Mastitis Vaccine - Pakistan Research Repository

ii

Laboratory and Field Evaluation of a Locally Prepared

Montanide® Adjuvanted Combined Hemorrhagic Septicemia-

Mastitis Vaccine

By

QUDRATULLAH

D.V.M., M. Phil. (CMS) (UAF)

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

CLINICAL MEDICINE & SURGERY

DEPARTMENT OF CLINICAL MEDICINE & SURGERY

FACULTY OF VETERINARY SCIENCE

UNIVERSITY OF AGRICULTURE,

FAISALABAD,

PAKISTAN.

(2016)

iii

To,

The Controller of Examinations,

University of Agriculture,

Faisalabad, Pakistan.

We, the Supervisory Committee, certify that the contents and form of thesis submitted

by Mr. Qudratullah (Regd. No. 2003-ag-2087) have been found satisfactory and

recommend that it be processed for evaluation by the External Examiner(s) for the award of

the degree.

SUPERVISORY COMMITTEE:

Chairman:

(Prof. (R) Dr. Ghulam Muhammad)

Member:

(Dr. Muhammad Saqib)

Member:

(Dr. Muhammad Qamar Bilal)

iv

DEDICATED

TO

The sweet memories of my late father Hon. Capt. (R) Muhammad

Tahir and Evere-praying affectionate Mother, sweet daughter (Nehal

Khan) and my caring wife (Tanzila)

v

DECLARATION

I hereby declare that the contents of the thesis “Laboratory and Field Evaluation of a

Locally Prepared Montanide® Adjuvanted Combined Hemorrhagic Septicemia-

Mastitis Vaccine” are product of my own research and no part has been copied from any

published source (except the references, standard mathematical or generic models/ equations/

formulae/ protocols etc.). I further declare that this work has not been submitted for award of

any other diploma/ degree. The University may take action if the information provided is

found inaccurate at any stage. (In case of any default, the scholar will be proceeded against as

per HEC plagiarism policy).

--------------------------------------

QUDRATULLAH (2003-ag-2087)

vi

ACKNOWLEDGEMENT

I would like to take this opportunity to sincerely thank all of the people who have helped me

with my Ph.D. work. I am grateful to Prof. (R) Dr. Ghulam Muhammad Department of

Clinical Medicine and Surgery and Chairman Supervisory Committee for generously

providing me a conducive environment that enabled me to concentrate on, and successfully

complete my Ph.D. studies. His supervision and able guidance in planning study program and

timely completion of work are highly appreciated. I also thank Dr. Muhammad Saqib,

Department of Clinical Medicine and Surgery for moral and technical help in research. I

thank sincerely, Dr. Muhammad Qamar Bilal for his suggestions, constructive criticism,

and interest in my academic pursuits. I am thankful to all colleagues, particularly Dr. Immad

Rashid for their help and moral support during the course of my studies. Dr. Muhammad

Latif, Director Farms and the staff of the Livestock Research Station responded cheerfully

whenever called upon to assist in research and data collection. I would like to appreciate the

help extended by staff members of Department of Clinical Medicine and Surgery, Nuclear

Institute of Agriculture and Biology (NIAB) Faisalabad, Ayub Agriculture Research

Institute, Faisalabad, and owners of private dairy farms Raja Dairy Farm, Rajawala,

Faisalabad; Bilal Rashid Dairy Farm, Aminpur Road, Faisalabad, and Mehar Anayat Dairy

Farm, Green View Colony, Faisalabad. Last but not the least deepest appreciation are

extended to my ever praying affectionate Father (Late) and Mother for their financial help,

moral supports, my Brothers, Sisters, Wife, and all the family members for their affection,

cooperation and amicable attitude. I feel this endeavor justifies their faith in me.

I am thankful to Dr. Siraj Ahmad Kakar, Chairman Board of intermediate and Secondary

Eductaion, Quetta and Dr. Habib-ur-Rehman Veterinary Officer, Quetta for their kind

support and encouragement throughout the research work.

I am also thankful to the Higher Education Commission, Islamabad, for financial assistance

provided under HEC indigenous 5000 Ph.D. fellowship Batch VII. Above all, I have enjoyed

tremendous health, motivation, intellect, wisdom, protection, and facilitating circumstances,

which made it all possible, for which I am forever grateful to Almighty Allah who gave me

the courage to complete this work.

Qudratullah

vii

CONTENTS

CHAPTER TITLE PAGE #

1 INTRODUCTION 1-3

2 REVIEW OF LITERATURE 4-27

3 MATERIALS AND METHODS 28-44

4 RESULTS 45-100

5 DISCUSSION 101-114

6 SUMMARY 115-119

LITERATURE CITED 120-139

viii

TABLE OF CONTENTS

CHAPTER NO. TITLE

Page No

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 4

2.1 Economic impact of animal diseases in developed and

developing countries

4

2.2 Mastitis 4

2.3 General aspects of hemorrhagic septicemia ( HS) 5

2.3.1 Carrier state 6

2.3.2 Transmission 6

2.3.3 Association of HS with other diseases 6

2.3.4 Pathogenesis 7

2.3.5 Rainy seasonal occurrence 7

2.3.6 Clinical signs and post mortem findings 8

2.3.7 Treatment. 8

2.3.8 Morbidity, mortality and case fatality rates 10

2.4 Prevalence and economic significance of HS in Pakistan 11

2.5 Hemorrhagic septicemia vaccines 13

2.6 Importance of transferrin binding protein A as an

Immunogen

18

2.7 Montanide® adjuvanted HS vaccines 19

2.8 Prevalence and economic significance of mastitis in Pakistan 20

2.9 Role of Staphylococcus aureus and Streptococcus agalactiae

in mastitis

21

2.10 Mastitis vaccines 22

2.10.1 Montanide® (Seppic, France) adjuvanted mastitis vaccines 23

2.11 Need for a combined HS–Mastitis vaccine (‘combo’ or

‘cocktail’ HS –mastitis vaccine)

24

2.12 Mastitis in heifers and justification of use of combined HS-

mastitis vaccine

26

3 MATERIALS AND METHODS 28

Phase I Preparation and Evaluation of Montanide® Adjuvanted

Combined Hemorrhagic Septicemia -Mastitis Vaccine in

Laboratory Settings

28

3.1 Collection and culture of blood samples for isolation of HS

pathogen

28

3.1.1 Sources and types of samples for isolation of P. multocida: 28

3.1.2 Isolation and biochemical and molecular confirmation of P.

multocida

29

3.1.3 Synthetic oligonucleotide primers 30

3.1.4 PCR Reaction 30

ix

3.1.5 Pathogenicity (virulence) of 3 candidate vaccinal isolates of P.

multocida

30

3.2 Sources of milk samples, isolation of candidate vaccinal S.

aureus and Str. agalactiae isolates and their partial

characterization.

31

3.2.1 Molecular confirmation of S. aureus 32

3.2.1.1 Detection of nuc gene by polymerase chain reaction (PCR) 32

3.2.1.2 Preparation of genomic DNA from S. aureus 33

3.2.1.3 PCR amplification 33

3.3 Detection of biofilm production trait of S. aureus 34

3.3.1 Qualitative determination by tube method (TM) 34

3.3.2 Qualitative determination by Congo red agar method (CRA) 34

3.3.3 Quantitative determination of biofilm production by

spectrophotometric method

35

3.3.4 Pathogenicity of 4 candidate vaccinal isolates of S. aureus 36

3.3.5 Pathogenicity of 2 candidate vaccinal isolates of Str. agalactiae 36

3.4 Immunogenicity testing of vaccinal isolates of P. multocida,

S. aureus and Str. agalactiae

37

3.5 Antimicrobial susceptibility testing of candidate bacterial

isolates (Antibiogram)

37

3.6 Selection of vaccinal P. multocida isolate 37

3.7 Selection of vaccinal S. aureus isolate 38

3.8 Selection of vaccinal Str. agalactiae isolate 38

3.9 Preparation of Montanide® adjuvanted combined HS-

Mastitis vaccine

39

3.9.1 Preparation of antigen of P. multocida 39

3.9.2 Preparation of antigens of S. aureus and Str. agalactiae 39

3.9.3 Preparation of combined HS-Mastitis vaccine 40

3.10 Evaluation of quality of vaccine 40

3.10.1 Sterility 40

3.10.2 Safety and side effects 40

3.10.3 Challenge- protection assay (Vaccination- challenge study) 40

3.10.4 Stability 41

Phase II Field Evaluation of Montanide® Adjuvanted Combined HS-

Mastitis Vaccine in Target Dairy Species

41

3.11 Animals, their management and treatment-based grouping 41

3.12 Evaluation parameters 43

3.12.1 Hemorrhagic septicemia 43

3.12.1.1 ELISA based antibody titers against P. multocida 43

3.12.1.2 Incidence of HS 43

3.12.1.3 Local and systemic reactions 43

x

3.13 Mastitis 43

3.13.1 ELISA based antibody titers against S. aureus and Str.

agalactiae

43

3.13.2 Incidence and prevalence of S. aureus and Str. agalactiae

mastitis

43

3.13.3 Mastitis screening tests based prevalence and incidence of

mastitis

43

3.13.4 Severity of mastitis in vaccinated and control animals 43

3.13.5 Isolation and identification of pathogens from clinically mastitic

animals

44

3.13.6 Milk somatic cell count 44

3.13.7 Effect of vaccination on milk yield 44

3.13.8 Vaccine efficacy 44

3.13 Statistical Analysis 44

4 RESULTS 45

PHASE-I 45

4.1 Pasteurella multocida isolates recovered from peripheral

blood, femur, and heart blood of HS affected animals

45

4.2 Cultural and biochemical characteristics of suspected

isolates of P. multocida

45

4.2.1 Biochemical reactions of P. multocida isolates 46

4.2.2 PCR based confirmation of P. multocida isolates 47

4.2.3 Antimicrobial susceptibility testing of 3 candidate vaccinal P.

multocida isolates 47

4.2.4 Pathogenicity of the 3 candidate vaccinal isolates of P.

multocida in mice

47

4.3 Staphylococcus aureus isolates recovered from peracute/acute

clinically mastitic quarters (n=37) of dairy cattle and buffaloes

48

4.3.1 Confirmation and characterization of 4 candidate vaccinal

S. aureus isolates

48

4.3.2 Antimicrobial susceptibility of candidate vaccinal S. aureus

isolates

50

4.3.3 Pathogenicity of the 4 candidate vaccinal isolates of S. aureus in

rabbits

50

4.3.4 Biofilm production by candidate vaccinal S. aureus isolates

(n=4)

51

4.3.4.1 Tube method 51

4.3.4.2 Spectrophotometric method 51

4.3.4.3 Congo red agar-method 52

xi

4.3.4.4 PCR based confirmation of S. aureus isolates 52

4.4 Streptococcus agalactiae isolates recovered from

peracute/acute clinical mastitis from dairy cattle and

buffaloes.

