the host species and also in the absence of adult insect vectors, for periods aslong as 9 to 12 months. Virus survival through the winter in this way is calledoverwintering but the mechanism involved, has not been satisfactory explained. Recently we hypothesised a possible overwintering mechanism based oncurrently available knowledge and a series of preliminary experiments asfollows:1] Ovine/bovine lymphocyte cultures (including γδ T cells) can be persistently

infected with BTV in vitro without apparent cytopathic effects or host cellprotein shut-down.

2] BTV infected γδ T cells can be isolated from experimentally infected sheep,during viraemic periods.

3] BTV-persistently infected γδ T cells can be converted to a productive, lyticinfection by co-culturing them with anti-WC-1 antibody or with certain skinfibroblasts.

4] Culicoides feeding induces skin inflammation in ruminants.5] Skin inflammation recruits activated γδ T cells into the inflamed areas. 6] BTV can be isolated from cultures of activated γδ T cells derived from skin

biopsy sites on previously infected sheep and cultured with IL-2, for at least

9 weeks after the termination of viraemia.7] Proteases in inflamed skin can cleave the outer capsid protein of BTV to form

infectious subviral particles (ISVP) which are >100 times more infectious tomidges than intact virus particles an event that may occur in midge bittenareas of skin.

Thus, inflammation induced by midge bites recruits BTV persistently infected γδT cells to the site of midge feeding where interactions with skin fibroblastsconvert the persistent infection to a lytic one. The released BTV is then cleavedinto the more infectious ISVP’s by inflammatory proteases thereby enhancingthe likelihood that an infection will be established in vector midges. To date, many of the components comprising this mechanism remain to beexplored and confirmed. These include the following:1] Where are persistently infected γδ T cells localised in the host during vector

free periods? 2] How do persistently infected γδ T cells (and possibly other cell types) remain

undetected by immune surveillance? 3] Is there a role in this mechanism for other lymphocytes subsets (i.e. CD4+

and CD8+ T cells also can be persistently infected with BTV in vitro)? 4] Are some species/breeds of ruminant more likely to support overwintering of

BTV than others? 5] Are some BTV types/strains more adapted to overwintering than others? 6] What is the molecular mechanism by which BTV fails to shut-down

persistently infected γδ T cells, and how does WC-1 signalling enhance BTVreplication?


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H.H. Takamatsu

C 1ABSTRACT BOOK

GLOBAL ISOLATES OF BLUETONGUE VIRUS SEGGREGATE INTO REGION-SPECIFICTOPOTYPES BASED ON PHYLOGENETIC ANALYSES OF THEIR NS3 GENESU.B.R. Balasuriya, S.A. Nadler, W.C. Wilson, A.B. Smythe, P. De Santis, N. Zhang and N.J. MacLachlanDepartment of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA; Phone (530) 752-1323; Fax(530) 752-3349; e-mail [email protected]


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There are at least twenty four bluetongue virus (BTV) serotypes worldwide, withvariation of each of the 10 individual gene segments giving rise to many geneticallydistinct virus strains within each serotype. Field isolates of BTV evolve both bygenetic drift and genetic shift, the former as a consequence of quasispeciesevolution and founder effect in vector insects and the latter as a consequence ofreassortment of individual virus gene segments in either the insect or ruminanthost. The global distribution of BTV parallels the location of competent insectvectors, however, it is clear that ecological factors can influence the distribution ofspecific virus serotypes and strains; for example only BTV serotypes 10, 11, 13 and17 currently occur in North America, and their distribution coincides with thedistribution of Culicoides sonorensis, whereas Culicoides insignis is the vector ofBTV serotypes 1, 2, 3, 4, 6, 8, 11, 12, 13, 14 and 17 in the Caribbean andCentral/South America. Furthermore, with the notable exception of BTV serotype2 that transiently incurred into Florida, there is little apparent movement of thevirus serotypes unique to each region between the two ecosystems, despite thelack of geographic boundaries and the rapid movement of millions of cattle betweenthe two regions under the North American Free Trade Agreement. The NS3 protein encoded by the S10 gene segment is associated with virus egressfrom infected cells. Previous studies suggest that the NS3 gene may co evolve withthe specific insect vectors of BTV that occur in different regions of the world, thus,phylogenetic analysis based on the NS3 gene may be useful in predicting the globalevolution of BTV strains, independent of their serotype. Phylogenetic analyses ofthe NS3 gene of a large number of BTV isolates from the Americas (North andCentral), Caribbean Islands, Australia, Asia (China and India) and Europe (Italy and

France) segregated the viruses into two distinct clades, regardless of BTV serotypeor year of isolation. One clade segregated into 2 distinct groupings, one ofAmerican/Caribbean isolates of BTV and the other of African isolates. The onlyexception was isolate 11US631 (identified as prototype US BTV serotype 11, butwith a very different sequence than the confirmed US prototype strain of BTV 11[Pierce et al., 1998]) that grouped with the African viruses, raising the possibilityof laboratory contamination of US serotypes with African serotype 11 as previouslysuggested by Meissner et al., (2001). The other clade contained three very distinctlineages representing viruses from Asia/Australia, Americas/Caribbean, andAfrica/Europe. The recent European BTV isolates from Italy (BTV4 and 9) andFrance (BTV2) were closely aligned with African viruses in the two different majorclades. Interestingly, one of the European viruses segregated with the SouthAfrican vaccine strain of BTV2.The phylogenetic analyses of the NS3 gene of global isolates of BTV clearlysegregated the viruses into groupings reflective of their geographic origins,regardless of serotype or year of isolation. The fact that the viruses segregated intodistinct Asian, American/Caribbean, and African/European groupings indicates thatthese viruses have not been recently circulated around the world by animalmovement; rather the viruses segregate according to their geographic origins andrespective Culicoides vectors. The distinct region-specific (topotype) groupings ofglobal isolates of BTV that were identified in this study by phylogenetic analyses ofthe NS3 gene suggest that unique strains of BTV arise as a consequence ofprolonged co-evolution of the virus with the different species of insect vectors thatoccur in different regions of the world.

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Bluetongue virus and Bluetongue disease C 2ABSTRACT BOOK

ISOLATION AND MOLECULAR CHARACTERIZATION OF BLUETONGUE VIRUS FROM SHEEPS.M. Byregowda, V.V.S. Suryanarayana, L. Muniyappa, G. Krishnappa, C. Renuka Prasad and Suguna RaoE-mail: [email protected]

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Bluetongue disease is known to cause a serious disease in sheep withsevere economic loss to the farming community. The disease is endemicin India and is seen between October and February, after the rainyseason. In a recent outbreak of the disease, the morbidity observed wasup to 60 % and mortality was up to 20%. Bluetongue virus was isolatedfrom the blood samples collected in heparin both by embryo inoculationas well as by direct cell culture inoculations in BHK21 cells. After twopassages in embryos, one passage in Aedes alboticus C36/9 was givenbefore adopting to BHK21 cells. In case of direct inoculation, thecharacteristic cytopathic effect (CPE) was noticed in fourth passage levelafter three blind passages.

The virus was confirmed by reverse transcriptase polymerase chainreaction (RT-PCR) using primers specific to VP7 genome, coding for majorgroup specific core protein and of NS3 genome which is also conservedamong BTV. The amplified regions of both VP7 and NS3 genome weresequenced and compared for homology among other genome sequencesavailable in the genebank. A partial fragment of genome coding for typespecific VP2 fragment of the virus was also sequenced and compared withthe published sequences. The possibilities of knowing the serotype bysequence homology of the VP2 genome with the genome sequencesalready available was explored. The virus isolate was later serotyped asBTV-23 by the ARC-Onderstepoort.

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CHARACTERIZATION AND SEROPREVALENCE OF BLUETONGUE VIRUSIN THE MAHARASHTRA STATE OF INDIAV.V. Deshmukh, S.G. Kale and M.B. GujarDepartment of Microbiology, College of Veterinary & Animal Sciences Maharashtra, Animal & Fishery Sciences University, Parbhani 431 402 ( Maharashtra), India. E-mail: [email protected]

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The purpose of present study was to characterize the bluetongue virusisolate and to study seroprevalence of bluetongue virus in cattle andbuffaloes in the Marathwada region of the Maharashtra State of India.Atotal of two passages in embryonated chicken eggs and three passagesin BHK-21 cell lines were made to study biological character ofbluetongue virus isolate. Lesions observed in dead embryos were ofedema , haemorhage and cherry red colouration. The cytopathic effect(CPE) of rounding, aggregation in grape like clusters and detachment inBHK-21 cells were typical of bluetongue virus. On Haemotoxylin & Eosin(H&E) staining of infected cells intracytoplasmic eosinophillic inclusionscharacteristic of bluetongue virus were seen.The bluetongue virusisolate was inactivated when exposed to a temperature of 56°C for

different intervals of time.The isolate also lost infectivity when kept in amedium of pH 3.0 for hour at 25°C.The virus was inactivated aftertreatment with 0.25 percent trypsin for different intervals of time.Treatment with 5 percent chloroform v/v had no effect on infectivity ofbluetongue virus isolate. Serum samples of cattle and buffaloes werecollected from slaughter houses and were tested by gel diffusion forseroprevalence of bluetongue. Over all 13.33 percent of largeruminants were positive for bluetongue antibody. Species wise 17percent cattle and 9 percent buffaloes were positive for blue tongueantibody. In conclusion, the characterization of bluetongue virus isolatewas done. Seroprevalence of bluetongue antibody in cattle and buffaloeswas recorded.

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REPLICATION OF EPIZOOTIC HEMORRHAGIC DISEASE VIRUS IN DH 82 CELLSE.W. Howerth, G.S. Parlavantzas and D.E. Stallknecht Departments of Pathology and Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, GA 30605. USA. 706-542-5833 (phone), 706-542-5828 (FAX), [email protected]

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Bluetongue viruses (BTV) and epizootic hemorrhagic disease viruses(EHDV) can be propagated in a variety of mammalian cell lines rangingfrom green monkey fibroblasts (Vero cells) to bovine nasal turbinate cells.However, in the ruminant host, these viruses infect and replicate inendothelium and monocytes/macrophages. Primary ruminant endothelialcell cultures that can be used to study virus-endothelial interactions areavailable and can be propagated and passed numerous times. On theother hand, continuous ruminant monocyte cultures are not available andin vitro studies of virus-monocyte interactions rely on primarymacrophage cultures that cannot be propagated. Dogs were shown to besusceptible to BTV infection when pregnant bitches died of bluetonguefollowing inoculation of a BTV contaminated vaccine. Thus, an availablecontinuous cell line of dog monocytes, namely DH82 cells, might besusceptible to these viruses and could prove useful in the study of virus-monocyte interactions. The objective of this study was to determine ifDH82 cells are susceptible to BTV and EHDV infection.Cultures of DH82 cells were inoculated with either 102.5 TCID50 of EHDVserotype 2 or 102.5 TCID50 of BTV serotype 10. Viral titers in culturesinoculated with both EHDV (104.8 TCID50/ml) and BTV (105.26 TCID50/ml)cells were significantly increased over controls (virus and media withoutcells) by post inoculation day 2. Titers peaked slightly on post inoculation

day 3 (BTV=105.6 TCID50/ml; EHDV=105.26 TCID50/ml), but remained highuntil termination on post inoculation day 5. Cultures were also infected with EHDV serotype 2 and examinedultrastructurally. At 24 hours post inoculation, infected cells had multiplecoated pits on their surface, some of which contained viral particles. Inaddition, a few viral particles and viral-associated macrotubules werepresent in the cytoplasm of some cells. By post inoculation day 3, virtuallyevery cell was infected and contained large viral matrices and enormousnumbers of viral-associated macrotubules. Surprisingly, lysis of infectedcells, as is seen in other cell systems (e.g. BHK-21 and bovine endothelialcells) at similar times post infection, was not observed (personalobservation). DH82 cells are susceptible to both EHDV and BTV infection resulting inrelatively high titers. This system allows for the infection of a largenumber of monocytes and could be useful for viral receptor studies.Infection also resulted in the production of large quantities of viral-associated macrotubules and this feature might be useful for structuralor functional studies of these tubules. DH82 cells are also used for theisolation of ehrlichial and anaplasmal agents of deer, and isolation ofthese agents could be confounded by the concurrent isolation of eitherEHDV or BTV.

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EXCRETION OF BLUETONGUE VIRUS IN CATTLE SEMEN -A FEATURE OF LABORATORY - ADAPTED VIRUSP.D. Kirkland, L.F. Melville, N.T. Hunt, C.F. Williams and R.J. Davis Elizabeth Macarthur Agricultural Institute, PMB 8, Camden NSW, 2570, Australia. Ph: 61-2-4640 6331; Fax: 61-2-4640 6429. Email:[email protected]

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The studies described in this presentation were designed to investigate factors thatmay be involved in the excretion of BLU virus in bovine semen. The study alsoinvestigated the possibility of persistent infection in these animals. These issues haveinfluenced international policy for the movements of both live animals and semenbetween countries, and sometimes, within countries.A series of experiments were conducted over a period of 4 years and involved bothyoung (2-4 years) and old bulls (5-15 yrs) that were both naturally andexperimentally infected. Several virus serotypes were also studied. In the NorthernTerritory, young bulls were exposed to natural infection with BLU viruses over threewet seasons. During this period bulls were infected with BLU 1, BLU 3, BLU 16 andBLU 20. In New South Wales (NSW), semen samples were examined from a largegroup of mixed age bulls that were naturally infected with BLU 1. Experimentalinfections in both young and old bulls (5-8 animals per group) employed both 'wild-type' and laboratory adapted viruses from serotypes 1 and 23. A total of 41 bullswere included in the studies of natural BLU infection and 54 bulls in experimentalinfections. On each occasion that a semen sample was collected, blood samples were alsocollected to monitor the onset and duration of viraemia. From day 7 afterexperimental infection, samples were collected twice weekly for 4 weeks then oncea week for a further 4 weeks. Samples were collected twice weekly from bullsthroughout the period of possible natural exposure. At approximately 8 weeks afterthe onset of infection, 9 bulls were treated with a high dose of corticosteroids toinduce immunosuppression. Sampling then continued weekly for a further 4 weeks.At the conclusion of the studies (between 8 -16 weeks) bulls were slaughtered and

spleen and a full range of reproductive tissues collected for virus isolation. Methods used to detect infectious virus in blood and semen included the inoculationof embryonated chicken eggs followed by passage in insect and mammalian cellcultures, direct passage in both insect and mammalian cell cultures, and inoculationof sheep. For both blood and semen, a large volume of sample was examined tomaximise virus detection. Serological methods (AGID, cELISA and VNT) were alsoemployed to monitor infection. There was no evidence of BLU virus in any of the semen samples collected fromnaturally infected bulls or experimentally infected young bulls. BLU virus wasdetected intermittently in semen from a number of old bulls infected with eitherlaboratory-adapted BLU 1 or BLU 23 virus. These detections occurred during orimmediately after the period of detectable viraemia. Virus was also detected in a fewsemen samples from very old bulls infected with 'wild-type' BLU 23. However thesesamples were collected during the period of viraemia and there was visible evidenceof blood in the semen. Viraemias varied in duration between 17 and 31 days.Following immunosuppression, there was no evidence of resurgence of viraemia, norexcretion of virus in semen, even in animals in which virus had been previouslydetected. When the bulls were slaughtered, virus was not detected in any tissues,even after sheep inoculation.It was concluded from these studies that, when semen is not contaminated withblood, the excretion of BLU virus is confined to old bulls that have been infected withlaboratory adapted virus. In this study, the duration of viraemia varied between17and 31 days and there was no evidence of persistence of the virus, even withlaboratory adapted strains.

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INVESTIGATION OF BLUE TONGUE DISEASE IN TAMIL NADU, INDIAA. Koteeswaran, T.G. Prabhakar and N. Daniel Joy Chandran Ph.D., Director, Centre for Animal Health Studies, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Chennai – 600 051, Tamil Nadu,India. Tel: 0091-44-2555 5151. Fax: 0091-44-2555 1577. E-mail: [email protected]

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Blue tongue (BT) is a vector borne viral disease of sheep and goat. Amajor outbreak of Blue tongue was reported in sheep goats in SouthernDistricts of Tamil Nadu, India during 1997. The morbidity and mortalitywere 70% and 50% respectively. The clinical sign recorded were fever,oedema of the face, hemorrhages and ulceration of the mucousmembrane. The tongue showed intense hyperemia, swollen, edematousand in severe cases became cyanotic. Due to dermatitis there was woolbreaks. Coronitis with hemorrhages of coronary brand of the hoof resultedin lameness. Incidence of Bluetongue in Tamil Nadu had been reportedsince 1987 with very low mortality and morbidity. In the present outbreak,due to continuous and incessant rainfall with high relative humidity whichwere conducive for multiplication of insect vectors, the disease assumeda severe form resulting in high morbidity and mortality.Blood collected from febrile animals in Oxalate-phenol-glycerin (OPG)were used for virus isolation in embryonated chicken eggs (ECE) by

intravenous route and yolk sac route. Those embryos that died betweendays 2 and 7 were examined and embryos that showed a “Cherry red”appearance were homogenized after removal of the head and debris bycentrifugation. The virus in the supernatant was passaged in BHK21 cellline and monitored for cytopathic effect. Approximately one third of cellsbecame rounded and detached by 72 hrs and all the cells finallyrounded; aggregated and fell off the glass. The specificity of BT virusinduced CPE was confirmed by florescent antibody test. The BT viruswas also confirmed by animal experimentation in sheep and the diseasecould be reproduced experimentally at 4th animal passage level. The BTisolates were sent to Institute of Animal Health, Pirbright, UK andOnderstepoort, South Africa for serotyping and the BT virus was foundto belong to BTV type 23. In subsequent outbreak of BT in Tamil Nadu,BT virus was isolated and was typed at Institute of Animal Health,Pirbright, UK as BTV type1.