53

4.4.1 Confirmation and characterization of candidate vaccinal Str.

agalactiae isolate

53

4.4.2 Seven-digit numerical biotype (biochemical) characteristics of

candidate vaccinal isolates

53

4.4.3 Antimicrobial susceptibility of candidate vaccinal Str.

agalactiae isolates

54

4.4.4 Pathogenicity of the 2 candidate vaccinal isolates of Str.

agalactiae in rabbits

55

4.4.5 Immunogenicity of the 3 candidate vaccinal isolates of P. multocida

in rabbits 55

4.4.6 Immunogenicity of the 4 candidate vaccinal isolates of S.

aureus in rabbits

56

4.4.7 Immunogenicity of the 2 candidate vaccinal isolates of Str.


56

4.5 Final selection of vaccinal isolates from amongst the

candidate vaccinal isolates of P. multocida, S. aureus and Str.

agalactiae for vaccine production

57

4.6 Evaluation of quality of vaccine 59

4.6.1 Sterility 59

4.6.2. Stability 60

4.6.2.1 Effect of temperature and duration of storage on the physical

stability of Montanide® adjuvanted combined hemorrhagic

septicemia-mastitis vaccine.

60

4.6.3 Safety and side effects 60

4.6.3.1 Comparative safety of Montanide® adjuvanted combined

hemorrhagic septicemia-mastitis vaccine in cattle and buffaloes 60

4.6.4 Challenge- protection assay (Vaccination- challenge study) 62

4.6.4.1 Vaccination challenge protection assay of Montanide®

adjuvanted combined hemorrhagic septicemia-mastitis vaccine

in mice.

62

4.6.5 ELISA- based antibody titers generated by Montanide®

adjuvanted combined HS-mastitis vaccine against P. multocida,

S. aureus and Str. agalactiae in cows and buffaloes

63

xii

4.7 Incidence of HS 65

4.7.1 Number of new HS cases in vaccinated and control cows,

buffaloes and calves during 180 days post- vaccination

65

4.8 Incidence and prevalence of S. aureus and Str.

agalactiae mastitis

68

4.8.1 Effect of Montanide® adjuvanted combined HS-mastitis

vaccine on quarter-based incidence of Str. agalactiae and S.

aureus IMIs in cows and buffaloes initially culture negative

and CMT/ SFMT negative

68


vaccine on quarter- based incidence of Str. agalactiae and S.

aureus IMIs in cows and buffaloes initially culture positive and

CMT/ SFMT positive

70


vaccine on quarter- based time points prevalence of Str.

agalactiae and S. aureus IMIs in cows and buffaloes initially

culture positive and CMT/SFMT positive

72

4.9 Mastitis screening tests based prevalence and

incidence of mastitis

75


vaccine on CMT and SFMT based quarter incidence of mastitis

in cows and buffaloes initially culture negative for S. agalactiae

and S. aureus and CMT/ SFMT negative

75


vaccine on CMT/SFMT- based quarter time points prevalence

of mastitis in cows and buffaloes initially negative for Str.

agalactiae and S. aureus plus CMT/SFMT negative

77


vaccine on CMT and SFMT based quarter incidence of mastitis

80

xiii

in cows and buffaloes initially culture positive for S. agalactiae

and S. aureus and CMT/ SFMT positive


vaccine on CMT/SFMT- based time points quarter prevalence

of mastitis in cows and buffaloes initially positive for Str.

agalactiae and S. aureus plus CMT/SFMT positive

82

4.10 Severity of mastitis in vaccinated and control

animals

85


vaccine on the severity of mastitis.

85

4.11 Milk somatic cell count 87


vaccine on the quarter milk somatic cell count (× 105

ml of

milk) in vaccinated–non-diseased, control-non-diseased,

vaccinated –diseased and control-diseased groups of cows.

87

4.12 Effect of vaccination on milk yield 91

4.12.1 Milk yield (Mean ±SE; L/24 hour) in vaccinated and control

cows and buffaloes at different post-vaccination days

91

4.13 Cost- benefit analysis of Montanide® adjuvanted combined

hemorrhagic septicemia-mastitis vaccine

95

4.13.1 HS related cost -benefit calculations 95

4.13.2 Mastitis related cost-benefit calculations 97

4.13.2.1

Mastitis control related benefits accruing from use of

Montanide® adjuvanted combined hemorrhagic septicemia-

mastitis vaccine

97

4.14 Calculation of vaccine efficacy 99

4.14.1 Calculation of vaccine efficacy for HS in cows, buffaloes and

calves

99

4.14.2 Calculation of vaccine efficacy for mastitis in cow sand

buffaloes

100

5 DISCUSSION 101

xiv

5.1 Preparation of combined HS-mastitis vaccine and its evaluation

against HS

101

5.2 Evaluation of combined HS-mastitis vaccine against mastitis 105

6 SUMMARY 115

LITERATURE CITED 120

APPENDICES 140

xv

LIST OF TABLES

Table No. Title Page No. 2.1 Cure rate of 9 different treatment protocols used to manage

an outbreak of HS (Pasteurella multocida serotype B2) that

occurred in the wake of SAT2 FMD infection in cow and

buffalo fattening calves in Alexandria Egypt.

9

2.2 Reported prevalence of mastitis in cows and buffaloes in

some selected studies conducted in Pakistan.

20

3.1 The oligonucleotide primer pair used for PCR based

confirmation of Pasteurella multocida

30

3.2 Sequence of nuc gene primers used for confirmation of S.

aureus and PCR product size

33

3.3 Composition of PCR reaction mixture used for amplification

of nuc (S. aureus specific) gene

34

3.4 Composition and preparation of Congo red agar medium

(Mathur et al., 2006). Ingredients per liter (L):

35

3.5 Selection of vaccinal isolate by biofilm detection methods

and other traits

38

3.6 Grouping of animals, their experimental treatments, and

HS/mastitis status at the time of initiation of field trial of

combined HS- mastitis vaccine.

42

4.1 Pasteurella multocida isolates recovered from peripheral

blood, femur, and heart blood of HS affected animals

45

4.2 Biochemical profiles of P. multocida isolates (n=3)recovered

from femur bone marrow samples and heart blood samples

46

4.3 Antibiotic susceptibility profiles of 3 candidate vaccinal isolates

of P. multocida against 11 antibiotics/antimicrobials

47

4.4 Pathogenicity of the 3 candidate vaccinal isolates of P. multocida in mice

48

4.5 Seven-digit numerical biotype (biochemical) profiles of 4

candidate vaccinal S. aureus isolates determined by using

API® STAPH

48

4.6 Antibiotic susceptibility profiles of 4 candidate vaccinal isolates of

S. aureus against 11 antibiotics/antimicrobials 50

4.7 Pathogenicity of the 4 candidate vaccinal isolates of S. aureus

in rabbits

51

4.8 Tube method-based scores of biofilm production by

candidate vaccinal S. aureus isolates (n=4)

51

4.9 Spectrophotometric method based optical densities (OD

values) for biofilm production of 4 candidate vaccinal S.

aureus isolates

52

xvi

4.10 Degrees of biofilm production trait of 4 candidate vaccinal S.

aureus isolates on Congo red agar (CRA)

52

4.11 Seven-digit numerical (biochemical) profiles of 2 candidate

vaccinal Str. agalactiae isolates determined by API® 20

STREP (bioMerieux, France)

54

4.12 Antibiotic susceptibility profiles of 2 candidate vaccinal

isolates of Str. agalactiae against 11

antibiotics/antimicrobials

55

4.13 Pathogenicity of the 2 candidate vaccinal isolates of Str.


55

4.14 Indirect ELISA-based serum antibody titers in rabbits to 3

candidate vaccinal isolates of P. multocida

56


candidate vaccinal isolates of S. aureus

56


candidate vaccinal isolates of Str. agalactiae

57

4.17 Composition of Montanide® adjuvanted combine HS-

mastitis vaccine.

57

4.18 Indirect ELISA-based serum antibody titers in rabbits to

vaccinal isolates of P. multocida, S. aureus and

Str.agalactiae

58

4.19 Effect of temperature and duration of storage on the physical

stability of Montanide® adjuvanted combined hemorrhagic

septicemia-mastitis vaccine

60

4.20 Comparative safety of Montanide® adjuvanted combined

hemorrhagic septicemia-mastitis vaccine in cattle and

buffaloes

61

4.21 Comparative safety of Montanide® adjuvanted combined

hemorrhagic septicemia-mastitis vaccine in mice

61

4.22 Vaccination challenge protection assay of Montanide®

adjuvanted combined hemorrhagic septicemia-mastitis

vaccine in mice

62

4.23 ELISA-based antibody titers generated by Montanide®

adjuvanted combined HS-mastitis vaccine against P.

multocida, S. aureus and Str. agalactiae in cows and

buffaloes

64

4.24 Number of new HS cases in vaccinated and control cows,

buffaloes and calves during 180 days post- vaccination period

66

4.25 Effect of Montanide® adjuvanted combined HS-mastitis

vaccine on quarter-based incidence of Str. agalactiae and S.

aureus IMIs in cows and buffaloes initially culture negative

and CMT/ SFMT negative

69


vaccine on quarter- based incidence of Str. agalactiae and S.

aureus IMIs in cows and buffaloes initially culture positive

and CMT/ SFMT positive

70

xvii


vaccine on quarter- based time points prevalence of Str.

agalactiae and S. aureus IMIs in cows and buffaloes initially

culture positive and CMT/ SFMT positive

73


vaccine on CMT and SFMT-based quarter incidence of

mastitis in cows and buffaloes initially culture negative for S.

agalactiae and S. aureus and CMT/ SFMT negative

75


vaccine on CMT/SFMT- based quarter time points

prevalence of mastitis in cows and buffaloes initially negative

for Str. agalactiae and S. aureus plus CMT/SFMT negative

78


vaccine on CMT and SFMT based quarter incidence of

mastitis in cows and buffaloes initially culture positive for S.

agalactiae and S. aureus and CMT/ SFMT positive

80


vaccine on CMT/SFMT- based time points quarter

prevalence of mastitis in cows and buffaloes initially positive

for Str. agalactiae and S. aureus plus CMT/SFMT positive

83


vaccine on the severity of mastitis in vaccinated–non-

diseased, control-non-diseased, vaccinated –diseased,

control-diseased groups of cows and buffaloes

85



ml of


vaccinated –diseased, control-diseased groups of cows

88



ml of


vaccinated –diseased, control-diseased groups of cows

88



ml of


vaccinated –diseased, control-diseased groups of buffaloes

90



ml of


vaccinated –diseased, control-diseased groups of buffaloes

90

4.37 Milk yield (Mean ±SE; L/24 hours) of vaccinated and control

cows and buffaloes at different post-vaccination days

94

4.38 Number of HS cases in vaccinated and control cows,

buffaloes and calves during 180 days post- vaccination period

and pecuniary losses based on market prices of affected

animals

96

xviii

4.39 Mastitis control related benefits accruing from use of

Montanide® adjuvanted combined hemorrhagic septicemia-

mastitis vaccine

98

4.40 Calculation of vaccine efficacy for HS in cows, buffaloes and

calves

99

4.41 Calculation of vaccine efficacy for mastitis in cows and

buffaloes

100

xix

LIST OF FIGURES Fig. No. Title Page No.

4.1 Indirect ELISA-based serum antibody titers in rabbits to vaccinal isolates

59

4.2 ELISA-based antibody titers generated by Montanide® adjuvanted combined HS-mastitis vaccine against P. multocida, S. aureus and Str. agalactiae in cows and buffaloes

65

4.3 Number of new HS cases in vaccinated and control cows, buffaloes and calves during 180 days post- vaccination

67

4.4 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on quarter-based incidence of Str. agalactiae and S. aureus IMIs in cows and buffaloes initially culture negative and CMT/ SFMT negative

69

4.5 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on quarter- based incidence of Str. agalactiae and S. aureus IMIs in cows and buffaloes initially culture positive and CMT/ SFMT positive

71

4.6 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on quarter- based time points prevalence of Str. agalactiae and S. aureus IMIs in cows and buffaloes initially culture positive and CMT/ SFMT positive

74

4.7 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on CMT and SFMT-based quarter incidence of mastitis in cows and buffaloes initially culture negative for S. agalactiae and S. aureus and CMT/ SFMT negative

76

4.8 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on CMT/SFMT based quarter time points prevalence of mastitis in cows and buffaloes initially negative for Str. agalactiae and S. aureus plus CMT/SFMT negative

79

4.9 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on CMT and SFMT based quarter incidence of mastitis in cows and buffaloes initially culture positive for S. agalactiae and S. aureus and CMT/ SFMT positive

81

4.10 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on CMT/SFMT- based time points quarter prevalence of mastitis in cows and buffaloes initially positive for Str. agalactiae and S. aureus plus CMT/SFMT positive

84

4.11 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on the severity of mastitis in vaccinated–non-diseased, control-non-diseased, vaccinated –diseased, control-diseased groups of cows and buffaloes

86

4.12 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on the quarter milk somatic cell count (× 10

5 ml of milk) in

vaccinated–non-diseased, control-non-diseased, vaccinated –diseased, control-diseased groups of cows

89

4.13 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on the quarter milk somatic cell count (× 10

5 ml of milk) in

vaccinated–non-diseased, control-non-diseased, vaccinated –diseased, control-diseased groups of buffaloes

91

4.14 Milk yield (Mean ±SE; L/24 hours) in vaccinated and control cows and buffaloes at different post-vaccination days

91

xx

LIST OF PLATS

Plate No. Title Page No.

1 Growth of a P. multocida on blood agar plate 46

2 Growth of a P. multocida on Casein-sucrose yeast agar 46

3 Seven-digit numerical biotype profile of a candidate vaccinal

S. aureus isolates determined by API® STAPH.

49

4 Latex agglutination test positive and negative reactions using

Staphytect plus® kit

49

5 Growth of a Str. agalactiae on a blood agar plate 53

6 Seven-digit numerical biotype profile of a candidate vaccinal

Str. agalactiae isolates determined by API® 20 STREP.

54

7 Montanide® adjuvanted combined hemorrhagic septicemia-

mastitis vaccine

58

xxi

LIST OF APPENDICES

App. No. Title Page No.

I Composition of casein-sucrose-yeast (CSY) agar and method

of its preparation

101

II Preparation of blood agar medium and collection of

defibrinated blood

101

III Preparation of culture media and composition 102

A Blood agar plates 102

B Staph-110 agar plates 102

C MacConkey Agar Plates 103

D Nutrient Broth 103

E Brain Heart Infusion Broth (BHI) 104

F Tryptone Soya Broth 104

IV Collection, processing and preparation of rabbit plasma for

tube coagulase test to differentiate coagulase positive and

coagulase negative Staphylococci

105

V Preparation of sterile milk whey 106

VI CAMP (Christie, Atkins and Munch-Peterson)-Esculine test 106

VII Somatic cell count 106

xxii

ABSTRACT The present study represents a maiden attempt to develop and evaluate a combined hemorrhagic

septicemia (HS) and mastitis vaccine in cows and buffaloes. The study was compartmentalized into

two phases. In phase I (laboratory settings), isolates of Pasteurella multocida, Staphylococcus aureus

and Streptococcus agalactiae isolated from field cases of HS and mastitis were scrutinized for

virulence/pathogenicity and immunogenicity in laboratory animals. Bacterin- toxoids of S. aureus and

Str. agalactiae were blended with prepared antigen of P. multocida, Montanide® ISA 201 VG,

thimerosal and sodium azide to prepare combined HS- mastitis vaccine that was evaluated for

sterility, safety and side effects under in vitro conditions and in vivo in cattle, buffaloes and mice. A

challenge-protection assay conducted in immunized mice indicated 100% survival of challenged

mice. The vaccine was physically stable in terms of pH, sedimentation, color, appearance, and

syringibility for 6 months observation period at 37°C. In Phase II (field evaluation), the combined

vaccine was evaluated in adult cattle and buffaloes and calves of cattle and buffaloes. To this end, a

total of 70 S. aureus and Str. agalactiae free lactating buffaloes (n=45) and cows (n=25), 50 lactating

cows (n=25) and buffaloes (n=25) positive for S. aureus/Str. agalactiae and dairy calves (buffalo

calves n=70; cow calves n=50) aged up to 1 year were treated with 2 doses of combined HS-mastitis

vaccine at 21 day interval and evaluated (where relevant) for 6 months in terms of ELISA based

antibody titers against P. multocida, S. aureus and Str. agalactiae, incidence of HS, local and

systemic reactions, incidence and prevalence of S. aureus and Str. agalactiae mastitis, severity of

mastitis, milk somatic cell count, milk yield, cost effectiveness and vaccine efficacy. ELISA based

antibody titers against P. multocida, S. aureus and Str. agalactiae were significantly (P˂0.05) higher

in vaccinated groups than in unvaccinated groups. Two cases of HS were recorded in vaccinated

animals vis-à-vis 7 cases in un-vaccinated animals. Incidence of S. aureus and Str. agalactiae over

180 days in vaccinated and un-vaccinated cows and buffaloes initially cultural –ve for these

pathogens was 3 and 10, respectively; the corresponding figures in groups initially culture +ve for

these pathogens being 2 and 12, respectively. Cumulative mean somatic cell counts in vaccinated

groups were significantly lower (P<0.05) than those in respective unvaccinated controls. Milk yield

was significantly higher (P<0.05) in vaccinated cows and buffaloes than in un-vaccinated controls.

Mastitis severity scores were significantly lower (P<0.05) in vaccinated groups than in unvaccinated

controls. The vaccine tested had a vaccine efficacy 84.78 and 90.25% against HS and mastitis,

respectively with a financial benefit worth Rs 2,060,300. In sum, Montanide® adjuvanted combined

HS-mastitis vaccine had preventative role against HS and both preventative and curative role against

S. aureus and Str. agalactiae associated mastitis. In view of the preliminary nature of the study,

additional work involving much larger number of cows, buffaloes and calves is clearly warranted.

1

CHAPTER 1

INTRODUCTION

Field surveys of major dairy animal diseases in Pakistan have indicated that

hemorrhagic septicemia (colloquially known as "gulghotoo") and mastitis (colloquially

known as "Sauroo") are the two most rife, endemic and economically important diseases of

buffalo and cattle (Khan et al., 1991; Anonymous, 1996; Hussain et al., 2005) Hemorrhagic

septicemia (HS) is an infectious, highly fatal bacterial disease caused by Pasteurella

multocida serotype B: 2 or Robert's type 1 (De Alwis, 1984; OIE, 2012). This organism was

also incriminated as the etiologic agent of a respiratory syndrome in buffaloes in Landhi

Dairy Colony, Karachi (Zahur et al., 2007). Accounting for annual losses of Rs. 2.17 billions

in Punjab only, this disease was ranked number one among the economically important

livestock diseases in Pakistan (Anonymous, 1996) and it was further appraised that even a

50% reduction in HS incidence would be sufficient to bridge the gap between growing milk

demand and supply. Milk demands will double itself by 2030 (Upton, 2001). Control of

infectious diseases of epidemic proportion ought to be the priority number one in the animal

sector in developing countries (Arye-Smith, 1971). Launching a National program for control

of HS has been identified as an animal health imperative in Pakistan (Afzal, 2009).

Vaccination using different types of vaccines (e.g. formalin-killed alum precipitated bacterin,

oil adjuvanted vaccines) is the single most important control measure against HS in Pakistan

and in other Asian , African, South European and Middle East countries where this disease is

endemic (Benkirane and De Alwis, 2002; Sotoodehnia et al., 2005). The vaccines are quite

effective when used as per the recommendations of manufacturers and extension agencies.

Alum precipitated bacterin is still the most commonly used HS vaccine in Pakistan but it

gives immunity for a short duration i.e. 3 months (Muneer et al., 2005) and its protective

efficacy is about 60% (FAO, 1998). Comparative field evaluation of alum precipitated (APV)

and oil adjuvanted HS vaccines has demonstrated a justification for the replacement of the

former by the later (Muneer et al., 2005).

Mastitis (inflammation or swelling of milk producing organ) is another

common dairy disease which although not fatal, causes colossal economic losses to our

resource-poor dairy farmers and milk processing industry (Khan et al., 1991; Blood and

2

Studdert, 1999; Deb et al., 2013). Research conducted over the past four decades has shown

that at the very least 20% of cows and buffaloes are afflicted with this disease. The huge

affected population of buffaloes and cattle not only sustains nearly a 25% reduction in their

milk yield (Arshad et al., 1995; Radostits et al., 2007) but the milk produced is also

unwholesome for human consumption as it contains pathogenic bacteria, toxins and other

harmful substances some of which are not destroyed by UHT treatment (Verdi, 1988;

Barbano, 1989; Heeschen, 2005) utilized by the milk processing industry in Pakistan. Control

of this disease is therefore, imperative for economically viable dairy farming as well as to

produce milk which conforms to the standards of WTO Accord.

Over the past nine years, researchers at the Department of Clinical Medicine and

Surgery, University of Agriculture, Faisalabad have demonstrated that a cost-effective

control of mastitis is an attainable dairy health objective with the use of locally prepared

mastitis vaccines containing two most prevalent mastitis pathogens (Staphylococcus aureus

and Streptococcus agalactiae) associated with mastitis in local dairy cattle and buffaloes

(Shakoor et al., 2006; Athar et al., 2007). Montanides® are ready-to- use mineral oil

adjuvants. These are reported to elicit superior immune response as compared to aluminium

hydroxide in cattle when used as an adjuvant in HS vaccine (Reddy et al., 1995). These

retain potency for longer period than conventional aqueous formulation following storage at

4°C and have no evidence of toxicity in cattle even after booster dose.

At present, a "do nothing" policy is in place virtually everywhere in Pakistan as far

as mastitis control is concerned. As mentioned earlier, the use of HS vaccine is a fairly well

established practice in Pakistan and the mastitis vaccination can conceivably be integrated

into the vaccination program already in vogue by developing a combined HS- mastitis

Montanide®vaccine. The use of a combined HS-mastitis vaccine would theoretically be better

than the use of either vaccine alone because:

1) Control of HS and mastitis in tandem with a single vaccine would be achieved,

2) Less frequent handling of animals required for vaccination against HS and mastitis.

3) Combined HS-mastitis vaccination may potentially be more protective and cost-

effective by virtue of its Montanide® adjuvant.

4) Initiation of control of mastitis against which a "do nothing" policy is in place

virtually throughout the country

3

The present study was therefore planned to prepare and evaluate a Montanide®

adjuvanted combined hemorrhagic septicemia-mastitis vaccine under laboratory settings and

under field conditions

Objectives:

a. To prepare and evaluate a Montanide® adjuvanted combined hemorrhagic septicemia-

mastitis vaccine in laboratory settings and experimental animals

b. To evaluate Montanide®adjuvanted combined hemorrhagic septicemia-mastitis

vaccine under field conditions.

c. To determine the cost-benefit ratio of hemorrhagic septicemia and mastitis control

through the use of a Montanide® adjuvanted combined hemorrhagic septicemia-

mastitis vaccine.