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BLUETONGUE VIRUS DOES NOT PERSIST IN NATURALLY INFECTED CATTLEL.F. Melville, N.T. Hunt, S.S. Davis and R.P. Weir Department of Business, Industry and Resource Development; GPO Box 3000 Darwin NT 0801; Australia. Ph: 61 8 8999 2251; Fax: 61 8 8999 2024; Email:[email protected]

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Takamatsu et al. (2003) reported that infectious BTV could be recoveredfrom ovine skin biopsies more than 9 weeks post infection. Following thisreport monitoring of cattle naturally infected with BTV1 was carried out toassess if these observations were applicable to cattle. The cattle were alsoexposed to insect attack throughout the observation period.Skin biopsies and blood samples were collected weekly from 16 cattlenaturally infected with BTV1. The blood samples were processed for virusisolation by embyronated chicken egg (ECE) inoculation and for serologyby BTV cELISA and BTV1 virus neutralisation. BTV1 was isolated from allanimals and serology confirmed infection with BTV1. A total of 171 skinbiopsy samples were collected and cultured in the presence of IL2 andepidermal growth factor (EGF). Sampling commenced either duringviraemia or up to 7 days after the last isolation of BTV1. Weekly samplingcontinued until 42-90 days and monthly sampling to 102-150 days after

the last isolation. Skin biopsy cultures were harvested 7-10 days afterprocessing and held at – 80°C prior to virus isolation by ECE inoculation.BTV1 virus was not isolated from the skin biopsy cultures. In 5 of these infected cattle lymphocytes were harvested from bloodsamples collected during viraemia to 28 days after the last isolation ofBTV1. Lymphocytes were isolated through Nyco-Prep and co-cultured inthe presence of IL2 and EGF with a primary bovine skin fibroblast cell line.The skin fibroblast cells were previously shown to support BTV1 growth.Co-cultures were harvested at 7 days and processed for virus isolation byECE inoculation. BTV1 was not isolated from the lymphocyte/skinfibroblast co-cultures.These results show that there is no evidence of persistent BTV infection oflymphocytes in naturally infected cattle and BTV cannot be isolated fromcultured skin biopsies from these animals.

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PHYLOGENETIC ANALYSIS OF BLUETONGUE VIRUS GENOME SEGMENT 6(ENCODING VP5) FROM DIFFERENT SEROTYPESK.P. Singh, S. Maan, A. R. Samuel, S. Rao, A. Meyer and P.P.C. MertensInstitute For Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey, GU24 0NF(UK). Telephone: 0044 1483 231094. Fax: 0044 1483 232448. E-mail :[email protected]

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95

Bluetongue virus (BTV) is an arthropod-borne agent (transmitted by biting midges ofCulicoides species), which can infect domesticated and wild ruminants. The viruscauses 'bluetongue' disease in sheep, which can be acute, with high mortality but ismore usually mild in cattle and goats. BTV is currently prevalent in the Mediterraneanregion, involving serotypes 1, 2, 4, 9 and 16, with strong evidence that the disease isgradually penetrating further north. The virus is also endemic in the Indian sub-continent involving as many as 21 different serotypes. Bluetongue virus is icosahedraland non-enveloped. It is composed of a core, containing the 10 dsRNA segments ofthe virus genome, three minor proteins of the transcriptase complex, VP1(Pol),VP4(Cap) and VP6(Hel) and two major protein components, VP3(T2) (sub-core shell)and VP7(T13) (core surface layer). Within the intact virion, the core is surrounded byan outer capsid layer containing 2 major proteins VP2 and VP5, which are morevariable than the core proteins and determined the virus serotype. VP2 and VP5 areprimarily involved in cell attachment and initiation of infection, and interact withneutralising antibodies. The segmented nature of the BTV genome makes it possiblefor different strains of the virus that infect the same cell, to exchange genomesegments by a process called reassortment. This may play a important role in theemergence of virus strains with novel combinations of serological and/or biologicalproperties. The present study is being undertaken to analyse the nucleotide sequenceof genome segment 6 from all 24 BTV serotypes, to provide more information onsequence variation in this gene and its correlation with virus serotype, as well as dataon the relative frequency of reassortment that occurs in the field.The viruses used in this study were grown in BHK-21 cells and their dsRNA waspurified using TRIZOL (Life Technologies). Full length cDNA was generated fromsegment 6 of different isolates and cloned in PGEMT- easy Vector. Clones were

screened by PCR using universal M13 F and R primers and by restriction endonucleasedigestion using BamH 1 and Apa 1. Plasmid DNA was sequenced using a BeckmanCapillary Sequencer. cDNA was also sequenced directly using BTV RNA-termini specificand gene specific primers. Nucleotide sequence data was aligned and analysed usingthe Bio Edit sequence alignment editor. Full-length segment 6 data of different BTVisolates were compared within and between different serotypes. The results of this study showed the level of genetic diversity in genome segment 6,among different BTV serotypes, as well as between isolates of the same serotype. TheBTV 1 isolate from Greece is closely related genetically to strains from India andAustralia but quite distinct from those from Africa. In contrast, BTV 2 from India isquite different to the European and African isolates, which are themselves closelyrelated. These data indicate the origins of the different European BTV strains and havehighlighted differences between the vaccine and field isolates of serotypes 1, 2 and 4,which may be sufficient to design RT-PCR primers to distinguish them. Sequence datafor BTV genome segment 6 will also help us to determine the origins of other virusstrains, increasing our understanding of BTV epidemiology and transmission.This database can also be used to facilitate primer design and RT-PCR conditionssuitable for the amplification of segment 6 from new BTV isolates. These methods(together with those that have also been generated for BTV genome segment 2, arebeing used as the basis for serotype specific RT-PCR assays, to improve the speed andreliability of BTV serotype determination. The sequences that are generated will beadded to those already available for the segments of different BTV serotypes in theinternational databases and listed at http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/btv_sequences.htm providing important diagnostic and research tools.

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POSSIBILITY OF BLUETONGUE VIRUS IN BOVINE SKIND.M. White and J.O. MechamUSDA, ARS, Arthropod-borne Animal Diseases Research Laboratory, PO Box 3965, Laramie, WY 82072, USA; voice: 307-766-3601; fax: 307-766-3500; e-mail:[email protected]

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The overwintering mechanism of bluetongue virus (BTV) has eludedresearchers for many years. It was recently proposed that ovine gamma-delta T-cells may become persistently infected with BTV, and serve as areservoir for infection of naïve vectors in the next transmission season(Takamatsu et al., Journal of General Virology 2003, v. 84 pp. 227-35).Since cattle are prevalent in the western United States (where BTV isendemic), this hypothesis was tested in bovines. In the winter of 2002, 56cattle from an endemic site in northern Colorado were age-selected such

that possible BTV exposure must have occurred in the summer of 2002,and were tested for the presence of anti-BTV antibody by ELISA; 55 wereseropositive, and one was seronegative. Naïve colony insects were fed onskin sites at day 0, then sequentially on separate sites for 4 days. Virusisolation and RT-nested-PCR from engorged insects and 6mm skin biopsysamples were performed for detection of viable BTV or BTV nucleic acid.Results will be presented and discussed.

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THE CHARACTERISATION AND MONITORING OF NEUTRALIZATION-RESISTANT VP2PHENOTYPES IN BTV-1 ISOLATES FROM NORTHERN AUSTRALIA COLLECTED OVER ATWENTY YEAR PERIODJ.R. White, V. Boyd, J.K. Brangwyn, C.J. Duch, L.I. Prichard, L. Melville, T.D. St. George and B.T. Eaton Australian Animal Health Laboratory, CSIRO, Division of Livestock Industries, PMB 24, Geelong, Victoria, 3220, Australia. Ph +61 3 5227 5216, fax +61 3 52275555. [email protected]

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Field isolates of bluetongue virus serotype 1 (BTV-1) collected from differentsites in Queensland and the Northern Territory between 1979 and 1986 showedsignificant variability in their susceptibility to neutralization by a panel of sevenmonoclonal antibodies (MAbs) specific for the VP2 protein of this virus. We hadearlier produced neutralization resistant variants of BTV-1 using these MAbsand via nucleotde sequencing, located specific amino-acid substitutions on theprotein associated with neutralization resistance. We were therefore interestedto fully sequence the VP2 coding gene of our collection of BTV-1 field isolatesboth from the 1979 – 86 period and from a later collection period (1996 –2001), to investigate the extent and nature of ‘drift’ in their VP2 amino acidsequence and whether there was any correlation with the particular sites thathad earlier been shown to be associated with neutralization resistance. We alsowanted to determine whether VP2 phenotypes showed significant sequencevariation between collection sites and between the two States in whichcollections were taken. Viruses collected at three separate sites in the Northern Territory during 1979and 1980 showed virtually identical amino-acid sequence to the prototypeAustralian BTV-1 isolate (CSIRO156). However, isolates from two sites inQueensland collected from 1981-1983 displayed a 3% variation in their aminoacid sequence compared to CSIRO156 and showed significant resistance toneutralization by six of the VP2-specific MAb panel. A further isolate from theNorthern Territory in 1986 still essentially conformed to the prototype sequenceyet showed resistance to neutralization by two of the MAb panel. When isolatesfrom the second collection period (1996 – 2001) were examined, the ‘resistant’

Queensland phenotype predominated in viruses collected at all sites in bothStates. The VP2 amino acid sequences of these viruses showed a high level ofhomology (>99%) and showed greater homology to the resistant phenotypeisolated in 1981 than to the prototype BTV-1 isolate. When the location of significant amino acid changes (compared to the prototypeBTV-1 isolate CSIRO156) present in the neutralization resistant isolates werecompared to those seen in our collection of neutralization escape variants, 5sites were found to be identical (ie. aa ‘s 219, 267, 270, 539 and 586) and afurther change was within two amino acids distance from a substitution seen inone variant (ie. 209 and 211 respectively). Of further interest were the resultsof a comparison between the prototype isolate amino acid sequence and thatof an attenuated virus isolate of BTV-1 (South Africa). This virus showed asimilar pattern of resistance to neutralization by the VP2-specific panel to thatpreviously seen with the neutralization resistant field isolates. In particular,four sites of significant amino acid difference were identical to those seen in theprevious comparison (ie. 219, 267, 270 and 586). Three other identical aminoacid changes were seen in both the neutralization resistant field isolates and theattenuated BTV-1 isolate (ie. aa’s 384, 395 and 435) but not seen in any of thevariant virus pools.Collectively, these data indicate that a VP2 phenotype originating in Queenslandhad become established in the Northern Territory at some period in the previoustwo decades and displaced the previously dominant phenotype, possibly in partdue to the selective pressure imposed by the immune response of host animals.

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DIAGNOSTICS

ABSTRACT BOOK

“VIRUS ISOLATION AND IDENTIFICATION” OR“VIRUS IDENTIFICATION AND ISOLATION”?B.T. Eaton Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation • 5 Portarlington Road • Geelong, Victoria 3220 • Australia •Tel: (61) 3 5227 5116. Fax: (61) 3 5227 5555. E-mail: [email protected]

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Traditionally, virus isolation has preceded virus identification andcharacterization. Thus, bluetongue viruses have been isolated from sheep,cattle and insects by a variety of methods prior to their biochemical,antigenic and biological characterization. In recent years however, wehave seen how molecular detection systems have revolutionized medicalinfectious disease diagnosis and have removed the requirement to isolatepathogens as a means of confirming clinical diagnoses. Pathogendetection methods that utilize PCR-based multiplexed technologiesprovide clinicians with the speed, sensitivity and discriminating power toidentify disease-causing organisms and respond rapidly to provideappropriate medical treatment. Application of PCR-based methods inmultiplexing formats also have the capacity to detect novel pathogens andvariants of existing pathogens. Detection and characterization ofveterinary pathogens will follow the same evolutionary path and to thatend, work is underway in a number of laboratories to develop theinfrastructure and databases required to permit the use of DNA-basedmolecular systems for detection and characterization of orbiviruses.Ultimately such systems based on RNA segment 2 will provide informationon virus serotype and those based on other segments such as RNA 3 will

provide topotype data. Mutliplexing approaches that utilise a number ofRNA segments will have the capacity to provide both serotype andtopotype information and identify a wide range of reassortants. However, many reasons remain to isolate pathogenic and non-pathogenicbluetongue viruses. While rapid identification of virus RNA in clinical orsurveillance samples and characterization to family, genus, serotype andtopotype level on the basis of nucleic acid sequence are very powerfuladjuncts to clinical and epidemiological studies, we do not yet knowenough about the molecular basis of pathogenesis and virulence or themolecular foundations of the complex antigenic basis of serotype towarrant relying on nucleic acid sequences solely for virus characterization.In addition, there are many situations where it will be necessary to uselive virus in cell culture, animal or vaccine studies. In addition the speedand multiplexing capacity of PCR-based technologies must not blind us tothe fact that these techniques do not detect live virus per se. Thus virusisolation will remain as an important component of bluetongue virusdiagnosis and research. Methods of virus isolation developed over manyyears will be summarised.

Diagnostics

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B.T. Eaton

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DiagnosticsABSTRACT BOOK

APPLICATION OF LABORATORY DIAGNOSTIC TESTSC. Hamblin Institute for Animal Health, Pirbright Laboratory, Ash Road Pirbright, Woking, Surrey, GU24 0NF, UK. Tel **(0) 1483 232441. Fax (0) 1483 232448. [email protected]

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The occurrence of bluetongue, particularly in countries that are normallyfree of disease, can have a devastating effect on international trade andanimal welfare. Although the clinician can make a presumptive diagnosiswith some degree of confidence, confirmation is inevitably sought from aninternationally recognised diagnostic laboratory. Traditionally, the laboratory confirmation of the clinical diagnosis hasrelied on the premise that virus and antibody bind together to form anantigen/antibody complex. As a consequence most of the currentlyavailable diagnostic tests have been designed with that in mind. The OIEManual of Standards for Diagnostic Tests and Vaccines describes severalserogroup and serotype specific immunological tests, as well as the newermolecular based assays such as PCR. Most of the immunological assaysmentioned have been available for many years and are well established.In recent years the sensitivity, specificity and ease of performance ofsome of these assay methods has been questioned and as a result someare either no longer routinely used or have been superseded by improvedmethodologies. Ideally the test of choice should be sensitive, specific,rapid, reliable, validated and accepted, easy to perform and economic in

terms of reagents and cost. When faced with either an outbreak of disease or an incursion of viruslaboratory diagnosticians must draw upon their experience and knowledgeto ensure that they apply appropriate tests that will satisfy theepidemiological situation, thereby confirming the identity of the virusand/or antibody. To help ensure that this is achieved, all availableinformation should be sent to the laboratory with the test samples. Forexample, have the animals been vaccinated, how frequently were theyvaccinated, when were they last vaccinated and against what serotypes.Other information relating to the number of animals affected, clinicalsigns, location, and previous outbreaks are also important. A successfullaboratory diagnosis is also dependent on the quality and type of sampleand the conditions under which they are submitted. This presentation will describe some of the advantages and limitations ofdifferent assays currently used and the difficulties in interpreting some ofthe results generated. Modifications and adaptations of test proceduresthat can be used when testing ‘difficult’ samples will also be discussed.

C. Hamblin

ABSTRACT BOOK

BLUETONGUE DIAGNOSIS BY REVERSE TRANSCRIPTION-POLYMERASECHAIN REACTIONS. Zientara, E. Bréard and C. SailleauAgence Française de Sécurité Sanitaire des Aliments –Alfort, 22 rue Pierre Curie, 94703, Maisons-Alfort, France. Tel : ++ 33 1 49 77 13 12 ; Fax : ++ 33 1 43 6897 62 E-mail: [email protected]

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Bluetongue virus (BTV) is the prototype of the member of the Orbivirusgenus within the family Reoviridae. The BTV serogroup contains 24serotypes. Diagnostic tests which are currently used for the detection ofbluetongue virus involve the isolation and growth of virus isolates in eggsor mice, followed by passage in tissue culture. The virus is subsequentlycharacterized by serological means, involving its reaction with referenceantisera, for example, agar gel immunodiffusion test or serumneutralization test. These procedures are time consuming and may fail todetect low levels of infectious virus or strains of BTV which fail to replicatein eggs, mice or tissue culture. The use of the ELISA for the detection ofantibodies to BTV in previously infected animals is faster but in theabsence of a highly sensitive technique and specific test for BTV antigen,these serological techniques do not allow to direct detection of the virus

itself in tissue sample. A number of procedures have been developed todetect the presence of BTV antigens or nucleic acids. Polymerase chainreaction (PCR) technique has appeared to be a powerful tool in the fieldof BTV diagnosis. Polymerase chain reaction techniques may be used, notonly to detect the presence of viral nucleic acid, but also to ‘serogroup’orbiviruses and provide information on the serotype and possiblegeographical source (topotype or genotype) of BTV isolates within a fewdays of receipt of a clinical sample such as infected sheep blood.Traditional approaches, which rely on virus isolation followed by virusidentification, may require at least 3-4 weeks to generate information onserogroup and serotype and yield no data on the possible geographicalorigin of the isolated virus. Moreover, PCR allows sometime todifferentiate between field isolates and vaccine strains.