4

CHAPTER 2

REVIEW OF LITERATURE

2.1 Economic impact of animal diseases in developed and developing countries

Animal diseases, in particular infectious diseases, have major adverse effects on

livestock productivity as well as animal welfare worldwide. The costs of animal diseases are

around 17% of turner-over of the livestock sector in the developed countries while it is 35-

50% in the developing countries (Bishop et al., 2010; Holden et al., 1996). Infectious

diseases pose a significant challenge to all animal production systems, which sustain

significant production losses from these diseases. Thus animal diseases are a significant

threat to food security worldwide. In addition, several infectious diseases do not respect the

species barriers and about 70% of all infections are common to man and animals (zoonosis).

Several options available to curtail the colossal economic losses associated with animal

diseases include immunization, chemotherapy, improved management, nutritional and

genetic improvement, diagnosis and removal of infected animals. Implementation of each of

these options can add to solving the problems of productivity losses and zoonosis caused by

these diseases (Bishop et al., 2010). Investment in animal health research has been shown to

pay rich dividend by alleviating poverty in developing countries (Perry et al., 2002).

According to Garcia et al. (2003), a comparison of average milk yield across different

countries would reveal that one New Zealand cow produces as much milk as 3 dairy animals

in Pakistan, whereas one American cow produces milk that is nearly equal to milk produced

by 7 Pakistani cows. This dramatic difference in the milk yield of Pakistani dairy animals vis-

à-vis dairy cows of advanced countries can be attributed to a verity of factors including

genetics, nutrition, management and diseases etc.

2.2 Mastitis

According to Bishop et al (2010), in terms of economic losses, mastitis is the single

most important disease in cows in developed counties and is also of major importance in

developing countries. Avenues of economic losses include milk loss, premature culling, and

cost incurred on replacements, occasional mortality, treatment costs and the cost of discarded

milk. Treatment of clinical cases not only involves the costs of antibiotics and other

therapeutic agents, but may also create opportunities of antibiotic and drug residues in the

5

milk of treated animals. Furthermore, in livestock production systems where farmers are

paid according to milk quality, elevated milk somatic cell count (SCC) as a result of clinical

and sub clinical mastitis may affect the profitability of dairying. Selection against subclinical

mastitis using SCC is now widely practiced in countries where dairying is well developed.

Thus, selection for resistance against clinical mastitis is one of the important components of

dairy cattle breeding programmes in Scandinavian countries.

Khan and Chaudhry (2001) analyzed the data on 2704 lactations of 993 Nili-Ravi

buffaloes to investigate the frequency and behavior of short and complete lactations.

Lactation milk yield up to 44 weeks (308 days) was included and lactations with less than 56

days were excluded from the analysis. Of 2107 lactations of less than 14 weeks durations, the

causes of drying could be determined from the farm record for 534 lactations. Mastitis

accounted for 31% of these lactations for drying during the course of shorter than normal

lactations. A meta-analysis of investigations conducted for one year period (1994-1995) on

economically important diseases of livestock in Punjab, Pakistan ranked mastitis at the 5th

slot. This disease was responsible for 8% of the total pecuniary losses inflicted by all

livestock diseases in this province (Khan et al., 1994-95).

2.3 General aspects of hemorrhagic septicemia (HS)

Hemorrhagic septicemia (HS) is an acute highly fatal, septicemic disease occurring

primarily in water buffaloes (Bubalus bubalis) and cattle but occasionally in other domestic

and wild mammals (De Alwis, 1992; Adlakha and Sharma, 1992). Water buffalo is far more

susceptible to this disease than cattle (De Alwis, 1999; Khan et al., 2006). The etiologic

agent of HS is a Gram negative bacterium, Pasteurella multocida (Tabatabaei et al., 2007;

Carter and De Alwis, 1989). Pasteurella multocida has at least five serotypes designated as

A, B, C, D and E. Serotypes B: 2 and E: 2 are two common serotypes of P. multocida

associated with HS in animals in Asia and Africa, respectively (Benkirane and De Alwis,

2002). In serotype classification, the letter is used to denote the capsular antigen that was

determined originally by Carter (1984) with the use of indirect hemagglutination test. The

numeral 2 denotes the somatic or O antigen that was determined originally by Namioka and

Bruner (1963) by using agar gel diffusion precipitin test developed by Heddelston and

associates.

6

Hemorrhagic septicemia (HS) is one of the most important diseases of bovines in

South Asian and Middle Eastern countries. Epidemiological studies conducted in India over a

period of approximately 13 years (1974-1986) indicated that HS was placed mortality wise

first and morbidity wise second as compared to 4 other epizootic diseases namely, foot and

mouth disease (FMD), rinderpest, anthrax and black quarter (Dutta et al., 1990).

2.3.1 Carrier state

Carriers animals are the source of the organism for other animals. Cattle and buffaloes

are considered to be the principal carriers of P. multocida, but other animals such as pigs,

sheep, goats and horses can also act as carriers. The incidence of HS has been shown to be

directly proportional to the number of carriers in the animal population. Thus in India in low

incidence areas, no carriers were detected, whereas in the moderate and high incidence areas,

the carrier rates were 1.9 and 5-6%, respectively (Adlakha and Sharma, 1992). Regular

vaccination has been shown to eliminate the carrier status. (De Alwis, 1984).

2.3.2 Transmission

Pasteurella multocida is spread by inhalation, ingestion of contaminated feed, water,

direct contact, and fomites etc. The causative agent of HS is mostly shed into the oropharynx,

lymphatic tissue associated with the upper respiratory tract, and periodically in nasal

secretions during stress (poor food supply, close herding, and wet conditions). HS pathogen

does not remain alive for log time, but can survive for a few hours or days in damp soil. The

favorable condition for transmission is rainy season and humid environment (OIE, 2012).

2.3.3 Association of HS with other diseases

Some other infectious diseases may predispose to occurrence of hemorrhagic

septicemia. Foot and mouth disease causes immunosuppression (Golde et al., 2014; Maddur

et al., 2010) and thus can precipitate an attack of HS or some other bacterial infections.

Elshemey and Abd-Elrahman( 2013) reported that 56.32% (n=2600) of 4616 cow and buffalo

fattening calves infected with SAT2 serotype of FMD virus showed signs of hemorrhagic

septicemia (P. multocida B2 serotype) with a case fatality rate of 13.92%. All of 5630 calves

had been vaccinated with an inactivated FMD vaccine containing antigens of serotype A and

O. Kumar et al. (2007) reported outbreaks of concurrent pasteurellosis (P. multocida

serotype B: 2) and classical swine fever that occurred in Indian Punjab. Overall mortality,

morbidity, and case fatality rate were 77.5, 88.2, and 87.8%, respectively, in pigs ≤3 months

7

of age, and 8.2, 20.5, and 40%, respectively in older pigs. More marked pneumonic lesions

were recorded in cases from which P. multocida was isolated.

2.3.4 Pathogenesis

Hemorrhagic septicemia is associated with P. multocida serotype B: 2. It is a Gram

negative bacterium that secretes lipopolysaccharide (LPS), also named as endotoxin, in blood

stream of the host on its lysis. All manifestations of the disease are due to these endotoxins

which lead to endotoxemia associated sepsis (De Alwis, 1992; Horadagoda et al., 2001).

Endotoxins induce many complex deleterious biological reactions in the host. They stimulate

a cascade of endogenous mediators such as the coagulation and arachidonic acid systems.

Endotoxin also activate polymorphonuclear cells, monocytes and macrophages. When

monocytes and macrophages sense the presence of endotoxin, they release special peptides

called cytokines.

Horadagoda et al (2002) studied the clinical changes along with acute phase

responses (including tumour necrosis factor; TNF α) in 6 buffalo calves following iv

administration of P. multocida B:2 endotoxin @ of 1µg /kg in 10 ml of phosphate - buffered

saline. There was a rapid onset of clinical signs (dullness, sternal recumbency, fever,

excessive salivation and respiratory difficulty) following administration of endotoxin. These

clinical signs lasted for about 12 hours. Serum TNF α levels increased rapidly within one

hour post inoculation of endotoxin, reaching their peak levels (8-150 ng/ml) at 1-2 hours post

inoculation and then declined rapidly to baseline values at 3-5 hours post inoculation. Other

acute phase responses included leukopenia, decreased serum levels of iron and zinc. In

addition, there was a delayed but prolonged increase in haptoglobin from 12 hours post

inoculution that reached its peak from 60 hours post inoculation. Control buffalo calves (n=3)

showed neither clinical signs nor the acute phase serum changes mentioned above. The

results of this study confirmed the notion that endotoxin has a cardinal role in the

pathogenesis of P. multocida B:2 associated pasteurellosis of buffalos, commonly knowns as

hemorrhagic septicemia.

2.3.5 Rainy seasonal occurrence

Although, occurrence of HS has been reported throughout the year (Dutta et al.,

1990), its incidence much higher in hot and humid weather (Sivakumar et al., 2012).

Chowdhury et al. (2014) recently reported an outbreak of HS in a buffalo herd in the month

8

of December. High humidity coupled with high environmental temperature and stressful

conditions prompts growth of the P. multocida and shortens the incubation period of the

pathogen by nearly 30 hours, resulting in HS outbreaks with high case fatality rate (De

Alwis, 1995).

2.3.6 Clinical signs and post mortem findings

Hussain et al. (2014) recorded an outbreak of pneumonic pasteurellosis (P.

multocida) in cattle and buffaloes in district Sahiwal of Pakistani Punjab. Clinical signs

observed included fever, anorexia, brisket edema, profuse salivation, mucopurulent nasal

discharge, protruded tongue with open mouth breathing, submandibular edema and dyspnea

with respiratory grunts. Necropsy of the dead animals reveled severe pneumonia with

consolidation of lungs, froth in trachea, clotted blood in heart, intense pleural adhesions,

accumulation of serosanguinous fluid in pericardial and peritoneal cavities. Acute HS with

severe respiratory distress has also been reported in wild ruminents (Dhoot and Upadhye,

2001)

2.3.7 Treatment

Owing to generally a peracute nature of the disease, treatment of HS is not effective,

and there is need to find effective therapeutic agents for the treatment of HS. This disease.

According to Shahzad et al. (2013), treatment was effective only when instituted at the early

stage of the disease. These workers also reported that ciprofloxacin, ceftiofur hydrochloride +

tylosin were the most effective antibiotics for treatment. Nonetheless, the workers did not

mention the cure rates of these antibiotics. Egyptian workers (Elshemey and Abd-Elrahman,

2013), treated an outbreak of HS that occurred in the wake of SAT2 FMD infection in cow

and buffalo fattening calves. Table 2.1 gives the cure rate of 9 different treatment protocols

used to manage this HS outbreak.

9

Table 2.1: Cure rate of 9 different treatment protocols used to manage an outbreak

of HS (Pasteurella multocida serotype B2) that occurred in the wake of

SAT2 FMD infection in cow and buffalo fattening calves in Alexandria

Egypt

Treatment

protocol

number

Treatment protocol No. of animals

Treated

Cure rate from H.S

No. of animals

that recovered

from HS

Percent cure

rate

1 Ceftiofur sodium 470 423 90

2 Lincspectin and

gentamycin.