Diagnostics

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S. Zientara

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Diagnostics D 1ABSTRACT BOOK

THE DIFFERENTIAL DIAGNOSIS OF BLUETONGUE VIRUS: A PCR APPROACHS. Anthony, S. Maan, A. Samuel, P.S. Mellor and P.P.C. MertensInstitute of Animal Health. Orbivirus Group, Ash Road, Pirbright, Woking, Surrey, UK GU24 ONF. Phone: + 44 (0) 1483 23 11 49. E-mail:[email protected]

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As bluetongue virus (BTV) persists and continues to spread within Europe, the threatthat it poses to international trade in livestock remains worryingly high. As a resultthere is a demand for increasingly rapid and reliable methods of BTV diagnosis andPCR is proving to be the method of choice for many new diagnostic tests. Currently,PCR is being used in many laboratories around the world for diagnosis andidentification of bluetongue virus. However, in most cases these tests have not beenfully validated for all BTV strains and may detect only closely related geographicaltopotypes. There therefore remains a need for a comprehensive, virus-speciesspecific RT-PCR assay, which can effectively and reliably identify all 24 serotypes ofbluetongue virus (including all of the different topotypes), with no significant crossreaction with members of other closely related orbivirus species.While many laboratories are starting to use PCR to diagnose BTV, methodologiessuch as ELISA and neutralisation assays remain the principal tools for laboratoryconfirmation. However, the implementation of a fully validated group-specific RT-PCR could have distinct advantages over existing methodologies, in terms of speed,sensitivity and availability of diagnostic reagents. Its establishment as a diagnostictest would provide important support, in primary screening for diagnostic samples,when used in conjunction with the existing tests. Equally, RT-PCR could provide animportant method for monitoring the epidemiology of the virus, e.g., detection oflow-level virus transmission between vector and host (for which current techniquesare unsuitable). Ideally a diagnostic RT-PCR assay would be specific for members ofthe BTV serogroup, would not cross-react with related orbiviruses and would besuitable for use on clinical samples of blood and tissue.

The development and validation of an RT-PCR assay that could be used routinelyto identify all serotypes and topotypes of BTV will be reported. The RT-PCRemploys terminal primers for genome segment 7 of BTV, as previously designedby Wade-Evans et al (1990). 'Touch-Down' PCR technology was used to ensurethat the PCR is highly specific for the BTV serogroup, and the 'b-actin system'was used as a control to monitor template stability during sample preparation.The assay can be used to detect all 24 BTV serotypes, including isolates fromeach of the topotypes that were available in the Pirbright reference collection upto March 2003 see: (http://www.iah.bbsrc.ac.uk/ dsRNA_virus_proteins/ReoID/viruses-at-iah.htm). Many of the isolates available in the referencecollection are currently being sequenced, which will serve to provide additionaldata for BTV segment 7, and if necessary, allow the design of better group-specific primers.Results from studies to optimise the RT-PCR assay for use on tissue-culturederived virus isolates as well as clinical samples, such as blood and tissue (e.g.spleen) will be presented. In particular an evaluation of the effect of samplestorage conditions on template stability and the subsequent detection of BTVdsRNA by PCR will be described. This has allowed us to make somerecommendations for sampling methods, to optimise virus detection of BTV inclinical samples. Using these methods studies on different sample types (includinginsects) have yielded encouraging results. These methods will be used in a morecomprehensive future study involving detection of BTV RNA in midges toinvestigate BTV epidemiology.


APPLICATION OF MOLECULAR BIOLOGICAL TECHNIQUES FOR RAPID DETECTION ANDDIFFERENTIATION OF BLUETONGUE VIRUS AND PALYAM ORBIVIRUSES SEROGROUP I.E. Aradaib (1), M.E.H. Mohamed (1) and B.I. Osburn (2)

(1) Molecular Biology Laboratory (MBL), Faculty of Veterinary Medicine, P.O. Box 32, Khartoum North, Sudan. (2) Department of Pathology, Microbiology andImmunology, School of veterinary Medicine, University of California, Davis, CA 95616.

Molecular Biology Laboratory (MBL), Department of Medicine, Pharmacology and Toxicology, Faculty of Veterinary Medicine, P.O. Box 32, Khartoum North, Sudan. E-mail:[email protected]

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Molecular biological techniques for rapid detection and differentiation ofBluetongue virus (BTV) and Palyam serogroup of orbiviruses weredescribed in this study. Using agarose gel and sodium dodecyl sulphate(SDS)- polyacrylamide gel electrophoresis (SDS-PAGE), the dsRNAgenome segments profiles of Sudanese BTV serotypes 1, 2, 4 and 16 anduntyped isolates of palyam viruses showed 10 bands, which ischaracteristic pattern of orbiviruses serogroup. The agarose gel showedidentical genome profiles for all isolates of BTV and palyam orbivirusesserogroup. However, SDS-PAGE system was able to detect geneticvariation between BTV and palyam viruses. Application of reversetranscriptase (RT) polymerase chain reaction (RT-PCR) assays resulted in

amplification of a 821 base pair (bp) PCR products and 520 bp PCRproduct from BTV and palyam virus RNAs, respectively. The result of thisstudy suggested that, the agarose gel electrophoresis and SDS-PAGEcould be used as supportive or complementary method to facilitatetentative diagnosis of orbivirus infection in susceptible animalpopulations. In addition, the SDS-PAGE could also be used to detectgenetic variations between serotypes of orbiviruses serogroup. However,RT-PCR assay could be used as a definitive diagnostic method forserogroup-specific detection and serotype-specific identification of theseorbiviruses serogroup.

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APPLICATION OF VARIOUS DIAGNOSTIC PROCEDURES TO EPIDEMIOLOGICALSITUATIONS ENCOUNTERED DURING ARBOVIRAL INFECTIONSUSING THE SIMBU SERO-GROUP AS EXAMPLEJ. Brenner, M. Malkinson and H. Yadin Kimron Veterinary Institute, 50250 Bet Dagan, Israel, Tel 972-3-9681688; Fax- 972-3-9681788; Email [email protected]

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The rationale behind the methodology employed to investigate a newdisease that appears in an area hitherto unaffected is basically differentto that applied in an endemic disease situation. Special considerationmust also be given to disease agents that appear and reappear at cyclicalintervals.In this study we present three separate approaches applicable in threedifferent epidemiological presentations. Each of which may be used

initially to determine both the identity of the causal agent and the natureof the disease. Special consideration will be given to situations where the disease appearsintermittently using a sentinel model. Although this latter approach isexpensive and time-consuming, it can yield excellent and reliable resultswhen applied correctly.

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DETECTION OF BLUETONGUE VIRUS BY DIRECT RT-PCR AND BY NON-RADIO ACTIVENUCLEIC ACID PROBES S.M. Byregowda, V.V.S. Suryanarayana, L. Muniyappa, G. Krishnappa, C. Renukaprasad and T.N. AthmaramE-mail: [email protected]

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Bluetongue is an arthropod borne viral disease of ruminants distributed inalmost all tropical and subtropical countries of the world. In India thedisease is endemic and accounts for greater economic loss. Detection ofthe disease and its agent using confirmatory tests are important for takingcontrol measures. Single tube reaction of Reverse transcriptasePolymerase Chain Reaction (RT-PCR) was standardized using the primersdesigned for NS3 genome where in Bluetongue viral RNA could bedetected from as little as 10µl of infected culture fluid with out extractingthe RNA. The polymerase buffer prepared in the laboratory used for bothRT reaction and PCR. The technique is found to be more useful in isolationstudies while passing in cell culture and confirmation of virus presence in

cell culture fluid. The test is simple, rapid and more economical whencompared to the commercial kits available for RT-PCR and eliminates theuse of hazardous reagents required for RNA extraction. The single tubereaction was also found to be efficient for detection of viral genome fromtotal RNA extracted from clinical samples like blood and tissue samples. Non-radio active DIG labelled nucleic probe of the NS3 genome wasprepared using random priming method and used to confirm the presenceof viral RNA in clinical samples as well as in cell culture fluid. The probecould detect efficiently the viral RNA extracted from clinical samples andcell culture fluid that were transferred on to a nitrocellulose membrane.The detailed methods of RT-PCR and probe preparation are discussed.

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BLUE TONGUE: AN OVERVIEW ON RECENT TRENDS IN DIAGNOSTICSH. DadhichDepartment of Veterinary Pathology, College of Veterinary an Animal Science, Bikaner-334001 India. E-mail: [email protected]

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Blue tongue is a viral disease of sheep and occasionally cattle, transmittedby Culicoides. Blue tongue virus (BTV) is an arthropod- borne Orbivirus inthe family Reoviridae. There is considerable genetic variability within theserogroup of BTV having atleast 25 serotypes worldvide. This arises bygenetic drift of individual gene segments when ruminants or the vectorsare infected with more than one strain. Until the 1940s this disease wasrecognized only in Africa, then following a major epidemic in 1956- 1957in Portugal and Spain, the disease was recognized in the US, the middleeast, Asia and later in Australia.Blue tongue virus replicates in haemopoeitic cells and endothelial cells ofthe blood vessels. Rarely, and only when the bull is viremic, blur tongueviruses may be recovered from semen.

These viruses are often difficult to isolate in the laboratory. The successof virus isolation is enhanced if blood is collected from animals showingearly clinical signs. Viremia is primarily associated with red blood cells andleucocytes and the viruses can coexist in infected animals with highconcentrations of neutralizing antibody. Serological techniques, most notably enzyme immunoassays, based onthe detection of a group antigen have been used where required in thecertification of animals as “blue tongue free”. However, intermediateserologic reactions have been a major problem. For accuracy in diagnosis,more sensitive and specific assays such as those based on antigensproduced by recombinant DNA technologies and the polymerase chainreaction (PCR) should prove useful.

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EVALUATION OF NEW PRIMERS FOR IDENTIFICATION OF BLUETONGUE VIRUSSEROTYPE 1 BY NESTED RT-PCRS. Dahiya, Ramesha, Minakshi, S. Verma and G. Prasad Department of Veterinary Microbiology, CCS Haryana Agricultural University, Hisar-125004, Haryana, INDIA. Tel. 91-1662-231171, Extn. 4539 (0), 91-1662-235508(R), Fax: 91-1662-234613, E-mail: [email protected] or [email protected]

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Bluetongue virus (BTV) is causing major epizootics in the tropical,semitropical and temperate regions of the world involving severalserotypes. Serotype identification of BTV isolates is important to theepidemiology of the virus, but current methods are time consuming andcumbersome. More recently PCR assays have been developed for quickamplification of desired gene/region for diagnosis, serotyping, restrictionenzyme analysis, cloning and sequencing. Keeping this in view, differentprimer pairs were evaluated for amplification of unique regions on theserotype-specific segment 2. The primer pair for BTV1A [FP: (2242-2256) 5’- TAC CTC TGT TCT TTC -3’, RP: (2933-2884) 5’- TGA TAG CGCGCG GAC CCA CGG TCG ACC GGG TCA TCT CGA GAG AAG TTT TG -3’]and BTV23A [FP (604-623) 5’- TTG TCC ACG CCG AGC GCG CA -3’, RP:(1262-1243) 5’- ATC GAA CAG GTT CAC TCG GC -3’] used in this studywere reported earlier. BTV serotype 1 primer pair successfully amplifiedsix Indian isolates of BTV1Ind yielding an expected PCR product of 691bpin 25 cycles. This primer pair failed to amplify Indian isolates ofBTV18Ind and BTV23Ind. BTV23A primer pair amplified 658bp amplicon

with serotype 23 only and not with BTV1Ind and BTV18Ind. Howeverthese primer pairs resulted in the amplification of non-specific fragmentswith some isolates that differed in molecular weight from the authenticPCR product. So we designed a new set of primer pair specific for BTVserotype 1. The genome segment 2 specific primers [FP: (1240-1271) 5’-ATG GTC GAG TTA ACC TGT TTG ATT ATG TC -3’, RP: (1844-1813) 5’- AATTCC ACG CCG TTG CAA GAT -3’] were designed using BTV1 genesequences available in GenBank. This primer pair successfully amplifiedcell culture grown six BTV1 Indian isolates from different geographicalregions yielding an expected PCR product of 604bp. This primer pairfailed to amplify Indian isolates of BTV18Ind and BTV23Ind. The serotypespecificity of these primers was tested using genomic dsRNA extractedfrom BHK-21 cell culture grown bluetongue viruses. However, the newBTV1 specific primer pair could not be evaluated against the otherserotypes because of their unavailability in the laboratory. The newprimers appear to have potential application for serotype 1 identificationby nested RT-PCR.

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THE MOLECULAR DIFFERENTIATION OF FIELD AND VACCINE STRAINS OF BLUETONGUEVIRUS SEROTYPE 2 (BTV-2), USING REAL-TIME PCR BY FLUORESCENCE RESONANCEENERGY TRANSFER (FRET) HYBRIDISATION PROBESG. Orrù, P. De Santis, F. Solinas, V. Piras, G. Savini and V. Caporale Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo Italy. Tel: +390861332234. Fax: +390861332251. e-mail: [email protected].

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As a consequence of the recent outbreaks of bluetongue disease amongst sheepin the Mediterranean basin, and following the subsequent vaccination campaign tocontrol its further spread and long-term maintenance, it has become mostimportant to develop rapid and sensitive methods that can reliably differentiatebetween field and vaccine strains of the causative virus. Although directsequencing is the gold standard for characterising known gene regions and formutation detection, it remains impractical for routine purposes. Recentlydeveloped instruments which couple the PCR method to fluorescent hybridisationprobes now allow target amplification and analysis without sample handling. Here we describe a new method for the differentiation of BTV-2 field and vaccinestrains. The method is based on the principal that the melting temperature of aDNA duplex gives information about the sequence, and allows even single basealterations in the amplicon to be identified. The RT-PCR, the generation of meltingcurves, and fluorescence detection, were all performed using the Light CyclerSystem (Roche). The primers (OG174-OG177) were designed (using VP2 genesequences available on the internet) to flank a region of about 180bp, whereappropriate sequence differences were identified by alignment using the ClustalWprogram. The Fluorescence Resonance Energy Transfer (FRET) method wascreated using separate 3’ and 5’ labelled probes (OG175LC-OG176FL) whichhybridised adjacent to the unlabelled complementary PCR strand. The primers andprobes were designed with the aid of the Oligo 6.0 program (MedProbe, Oslo,Norway). After RT-PCR, the reaction is cooled automatically, and the subsequentslow heating (0.1-0.2°C), as an addendum to PCR, causes denaturation of theprobe/target duplex (melting curve), and so allows for the identification of the

sequence differences. For the real time RT-PCR, the Master Hybridisation Probesreaction mix (Roche), RNA template, primers and FL-LC probes, were used in afinal volume of 20 µl. The analysis of the melting curves derived by the FRET real-time PCR, was done using the Light Cycler Data Analysis program (Roche). To assess the diagnostic value of the method, a vaccine strain of BTV-2(Onderstepoort Biological Products, South Africa) was first compared against afield strain of BTV-2 (isolated during an outbreak in the year 2000 in Sardinia).The resultant melting curves distinctly reveal the two strains to have differingmelting average peak values of 47.8°C ± 0.6°C and 60.5°C ± 0.6°C, respectively.The ability of the method to reliably identify all the BTV-2 strains was tested usingan array of 16 BTV-2 field strains isolated during outbreaks in various Italianregions between 2000 and 2002. The sensitivity of the method has beencalculated using a titred BTV-2 culture, and resulted in a lower limit of detection(LOD) of 102 copies of RNA. The specificity was tested against other serotypes (i.e.BTV-1, BTV-4, BTV-9 and BTV-16) that had cycled also during these recentoutbreaks of BT in the Mediterranean Basin. The method was clearly able to differentiate the BTV-2 strains of vaccine virusfrom all wild-type strains of the same serotype tested, and all had melting peakswithin the predicted range and size. No fluorescence was detected against BTVserotypes -1, -4, -9 and -16. The usefulness of this system was confirmed furtherwhen 15 field strains of BTV-2, isolated from vaccinated and infected animals,showed 10 to be of the field wild type, and five to be of the vaccine strain. Thismethod can thus be used also to identify vaccinal strains of BT in areas wheredifferent serotypes of wild-type virus are circulating simultaneously in ruminants.

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THE FIRST BLUETONGUE VIRUS ISOLATION IN YUGOSLAVIAB. Djuricic, G. Jermolenko, B. Milosevic, S. Radojicic, Z. Debeljak and A. Tomic E-mail: [email protected]

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Catarral fever in sheep (Bluetongue) is a disease until recentlyundetected in Yugoslavia. It is an acute viral disease of sheep, goatsand cattle, to which deer are also susceptible. It appears as enzootic,in the form of infections with natural foci, and is transmitted byhaematophagous insects (mosquitos of the Culicoides genus). Theworldwide incidence of the disease is connected with the movementsand spreading of the carriers. Cattle, being the natural reservoir of thevirus for long periods of time, suffer less from the disease itself, but arealso crucially important in filed epizootiological research.Immunoprophylaxis is an unpopular method for protection, prevention

and eradication of the disease, so the main attention is paid to generalmeasures, namely detection and removal of reactors, as well asdestroying the Culicoides genus mosquitos. Correct diagnosis andtimely detection is of vital importance for controlling the disease.During the first incidence of the Bluetongue disease in Serbia andMontenegro (in 2001) we carried out field clinical research in order torecognize the disease and we collected sample materials for laboratoryresearch. The virus was isolated, and the results verified by theBluetongue reference laboratory in Pirbright, UK (strain 9). Hisprocedure is usually performed by the current OIE standards.