350 280 80

3 Cefotaxime sodium. 905 815 90.05

4 Tylosine and

gentamycin

150 105 70

5 Tultrathromycin 170 85 50

6 Sulpha +

Trimethoprim

70 14 20

7 Oxytetracycline LA 200 40 20

8 Amoxycillin 75 0 0

9 Florfenicol 210 0 0

Adapted from Elshemey and Abd-Elrahman (2013)

Hussain et al. (2014) treated sick animals suffering from pneumonic pasteurellosis

with antipyretic, steroids and antibiotic drugs (nature of drugs used, their dosages and

duration of treatment not stated by the authors). However, the treated cows and buffaloes did

not show improvement in signs and death ensued in a very short period (2-4 hours) in per

acute cases.

10

Ashraf et al. (2009) compared florfenicol, florfenicol + flunixin meglumine,

amoxicillin and amoxicillin + flunixin meglumine for the treatment of HS in buffalo calves (a

total of 40 buffalo calves aged 6-18 months). The recovery rate (survival) in calves treated

with florfenicol + flunixin meglumine was 90%, whereas in calves treated with amoxicillin +

flunixin meglumine was 60%. Calves treated with florfenicol alone had a recovery rate of

80%, whereas 50% of the calves treated with amoxicillin alone survived. Chowdhury et al.

(2014) treated HS affected buffaloes with (a) Inj. Ceftiofur sodium @ 1gm I/M for 5 days,

(b) Inj. Meloxicam @ 20 ml I/M for 5 days, (c) Inj. Prednisolone @10 ml I/M for 5 days (in

tapering doses). The buffaloes with early stage of disease responded to the treatment.

Raza et al. (2000) investigated a total of 50 animals (39 buffaloes and 11 cattle)

suffering from hemorrhagic septicemia (HS), selected from the field and divided into 3

groups (A, B and C). Group A (20 animals) was treated with protocol A i.e. norfloxacin

(Norfloxillin; Tarobina) + diclofenac sodium +Novacoc forte (Richter Pharma). Group B (20

animals) was treated with protocol B which was essentially the same as protocol A except

that Novacac forte was omitted. Group C (10 animals, control) was treated with one of

conventional therapies of HS, i.e gentamicin + dipyron. The disease severity index of

experimental animals under each treatment protocol was recorded at 0, 12, 24, 48, and 72

hours from the start of treatment. All treatments were repeated at 12, 24, and 48 hours after

start of treatment as the sitaution warranted. The survival percent was 85, 80 and 30 amongst

animals treated respectively with protocol A, B and C. Three animals in group A and 2

animals in group B died after recovering completely. Excluding these animals from the

recovered ones, net survival (percentage) was 70, 70, and 30 in groups A, B and C,

respectively. In terms of reduction of severity of disease, there was significance difference

(P≤ 0.05) between protocol C and A, between protocol C and B at 12 and 48 hours after

treatment but there was non-significant difference at hours 24. Both treatment protocols A

and B exploiting the use of a quinolone (norfloxacine) plus a non–steroidal anti-

inflammatory drug (diclofenac sodium) with or without a toxin neutralizer and circulatory–

stimulant (Novacoc forte) were more effective than the conventional treatment i.e gentamicin

+ dipyron for the treatment of HS.

11

2.3.8 Morbidity, mortality and case fatality rates

Hemorrhagic septicemia or pasteurellosis is probably the most serious disease of

buffaloes and there are outbreaks which cause heavy mortality and morbidity (Dhillion et al.,

1971; Saini et al., 1991; Joshiet et al., 1987; Dhand et al., 2002). The death rate in buffaloes

is three times more than in cattle (Bain et al., 1982). Field observations of 9 districts of

Punjab, Pakistan showed 9% mortality, 11% incidence, and 87% case fatality rates of

hemorrhagic septicemia in buffalo, whereas these values were 2.5, 4, and 62% in cattle

(Sheikh et al., 1996)

Taking cues from the previously reported observation that fever increases the survival

rate in reptiles, Kluger and Vaughn (1978) investigated the relationship between fever and

survival in rabbits infected with Pasteurella multocida. A statistically significant correlation

was recorded between the fever magnitude and survival. As fever increased by 2.25 ºC, the

survival rate increased.

2.4 Prevalence and economic significance of HS in Pakistan

Hemorrhagic septicemia is a common and important infectious disease of buffaloes

and cattle in Pakistan (Khan et al., 1991; Ashraf et al., 2011). With a mortality rate near

70% (May et al., 2001), this disease was responsible for causing pecuniary losses worth Rs

2.17 billion per annum in 1996 in the Punjab province alone (Anonymous, 1996). Owing to

an increase in animal population (cattle n=41.2 millions; buffalo n= 35.6 millions) in 2014-

2015, and escalation of prices of animals, the current monitory losses may be at least 2

magnitude higher than these figures. Using an active surveillance system, Khan et al. (1991)

reported an estimated economic loss of Rs 689900 due to HS in 10 randomly selected

villages of Tehsil Lahore. During 2000-2005, field workers of FAO project entitled “Support

for Emergency Prevention and Control of Main Trans-boundary Diseases in Pakistan”

conducted a Participatory Disease Surveillance. An active surveillance of trans-boundary

animal diseases and other important diseases throughout the country (10% villages were

randomly selected throughout Punjab, Sindh, Khyber Pakhtunkhwa, Azad Jammu and

Kashmir and Northern Areas) was conducted by 17 teams comprising of 5 active field

veterinarians each. The highest prevalence (49%) of HS was recorded in district Khanewal,

Punjab. On the other hand, the lowest prevalence (0.78%) was recorded in Jamshaid Saddar

town of Karachi. The highest prevalence (75.64%) was recorded in Faisalabad district, while

12

lowest (1.53%) in Bajaur agency (Farooq et al., 2007). Based on information gleaned from

veterinary officers in 9 districts of Pakistani Punjab, Sheik et al. (1996) documented 9%

mortality, 11% incidence, and 78% case fatality rates of hemorrhagic septicemia in buffalo,

whereas these values were 2.5, 4, and 62% in cattle. Disease incidence was higher in 0 to 24

month old animals and groups of less than 10 animals. Farooq et al. (2011) determined the

sero-surveillance, morbidity, mortality and case fatality rate of HS in cattle and buffaloes in

Dera Ghazi Khan district, Punjab, Pakistan. The average geometric mean titers (GMT)

recorded against HS in diseased buffaloes and cattle were 5.7 and 6.1, respectively. The

morbidity, mortality and case fatality rates respectively were 57.58, 52.30 and 90.83% in

young buffalo calves. The corresponding values for adult buffaloes were 3.17, 1.92 and

60.65%, respectively. In case of young cattle calves, morbidity, mortality and case fatality

rates were 8.63, 5.27 and 61.11%, respectively; the corresponding values in adult cattle being

4.83, 2.18 and 45.23%, respectively. This study demonstrated that the mortality, morbidity

and case fatality rates due to HS were higher in young calves than in adults both in buffaloes

and cattle. It also reinforced the notion that buffaloes are more susceptible to HS than cattle.

Recently, Khan et al. (2011) reported an outbreak of HS in buffalo calves (aged 6-11 months;

n=54) kept at the Livestock Experimental Station, University of Agriculture, Faisalabad,

Pakistan. This outbreak occurred in August and September, 2009. Morbidity and mortality

rates in this outbreak were 100% and 31.48%, respectively. Mortality peaked on 8th

day

despite institution of a theoretically effective treatment (Amoxicillin long acting, ceftiofur

sodium, prednisolone acetate + dexamethasone sodium phosphate and meloxicame). It is

pertinent to note that this outbreak occurred despite vaccination in June with a reputed oil

based HS vaccine inoculated as per the manufacturer‟s recommendation.

Khan et al. (2006) reported the findings of a comparative sero-surveillance and

clinical epidemiological study conducted on HS in cattle and buffaloes in district Malakand,

NWFP (current name of the province is Khyber Pakhtoon Khwa). The average geometric

mean titer (GMT) recorded against HS in buffaloes ranged from 4.12-46.98, whereas in cattle

the corresponding values ranged from 4.45-46.40. In young buffalo calves (the age not

mentioned by the authors), mortality rate, morbidity rate and incidence rate, were 21.19,

95.25 and 22.25%, respectively. In adult buffaloes, the corresponding values were morbidity,

mortality and case fatality rates were 5.49, 1.65 and 30%, respectively. In case of young

13

cattle calves, mortality, morbidity, and case fatality rates were lower than those in young

buffalo calves, being 1.77, 3.94 and 45%, respectively. The values for morbidity, mortality

and case fatality rates in adult cattle were 2.51, 0.39 and 15.79%, respectively.

Ahmad and Naz (2000) reported HS incidence of 6.8% in buffalo calves maintained

at the Livestock Experiment Station, Bahadurnagar, Okara, Pakistan. Riaz et al. (1992)

conducted an active surveillance from September 1989 to August 1990 in district Gujrat,

Pakistan. A total of 1025 farmers were interviewed. HS incidence of 1.31% in cattle and

7.24% in buffaloes was recorded.

With the establishment of Directorate for Animal Disease Surveillance and Reporting

System in Punjab under the administrative control of Director General (Ext), Livestock and

Dairy Development, Punjab, the disease incidence reports communicated by field

veterinarians through District Livestock Officers/ District Disease Reporting Officers across

Punjab during the month of July to September, 2009 were incorporated to prepare the first

quarter report on the distribution of notifiable animal diseases in the province (Anonymous,

2009). FMD (foot and mouth disease) PPR (Peste des petitis ruminants), CCPP (contagious

caprine pleuropneumonia), ET (enterotoxemia), HS, BQ (black quarter) and ecthyma are the

diseases among all the contagious diseases that were reported during the reporting period. A

total of 208 outbreaks of 23 contagious diseases were reported for different diseases

including HS (82), FMD (38), ET (19), PPR (12), BQ (9), and CCPP (9) as main prevalent

diseases in the reported area. HS, FMD, ET, PPR, BQ and CCPP respectively affected 247,

148, 74, 50, 9 and 159 heads of animals while 95, 22, 16, 13, 5 and 12 died, respectively.

Prevalence and incidence of HS, FMD, ET, PPR, BQ and CCPP were found to be higher than

other contagious diseases. HS and BQ were reported from Faisalabad, Kasur, Rahim Yar

khan and Chakwal, DG Khan, Rahim Yar Khan, respectively. FMD was found to be present

in district Bahawalpur, Dera Ghazi Khan and Faisalabad. FMD is a disease that affected

highest number of animal heads in Punjab with a low mortality rate. PPR and ET were

reported from district Bahawalpur, DG Khan, Faisalabad, Lahore and Bahawalpur, Chakwal,

DG Khan, and Faisalabad, respectively. It was concluded that HS, FMD, ET, PPR, BQ and

CCPP were the diseases of main concern in the reported areas of Punjab during the months of

July to September, 2009.

14

Khan (2013) reported that no case of HS was brought to the notice of District Diagnostic

Laboratory Rahim Yar Khan during the period from May – July, 2012.

2.5 Hemorrhagic septicemia vaccines

Use of vaccines for the control of pasteurellosis in animals is since long in vogue.

Shilston (1923) while reviewing the information on hemorrhagic septicemia (pasteurellosis)

(that was published in the book edited by Wooldridge, 1923) cited several authors and

investigators who used pasteurellosis vaccines for the control of this group of infectious

diseases associated with P. multocida. For want of availability of these archaic reports, the

work of some of these investigators has been reproduced almost verbatim from Shilston

(1923) in the ensuing paragraph Oreste and Armmani (1887) attenuated the Bacillus.