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ABSTRACT BOOK

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Diagnostics D 9

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The occurrence of Bluetongue (BTV) in Italy in 2000 following its appearance in 1998in some surrounding Mediterranean Countries, prompted an increase in surveillancemeasures throughout the Italian territory, including the screening of sentinel cattlesera to detect BTV antibodies, in case of virus circulation. An enzyme-linkedimmunosorbent assay (ELISA) to detect immunoglobulins to BTV was developed,standardized, and characterized as a rapid and sensitive way to detect BTV antibodiesin susceptible animals, by the National Reference Centre for Exotic Diseases, locatedby the Istituto Zooprofilattico Sperimentale Abruzzo and Molise (IZSAM). Thisdiagnostic kit, using a competitive ELISA (cELISA –IZSAM), standardised andvalidated, has been distributed to the Italian veterinary diagnostic laboratoriesnetwork, constituted by the ten Istituti Zooprofilattici Sperimentali, and theirprovincial sections. Each laboratory used the IZSAM kit to carry out the screening ofanimal sera. The IZSAM has also organised a ring test, which involved twenty sevenlaboratories belonging to the aforementioned network, in order to compare theresults obtained, and to evaluate the kit sensitivity and specificity.The main goal of the ring test was to assess each laboratory performance, as faras the identification of true BT positive and negative samples was concerned.Moreover the performances of the commercial kit currently used and of the IZSAMc-ELISA test were compared by means of the McNemar χ2 and the Cohen K index.Sensitivity and specificity values were calculated using data generated by all theparticipating laboratories. One set of thirty samples was prepared, coded and tested for homogeneity andstability before dispatching. The set included positive and negative ovine sera, thepositive ones provided by animals involved in outbreaks occurred in Italy; negativeones were collected in disease free herds.

Results were statistically analysed. Each tested sample was classified as correct orwrong; the probability that the laboratory actually provides correct results wasestimated through a beta distribution (s+1; n-s+1), where “s” is the number of thecorrect results provided by each lab and “n” is the total number of results providedby the lab.Participating laboratories obtained better results when using the IZSAM c-ELISA kit;the IZSAM test detected 805 results out of the 809 useful ones, while the commercialkit detected only 757 correct results. Moreover, although the specificity distributionsof the two tests are perfectly overlapping, with a value of 99,7% for the IZS AM kitand 99,3% using the commercial kit, the sensitivity is significantly different andequal to 99,3% using the IZS AM kit when compared to the to 88,4% with thecommercial kit The c-ELISA-IZS AM proved to be more specific and sensitive than commercialavailable assays in detecting antibodies in sheep, cattle and goat sera.The cELISA-IZS AM kit has also been distributed to other international laboratories; the resultsobtained by 4 c-ELISA kits (the one developed by the IZSAM and the other ones usedby the research institutions taking part in the project) were compared. Also in thiscase, the IZSAM c-ELISA detected the highest number of positive samples, beingtherefore characterised by a greater sensitivity respect to the other c-ELISA kits. The high level of sensibility characterising the IZSAM c-ELISA plays a crucial role inthe diagnosis of a diseases that must be eradicated from the national territory, as thedetection of any infected animal in a flock is vital, in order to eliminate the disease.In this paper results obtained by the Virus Department, Institute of Infectiousdiseases, Centre of Athens Veterinary Institutions, Ministry of Agriculture, Greece arealso presented.

BLUETONGUE LABORATORY DIAGNOSIS: A RING TEST TO EVALUATE SEROLOGICALRESULTS USING A COMPETITIVE ELISA KITR. Lelli(1), M.T. Mercante(1), M. Tittarelli(1), O. Mangana(2), K. Nomikou(2), O. Portanti(1), G. Giovannucci(1), B. Di Emidio(1), M. D’Ancona(1), A. Conte(1) and V. Caporale(1)

(1) Istituto Zooprofilattico Sperimentale Abruzzo e Molise – Teramo - Italy (2) Virus Department, Institute of Infectious and Parasitic Diseases, Centre of AthensVeterinary Institutions, Ministry of Agriculture, Greece

Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo, Italy - Phone +39 0861 332216; Fax n. +39 0861 332251; e-mail: [email protected]


RT-PCR BASED ASSAYS AND SEQUENCING FOR TYPING EUROPEAN STRAINS OFBLUETONGUE VIRUS AND DIFFERENTIAL DIAGNOSIS OF FIELD AND VACCINE STRAINSS. Maan, N. Maan, K. Singh, A. Samuel and P. MertensDepartment of Molecular Biology, Institute for Animal Health, Pirbright laboratory, Ash Road, Pirbright, Surrey GU24 ONF. Phone +44(0) 1483 232441; e-mail:[email protected]

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Bluetongue virus (BTV) infects cattle, goats, deer and other domestic and wildruminants, causing an economically important disease in sheep (classifiedwithin OIE 'list A'). Bluetongue viruses have a ten-segmented double strandedRNA genome and are classified within the species Bluetongue virus, theprototype species of the genus Orbivirus within the family Reoviridae). Thevirus exists as 24 distinct serotypes that are currently identified by serumneutralisation assays. It is transmitted by certain species of biting midges(Culicoides sp.) and has a global distribution, between latitudes 50o North and30o South, that is largely controlled by the distribution and activity of thesevector insects. Within Southern Europe and North Africa the major vectorspecies is thought to be C. imicola. Since 1998 four BTV serotypes (2, 4, 9 and 16) have caused disease inSouthern Europe. More recently (in 2001), BTV-1 was also isolated in Greece.Recent outbreaks of disease in Bulgaria, Serbia and Croatia reflect a northwardmovement of BTV within Europe, beyond the range of C. imicola and indicatethe involvement of novel insect vector species.The virus has two major outer capsid proteins, VP2 and VP5, encoded by genomesegment 2 and segment 6 respectively. VP2 is the most variable of the virionstructural proteins and contains the majority of the neutralising epitopes. As aconsequence variation in genome segment 2 can exert a major influence on BTVserotype. However, there was an absence of data, or only partial sequence datawas available, for segment 2 of many BTV serotypes, making it impossible to

design primers that could be used for RT-PCR based typing assays. Representative segment 2 sequences are now available for all twenty-fourBTV serotypes (see Abstracts by S. Maan et al). cDNA products and sequencedata have been obtained for genome segment 2 of multiple European isolatesof BTV 1, 2, 4, 9 and 16 as well as additional isolates from other geographicalregions. This has facilitated the design of homologous, 'serotype-specific'primers that can be used to amplify and identify different strains of theseserotypes, distinguishing them from other BTV types, in less than 24 hrs. Primers for types 1 & 2 have been tested against reference strains of the 24 BTVserotypes and were shown to be specific for the homologous BTV type. However,during the testing of these primers with field stains of BTV from variousgeographic regions, it became clear that they are less efficient with some strainswithin a serotype than others. Additional primers were therefore designed toensure the efficient amplification of genome segment 2, from different topotypesof each serotype. Using the BTV genome segment 2 and segment 6 sequencedatabase that we have generated (see abstracts by Maan et al and Singh et al)primers were also designed for use in assays to differentiate field strains fromvaccine strains of the European BTV serotypes. The results from these assays willbe presented. The type specific RT-PCR primers that have already beenevaluated for their intra-typic and inter-typic specificity, are listed on a web site(www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm),for useand evaluation by other researchers.

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COMPARATIVE EVALUATION OF SEROGROUP SPECIFIC VP3, NS1 AND VP7 GENEBASED NESTED RT-PCR FOR THE DETECTION OF BLUETONGUE VIRUSESRamesha, S. Dahiya, Minakshi and G. Prasad Department of Veterinary Microbiology, CCS Haryana Agricultural University, Hisar-125004, Haryana, INDIA. Tel. 91-1662-231171, Extn. 4539 (0), 91-1662-235508(R), Fax: 91-1662-234613, [email protected]

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Bluetongue (BT) disease is endemic in India and economically veryimportant to Indian livestock industry. Till recent past, BT diagnosis wasmainly based on conventional approaches such as clinical profile, virusisolation and immunological methods. However, due to certain drawbacksinherent to these approaches, more sensitive, specific and rapid methodsbecame necessary for epidemiological investigations, monitoring ofvaccination program and certification of livestock and their germplasm forexport and import purposes. Keeping this objective in mind, nested RT-PCR assay targeting three serogroup specific genes NS1, VP7 and VP3 wasevaluated and compared to determine the threshold sensitivity limit of theassay. The NS1 and VP7 gene specific primers were designed usingrespective gene sequences of bluetongue virus (BTV) available inGenBank (NS1 FP: (19-39) 5’-GTT GGC AAC CAC CAA ACA TGG-3’, NS1RP: (384-361) 5’- TCC CAC TTT TGC GGT AAT CCT CAA-3’; VP7 FP: (1-23) 5’-GTT AAA AAT CTA TAG AGA TGG AC-3’, VP7 RP: (340-321) 5’-TCATTC GCA GCA YCA GTT GT-3’) whereas VP3 gene specific primers (VP3 FP:((1055-1082) 5’- CCT GAT GTT TCC AGG ACA AAT TAT ACT C-3’, VP3 RP:(1434-1410): 5’-TAT GTA ACG CTG AGC ATG TAC GTA G-3’) used in thestudy were reported earlier. All the three sets of primers successfullyamplified respective gene segments yielding expected PCR products of366 bp, 340 bp and 380 bp respectively in 25 cycles using genomic dsRNA

extracted from BHK-21 cell culture grown bluetongue viruses. Theserogroup specificity of these primers was tested by using the dsRNAextracted from eight Indian isolates of BTV belonging to three differentserotypes (five isolates of BTV-1, one isolate each of BTV-18 and BTV-23)isolated from different geographical regions with diverse climaticconditions, as template for the nested RT-PCR assay. All the three set ofprimers aptly yielded the expected amplicons. These nested primers werealso tested to amplify the desired size fragments of serogroup specificgenes of BTV in spiked sheep blood and buffalo semen samples. SinceEHDV has not been reported in India, the primer sequences of BTV werematched for the presence of complementary sequences by using therespective gene sequences of EHDV available in the GenBank. Thesequence analysis revealed no homologous sequences. Hence, theseprimers could be used for the detection of BTV directly in clinical samplesincluding blood and semen collected from different host species. Thethreshold sensitivity limits of VP3, NS1 and VP7 gene specific nestedprimers were found to be 10 fg, 1 fg and 10 fg, respectively for thegenomic dsRNA quantified spectrophotometrically. This study suggestedthat the NS1 gene specific primers are more sensitive for the detection ofBTV, hence suitable for RT-PCR based diagnosis of BT.

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THE FREQUENCY OF THE CROSS REACTION BETWEEN THE IBARAKI VIRUSAND BLUE TONGUE VIRUS INFECTED SERUM IN AGAR GEL IMMUNO-DIFFUSSION TESTS. Shimizu(1), I. Toyota(2), T. Arishima(3) and Y. Goto(1)

(1) National Institute of Animal Health Japan, (2) Animal hygiene service station of Nagasaki prefecture, (3) Animal hygiene service station of Saga prefecture

Immunopathology Section, National Institute of Animal Health, 3-1-5, Kan-non-dai, Tsukuba, Ibaraki 305-0856, Japan. Tel: +81-298-38-7833 or -7713, Fax: +81-8-38-7833 or 7880, E-mail: [email protected]

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Ibaraki virus belongs to epizootic hemorrhagic disease (EHD) serogroupvirus is endemic in Japan, and it well is known that cross reaction betweenthe blue tongue (BT) virus and EHD serogroup virus. The agar gelimmunodiffusion test (AGID) is performed for the serodiagnosis of BTinfection in Japan. But, it is not clear that the frequency of cross reactionbetween Ibaraki virus and BT virus infected serum in AGID. We aimedmaking clear the cross reaction between Ibaraki virus infection and BT virusinfection in AGID. Furthermore we evaluated competitive ELISA (C-ELISA)to BT virus for the cross reaction between Ibaraki virus and BT infection. For the check of cross reaction in Ibaraki virus infected serums, fortyserums from Ibaraki virus infected cattle in 1997 in Hyogo prefecture inJapan were checked the titers of Ibaraki virus neutralization, the titers ofAGID to Ibaraki virus, the titers of AGID to BT virus, the titers of BT virusneutralization and % inhibition of C-ELISA to BT. And for the check of crossreaction in BT infected serums, seventy seven bovine serums and onehundred twenty five two ovine serums from BT infected herd in Tochigiprefecture in Japan in 1994 were checked the titers of AGID to BT virusneutralization, the titers of Ibaraki virus, the titers of AGID to BT virus and% inhibition of C-ELISA to BT. Ibaraki virus has not been epidemic in Tochigiprefecture. In Ibaraki virus infected serums, each positive rate between titer of Ibarakivirus neutralization and the titer of AGID to Ibaraki virus, the titer of AGID

to BT virus, the titer of BT virus neutralization and % inhibition of C-ELISAto BT were, 90%, 42.5 %, 2.5 % and 0 % respectively. Thus, 42.5 % ofIbaraki virus positive serum, which were negative in BT virus neutralization,showed positive in BT AGID. This indicates that serums from Ibaraki virusepidemic area, which are negative in BT infection, would highly showpositive by BT virus AGID. C-ELISA to BT showed one serum 2.5 % positivein Ibaraki virus positive serums. These results indicate that C-ELISA shouldbe applied for the survey of BT where Ibaraki virus is epidemic. In bovine serums from Tochigi (77 serums), 19 was negative (less than1:2) and 58 was positive (more than 1:2) in BT virus neutralization. OneBT negative bovine serum and five BT positive bovine serums showedpositive line in Ibaraki AGID. The rates of cross reaction in bovine samplebetween BT virus and Ibaraki virus was 7.8 % (6/77). In ovine serums fromTochigi (125 serums), 60 was negative (less than 1:2) and 65 was positive(more than 1:2) in BT virus neutralization. One BT negative ovine serumand ten BT positive ovine serums showed positive line in Ibaraki AGID. Therates of cross reaction in ovine sample between BT virus and Ibaraki viruswas 8.8 % (11/125). These results indicate that the cross reaction between Ibaraki virus and BTin AGID will frequently happen and, more specific serodiagnosis, such as C-ELISA should apply at the area where EHD virus is epidemic.

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BLUETONGUE IN BOSNIA: COMPARISON OF cELISA AND STANDARD AGID TESTSL. Velic, R. Velic, T. Bajrovic, B. Dukic, D. Camo and N Fejzic Institut of Epidemiology, Faculty of Veterinary Medicine, University of Sarajevo, Bosnia and Herzegovina, Zmaja od Bosne 90, 71 000 Sarajevo, BiH. E-mail:[email protected]

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At the and of August 2002, clinical symptoms of Bluetongue (fever 39-41ºC, muco-purulent or bloody nasal discharge, oedema of the lips andintramandibular space, foot lesions including laminitis and coronitis insome cases, diarhea and dysentery) appeared in pramenka sheep flocksin North-Eastern Bosnia. A total 9 599 field sera (ovine 8 967, bovine 632)from 40 communities of FBiH were tested for presence of anti-bluetonguevirus antibodies by using competitive ELISA and standard AGID test. BTV

seropositive reactions were obtained in 187 (1,94%) by cELISA and 141(1,53%) cases by using AGID test.Complete agreement was recordedbetween the cELISA and AGID test results for bovine sera.The results ofour study indicate that the ability of cELISA to detect anti-bluetonguevirus antibodies in ovine sera was superior to that of the AGID. All positiveanimals sera collected from river areas of FBiH.

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FIELD-DEPLOYABLE REAL-TIME PCR DETECTION OF BLUETONGUE ANDEPIZOOTIC HEMORRHAGIC DISEASE VIRAL RNAW.C. Wilson and J.O. Mecham Arthropod-Borne Animal Diseases Research Laboratory USDA, ARS P.O. Box 3965, University Station Laramie, WY 82071-3965. Ph: (307)766-3622; Fx: (307)766-3500; Em: [email protected]

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Nucleic acid sequence information from molecular evolution studies ofbluetongue virus (BTV) and related epizootic hemorrhagic disease virus(EHDV) strains has resulted in a large database of genomic information.Published sequence data and sequence data from our laboratory wereused to design real-time field deployable RT-PCR assays for the detectionof BTV or EHDV viral RNA. The assays use standard RNA extraction andTaqMan chemistries and the entire process can be completed in <= 4hours. The reaction conditions have been adapted to run on a field-

deployable (R.A.P.I.D.) instrument from Idaho Technologies, Inc. Thisinstrument consists of a 50-pound backpack containing everythingneeded to run the assays. The current assays are specific for U.S.serotypes of BTV and EHDV; however, new designs based on newsequencing information are being evaluated to improve specificity andsensitivity. This new technology greatly enhances the speed of virusdetection and the ability to monitor disease outbreaks.

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BLUETONGUE CONTROLUSING VACCINES

ABSTRACT BOOK

THE USE OF VACCINATION IN THE CONTROLOF BLUETONGUE IN SOUTHERN AFRICAB. Dungu and T. SmitOnderstepoort Biological Products, Private Bag X07, Onderstepoort 0110, South Africa. Email: [email protected]

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The eradication of bluetongue (BT) virus from endemic regions of Africa isvirtually impossible due to the role played by widely distributed Culicoidesspp. midge vectors and the presence and ubiquitous distribution ofreservoir species, both known and unknown. Fortunately, most indigenousbreeds of sheep in sub-Saharan Africa are resistant to the disease.Strategies for the control of BT depend on whether they are aimed atoutbreaks of the disease in endemic areas or in areas where the disease isnot usually present. In the latter case, the aim is usually eradication,whereas in endemic areas attempts can only be made to limit theoccurrence of the disease and its economic impact, through vaccination.The initial BT vaccine was developed more than 50 years ago in SouthAfrica and has been improved over that time to currently include 15 of the21 serotypes known to occur in Southern Africa. The current vaccineconsists of live attenuated, cell-adapted, plaque-purified viruses in threepentavalent vaccines, which are administered separately at 3-weekintervals. After 2 to 3 annual immunizations, most sheep are immune to allserotypes in the vaccine. An average of 9 million doses are used annually in South Africa, while alimited number of sheep are vaccinated in other Southern African countries.A number of concerns about the current vaccine have been raised in recent

years. Some attenuated BT vaccine strains may be teratogenic, resulting inbrain defects in the fetus, when administered during the first half ofpregnancy. The theoretical risk of reassortment and recombination between attenuatedand virulent strains has not yet been demonstrated or observed in the field,nor has the risk of transmission of attenuated viruses by vector midges ortheir release in the environment. The risk of reassortment in the field isfurther minimized by the long interval between the recommendedvaccination period (late winter, early spring) and the BT season (summer),which would make the incidence of co-circulating vaccine and virulent wild-type viruses highly unlikely. The virus titer in vaccinated animals (a vaccine dose has a titer of104.5pfu/dose) at the height of vireamia is normally lower than the 103

pfu/ml of blood, which is regarded as the minimum level required for thetransmission of the virus by blood-sucking insects. It is therefore highly unlikely that vaccination with the current vaccine,which has been successfully used for decades in endemic regions ofSouthern Africa, could contribute to the transmission or release of theviruses to the environment through vector midges.

Bluetongue control using vaccines

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B. Dungu

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Bluetongue control using vaccinesABSTRACT BOOK

AN EXPERIENCE FROM THE MEDITERRANEAN ISLANDS IN EUROPEG. Gerbier(1), F. Roger(1), P. Hendrikx(2), S. Zientara(3), F. Biteau-Coroller(1), C. Grillet(1), T. Baldet(1) and Albina E.(1)

(1) CIRAD-EMVT, Animal Health Programme, TA 30/G, Campus International de Baillarguet, 34398 Montpellier Cedex 5 (France), (2) DGAL (France), (3) AFSSA(France)

E-mail: [email protected]

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Following the emergence of bluetongue (BTV-2) in the island of Corsica (France)in 2000, annual vaccination campaigns were conducted between 2001 and 2003.Only the ovine population was vaccinated with the South African BTV-2 liveattenuated vaccine. Despite the vaccination campaign, outbreaks were reportedfrom several areas in 2001. No more outbreaks occurred in 2002, but viralcirculation was revealed in different areas of the island through serologicalconversion in sentinel cattle. This appeared to be associated with an insufficientlevel of vaccination. A cross-sectional survey was conducted in 2001: around 60%of the vaccinated sheep had antibodies. In 2002, the estimation of the level ofvaccination was close to 90%. The prevalence in the other ruminant populations(mainly bovine) was estimated at 50% for the whole Island. Finally, the overallcoverage – taking into account the census data – was estimated at 70% (+/- 5%).The immune coverage was probably not regularly distributed across the Island andcould explain the occurrence of viral circulation in various areas.A key element of the success of vaccination campaigns is the cooperation offarmers. Efficacy of the vaccination and the absence, or a low level of side effects,are then necessary. The efficacy of the vaccination is described based onexperimental animals and field studies. In the literature, some attenuatedbluetongue vaccine strains were shown to cause abortion or to be teratogenic insheep when administered during the first half of pregnancy. Corsican farmers(mainly sheep breeders) reported a decrease in ram fertility. However, noquantitative studies have been done to assess these negative side-effects. Using published and available data, the strategies of vaccination inMediterranean islands are examined and compared with the Corsican one. Four

main strategies for vaccination have been identified: the prevention of theestablishment of BT in an area, the reduction of the number of clinical cases, theeradication of the infection and the immunisation of animal that will beintroduced in an infected area. Given that the large-scale herd immunitythreshold (HIT) is not known for bluetongue, the level of vaccination to achievethe eradication of the disease is unknown. The absence of clinical cases inCorsica since November 2001 demonstrates that vaccination is efficient to avoidclinical cases. Nevertheless it is unclear if eradication has been achieved and ifvaccination against BTV2 should be stopped. Two elements have to be taken intoaccount: firstly, the strong connexion of the Corsican epidemiological situationwith the one in Sardinia (where BTV4 appeared in August 2003) and secondly,the possibility that BTV2 could persist in some places in Corsica still threateninglivestock if the level of protection should decrease. Studies in small islands are attractive because a restricted and limited area allowsinvestigating epidemiological patterns in a laboratory-like environment.Nevertheless, conclusions drawn in this environment are specific as thesusceptible population in some of the Mediterranean islands is limited so thedisease can die out more easily. Comparison between Balearic islands on one sideand Corsica, Sardinia and Sicily on the other side should be pursued to understandthe contrasts between the 2 situations. Mathematical models for determining thebasic reproductive number (R0) of BT appear essential in order to help decisionmakers. Precise field appraisals of prevalence (natural infection) in cattle andgoats, and vaccination coverage in sheep have to be carried out through cross-sectional studies to determine the efficacy of control measures.

G. Gerbier

ABSTRACT BOOK

BLUETONGUE VACCINATION IN EUROPE: THE ITALIAN EXPERIENCEC. Patta, A. Giovannini, S. Rolesu, D. Nannini, P. Calistri, U. Santucci and V. Caporale Istituto Zooprofilattico Sperimentale della Sardegna, via Duca degli Abruzzi 8. 07100 Sassari (Italy). Phone: +39 079 289200; e-mail: [email protected]

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The penetration of bluetongue in Italy in August 2000 caused heavyeconomic losses, partly due to the disease itself, but mostly due to aderangement of the national trade structure.In order to limit direct losses and the virus circulation, the Italian Ministryof Health ordered on 11th May 2001 the vaccination of susceptible animalsof all domestic ruminant species (i.e. sheep, goats, cattle and waterbuffaloes) in infected and surrounding areas. The vaccination strategyderived from a risk assessment that suggested the possibility to preventdirect economic losses and to significantly reduce virus circulation. The vaccination of the target populations started in January 2002.Nevertheless, in July 2002 when the new epidemic peak started, differentproportions of vaccinated populations were achieved in the variousregions. The different levels of vaccination had clear consequences on thedisease and infection spread. In those regions where more than 80% oftarget populations were properly vaccinated, the disease disappearedalmost completely and virus circulation was significantly reduced. Theimportance of the reduction of the extension of virus circulation (i.e.extension of infected areas) was immediately evident in areas affected bythe less virulent BTV9 where, despite the virtual absence of clinicaldisease, the trade of animals to other areas was forbidden. Also areasaffected by the highly virulent BTV2 started to appreciate the importanceof reducing virus circulation as soon as the vaccination was able to

eliminate the clinical disease and the animal movement still remainedforbidden.The main consequence of the reduction of virus circulation consequent tovaccination, documented by the serological surveillance system, was asignificant decrease of the extension of the areas under movementrestrictions. Subsequently, the analysis of available surveillance data andsome specific risk assessments allowed a progressive release of themovement restrictions, also in areas where the infection was still present,but most of the population was properly vaccinated.The field evidence of the effectiveness of the adopted strategy (i.e.vaccination of all domestic ruminants) has also been supported by a greatdeal of experimental and field studies. Such studies were aimed to (1)evaluate level of herd and individual immunity and resistance to challengeconferred by vaccination and (2) quantify the frequency and severity ofadverse effects of vaccination.Ongoing research is focusing on the ability of vaccination to suppress orreduce the viremia following a natural challenge by a virulent strain ofBTV. These studies assess the safety of trade of vaccinated animalsoriginating from areas where virus circulation is still documented andcould be conducive to the reversal of the current policy about theinternational trade of BT vaccinated animals.

Bluetongue control using vaccines

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C. Patta

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Bluetongue control using vaccinesABSTRACT BOOK

GENETICALLY ENGINEERED STRUCTURE-BASED VACCINEP. RoyDepartment of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK. E-mail: [email protected]

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Control of Bluetongue virus disease is difficult due to the multiple serotypes ofthe virus. In addition, viral genome is made up of 10 segments allowingexchanging the genes randomly between different viruses. This may causegeneration of infectious virus with mixed genes. Also since viruses with RNAgenome have high frequency of mutations attenuated live virus vaccine couldhave breakthroughs and mutate to virulent strains. In addition, virus vaccineproduction has to be undertaken in containment laboratories providing addedcosts both for the production and for the safety and efficacy testing of vaccinelots.Recent protein expression technology has provided novel approaches fordevelopment of intrinsically safe vaccines. The technology involves the synthesisof immunogenic proteins and particles that elicit highly protective immuneresponses. Successful vaccine development requires systems where theengineered products mimic the authentic proteins, not just in terms of theirprimary amino acid sequences but specifically in terms of their three dimensionalstructures, i.e., the products must be as authentic as possible. We have utilizedone such protein engineering systems to synthesise individual Bluetongue virusproteins and core- (single coat) and viral-like (double coat) multiproteinstructures (VLPs, CLPs) at high level. At structural level these synthetic particlesessentially mimic the virus particles, but do not contain any genetic materials. The immune responses of these synthetic proteins (subunits) and empty particles(non-replicating) have been tested both in vitro virus neutralising tests and invivo animals (BTV susceptible sheep). Based on this initial data, a series ofclinical trials have been undertaken using animals (from 50 to 200 sheep in each

trial). When one unpurified recombinant protein (virus neutralisation protein VP2)was used for vaccination of sheep, 50 µg/dose could afford protection in sheepagainst virulent virus challenges, while much less of this same protein wasneeded for complete protection when mixed with the second virus outer coatprotein, VP5. In contrast, vaccination with 10 µg VLPs in several trials (eachincluding 100-200 sheep) gave long lasting protection (at least 15 months,maybe longer) against homologous BTV challenge. Cross-protection was alsoachieved depending on the challenge virus and the amounts of antigen used forvaccination.Limited vaccination trials with CLPs that contain only the two conserved proteinsVP3 and VP7 had also been undertaken against homologous and heterologousBTV challenges. It was clear that CLPs could provide either partial (with onlyslight fever) or complete protection (depending on dose) against homologous andheterologous virus challenges. Animals showed strong group specific antibodyresponse, but no neutralising antibodies. Since CLPs are conserved across thevarious serotypes, CLPs could have potential for candidate vaccine which may atleast mitigate the Bluetongue disease and inhibit the virus spread.BTV CLPs and VLPs offer particular advantages as potential vaccines overother systems. The large quantities of CLPs and VLPs can be produced dueto the high expression capabilities of baculovirus vectors (produced inserum-free medium), and can be purified using a one-step generic protocolbased on the physical proprieties of the particle. More importantly, theseparticles are devoid of any detectable amount of insect, or baculovirusproteins or nucleic acids and thus pose no potential adverse effects.

P. Roy

Bluetongue control using vaccines E 1ABSTRACT BOOK

THE EFFECT OF VACCINATION AGAINST BLUETONGUE ON MILK PRODUCTION ANDQUALITY IN CATTLE VACCINATED WITH LIVE-ATTENUATED MONOVALENT TYPE 2 VACCINEA. Giovannini, A. Conte, G. Panichi, P. Calistri, M. Dessì, F. Foddis, A. Schintu and V. CaporaleIstituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo (Italy). Phone: +39 0861-332233; Fax: +39 0861-332251; e-mail: [email protected]

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After the first epidemic wave of bluetongue in Italy, during summer –autumn 2000, which involved 3 Italian Regions (Sardinia, Sicily andCalabria), the Italian Ministry of Health decided on May 2001 to vaccinateall domestic susceptible populations in infected and neighboring Regions.The decision to vaccinate all susceptible species originated from a riskassessment (Giovannini et al., 2003b) that indicated the need toimmunize at least 80% of all susceptible livestock to be able tosignificantly reduce the virus circulation.The vaccination campaign was performed using a live-attenuatedbluetongue monovalent vaccine type 2 produced by the OndersterpoortBiological Products (OBP). This vaccine may cause transient viremia andhyperthermia (39,4°C e 39.8°C). Some Authors have also describedabortion and teratogenic effects in sheep experimentally vaccinatedduring the first three months of pregnancy.

Following the start of the vaccination campaign, losses due to vaccinationwere notified in various Italian regions, including, besides the type oflosses already reported in the literature, also a decrease in milkproduction.The Authors compared the quantity and quality of milk produced by avaccinated cattle population. Milk production data of years 1999-2001(before the start of vaccination campaigns) were compared to datareferring to years 2002-2003, in order to verify whether significantvariations occurred after vaccination of bovine animals. Variables usedwere: (1) for milk quantity, the average individual monthly milkproduction, (2) for milk quality, the average monthly value of fat (%),protein (%), cells and microbial count.No significant effect of vaccination was detected neither on monthly milkproduction nor on milk quality.

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EFFECTS OF BLUETONGUE BIVALENT BTV2 - BTV9 VACCINE ON REPRODUCTIVEPERFORMANCE IN CATTLE: A CASE STUDY IN CALABRIA REGION (ITALY)G. Lucifora, P. Rossi, P. Calistri and A. GiovanniniIstituto Zooprofilattico Sperimentale del Mezzogiorno, Sezione Diagnostica di Cosenza, Via Panebianco 301, 87100 Cosenza (Italy). Phone and Fax: +39 0984-33135e-mail: [email protected]

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After bluetongue appeared in 3 Italian Regions (Sardinia, Sicily andCalabria) during summer – autumn 2000, the Italian Ministry of Healthdecided on May 2001 to vaccinate all domestic susceptible populations ininfected and neighboring Regions. The Ministry of Health approved the useof vaccine, not only in sheep, but also in goats and cattle, with the aim ofcreating a resistant population able to reduce the virus circulation.During 2002, the live-attenuated bluetongue bivalent vaccine BTV2-BTV9,produced by the Onderstepoort Biological Products (OBP), Onderstepoort,South Africa, was utilized in Calabria.A large bovine herd (more than 900 animals with an average number of390 cows) was followed for 4 months after the vaccination in order toverify any abnormality during parturitions (stillbirth, placental retention,etc.). In particular, 111 cows (89 vaccinated and 22 not vaccinated) gave

birth during the 4 months study. In 26 cases abnormalities duringparturition were observed.The Authors checked whether abnormalities during parturitions wereassociated with vaccine, considering the time span between vaccinationand parturition also. No association was found between vaccination ofcows and problems during parturition (Pearson’s chi-square = 0.517,P> 0.05).Furthermore, data were gathered in 4 different group according to thenumber of days from vaccination to parturition (less or equal to 30 days, 31- 60 days, 61 – 90 days, more than 90 days). Even considering the differenttime passed from vaccination, no association was found between vaccinationand problems during parturition.

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FIELD VACCINATION WITH BIVALENT MODIFIED-LIVE VACCINE AGAINST BLUETONGUEVIRUS SEROTYPES 2 AND 9 IN CATTLE: EFFECT ON MILK PRODUCTIONF. Monaco, N. De Luca, D. Morelli, M. Pisciella, S. Palmarini, M. Di Giandomenico and G. SaviniIstituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario 64100, Teramo, Italy. Telephone +390861332219; fax number+390861332251; e-mail: [email protected]

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To evaluate the effect of the vaccination against Bluetongue virus (BTV)serotypes 2 and 9 on milk production in cattle, 30 cows at various stagesof gestation were vaccinated with live modified bivalent vaccine againstBTV serotypes 2 and 9 produced by the Ondersterpoort BiologicalProducts, Ondersterpoort, South Africa while another group of 30pregnant cows was used as unvaccinated control. All animals were bledthree times a week for two months. The viremic titers were determinedand, at the same time, the daily milk production from each animal was

quantitatively and qualitatively evaluated. Twenty-seven of the 30vaccinated cows showed BTV viraemia and detectable titers wereobserved from the 4th up to the 35 th day post inoculation (pv). No effectsof BTV vaccine on milk production, somatic cell count, pH, milk fat, proteinand lactose content were observed. It is concluded that bluetongue livemodified bivalent vaccine against BTV serotypes 2 and 9 does notinterfere with cow milk production.

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VACCINATION WITH MONOVALENT MODIFIED-LIVE VACCINE AGAINST BLUETONGUE VIRUSSEROTYPES 2 IN CATTLE: INNOCUITY, IMMUNGENICITY AND EFFECT ON PREGNANCYF. Monaco, S. Bellini, B. Bonfini, M. Zaghini, A. Pini and G. SaviniIstituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario 64100, Teramo, Italy. Telephone +390861332219; fax number+390861332251; e-mail: [email protected] mail: [email protected]

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To evaluate the immunogenicity, innocuity and possible theratogeniceffects of the monovalent live modified vaccine against bluetongue virusserotypes 2 manufactured by the Ondersterpoort Biological Products,Ondersterpoort, South Africa, 16 cows were selected at different stage ofpregnancy. Fourteen were vaccinated with 2 ml of monovalent vaccine andtwo were used as unvaccinated control. After immunization, ninevaccinated and two unvaccinated animals were maintained in the fieldwhereas 5 were kept inside an insect proof stable under controlledenvironment. The animals in the field were bled once a week for twomonths whereas those under controlled environment three times a week.Samples were tested for the presence of BTV and both, c-ELISA andserum-neutralisation (SN) antibodies. Intravenous egg inoculationfollowed by two blind passages in VERO cells was used to isolate BTV2from EDTA blood samples and from viraemic animals virus titres weredetermined. The viraemia, never higher than 102.8 TCID50/ml, were

detected in the circulating blood of 9 animals. It appeared one week aftervaccination and lasted for 3 weeks reaching the highest peak on day 14pv. In the period considered, none of the vaccinated animals showedclinical symptoms that could be attributed to the BT virus and after 3weeks all animals had developed a serological reactivity to BTV2. c-ELISAantibodies were detected starting from day 7 pv while SN antibodies wereobserved starting from the 21 st pv.All pregnant cows completed their gestation: 13 gave birth to healthycalves, while one of the field group, vaccinated at the 6th month ofgestation, delivered a calf with prosencephalic hypoplasia, likely to havebeen developed during the foetal organogenesis before the vaccination.The results obtained allow us to conclude that the attenuated BTV2 strainused in this study is harmless, immunogen and does not cross theplacental membrane.