Bubalisepticus (P. multocida) by passage through pigeons and employed the blood of these

birds after they had died from the inoculation of the organism, for the immunization of the

buffaloes. Three injection of 0.1 ml of the blood were given. By growing the organism at

temperature of 300C to 32

0C, reduced the virulence of P. multocida so that the cultures could

be employed to give protection to the buffaloes and sheep. Lignieres (1902) prepared a

polyvalent vaccine with strains of the hemorrhagic septicemia bacillus from cattle, sheep,

horses, pigs, fowls, and dogs. Cultures of the organisms of uniform virulence were kept at a

temperature of 420

to 430 C. for five days to produce the first vaccine and for two days for the

second vaccine. The vaccines were injected subcutaneously in doses of from 0.15 to 1 ml

according to the size of the animals, at interval of 12-15 days and were assessed protective

against the disease in all species of animals for a period of about one year. Mohler and

Eichhorn (1912) employed vaccines prepared by Lignieres method of protecting the buffalo

herd in the Yellowstone Park against hemorrhagic septicemia which was causing a heavy

mortality. The organisms were injected subcutaneously with 1 ml of each vaccine at an

interval of ten days, and no further cases of disease occurred during the succeeding 12

months. These investigation also tested the immunization power of vaccines prepared in the

same way as those of Lignieres but to which 5% carbolic acid had been added, thus making

them dead vaccines. They found that the carbolized vaccine gave protection against an

inoculation of the virulent organism, but, as judged by complement fixation test of serum of

the treated animals, this protection was of shorter duration than after injections of

uncarbolized vaccines. Holmes tested the immunity conferred by dead vaccines prepared in

15

various ways from cultures of P. multocida. Broth cultures of a virulent strain of the

organism heated to 650

C. for half an hour and carbolized was found to protect young cattle

and buffaloes, when injected subcutaneously in dose of 5-10 ml for a period of 6 weeks

ageist an inoculation of the virulent organism. The vaccine was considered harmless, only

causing in some cases slight swelling which disappeared in a few days, protection was not

established until four days after the injection of vaccine. After 150,000 doses of vaccine were

employed annually in India as a prophylactic measure in areas where the disease is enzootic

at periodic, inoculations, being carried out at the beginning of the winter rains and monsoon

season. Muktesar Laboratory in India adopted serum-live culture inoculation method for the

control of HS. This method is briefly as follows: Buffaloes and hill cattle are first injected

subcutaneously with a protective dose of immune serum and 4 hours later receive 0.1 ml of

48 hours old broth culture of virulent hemorrhagic septicemia bacilli; the initial protection

may also be conferred by an injection of dead vaccine followed in ten days by 1ml of virulent

culture. A temperature reaction and local edema usually follows the inoculation of the

culture; when this has subsided, after 8-10 days an injection of 1ml broth culture was given.

Further injections of 5, 25, 100 and 500 ml of culture were given at intervals of ten days and

the animals were given at intervals of 10 days. The animals are then bled for serum; the

injections and bleedings were repeated, the dose of culture being gradually increased up to

100 ml. An injection of the serum confers an immediate immunity lasting for a period of 3-4

weeks. It has been very effective when employed during outbreaks of hemorrhagic

septicemia, the mortality being at once arrested; the serum also possesses some curative

power.

Aggressins were one of the earliest immunizing agents used against HS, blackleg,

anthrax and certain other human and animal diseases. These are the substances (including but

not limited to capsular material, bacterial protein, secretions, excretions, enzymes and toxins)

secreted and excreted by certain organisms under favorable conditions of growth, which have

the property of inhibiting phagocytosis by a specific action on the leucocytes and reticulo-

endothelial system. Owing to their deleterious effect on the tissues of the host and leucocytes

and reticulo-endothelial system, they may facilitate the rapid development of normally

sublethal infections of disease- producing organisms, resulting in death. Injection of

aggressins into the body causes the production of specific anti-aggressins (Scott, 1931).

16

Gochenour, (1924) cited by Scott, (1931) developed an aggressin for the immunization of

animals against hemorrhagic septicemia. The hemorrhagic septicemia aggressin is probably

the least efficient, due to the peracute nature of the disease. Losses from shipping fever were

reportedly nearly twice as great among cattle vaccinated with HS aggressins, or other

vaccines including bacterins at the stock yards or on arrival at the farm as among the cattle

not treated with these immunizing agents (Scott, 1931).

Currently, vaccination against HS is the single most important control intervention in

countries plagued by this disease. A variety of vaccines containing either whole local isolates

of P. multocida or their components have been developed and tested under control and filed

conditions (Verma and Jaiswal, 1998; Shivachandra et al., 2011). A few of these vaccines

have found their wide spread field use. One of the hallmarks of these vaccines is a

considerable variation in duration of immunity conferred (Shivachandra et al., 2011). Plain

broth formalin killed bacterins, alum precipitated and aluminium hydroxide gel adjuvanted

vaccines have been used until recently. Their immunity lasts for 4-6 months and their

protective efficacy is about 60% (FAO, 1998; Verma and Jaiswal, 1998; Tasneem et al.,

2009; Shivachandra et al., 2011). Qureshi and Saxena, (2014) recently reported that cattle

vaccinated with conventional alum precipitated HS bacterin did not develop and sustain

adequate levels of antibody for long duration. Owing to their shorter duration of immunity,

these vaccines are now being supplanted by oil adjuvant vaccines that give both a higher

degree of protection and a longer duration of immunity up to a year (Verma and Jaiswal,

1998; Tasneem et al., 2009). Oil adjuvant HS vaccines have the drawback of high viscosity

and consequently difficulty in injecting (De Alwis, 1984). In order to address these

difficulties, a double emulsion and multiple emulsion vaccines endowed with thin viscosity

and long stability have been developed that gave immunity similar to oil adjuvant vaccines

(Muneer and Afzal, 1989; Muneer et al.,1994; Verma and Jaiswal, 1997; Verma and Jaiswal,

1998). A score of studies have investigated live streptomycin-dependent P. multocida (Wei

and Carter, 1978; De Alwis et al., 1980; Lu and Pakes, 1981; Verma and Jaiswal, 1998;

Dagleish et al., 2007; Ataei et al., 2009) and P. multocida + P. haemolytica vaccine (Catt et

al., 1985). De Alwis et al. (1980) investigated a live mutant strain of P. multocida that was

administered as a single dose containing 1010

to 1011

viable organism. This vaccine

immunized 75 % of cattle and 100% of buffalo. The number of live microorganism used in

17

this vaccine is seemingly so high that it precludes the practical utility of this vaccine in the

immunological control of HS. In a subsequent study, De Alwis, (1981) reported that natural

P. multocida infection of buffalo calves give considerably higher immunity than that

conferred by vaccine which can be interpreted to mean that a live vaccine, using a suitable

mutant, may prove superior to the existing ones(Adlakha and Sharma, 1992).A live HS

vaccine containing P. multocida serotype B: 3, 4 was administered by intranasal aerosol in

Myanmar (Burma) to more than 15 million cattle and buffaloes between 1989 and 1999

(Myint et al., 2005). Field observations of veterinary officers in 9 districts of Pakistan,

Punjab have pointed out the occurrence of field outbreaks of HS despite vaccination with

alum- precipitated P. multocida bacterin prepared in public sector (Sheik et al., 1996). In

order to obtain maximum protection from HS vaccine, it is recommended that the vaccines

be prepared from P. multocida strains circulating in the regions of intended use. FAO

recommends a live avirulent HS vaccine prepared from a P. multocida serotype B: 3 of

fallow deer origin for use in Southeast Asia (Carter, 2005). Despite vaccination and

improved management, occurrence of disease outbreaks is a regular feature each year,

especially in endemic areas of the country (Afzal and Muneer, 1990). Pakistani workers

(Shahzad et al., 2013) reported an outbreak of HS in buffalo calves that had been vaccinated

with alum precipitated HS vaccine two months earlier. Arif et al. (2013) recently

documented that subunit or whole bacterin based P. multocida vaccines were less

immunogenic than an anti-idiotypic vaccine prepared against P. multocida B: 2.

Hemorrhagic septicemia is predominantly a disease of young cattle and buffaloes.

How soon after birth, the calves should be vaccinated against this disease is a very pertinent

question in the immunization program of dairy animals. Japanese workers (Otomaru et al.,

2015) in a bid to answer this question investigated the dynamics and duration of antibody

titers against P. multocida in Japanese black calves. To this end, 20 unvaccinated calves from

two Japanese black breeding farms in Japan were investigated. Serum samples were collected

from these calves at 1, 4, 8, 12, 16 and 20 weeks after birth. Similarly, serum samples were

also obtained from their dams once at 1 week after calving. Serum antibody titers against P.

multocida in calves at 1 week of age correlated very well with those in their dams at 1 week

after calving. Maternally derived antibody titers against P. multocida in experimental calves

fell to their lowest values at 4 weeks of age. One of the findings relevant to immunization

18

against P. multocida was that calves started producing antibodies against this organism by

themselves between 4 and 8 weeks of age. In the light of the findings of this Japanese study,

vaccination against P. multocida can probably be started after the calves have attained an age

of 4 weeks.

Nadeem et al. (2010) conducted a study to determine the effect of prophylactic

application of levamisole in hemorrhagic septicemia (HS) vaccinated buffalo calves. A total

of 60, 5 month old buffalo-calves were randomly selected from the Livestock Production

Research Institute Bhadurnagar (LPRI) Okara, Pakistan and divided into 3 groups (G1, G2

and G3) of 20 calves each. Animals in G1 group were vaccinated with 3ml (I/M) injection of

a commercially available oil based HS bacterin (NIAB-HS™) while animals in group G2

received levamisole @ 0.5mg/kg body weight two days prior to vaccination with this

bacterin. Group G3 served as unvaccinated and non-medicated control. IHA based geometric

mean titers (GMT) against P. multocida and Lymphocytes Proliferation Assay (LPA) were

used as evaluation criteria. Geometric mean titers (GMT) were significantly higher (P>0.05)

in calves of group G2 (21.5) as compared to GMT values (14.79) recorded in calves of group

G1. Similarly, the values (0.317±0.041) of LPA in calves of group G2 were significantly

higher than the corresponding values (0.043±0.002) recorded in calves of group G1. The

values of GMT and LPA in non–vaccinated, non–medicated calves (group G3) were 2.5 and

0.043±0.002, respectively. It was concluded that prophylactic application of levamisole helps

in mounting a better humoral and cellular immune response to P. multocida vaccine.

2.6 Importance of transferrin binding protein A as an immunogen

IROMPs (Iron regulated outer membrane proteins), in particular TbpA (transferrin

binding protein A) has been proposed as a vaccine candidate immunogen for P. haemolytica,

Neisseria meningitides, and Haemophilus influenza. The rationale for targeting TbpA as an

immunogen is three folds (Singh et al., 2011). Firstly, this is one of the important IROMPs

involved in acquisition of iron from the host and is essential for overcoming the iron

restriction imposed by the host iron binding i.e. transferrin. Secondly, it is present at the cell

surface of the bacterial cell and expressed by bacteria when growing inside host body in

response to iron depleted conditions. Its expression is enhanced by incorporation of iron

chelators like 2, 2-dipyridyl in the culture media (Srivastava, 1998). Transferrin binding

protein is one of the important virulence factors of P. multocida. Thus, Veken et al. (1996)

19

reported a positive association of TbpA with HS causing strains of P. multocida in bovine

whereas the strains of non HS serotypes did not express this protein. Singh et al. (2011)

evaluated the potential of TbpA as a DNA vaccine against HS in a mouse model. The TbpA

gene of P. multocida serotype B:2 was cloned in a mammalian expression vector alone and

along with murine IL2 gene as immunological adjuvant to produce monocistronic and

bicistronic DNA vaccine constructs, respectively. The immune response to DNA vaccines

was determined on the basis of serum antibody titers and lymphocyte proliferation assay.