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VIROLOGICAL AND SEROLOGICAL RESPONSES IN CATTLE FOLLOWING FIELD VACCINATIONWITH BIVALENT MODIFIED-LIVE VACCINE AGAINST BLUETONGUE VIRUS SEROTYPES 2 AND 9F. Monaco, N. De Luca, P. Spina, D. Morelli, I. Liberatore, R. Citarella and G. SaviniIstituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario 64100, Teramo, Italy. Telephone +390861332219; fax number+390861332251; e-mail: [email protected]

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Following the bluetongue epidemic the Italian government began avaccination campaign involving all domestic animals (cattle, sheep andgoats) of affected and adjacent areas to create a resistant population andreduce virus circulation. In accordance with the serotypes present in theaffected areas, BTV2 monovalent or BTV2 and BTV9 bivalent modified livevaccines have been used. The vaccines are manufactured by theOndersterpoort Biological Products, Ondersterpoort, South Africa and arerecommended for sheep only. As a consequence very few data areavailable on cattle vaccination under field conditions. To evaluate theviraemic curve and the antibody response after vaccination, 30 cattle atvarious stages of pregnancy were selected and vaccinated versusbluetongue virus serotypes 2 and 9. The animals were bled three times a

week for two months and samples were tested for the presence of BTVsand both, c-ELISA and serum-neutralisation (SN) antibodies. Intravenousegg inoculation followed by two blind passages in VERO cells was used toisolate BTV2 and BTV9 from EDTA blood samples and from viraemicanimals virus titres were determined. Bluetongue virus titers weredetected in the circulating blood of 27 animals starting from the 4th up to35th day after vaccination (pv). The highest peak of viraemia was reachedon day 9 pv with viral titers of 104,5 TCID50/ml on average. c-ELISAantibodies were detected in all animals starting from the 9th pv while SNlow titers were observed starting from the 18th pv. Seventeen animalsserum-converted for BTV2 and 27 for BTV9.

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DEVELOPMENT AND EVALUATION OF INACTIVATED VACCINES FOR BLUETONGUE IN INDIAM.A. Ramakrishnan, A.B. Pandey, K.P. Singh, R. Singh, S. Nandi and M.L. MehrotraResearch Associate, AINP on H.S., Division of Bacteriology and Mycology, I.V.R.I., Izatnagar, U.P. (243122), India. Email: [email protected]

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An Indian isolate of Bluetongue virus (BTV) serotype 18 (Bhopal isolate)was passaged in Vero cell culture and inactivated by binary ethylenimine(BEI) or hydroxylamine and adjuvanted with aluminium hydroxide(Al(OH)3) gel and/or saponin for vaccine development. The inactivatedvirus batches were passaged three times in Vero cell culture to testinactivation efficiency. The efficacy of all the six vaccines was carried outin sero-negative adult sheep. Animals were immunized with two shotsof vaccines at the interval of 21 days, followed by challenge with 104.5

TCID50 of homologus virus on 42 days. The efficacy of vaccines wasassessed by two criteria, viz., reduced duration of post challengeviremia and reduction in clinical reaction index (CRI). The humoralresponse of sheep after vaccines and challenge was assessed by agargel immuno diffusion (AGID) test and microtitre serum neutralizationtest (MTSN). In vivo delayed type hypersensitivity (DTH) response usingphytohaemagglutinin was used to measure the cell mediated immuneresponse. The virus was either inactivated with 0.02M BEI or 0.2 Mhydroxylamine. The inactivated viruses were blind passased in Vero cell

culture three times. No cytopathic effect was developed. No untowardeffects or clinical signs of BT disease were observed after vaccination.Following challenge, clinical signs were scored as clinical reaction index(CRI). All the vaccinated animals had significant reduction (P<0.01) inCRI and duration of viremia. Total leucocyte counts (TLC) and packedcell volume (PCV) were significantly (P<0.01 to P<0.05) reduced incontrol animals after challenge. All the vaccinated animals developedgroup specific non-neutralizing antibodies. Before challenge,neutralizing antibody could be detected in the sheep vaccinated with BEIinactivated BTV adjuvanted with saponin but not in other groups.However, after challenge, neutralizing antibody could be detected fromboth control and vaccinated animals as early as 7 days. There wassignificant reduction in mean skin thickness was observed in challengeanimals but not in vaccinated animals. Neutralizing antibody did notprevent the viraemia in vaccinated animals. These findings indicatedthat strong non-neutrilizing antibodies and/or CMI response might havea role in protection against BTV.

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BLUE TONGUE CONTROL USING VACCINES: EMILIA ROMAGNA EXPERIENCEA. Santi(1), L. Loli Piccolomini(1), P. Viappiani(2), M. Tamba(3) and I. Massirio(1)

(1) Regione Emilia Romagna, Servizio Veterinario e Igiene degli Alimenti; (2) Azienda USL Reggio Emilia, Servizio Veterinario; (3) Istituto ZooprofilatticoSperimentale della Lombardia e dell’Emilia Romagna, Osservatorio Emiliano Romagnolo di Epidemiologia Veterinaria.

Servizio Veterinario e Igiene degli Alimenti, Direzione Generale Sanità e Politiche Sociali, Regione Emilia Romagna, Via Aldo Moro, 21, 40128 Bologna. Tel. 0516397380; fax 051 6397064; e-mail: [email protected]

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A national vaccination program aimed at promoting immunisation againstBlue Tongue started in 2001 in Italy, involving many Regions of theCountry. To date the third annual vaccination has been completed.Aims of the vaccination program were to protect the bovine, ovine andgoat population from the illness and to create an immune covering on allthe sensitive population, able to stop the viral circulation. The vaccinationof at least the 80% of the sensitive population has been considered asnecessary. Emilia Romagna Region was involved in the national vaccination programin 2003: some Councils of three Province (Parma, Reggio Emilia andModena) bordering with the Tuscany have been chosen for vaccination ofall sensitive animals: bovine, ovine and goat, using the monovalent type2 vaccine, with a preventive purpose. Such prophylactic measure aims tocreate a barrier to the possible passage of the illness in the North of theCountry, still free from the virus.

The purpose of the study was to evaluate the efficacy of the measuresettled by the Regional Veterinary Service to reach the goal and toevaluate the costs of the annual vaccination, considering the largenumber of animals involved.The vaccination of about 40.000 bovines and 8.000 ovi-caprine requiredresources and time. During the first four months of the year 2003, theinvolved Local Health Units have had to concentrate all their availableresources in such activity. Costs sustained by the Reggio Emilia LocalHealth Unit were specifically analysed. At the end of operations, more than 95% of sensitive animals had beenvaccinated. Few cases of abortion in bovine or sheep were signalled, butthe Blue Tongue virus has never been isolated from the foetus or placenta. Thanks to the co-operation of the Veterinary Services of the Provincesinvolved, the vaccination has been concluded in time, without theengagements of additional staff.

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EFFICACY AND SAFETY STUDIES ON A LIVE AND INACTIVATED VACCINEAGAINST BLUETONGUE VIRUS SEROTYPE 2 (BTV2) B. Di Emidio, P. Nicolussi, C. Patta, G.F. Ronchi, F. Monaco, G. Savini, A. Ciarelli, and V. CaporaleIstituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario 64100, Teramo, Italy. Telephone +390861332219; fax number+390861332251; e-mail: [email protected]

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A batch of vaccine was prepared from a Bluetongue virus serotype 2(BTV2), isolate 1486/A/00 collected in Sardinia in August 2000. It waspurified through a molecular cut cassette membranes, inactivated with ß-propriolactone and adjuvated with MONTANIDE®‚ ISA 206 (Seppic). At thetime of inactivation the isolate titre was 107.8 TCID50/ml. The vaccine was then tested for sterility, toxicity and safety according toEuropean Pharmacopoeia in laboratory and target animals.Immunogenicity was assessed by injecting subcutaneously 2 ml each to10 sheep, 10 goats and 5 ml to 10 bovine. A booster dose was inoculatedafter 14 days. To assess the presence of protective antibodies bloodsamples were collected at days 0, 14, 60. In goats samples were collectedalso at days 137, 285 and 365.No outward effects were reported following vaccination. Fourteen daysafter the booster dose, all vaccinated animals showed serum neutralizingBT antibody titres that, 60 days after vaccination, ranged from 1/20 to

1/1280. Similar antibody titres were detected in goats one year aftervaccination. One hundred thirty eight days after vaccination sheep werechallenged by injecting subcutaneously 1 ml of 105.6TCID50/ml of pureBTV2 Italian strain. Four unvaccinated animals were also inoculated andused as control. At the time of the challenge, the antibody level ofvaccinated sheep ranged from 1/10 to 1/160.Starting from day 6 post challenge, control animals showed fever withtemperature ranging between 39.9 and 40.6°C that lasted 48 hours onaverage. BTV2 was also isolated from the blood of control animals startingfrom the 4th up till the 20th day after challenge. Conversely neither fevernor viraemia were detected in the vaccinated animals. In order to confirm these promising results and to evaluate the minimumprotective dose of the vaccine a new trials with a larger number of animalsincluding all target species has been planned and is already in progress.

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FIELD VACCINATION WITH BIVALENT MODIFIED-LIVE VACCINE AGAINST BLUETONGUEVIRUS SEROTYPES 2 AND 9 IN SHEEP: EFFECT ON MILK PRODUCTIONG. Savini, F. Monaco, A. Facchinei, C. Pinoni, S. Salucci, F. Cofini and M. Di Ventura Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario 64100, Teramo, Italy. Telephone +390861332219; fax number+390861332251; e-mail: [email protected]

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Complaints on the potential side effect of the bivalent live-modifiedvaccine used in Central-Southern Italy to control the spreading of thebluetongue virus (BTV) serotypes 2 and 9, induce the authors to study theeffect of immunisation on milk production. Thirty four of 40 Comisanacross breed sheep were vaccinated with bivalent modified live vaccineagainst bluetongue virus serotypes 2 and 9 produced by theOndersterpoort Biological Product, Ondersterpoort, South Africa, whereas6 animals were used as unvaccinated control. All animals were bled twicea week for two months. The presence and titres of the BT viruses in the

blood were determined and, at the same time, both somatic cell, pH, fat,protein and lactose content of the milk and the quantity of the milkproduced were measured. Starting from the 3rd up to the 20 th day postvaccination (pv) vaccine virus was isolated from blood samples ofvaccinated animals and peak titres were observed on day 6 pv. Milkproduction was reduced in the vaccinated group, compared to the controlfrom the 8th to the 14th day pv with the negative peak on day 9 pv.Conversely, no differences were observed on somatic cell count, pH, milkfat, protein and lactose content.

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LIVE MODIFIED MONOVALENT VACCINE AGAINST BLUETONGUE VIRUS (BTV) SEROTYPE 2:IMMUNITY STUDIES ON COWS G. Savini, F. Monaco, R. Citarella, G. Calzetta, G. Panichi, A. Ruiu and V. Caporale Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario 64100, Teramo, Italy. Telephone +390861332219; fax number+390861332251; e-mail: [email protected]

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To determine the efficacy of vaccination against bluetongue virus serotype2 in protecting cattle against Bluetongue virus (BTV) infection, a group of30 cows, vaccinated 7 months earlier with the modified live-attenuatedmonovalent vaccine against BTV2 produced by the OndersterpoortBiological Product, Ondersterpoort, South Africa, were challengedsubcutaneously with 2x105.7 TCID50/ml of BTV serotype 2 (BTV2) fieldisolate. All these animals came from the same cattle population of theOristano Province of Sardinia. Eight unvaccinated calves, from apopulation free of BT infection, were also used in this study. Four wereinoculated with BTV2 as described for the Sardinian group and used as apositive control, four were used as negative control to verify the possibleunwanted circulation of BT virus in the experimental groups but for thevirus inoculated to the animals challenged. All the animals were bled threetimes a week for two months and samples were tested for the presenceof BTV2 and C-ELISA and serum-neutralisation (SN) antibodies.Intravenous egg inoculation followed by two blind passages in VERO cellswas used to isolate BTV2 from EDTA blood samples. From the blood of

viraemic animals virus titres were also determined. Twenty nine of the 30vaccinated cows showed ELISA and SN antibodies after immunisationwhile one was negative. At the time of challenge 11 animals had no SNcirculating antibody while the rest had low titres ranging from 1:10(no.=11) to 1:20 (no.=6) but animals which titres of 1:40 and 1:80respectively. No animal showed sign of disease after challenge and no BT virus wasrecovered from the blood of 29 vaccinated animals showing antibody aftervaccination. Starting from day 9 after inoculation, BTV2 was isolated fromthe blood of the sole vaccinated cow which did not show any serum-conversion after immunisation. Viraemia in the latter animal behavedsimilarly to that observed in the positive control group and lasted till the21st day after challenge. Neither BT virus nor antibody were detected inthe blood samples collected from the negative control group. According tothese observations it is suggested that the modified live-attenuatedmonovalent vaccine against BTV2 might protect antibody positive animalsagainst infection by BTV2 inoculated 7 months after vaccination.

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NEUTRALISING ANTIBODY RESPONSE IN CATTLE AFTER VACCINATION WITHMONOVALENT LIVE-MODIFIED VACCINE AGAINST BLUETONGUE VIRUS (BTV) SEROTYPE 2 G. Savini, F. Monaco, P. Calistri, G. Panichi, A. Ruiu, A. Leone and V. Caporale Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario 64100, Teramo, Italy. Telephone +390861332219; fax number+390861332251; e-mail: [email protected]

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In Bluetongue virus (BTV) infected areas mortality and livestockmovement restrictions cause considerable economic losses. The Italiangovernment is carrying out a compulsory BTV vaccination campaign sinceMay 2001. In an attempt to reduce direct losses due to disease andindirect losses due to virus circulation the campaign, carried out ininfected and adjacent areas, included all susceptible domestic ruminants.During the first year campaign, Sardinia vaccinated 98.6 % of the totalsheep and goat population and 88.1% of the cattle population with live-modified monovalent vaccine against bluetongue virus serotype 2 (BT2monovalent vaccine), manufactured by the Ondersterpoort BiologicalProducts (OBP), Ondersterpoort, South Africa. It was the first time thatthe BT2 monovalent vaccine has been used in cattle and information onthe use of the vaccine in this species were missing. ELISA (VMRD, USA) and serum-neutralizing (SN) antibodies responseafter vaccination under both, field and experimental conditions and theduration of the colostral antibodies in calves born from vaccinated cowswere evaluated. To this end 1,005 animals of various breed and age were

selected at random from 10 herds of the Oristano province (Sardinia).The animals selected were vaccinated against BTV serotype 2 betweenJuly and August 2002, during the first year of the vaccination campaign.All animals were monthly bled for three months after the vaccination andsamples tested for the presence of both BTV ELISA and SN antibody.Serology results of field vaccinated animals were compared with thoseobtained by vaccinating 5 animals under experimental conditions. Toassess the duration of colostral antibodies in calf born from vaccinateddams, sera of 47 calves were ELISA and SN tested. Calves were dividedinto 3 age groups: group A included 22 calves 1 to 25 day old, group B,13 calves 26 to 39 day old and group C, 12 calves 40 to 60 day old.Sixteen (1.09%) of the 1,005 animals vaccinated under field conditionsdid not show antibody response whereas antibody were detected in allcows vaccinated under experimental conditions. Both groups showed thehighest median titres of 1:160 after 2 months. Calves included in groupsA and B showed antibody titres (68,2% and 46,1%, respectively) whereasanimals of the group C were serologically negative.

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SEROLOGICAL RESPONSES IN CATTLE AND SHEEP AFTER INFECTIONOR VACCINATION WITH BLUETONGUE VIRUSG. Savini, M. Tittarelli, B. Bonfini, M. Zaghini, M. Di Ventura and F. Monaco Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario 64100, Teramo, Italy. Telephone +390861332219; fax number+390861332251; e-mail: [email protected]

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To evaluate the serological response after different Bluetongue virus(BTV) vaccine combinations (Ondersterpoort Biological Products,Ondersterpoort, South Africa) and to assess the accuracy of the diagnosticprocedures commonly used for detecting BTV antibodies in theseconditions, data obtained from various experimental and field studieswere assembled and analysed. C-ELISA (IZSAM, Teramo, Italy) andserum-neutralizing (SN) antibodies response after vaccinations withmonovalent live modified vaccine against bluetongue virus serotype 2 incattle and sheep, monovalent live modified vaccine against bluetongue

virus serotype 9 in sheep, bivalent live modified vaccine against serotypes2 and 9 in cattle and sheep were evaluated. The data were compared tothe serology observed in the same species after infection with BTV2 orBTV9 Italian field isolates. C-ELISA constantly detected antibodies earlierthan serum-neutralization despite the species and BTV serotypes used.The highest and quickest response however resulted when sheep wereinfected with field isolates. In both species high SN titres were alsoachieved after immunization with monovalent vaccines while bivalentvaccines gave origin to the lowest and slowest serology.