Mice vaccinated with DNA vaccines showed significantly higher antibody titers and cell

mediated immune response than the unvaccinated mice. The bicistronic DNA vaccine

elicited a superior immune response and protection following challenge as compared to

monocistronic construct. These investigators concluded that DNA vaccine offers the promise

of an effective immunological approach for the control of HS.

2.7 Montanide® adjuvanted HS vaccines

In a bid to develop a stable, easily injectable HS vaccine with a long duration of

immunity, Pakistani workers (Muneer and Afzal, 1989) prepared and evaluated two oil-

adjuvant vaccines viz. water-in-oil emulsion (final product incorporating Marcol 52,

Montanide® 103 and antigen in ratios of 6:1:3) and double emulsion (Marcol 52, Arlacel A

and Tween 80 plus antigen) of P. multocida Robert's type 1. These preparations were

evaluated on only 10 buffalo calves aged ≥ 4 months which showed sustained antibody

response throughout the entire duration of experiment (=240 days). The pitfall of small

number of test subjects (buffalo calves) in this study were addressed by Muneer et al. (1994)

by evaluating three oil adjuvant vaccines of P. multocida 6: B. in terms of level and duration

of antibody levels in buffalo calves (n=45). The three vaccines evaluated were (a) water-in-

oil emulsion (= single emulsion; OAV1) containing Marcol 52, Montanide®

888 and antigen

incorporated at a ratio of 6:1:3, (b) double emulsion (OAV2) formulated to contain Marcol

52 (63%), Arlacel A (7%) and Tween 80 (1.5%) + antigen (28.5%), (c) vitamin E adjuvant

vaccine (OAV3) incorporating Vit. E (62%), Montanide® 888 (8%) and antigen (30%).

ELISA-based P. multocida antibody titers elicited by these vaccines remained substantially

higher than those recorded by using traditional alum precipitated bacterin following

sensitizing and booster vaccination spaced 2 months apart. All three vaccines induced

immune response in buffalo calves that persisted beyond 270 days post-vaccination. On

20

experimental challenge at 6 and 12 months after booster vaccination, the calves vaccinated

with OAV1, OAV2, and OAV3 withstood experimental challenge with virulent P. multocida

(6×108 cfu) given subcutaneously. Also, there was no untoward reaction like pyrexia or

swelling at the inoculation site. Contrarily, calves dose with alum precipitated bacterin (the

traditional HS vaccine) showed signs of HS on post-vaccination challenge at 6 and 12

months, albeit they also recovered. Unvaccinated animals (= control groups) died after

showing overt signs of HS on challenge at both 6 months and 12 months post vaccination.

Sotoodehnia et al. (2005) developed a Montanide® oil ISA-70 adjuvanted HS vaccine that

was injected at the dose rate of 3 ml (2 mg dry weight/ml) into 5 calves by intramuscular

route. The sera collected were tested by passive mouse protection test (PMPT). The results of

PMPT showed 100% protection up to 150 days and 66 to 83% up to 200 days post-

vaccination. In active mouse protection test (AMPT), 4 log of protection was recorded. The

investigators concluded that immunity induced by this Montanide®

HS vaccine was

protective for calves against HS. Multiphasic emulsions (e.g Montanide®

ISA 201)

reportedly induce long term immunity and protected cattle against hemorrhagic septicemia

for 1 year after only one vaccination (Reddy et al., 1995).

2.8 Prevalence and economic significance of mastitis in Pakistan

Mastitis is one of the most prevalent and economically important diseases of dairy

animals in Pakistan. Table 2.2 depicts animal, clinical, subclinical, quarter, and blind quarter

based prevalence of mastitis in some selected studies in Pakistan.

Table 2.2: Reported prevalence of mastitis in cows and buffaloes in some selected

studies conducted in Pakistan

Prevalence

(%)

No. of animals

investigated

Species

(cows/buffaloes)

Locale of

study

Reference

33.334

200 Buffalo Hyderabad Soomro et al. (1997)

77.981,58.75

3 300 Buffalo Attock Bachaya et al. (2005)

274,4

2,10

5

364,5.5

4, 8

5 50

50

Buffalo

crossbred cows

Faisalabad Khan and Muhammad

(2005)

291,7

2,32

3

411,12

2,24.75

3 100

100

local cows

crossbred cows

D. I. Khan Akhtar et al. (2012)

36.384,24.60

2

33.674,18.21

2 382

291

Buffalo

Cow

Tehsil

Burewala

Hameed et al. (2012)

1= Animal based; 2= Clinical signs based; 3=Quarter based; 4=Subclinical based; 5=Blind

quarters based.

21

Hussain et al. (2005) reported the findings of a farmers‟ participatory surveillance

survey of livestock diseases in Islamabad capital territory. They used the technique of

proportional piling to estimate the relative prevalence of livestock diseases in the area. In the

execution of the study, 100 beans (or pebbles in some cases) were dispensed to the farmers

and they were asked to make piles according the relative incidence of 5 most prevalent

diseases. The results indicated that HS, FMD and mastitis were the 3 most common disease

conditions observed by the participating farmers. Gender based stratification of the disease

incidence data revealed that 15.8 and 16.3% of the male and female livestock keepers,

respectively in Islamabad capital territory had noticed occurrence of mastitis in their cows

and buffaloes.

A recently reported study conducted under Pakistan Strategy Support Program of

USAID by Ashfaq et al. (2014 ) estimated and evaluated on the basis of prevalence of main

livestock diseases that effect on diary animal productivity and farm incomes in five tehsils of

district Faisalabad. Three villages were selected from each tehsil and ten livestock farmers

from each village picked randomly to collect information. Farmers categories into three on

the basis of adult cows and buffaloes owned were as follows: small (1-3 animals), medium

(4-6 animals), and large farmers (more than 6 animals). The study focused on the negative

impact on milk production and farm incomes due to mastitis, parturient hemoglobinuria,

FMD, and tick infestation. The morbidity or mortality rate, incidence rate, and case fatality

rates of each disease were determined. The economic losses related with these diseases were

appraised. In addition, economic returns associated with controlling these 4 diseases were

calculated in the form of benefit-cost ratios. Overall, 9.01% of total economic losses

associated with these 4 diseases were caused by mastitis. Small, medium and large farmers,

respectively sustained 14.09, 8.34 and 9.59% of the total economic losses caused by 4

diseases selected for survey information. Riaz et al. (1992) conducted an active surveillance

from September 1989 to August 1990 in district Gujrat, Pakistan. A total of 1025 farmers

were interviewed. Mastitis incidence of 4.04% in cattle and 5.48% in buffaloes was recorded.

A meta-analysis of investigations conducted over a one year period (1994-1995) on

economically important diseases of livestock in Punjab (Pakistan) ranked mastitis at the 5th

slot, accounting for 8% of the total pecuniary losses inflicted by all livestock diseases in this

providence (Khan et al., 1994-1995).

22

2.9 Role of Staphylococcus aureus and Streptococcus agalactiae in mastitis

In countries lacking application of mastitis control programs, contagious organisms

(S. aureus and Str. agalactiae) are the two most predominant mastitis pathogens (Pankaj et

al., 2013). Collectively, these two pathogens are responsible for nearly 75% cases of this

disease in cows and buffaloes (Allore, 1993). Pakistani worker (Shakoor, 2006) while

reviewing the reported prevalence of pathogens associated with mastitis in Pakistani studies

(n=16, conducted between 1966-2002) concluded that S. aureus was the most prevalent

mastitis pathogen in buffaloes and cows followed by Str. agalactiae , E. coli, and mixed

infections.

2.10 Mastitis vaccines

High prevalence, poor response to antibiotics, development of antibiotic resistance

and consumers concern about residues of antibiotics/antibacterials in milk and meat of the

treated animals have spurred interest in the control of mastitis through vaccination

(Tollersrud, 2001). The idea of use of mastitis vaccine for control and treatment is not new.

According to Wooldridge, (1923), vaccines of various kinds e.g. stock vaccines containing a

mixture of streptococci, staphylococci, and Bacterium pyogenes, were in wide use in 1920s

to prevent or treat bovine mastitis. This author also stated that in herds in which there has

been a series of clinical cases, favorable results have been reported from the use of so called

stall-specific vaccines (autogenous vaccines).The production of autogenous mastitis vaccines

entails isolation of the organism (s) responsible for the outbreak from several cows, its mass

cultivation followed by treatment of the whole of the milking herd with this vaccine made

therewith. In order to prevent outbreaks, it was recommended that doses of dead vaccine

should be given during the dry period. In the subsequent period, investigations by French

(Richou and Thienlin., 1955), and English workers Derbyshire et al. (1961), demonstrated

that use of staphylococcal mastitis vaccines had a definite increase in immunity against

certain strains of staphylococci as reflected by a decrease in the spread of this infection

coupled by a clear reduction of the number of acute cases of mastitis. An equal effect against

all strains of staphylococci was, however, not noticed. Notwithstanding this limited success,

as emphasized by Derbyshire et al. 1961) further research into mastitis vaccinology is clearly

warranted. (Heidrich and Renk, 1967). During the past 3 decades, a variety of mastitis

23

vaccines were investigated and a few of these are now commercially available (Ahmad,

2009; Pereira et al., 2011; Rashid, 2011). Monovalent and polyvalent mastitis vaccines

developed and evaluated at the Mastitis Research Lab. Department of Clinical Medicine and

Surgery, University of Agriculture, Faisalabad, Pakistan were found effective in mastitis

control and treatment (Shakoor, 2006; Athar, 2007; Yousaf et al., 2009; Ahmad, 2009;

Rashid, 2011). Recently, Leitner et al. (2011) reported the efficacy and safety of a

recombinant Montanide® ISA 206 S. aureus mastitis vaccine in dairy cows. This vaccine

was based on Target of RNAIII Activating Protein (TRAP), which is a membrane associated

167AA protein constitutively expressed in all strains of S. aureus and coagulase negative

staphylococci. This recombinant TRAP (rTRAP) S. aureus mastitis vaccine was safe,

immunogenic and elicited a humoral immune response that remained high for at least 160

days post booster shot.

2.10.1 Montanide® (Seppic, France) adjuvanted mastitis vaccines

Montanide® ISA are fairly a new generation of ready-to-use adjuvants which allow

the manufacture of different type of emulsions: water in oil (W/O), oil in water (O/W), or

water in oil in water (W/O/W). They can be based on mineral oil, non-mineral oil or their

mixture. Adjuvants designed for veterinary and human vaccines were reviewed by

Aucouturier et al. (2001). According to these authors, W/O/W (e.g. Montanide® ISA 201

VG, a multiphasic emulsions) can induce long and short term immune response. Low

viscosity (thus an easy syringibility) is another desirable attribute of this class of Montanide®.