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VIROLOGICAL AND SEROLOGICAL RESPONSES IN SHEEP FOLLOWING FIELD VACCINATIONWITH BIVALENT LIVE-MODIFIED VACCINE AGAINST BLUETONGUE VIRUS SEROTYPES 2 AND 9 G. Savini, F. Monaco, P. Migliaccio, C. Casaccia, S. Salucci and M. Di Ventura Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario 64100, Teramo, Italy. Telephone +390861332219; fax number+390861332251; e-mail: [email protected]

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To evaluate the viraemia and the antibody kinetic in sheep after bivalentmodified live bluetongue virus (BTV) immunisation, a group of 44 sheepwere vaccinated with the bivalent modified live vaccine against BTVserotypes 2 (BTV2) and 9 (BTV9) produced by the OndersterpoortBiological Product, Ondersterpoort, South Africa. The animals were bledthree times a week for two months and samples were tested for thepresence of BTVs and both, C-ELISA and serum-neutralisation (SN)antibodies. Intravenous egg inoculation followed by two blind passages inVERO cells was used to isolate BTV2 and BTV9 from EDTA blood samplesand, from viraemic animals, virus titres were also determined. Bluetongue

virus was detected in the blood of 39 animals starting from the 3rd up tothe 21st day after inoculation (pv). The highest pick of viraemia wasreached on the 7th day pv with titres of 105,3 TCID50/ml on average. c-ELISA antibodies were firstly detected on day 6 pv and by the day 16th allanimals were positive. Conversely, 4 of the 44 inoculated animals did notevidence serum-neutralising antibodies against both serotypes, andanother 4 versus BTV9. However in those animals with SN antibodies, thetitres were very low and often on the border line between the negativeand positive threshold.

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SEROLOGICAL AND PROTECTION RESULTS OBSERVED IN A VACCINATION-CHALLENGETEST USING LIVE AND INACTIVATED VACCINES AGAINST BTV2 IN SHEEPS. Boutrand(1), H. Coupier(1), M.F. Ogier (1), N. Reversat(1), K. Mure-Ravaud(1), P. Dubourget(1), C. Schumacher(1), P. Russo(2),J.M. Guibert(2), M. Pépin(2), E. Bréard(3), C. Sailleau(3), S. Zientara(3), C. Hamblin(4) and M. Lombard(1)

(1) Merial S.A.S. 29, avenue Tony Garnier – BP7123 69348 Lyon cedex 07 – France (2) AFSSA Sophia Antipolis BP111 F-06902 Sophia Antipolis cedex– France (3)AFSSA Maisons-Alfort 22 rue Pierre Curie - BP 67 94703 Maisons Alfort – France (4) IAH Pirbright Laboratory, Ash Road, Pirbright, Surrey GU24 ONF - EnglandE-mail: [email protected]

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Bluetongue (BT) is an insect-borne viral disease due to an Orbivirus (Blue Tongue Virus–BTV-) which affects mainly sheep. The disease was reintroduced recently inMediterranean countries. Live attenuated vaccines have been used with success in severalcountries. However due to the residual pathogenicity of live vaccines, interest fordeveloping efficient inactivated vaccines is strong. In this context, efficacy and safety ofinactivated vaccines have been evaluated by general and local examination in sheep,serological follow-up and challenge and compared to the efficacy of a live vaccine currentlyused in Europe.Inactivated, chromatography purified, concentrated and adjuvanted vaccines have beenprepared at a pilot scale from a field BTV2 isolate collected in Corsica in July 2001. Fivebatches (A, B, D, E and F) containing different antigen payloads were tested by challengingvaccinated sheep.Forty five animals, randomly allocated to 9 groups of 5 animals each, were used in thestudy: one control group (group 1), 2 groups (groups 5 & 9) with two injections (s/c) ofinactivated vaccines at one month interval and the last injection one week beforechallenge. The 6 remaining groups received one injection 28 days before challenge. Oneout of the six groups (group 2) was injected with a live vaccine dose. Following vaccination, the sheep were monitored for 14 days post-injection by recordingrectal temperatures, systemic and local reactions.Sheep remained healthy after vaccination without any increase of rectal temperatureseven after the booster. A slight transient local reaction was attributed to the presence ofsaponin, an adjuvant of the vaccine. Protective antibodies were detected by a viral neutralization test (VNT) on sera collectedat D0, 7, 14, 21 and 28 following vaccination. At the time of the challenge, the antibodylevel of all vaccinated sheep reached at least 2 log10. A strong anamnestic response(around 1 log10) was observed after the second injection of inactivated vaccine in twogroups.

The challenge test was based on the requirements of the OIE Manual of Standards (1),and a clinical reaction index was calculated, as previously described by H. Huismans (2)and modified by C. Hamblin (3).The challenge strain was produced from red blood cellscollected at the peak of the viremia from two previously infected sheep, injected by theintravenous route with a pooled suspension of embryonated eggs inoculated with a pre-tested BTV2 strain isolated in Corsica. In a previous study to validate the challenge strain, whatever the injection route(intravenous, intradermal, or both), challenged animals presented serious clinical signs ofBT including fever, oedema, general congestion of the skin, nasal discharge, lameness,apathy and/or depression ; death was observed in 20% of infected animals.For the vaccination-challenge trial, the intradermal route was selected. The 45 sheep werechallenged with 1mL of challenge strain injected in at least 4 points, in the skin of theinner part of the hind leg. Post-challenge follow-up lasted one month.Protection of the vaccinates was reported in all groups. All animals remained healthywithout hyperthermia. In the control group, 80% of the sheep were severely affected and the remaining one didnot develop signs of BT. Hyperthermia occurred on days 6, 7 and 8 post challenge; generalcongestion of the skin and oedema were observed from day 4. Lameness occurred at day8. Apathy and depression were also reported. One out of the four affected control sheepdied on day 12. Additional studies are in progress to evaluate viremia after challenge in all animals.Conclusion: Both inactivated and live vaccines induce high level of seroneutralizingantibodies and protect animals against the clinical signs of disease. Selected antigenpayloads and number of injections (1 or 2) have not induced differences in observedprotection after challenge. Further studies are necessary to assess if vaccines are able toavoid viremia after viral challenge.

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EFFECT OF LEVAMISOLE ADMINISTRATION ON BLUETONGUE VACCINATION IN SHEEPC. Stelletta(1), V. Cuteri(2), L. Bonizzi(3), A. Frangipane di Regalbono(4), F. Orsi(5), L. Nisoli(6), D. Lulla(6) and M. Morgante(7)

(1) Facoltà di Medicina Veterinaria, Università di Perugia; postal address: C/O Dipartimento di Scienze Cliniche Veterinarie, Facoltà di Medicina Veterinaria, Universitàdi Padova, via Romea 16 Agripolis, 35120 Legnaro (PD); Tel. +39 0498272508; Fax +39 0498272954; e-mail: [email protected]; (2) DipartimentoScienze Veterinarie,Università di Camerino; (3) Dipartimento di Sanità pubblica, Patologia Comparata e Igiene Veterinaria, Università di Padova; (4) Dipartimento diScienze Sperimentali Veterinarie, Università di Padova; (5) Libero professionista, Viterbo; (6) Bayer S.p.A., Sanità Animale; (7) Dipartimento di Scienze ClinicheVeterinarie, Università di Padova.

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Levamisole is an anthelmintic that stimulate the parasympatic andsimpatyc ganglia in susceptible worms. It is also an immunostimulantwhen administrated at repeated dose of 2.5 mg/kg before a vaccinationin different species (horse, pigs and salmon). Immostimulating effects arenot well understood. It is believed it restores cell mediated immunefunction in peripheral T-lymphocytes and phagocytosis by monocytes.Furthermore, it seem to stimulate the production of IL-2 and lysozime,stimulate the lymphocyte blastogenesis and increase the level of specificimmunoglobulin in colostrum of vaccinated animals. In order to assess theeffect of levamisole (CITARIN® L 10%, Bayer) administration onbluetongue vaccination in sheep four groups of pregnant sheep (8 sheepeach group) were used. Group A: vaccine (bluetongue vaccine type 2, Bio-Onderstepoort Production/South Africa). Group B: Levamisole + vaccine.Group C: Levamisole. Group D: Control. Levamisole was administratedtree time weekly at dose of 6.5 mg/kg at the first time and, 2.5 mg/kg atthe other time. The last administration in conjunction with vaccination inthe group B. Blood and faeces sampling were performed tree time on allsheep. Sample 1: at the first administration of levamisole. Sample 2: at

vaccination. Sample 3: 48h after vaccination. Sample 4: tree weeks aftervaccination. Faeces samples were used for quantitative and qualitativeparasitic analysis, blood samples for complete haematology and antibodytest for bluetongue (Blue tongue virus antibody test kit, cELISA, VMRD,Inc.). Results show significant differences in response to the anthelmitictreatment and vaccination among groups. There is a significant decrease(P<0,001) between groups B and C in the strongylous’s eggs number(Nematodirus spp., Strongyloides papillosus and other species). While atthe beginning of the trial (sample 1), animals were serologically negativefor bluetongue antibody test, after vaccination, animals showed differentincidence of seroconvertion in treated groups. Group B animals showedhigher seroconvertion percentage (P<0,001) then Group A (87.5 % and50 % respectively). Group B shows an increase of monocytes number atthe sample 3 (48 h after vaccination) compared to other groups. Lightincrease of lymphocytes number were noticed in groups A and B atsample 4. In conclusion the results seem to demonstrate aimmunostimolating effect on bluetongue vaccination in sheep.

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ABSTRACT BOOK

BLUETONGUE VIRUS AND TRADE ISSUES -THE NORTH AMERICAN PERSPECTIVER. DeHaven Deputy Administrator, Veterinary Services, U.S. Department of Agriculture, Animal and Plant Health Inspection Service, 14th and Independence Avenue,Washington, DC. Telephone: 202 720 5193, Facsimile: 202 690 4171, e-mail: [email protected]

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The geographical distribution of bluetongue virus is governed by definablevirus-vector-ecologic/environmental relationships. The disease can onlybe transmitted by competent vectors. In the United States, the NewEngland States (Maine, Vermont, New Hampshire, Massachusetts, RhodeIsland, and Connecticut) and the northern tier of States from Maine toMontana are bluetongue-free because they are vector-free. Likewise, theeastern Provinces of Canada are free of both the vector and the disease.In Mexico, different virus-vector ecosystems exist in the northern and

southern regions of the country.Historically, significant trade in cattle has occurred between Canadaand the United States and the United States and Mexico. Althoughunrestricted year round movement of cattle from bluetongue endemicareas to vector-free and bluetongue-free areas occurs, virus has neverbeen isolated from resident cattle in such bluetongue free areas in theUnited States. This paper discusses current bluetongue-relatedrequirements for trade within North America and elsewhere.

Control and trade issues

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R. DeHaven

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Control and trade issuesABSTRACT BOOK

EU POLICY FOR THE CONTROL AND ERADICATION OF BLUETONGUEJ. Février DG SANCO, Unit E2, F 101/37, Rue Froissard 101, 1049 Bruxelles, BELGIUM • Tel: 32.(0)2.296.58.72, Fax: 32.(0)2.295.31.44, e-mail: [email protected]

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When in 1998 bluetongue reappeared on the European territory the rulesapplying to control this vector born disease had to be reassessed. To thisend a draft Directive has been submitted to the Council as soon as 1999and adopted in 2000 just in time to face the unprecedented situationwhich occurred in late summer that year.Although based on the existing rules applying to African Horse Sickness,flexibility has been considered for the establishment of restricted zonesand movements of live animals in order to deal with local situations.Later on, following the evolution of the outbreak, a number ofimplementation provisions have been adopted in the frame of this newDirective as regard protection and surveillance zones and movementsapplicable to movements of animals in and from those zones.Previously, following the risk assessment of the Scientific Committee, the

Commission considered the option of a vaccination policy with the livevaccine available on the market.Studies have been in other respects conducted on request of theCommission to test the safety and the potency of the vaccine on sheep,cattle and goatsAs soon as July 2000 a bank of vaccine had been set up which allowed aquick and successful action in the Balearic Islands. Later on the Commission supported the vaccination option whenever thenational authorities have adopted this policy.In addition Commission modified the rules applying to financialcontribution of the Community to measures relating to bluetongue inorder to cover not only emergency situations but also long termsurveillance and control actions.

J. Février

ABSTRACT BOOK

SOUTH AMERICA PERSPECTIVEI.A. Lager Instituto de Virologia, INTA-Castelar, CP 1712, Hurlingham, Buenos Aires, ARGENTINA. Telephone: 54-11-4621-1447; Fax: 54-11-4621-1743; e-mail:[email protected]

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Bluetongue virus (BTV) infection is endemic in most of the SouthAmerican (SA) countries but only four outbreaks of the disease have beenreported. Up to date SA has to deal with Foot and Mouth Disease (FMD),through control and eradication campaigns and a big effort isconcentrated in this issue. Nevertheless BTV is slowly becoming intoconsideration in the livestock sanitary police of the region. BTV diagnosis has improved in SA in the last years. This is very importantfor the control of the infection, in trade issues and also in the differentialdiagnosis of vesicular diseases which are the main problem in the region.All the SA countries and Mercosur have import-export BTV regulationsconcerning livestock, animal products and germ plasm trade that arebased in the OIE International Zoosanitary Code.The control measures applied by 8 of the 13 SA countries and reported toOIE are: “notifiable disease” and “precautions at the border” and only twohave screening, surveillance and/or monitoring.SA has limited information on virus reservoir, efficient vector/s andsusceptible hosts witch are the factors to be consider at the control of

such an insect-borne viral disease. In Argentina an epidemiological surveillance program is taking place. Thisprogram included two serological surveys in 1995/6 and 1998 and asentinel animal monitoring project in 1999/01. The results showed thatthe infected area involved Misiones Province and two Departments ofCorrientes Province, four strains of BTV were isolated corresponding toserotype 4 and Culicoides insignis was the predominant vector in thatregion. A new project starting this year will provide information about theincidence of the BTV infection in bovines and ovines, the seasonalincidence of the virus and vector/s, the serotypes of the BTV isolationsand the Culicoides species efficient as vectors in the infected area. Theintroduction of positive animals in free of infection areas will be monitoredfor determining if they can be a source of infection for the native animalsand how long they remain seropositive. Therefore, although there is work to be done to achieve better results inany control strategy in SA, awareness of the problem is increasing andmeasures are taken to improve the situation.

Control and trade issues

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I.A. Lager

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Control and trade issuesABSTRACT BOOK

OIE INTERNATIONAL STANDARDS FOR BLUETONGUEA. Schudel, D. Wilson and J. PearsonOffice International des Epizooties, 12 rue de Prony, 75017 Paris, France, Tel. 33 1 44 18 88, Fax 33 1 42 67 09 87, e-mail [email protected]

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Preventing the spread of disease through international trade is one of theprimary objectives of the Office International des Epizooties (OIE), theWorld Organization for Animal Health. This is accomplished byestablishing International Standards that facilitate trade while minimizingthe risk of introducing diseases such as bluetongue (BT). The OIEStandards for BT are contained in the Terrestrial Animal Health Code (theCode) and the Manual of Diagnostic Tests and Vaccines for TerrestrialAnimals (the Manual).These Standards include procedures for prompt reporting of BT outbreaksrequirements that should be met for a country or zone to be defined asfree of BT; recommendations for the safe importation of live animals,semen and embryos into a BT free country or zone; and the GeneralProvisions that countries should meet to reduce the risk of spread of BTthrough trade. The Manual describes in detail the various tests for thediagnosis of BT. It provides a list of prescribed tests; these are the teststhat are required by the Terrestrial Animal Health Code for the testing of

animals in connection with international trade.There are 24 types of BTV and infected countries have the right to restrictimports from countries that have different types of BTV. However, thisshould only be done if a surveillance and monitoring program hasconfirmed that the other types are not present.Zoning for an arbovirus is difficult to apply but zoning for vectors ispracticable. Some countries have demonstrated that there is no evidenceof infection in their country or parts of their country even though therehas been unrestricted animal movement between endemic zones and freezones. This freedom is due to the absence of vectors in the free zone.Based on this observation, free countries and zones can be established ifan appropriate surveillance and monitoring program is in place to definetheir boundaries. Consequently, there have been extensive changes in theCode to allow the establishment of BT free countries and zones andseasonally free countries and zones to provide the bases for safe trade,while minimizing the risk of the introduction of BT.

A. Schudel

Control and trade issues F 1ABSTRACT BOOK

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SUSCEPTIBILITY AND REPELLENCY OF CULEX PIPIENS AND FIELD POPULATION OFCULICOIDES IMICOLA TO THE PYRETHROID LAMBDA-CYHALOTHRINY. Braverman(1), A. Wilamowski(2), A. Chizov-Ginzburg(1) and H. Pener(2)

(1) Kimron Veterinary Institute, P.O.B. 12, Bet Dagan 50250, Israel, Tel: +972 3 9681 634, Fax: +972 3 9681 753, E-mail: [email protected] (2) Laboratoryof Entomology, Ministry of Health, Jerusalem, Israel.

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The basic efficacy of lambda-cyhalothrin was tested in the laboratoryagainst newly colonized adult Culex pipiens and field-collected Culicoidesimicola. C. imicola was found to be more susceptible (LD50=0.0098%)

than Cx. pipiens (LD50=0.0233%); the efficacy against both species wasdefinitely higher than that of cyhalothrin. Lambda-cyhalothrin showedrepellency for C. imicola for up to 1 h.


SANITARY MANAGEMENT OF LARGE ANIMAL POPULATION MOVEMENT IN ITALY:THE CASE OF TRANSHUMANCE AND BLUETONGUE RISKD. Nannini, P. Calistri, A. Giovannini, M. Di Ventura, M.A. Cafiero, G. Ferrari, U. Santucci and V. Caporale Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”,Campo Boario, 64100 Teramo (Italy). Phone: +39 0861-3321; Fax: +39 0861-332251;e-mail: [email protected]

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Transhumance in Central Italy is more than two thousand year old husbandrymethod involving long-range seasonal movement of sheep, goats and cattle fromthe lowlands of Apulia and Latium Regions, to summer pastures in the mountainsof Abruzzi and Molise Regions. The massive transfer of livestock is due to lack ofgrazing land and drinking water in the plains of Apulia and Latium during summer.According to European legislation a 100 Km of radius protection zone and a 150 Kmof radius surveillance zone around the infected holding must be enforced. Movementof ruminant animals from these zones is strictly forbidden. In early 2001 summerItalian veterinary services were called to manage the risk connected to ruminantmovement from surveillance zones given the absolute impossibility of stoppingtranshumance. At the time the disease was present in three Italian regions: Sardinia,Sicily and Calabria. In that period neither an effective surveillance system norvaccination plan were implemented in Apulia although the region was included in thesurveillance zone. Movement of susceptible animal from Apulia towards the noninfected Abruzzi and Molise regions was impossible according to regulations. Animalowner started public unrest due to the impossibility of feeding and watering thousandsof animals. Police authorities and political leaders asked veterinary service for asolution to avoid further social unrest and animal suffering. The General Directorate ofVeterinary Public Health, Food and Nutrition of the Ministry of Health, organized a adhoc surveillance plan involving both Apulia and Abruzzi, based on serological,virological and entomological controls, in order allow movements while mitigating therisk of possible infection spread. The plan was implemented during May and June 2001and more than 8,000 animals moved from the Apulia surveillance zone to the infectionfree summer pastures in the Abruzzi and Molise regions.

In early summer of 2002 eight Italian regions were infected (Sardinia, Sicily,Calabria, Basilicata, Apulia, Campania, Latium and Tuscany) while a nationwidesurveillance system and vaccination in infected regions were implemented. In theprovinces were vaccination was compulsory derogation of the animal standstillwere allowed only if when more than 80% of susceptible animal populations werevaccinated. A large population of transhumant animals exists in the Latiumprovinces of Rome and Viterbo. The vaccination objective of covering more than80% of the susceptible domestic ruminant population was not achieved in neitherprovince and a certain degree of virus circulation was detected in some territoriesof the two provinces, when transhumance was due to start.Political leaders asked again the veterinary authority for a solution given thenecessity of avoiding, also in this case both social unrest and animal suffering. A specific control plan to allow transhumance from Latium towards Abruzzi,Marche and Umbria regions was, therefore, designed and implemented with theobjective of allowing either the movements of the whole transhumant populationor to reduce the number of animals that could not move to a size that could besustained by the meager feeding watering and housing resources available duringthe summer season in the winter pastures. Consequent to the result of thecontrols carried out between May and June 2002 the movement of more than32,000 animals was authorized. Twenty one flocks for a total of 12,000 sheepwere not allowed to move and regional authorities provided financial aid forfeeding, watering and housing the animals. Neither in 2001 nor in 2002 themovements of transhumant animals spread the infection in the free areas.

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DATA MANAGEMENT AND ANALYSIS SYSTEMS FOR BLUETONGUE VIRUS ZONINGIN AUSTRALIAA.R. CameronDirector, AusVet Animal Health Services, 140 Falls Road, Wentworth Falls, NSW 2782 Australia. Email: [email protected]; Phone: +61 2 4757 2770; Fax: +612 4757 2789

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In many countries where bluetongue virus (BTV) is present, environmental and otherfactors influence the distribution of the virus and its vectors, resulting in free and infectedareas which can change over time. OIE has introduced the concept of zones to facilitatesafe trade from those areas of a country that are free from the virus. Substantiation ofzone boundaries and zone status requires effective monitoring systems to detect changesin the distribution of BTV, in response to changing climatic or environmental conditions. Since the 1970’s, Australia has monitored virus and vector distribution using acombination of sentinel animals for serosurveillance and virus isolation and vectorcollections at sentinel and other sites supplemented with cross-sectional surveys.Specimens from the field are analysed in a national network of laboratories. Novelapproaches are required to rapidly collect, analyse, interpret and disseminate the largequantity of data generated by the monitoring program from laboratories in different partsof a very large country with a wide range of environments. This paper describes thesystems developed in Australia to manage BTV monitoring data, and to rapidly generateup-to-date zone maps in response to shifts in the distribution of the virus.The system uses a real-time on-line database for data storage and reporting. Virus andvector monitoring data are submitted from laboratories using the Internet and is mergedinto the database after detailed automated quality and consistency checking. Full reportsof raw monitoring data, analysed data, and textual descriptions of environmental or otherrelevant factors are available for immediate viewing or download via the Internet. A web-enabled GIS provides real-time automated mapping of the monitoring data, and allowsusers to view sentinel herd or vector trapping results along with the current BTV zoneboundaries, roads, railways, towns, rivers etc. In addition, several levels ofadministrative boundaries are available, including individual property boundaries. Userscan zoom in to determine if a specified property is inside or outside a zoning boundary,to an accuracy of about 5 metres.

Zone boundaries are determined on the basis of monitoring results, geographical andenvironmental factors, and the outputs of a temporo-spatial model of vector and virusdistribution. Three zones are defined – the free zone, the surveillance zone, and thezone of possible transmission. Boundaries are determined such that the boundarybetween the zone of possible transmission and the surveillance zone is at least 50 kmfrom the nearest identified BTV activity. The surveillance zone is at least a further 50km wide providing a separation of at least 100 km between the free zone and any areaof known activity. Where possible, zone boundaries follow clearly defined administrativesubdivisions or property boundaries. All of a property must lie within the free zone inorder to be classified as part of that zone. In some cases, the boundaries followgeographical barriers to vector spread, such as mountain ranges. Using desktop GIStools, it is usually possible to define new zone boundaries and update them on the website in less than a day. An automated email distribution list is employed to informregistered users of zone updates.The protocols governing changes to zoning boundaries are clearly defined. Any indicationof BTV activity within less than 100 km of the current free zone requires immediate re-drafting of the zone boundaries. Decreases in the distribution of BTV resulting indemonstrable lack of activity in an area for at least two years also result in changes tozone boundaries to expand the free zone. These reviews occur after a meeting of expertswho consider all the results and other factors.The system described demonstrates how advanced information management tools can beused to maximise the effectiveness of a BTV monitoring program. The system supportsthe definition of timely and accurate zone boundaries, providing a high degree ofconfidence as well as providing a strong scientific basis to negotiation of health protocolsto support trade. The current Australian BTV zones are displayed athttp://www.namp.com.au.

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THE IMPACT OF CURRENT AND PROPOSED CHANGES TO OIE GENERAL GUIDELINESFOR BLUETONGUE SURVEILLANCEA.R. CameronDirector, AusVet Animal Health Services, 140 Falls Road, Wentworth Falls, NSW 2782 Australia. Email: [email protected]. Phone: +61 2 4757 2770. Fax: +612 4757 2789

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Surveillance for bluetongue virus (BTV) infection poses a number of challenges. Complicatingfactors include seasonal, climatic and environmental variations in vector and virus distribution,and the persistence of antibodies in animals moving from one area to another. If surveillanceis being undertaken to estimate the risk of infection, traditional tools such as randomised crosssectional surveys are less useful, as they are generally only able to provide estimates of sero-prevalence. Estimates of incidence are more likely to be of value, but are often more difficultto measure with reasonable levels of precision. This is particularly the case in areas wheretransmission occurs at low levels such as at the margins of endemic areas.If, on the other hand, the purpose of surveillance is to substantiate zone or national claims ofdisease freedom, traditional cross-sectional survey approaches used in isolation are nowrecognised as often being expensive and inefficient. Combination of survey data with otherexisting sources of evidence may be able to generate the same level of confidence at lowercost. Surveillance systems need to be flexible enough to take into account available sources ofevidence, as well as differences in production systems and environmental conditions.Over the last four years, the OIE, through ad hoc groups has been involved in a process ofrevising its guidelines for general surveillance. Separate guidelines have been developed foraquatic animals and terrestrial animals, both based on the same set of principles. At the timeof writing, the revised aquatic animal guidelines had been endorsed by the 2003 GeneralSession of the OIE, and the draft terrestrial animal guidelines were under consideration forpossible submission to the 2004 General Session. If endorsed by OIE members, theseterrestrial guidelines will have an impact on the approach to surveillance for all OIE listeddiseases, including bluetongue. When planning long-term approaches to bluetonguesurveillance, it may therefore be useful for member countries to be aware of some of theprinciples in the current draft guidelines.The guidelines deal with surveillance both to demonstrate freedom from infection and toestimate the distribution and occurrence of infection. They also provide guidance on the issues

of historical freedom from infection, and the relationship between surveillance and risk analysis.Some of the important features of the draft guidelines are described here. Most importantlythey are non-prescriptive. Any approach to surveillance may be used, as long as themethodology used to collect and analyse the data is scientifically valid and justifiable. Theguidelines also provide a list of critical elements that must be considered and documented.These include, for instance, definitions of the population, cases and outbreaks, considerationof any tests used (including guidelines to documenting the test's performance especially withregard to precision, sensitivity and specificity), sampling methods, sample size calculation anddata analysis methods. In all cases, full transparency should be achieved through appropriatedocumentation.While allowing considerable flexibility in the surveillance methodologies used, the guidelinesare much more specific about the required standards for the outputs of surveillance, including,for instance, the level of confidence required to demonstrate freedom.A relatively new inclusion in the area of surveillance is the requirement for demonstrablequality control systems. These may be relatively simple, but should document both theestablished protocols for surveillance, and be able to detect and document any departures fromthese protocols.The guidelines specifically acknowledge the value of non-random approaches to surveillance,and the benefits of the analysis of multiple data sources to provide evidence of disease statusor distribution.If adopted, these guidelines are likely to have two major effects. Firstly, there is the opportunityto develop more effective and more affordable approaches to surveillance, closely matched tothe differing needs and practical constraints of different member countries. On the other hand,without prescriptive guidelines, there will be a requirement for greater skilled input into thedesign, documentation and assessment of surveillance systems. For bluetongue, a range ofdifferent approaches to surveillance may be available to produce equally acceptable outputs.

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RISK ANALYSIS ON THE INTRODUCTION INTO FREE TERRITORIESOF VACCINATED ANIMALS FROM RESTRICTED ZONESA. Giovannini, A. Conte, P. Calistri, C.E. Di Francesco and V. Caporale Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 Teramo (Italy). Phone: +39 0861-332282; Fax: +39 0861-332251; e-mail: [email protected]

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Since August 2000, after the first reporting of Blue tongue in Italy, animaltrade between infected and free areas has come to a complete standstill.Before the occurrence of BT, cattle left Sardinia, Sicily and the southernregions to be fattened and slaughtered in Northern Italy - during thetracing of animals that had left infected areas, in 2000, it was shown thatonly for Sardinia 10,957 bovine animals had left the Island from June toAugust 2000 and were scattered throughout continental Italy. Also mostreform cattle were sent from the south and the Island to Northern Italyfor slaughter. Long term standstill, therefore, not only leads to greateconomical losses and social consequences, but without compensation, isalso almost impossible to enforce indefinitely.In May 2001, the Italian Ministry of Health decided to both restrict animalmovements and to vaccinate all domestic susceptible populations ininfected and neighbouring regions, in order to reduce virus circulation.Authorities, furthermore, implemented an epidemiological surveillance

system to both monitor infection spread and manage movement control.The vaccination campaign carried out in the various regions obtaineddifferent levels of herd immunity, varying from virtually 0% up to about95%. Proper implementation of the vaccination campaign and levels ofherd immunity more than 80% were conducive to reduction of the clinicaldisease (i.e. Sardinia and Tuscany).Italian authorities and the European Commission decided to adopt a policyof risk management allowing some derogation from standstill. This paperpresents the risk assessment that supported the Italian and Europeandecision to adopt a risk management policy.Risk assessment indicates that, when more than 80% of susceptiblepopulation in the territory of origin is vaccinated, the movement ofvaccinated animals towards free areas poses an acceptable risk that canbe mitigated further adopting ancillary control measures.

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IMPLEMENTATION OF A NEW CONTINGENCY PLAN FOR BLUE TONGUE DISEASE IN ITALYG. Filipponi(1), R. Lelli(1), C. Carteny(2), U. Santucci(2), P. Calistri(1), C. Weiss(1) and V. Caporale(1)

(1) Istituto Zooprofilattico Sperimentale Abruzzo e Molise “G. Caporale” – Teramo - Italy (2) Ministero della Salute – Direzione Generale Sanità Veterinaria e degliAlimenti – Roma - Italy

Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Campo Boario, 64100 -Teramo Italy - Phone: +39 0861 332216, Fax: +39 0861332251, e-mail: [email protected]

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In 2000 Bluetongue virus (BTV) made its first appearance in Italy, followingits previous occurrence in some Mediterranean Countries. At that time, theItalian decision system activated and operating in an emergency situationinvolved local, regional or central levels (depending on the size of theoutbreak), as stated in the Italian Foot and Mouth Disease contingency plan,dating back to 1991. A relevant degree of freedom characterised theinteractions among the different levels of the system, considerably hinderingany coordination effort. Bluetongue, due to its complex epidemiology,represented therefore a serious challenge for the efficacy of the system inplace: being indeed a vector borne disease, its spread is affected by severalfactors, whose the most difficult to monitor are the vector’s ecology and theenvironmental conditions affecting vectors’ distribution and survival;seasonal variations in farming practice such as transhumance furthercomplicate disease control strategies. Given these assumptions, the ItalianMinistry of Health considered the local, or even regional, approach in themanagement of outbreaks, unable to prevent the further spread of thedisease. Meanwhile, at the end of the year 2000, the Council of the European Unionadopted Council Directive 2000/75/EC, laying down specific provisions for thecontrol and eradication of Bluetongue, including minimum criteria applicable tocontingency plans. According to both this Directive and the EU guidelines oncontingency plans for epidemic diseases, the Italian Ministry of Health decided

to implement a new contingency plan for Bluetongue Disease. The plan wasdeveloped by the National Reference Centre for Exotic Diseases of Animals(CESME), located by the Istituto Zooprofilattico Sperimentale Abruzzo andMolise (IZSAM), in collaboration with the Italian Ministry of Health.The new contingency plan was drafted according to the EU requirements. Itsmost innovative feature is the description of an integrated approach tocontrol BT epidemics. This integrated system is based upon some criticalpoints, the most important of which are the simplification of technicalprocedures for the modification of existing regulation to promptly react tothe changes in the epidemiological patterns of the disease in the Italianterritory, the structure of the chain of command (much shorter than in theprevious contingency plans, allowing a quicker response in case ofepidemics occurrence), the creation of both the National Disease ControlCentre (NDCC) and the Local Disease Control Centres (LDCCs), whoseorganization, responsibilities and operation are described in detail. The BTcontingency plan is further integrated and completed by an ad hocinformation system, operating also a decision support system both at localand central levels and of which an important component is the GIS and bya specific contingency manual. The activities carried out to define the Italian contingency plan forBluetongue disease and critical points of implementation are brieflydescribed.

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INTERATED MANAGEMENT OF CULICOIDES SP (DIPTERA: CERATOPOGONIDAE)OF DOMESTICATED ANIMALS OF MARATHWADA REGIONB.W. Narladkar, P.D. Deshpande, V.P. Vadlamudi and P.R. Shivpuje Assistant professor of Parasitology, College of Veterinary and Animal sciences, Maharashtra Animal and Fishery Sciences University PARBHANI – 431 402Maharashtra State (INDIA). E-mail: [email protected]. Telephone: 0091 – 2452 – 229890 (R). Fax: 0094 – 2452 – 226188

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Consecutive studies for two years on Culicoides insects from 18 sites ofMarathwada region revealed the prevalence of Culicoides peregrinus(Kieffer, 1910), C. schultzei (Enderlein, 1908) C. actoni (Smith, 1929)species.The relative efficacy of two herbal pesticides containing Azadirachtin(NeemAzal –T/S and Neemarin) and two biological pesticides, viz. Bacillusthrungiensis var. israelensis H-14 (Bacticide), Bacillus sphericus serotypeH-5a, 5b strain B-101 (Sphericide) in the control of larval Culicoides wereevaluated. The LC50 values of the above four products were 18.57 ppm,

23.44 ppm, 8433 ppm (0.84 %) and 9840 ppm. (0.98 %), respectively.The hebal products containing Azadirachtin also proved as ovipositiondeterrent and ovicidal against Culicoides midges.A filler trial was conducted on the utility of guppy fishes (Poeciliareticulata) as larvivorus against Culicoides larvae, and results indicatedthat one guppy fish was found to consume as much as 60 ± 5 Culicoideslarvae within 24 hrs. time. However, these fishes could not survive indrainage channel containing organic matter more than 4 per cent on drymatter basis.

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