After vaccination, the antigen in the external water phase is immediately available to the

immune system like aqueous formulations while antigen entrapped in the internal aqueous

phase is slowly released like W/O emulsions. Water in oil in water (W/O/W) emulsions elicit

superior immune response as compared with aluminium hydroxide in cattle. They are well

tolerated and induce a strong stimulation of cellular immune response. According to

Aucouturier et al. (2001) screening studies in mice with viral, bacterial or parasitic antigens

indicated that W/O/W emulsions induce higher IgG2a antibody levels than other type of

emulsions. IgG2 is the most desirable antibody for protection against S. aureus mastitis

(Watson, 1975) because neutrophils have receptors for this antibody. Tollersrud et al. (2002)

reported that S. aureus capsular polysaccharide type expressed on the surface of

formaldehyde inactivated whole cells emulsified in the Montanide® gave a strong and long

24

lasting immune response. Tollersrud, (2001) reported that Montanide® ISA-70 enhances a

stronger immune response to α, β-toxins than carbopol vaccine tested in sheep. Yousaf et al.

(2009) evaluated the effect of a Montanide®

ISA 206 adjuvanted S. aureus bacterin-toxoid on

the prevalence and incidence of mastitis in cows. A total of 60 California Mastitis Test and

surf field mastitis test negative cows in their first and second month of lactation were

selected and assigned randomly to 3 groups (C1, C2, and C3). Montanide® ISA-206

adjuvanted S. aureus bacterin –toxoid (in 5 ml doses) was injected I.M twice with 4 weeks

spacing to animals of group C1 and C2. Levamisole (2.5mg/kg body weight) was

administered per os to cows of group C2 after first and second shots of vaccine. Cows in

group C3 served as control (non -vaccinated and non- medicated with levamisole).

Prevalence and incidence rates of mastitis were determined. The results showed that

maximum number of quarters were found positive in group C3 with maximum cumulative

prevalence (27.5%) while minimum cumulative prevalence (11.25%) was noted in group C2

followed by group C1 (13.75 %). There was no significant variation in cumulative incidence

between group C1 and C2. Montanide® ISA-25 and ISA-206 (Seppic, France) as adjuvants in

cattle and pigs are much better than conventional aqueous formulations to retain potency and

to enhance the antibody response (Barnett et al., 1996). No toxicity (even after booster dose)

by the use of these vaccines can be seen (Castrucci et al., 1993). It is reported that

Montanide® adjuvanted vaccine is less viscous, less irritant and easily injected than

conventional Freund‟s adjuvanted vaccine (Cook et al., 1990).The Montanide® adjuvanted

vaccine elicited superior and rapid immune response at any time period which was

maintained for longer time ( Patil et al., 2002). Ahmad and Muhammad, (2008) demonstrated

that a locally prepared Staphylococcus aureus and Streptococcus agalactiae aluminium

hydroxide adjuvanted mastitis vaccine was immunogenic when tested in rabbits.

2.11 Need for a combined HS–Mastitis vaccine (‘combo’ or ‘cocktail’ HS –mastitis

vaccine)

Combined vaccines („Combo vaccines‟) are vaccines containing combination of two

or more vaccinal antigens from different pathogens blended in one vaccine vial/bottle. These

vaccines usually target more than one disease. The use of combination vaccines is a

pragmatic strategy to overcome the constraints and problems associated with multiple

25

injections of individual vaccines. Combined vaccines containing bivalent, trivalent as well as

polyvalent microorganisms (bacterial and /or viral) including P. multocida have been

developed to reduce the number of vaccine doses and reduce the cost of production as well

as to expand the spectrum of protective immunity against multiple infections (Verma and

Jaiswal,1998; De Alwis,1999). Combined vaccines targeting HS, black quarter, FMD,

infectious bovine rhinotracheitis, adeno virus type-3 parainfluenza and some other

respiratory pathogens have been developed and tested.(Srivastava et al., 1975; Osman et al.,

1990).

The use of combined vaccines lessens the cost for extra health care visits, facilitates the

addition of new vaccines and diseases into immunization programs (CDC, 2011). Geering

and Lubroth, (2002) opined that during visits by FMD vaccination teams to livestock farming

communities, it may be desirable to combine FMD vaccination with vaccination against

other diseases (e.g. HS, contagious bovine pleuropneumonia, sheep and goat pox). Combined

vaccination not only conserves resources but is also helpful in achieving a higher level of

farmer cooperation in the FMD control program. According to Hussain et al. (2005), HS and

mastitis are among the 5 most important diseases that affect the local animals in Pakistan. An

immunization program based on the use of combined HS- mastitis vaccine may potentially

be associated with the following advantages:

Less frequent handling of the candidate vaccinate cows and buffalos

Inclusion of mastitis in the vaccination program. By and large a „do nothing‟ policy is in

place in Pakistan as far as mastitis control is concerned.

Less cost of vaccination

Efficacy of a bivalent vaccine viz. FMD and HS was compared to individual FMD

and HS vaccines in rabbits by Altaf et al. (2012). The investigators concluded that this

combined vaccine induced higher antibody titers against both FMD and HS than the

individual FMD (prepared from O serotype) and HS vaccine (alum precipitated). Srinivasan

et al. (2001) compared the serological response of cattle to a combined FMD, rabies, HS,

black quarter vaccine to individual component vaccines containing FMD, rabies, P.

multocida, and Clostridium chauvoei antigens. A statistically non- significant variation in the

serological response was elicited by individual component vaccines and combined vaccine

26

containing antigens of all four disease pathogens. As far as could be ascertained from the

available literature, a combined HS-mastitis vaccine has not been evaluated thus far.

According to Chakrabarti, (2002), a combined H.S-black quarter vaccine is available in

India. It is a combination of inactivated antigen of P. multocida type-I and Clostridium

chauvoei. This is stabilized and adjuvanted with BAIF aluminium hydroxide gel aluminox.

The manufacturer claims that this vaccine protects animals against both hemorrhagic

septicemia and black quarter.

In Pakistan, because of extremely small herd size (more than 80% animals kept in

groups of 3-4 animals/family; Jost, 1980; Tuefel, 1998), widely rampant poverty and

illiteracy, lack of any milk quality premium program adoption of the standard mastitis control

practices i.e. post milking antiseptic teat dipping and dry period antibiotic therapy as

advocated by the National Mastitis Council Inc, USA (Nickerson, 1994) is conceivably

difficult. Even on well organized private dairy farms and those in public sector, mastitis

control is not in place. Against this backdrop, vaccination against the common mastitis

pathogens holds the promise of an adjunct/alternative mastitis control strategy (Koiranen,

1977).

2.12 Mastitis in heifers and justification of use of combined HS-mastitis vaccine

Future of a dairy herd hinges to a considerable extent on the milk producing potential

of replacement animals i.e. heifers. Investment in general management and health of the

heifers is reflected in good herd replacements. Managmental and health deficiencies during

the period intervening between calf birth and its first calving can drastically decrease the

milk yield of heifer as she enters the lactating herd. On many farms, calves suffer from the

vice of suckling each other‟s navel and developing udder .The occurrence of subclinical

intramammary infections (IMI) in non-lactating heifers was once considered rare but in

recent years, studies have indicated that the prevalence of IMI in heifers can be as high as

97% in some herds (Trinidad et al., 1990b). These infections are important because

infections acquired during the prepartum period may damage developing secretary tissue,

increase the SCC (Trinidad et al., 1990a; Trinidad et al., 1990b; Nickerson et al., 1995;

Oliver et al., 2003) and reduce lifetime milk production (Oliver et al., 2003, De Vliegher,

2004). Therefore, the control of IMIs in heifers is receiving increased attention.

Staphylococci (primarily coagulase-negative Staphylococci but also Staphylococcus aureus)

27

are the most frequent isolates recovered from heifers but environmental pathogens such as

Streptococci are also frequently recovered (De Vliegher, 2004). Use of a combined HS–

mastitis vaccine incorporating antigens of P. multocida, S. aureus and Str. agalactiae in

heifers may not only potentially reduce the incidence of HS but also reduce the incidence and

prevalence of S. aureus and Str. agalactiae associated mastitis.

Suboptimal immune response to bovine vaccines is an important problem and several

interventions can be used to overcome this problem. Arthington and Havenga, (2011)

demonstrated that an injectable trace minerals supplement (MultiMi®, Fort Collins,

Colorado, USA) containing 15, 40, 10 and 5 mg/ml of CU, Zn, Mn ( all as disodium EDTA

salts),and Se ( as Na selenite) when administered concurrently with a bovine viral vaccine

(Titanium® 5; AgriLabs, St. Joseph, Missouri, USA) containing bovine herpes virus

(BHV1),bovine viral diarrhea virus (BVDV1), BVDV-2, bovine parainfluenza virus type

3,and bovine respiratory syncytial virus did not impair humoral immune responses in beef

calves. In addition, concurrent administration of injectable trace minerals supplement and

BHV-1 vaccine enhanced the production of neutralizing antibodies to BHV-1 in previously

naïve beef calves. No such investigation seems to have been conducted on hemorrhagic

septicemia vaccine.

28

CHAPTER 3

MATERIALS AND METHODS

The present study is the first-ever attempt towards development and evaluation of a

Montanide® adjuvanted combined hemorrhagic septicemia -mastitis vaccine in dairy

buffaloes and cattle. It was executed keeping in view the animal welfare concepts of 3R‟s.

The objectives of the study were pursued by compartmentalizing the entire work into

following 2 phases

Phase I: Preparation and evaluation of Montanide® adjuvanted combined hemorrhagic

septicemia-mastitis vaccine in laboratory settings

Phase II: Field evaluation of Montanide® adjuvanted combined HS-Mastitis vaccine in

target dairy species

Phase I: Preparation and Evaluation of Montanide® Adjuvanted

Combined Hemorrhagic Septicemia-Mastitis Vaccine in

Laboratory Settings

3.1 Collection and culture of blood samples for isolation of P. multocida

3.1.1 Sources and types of samples for isolation of P. multocida

Peripheral blood samples of 5 buffalo calves and 2 adult buffaloes suffering from HS

were collected aseptically and processed for the isolation of P. multocida as per OIE, 2012.

Briefly, in each case, 0.2 ml of collected blood sample was eluted into 2 ml sterile normal

saline and an aliquot was cultured on casein-sucrose-yeast (CSY) agar plate containing 5%

calf blood and blood agar plates within 3 hours of collection. The inoculated plates were

incubated at 370C for 48 hours. The composition and method of preparation of casein-

sucrose-yeast (CSY) agar is given in the Appendix- I.

Isolation of P. multocida was also attempted from the femur of 4 buffaloes that died

of HS during the past 48-72 hours. To this end, the femur of the carcass was exposed with the

help of a necropsy knife, followed by swabbing with methyl alcohol, split opening of the

bone and aseptic aspiration of approximately 2 grams of the bone marrow. The bone marrow

sample was admixed in 10 ml of sterile normal saline in a sterile caped test tube. The tube

containing bone marrow–normal saline admixture was vigorously shaken for 2 minutes.

29

Finally, 0.1 ml aliquots of bone marrow-normal saline admixture were cultured on casein-

sucrose-yeast (CSY) agar plates containing 5% calf blood (OIE, 2012; Ali et al., 2000) and

blood agar plates.

In addition to peripheral blood samples and femur marrow samples, isolation of P.

multocida was also attempted from the heart of 4 additional buffalo calves immediately after

their slaughtering prompted by their moribund condition with overt clinical HS (De Alwis,

1992). Following slaughter, the heart was removed, washed with clean water, placed in a

sterile tray, and swabbed thoroughly with 70% alcohol. A sterile 18 gauge needle attached to

a disposable sterile 20 ml syringe was pierced into the left ventricle to withdraw about 5 ml

of blood that was mixed with 5 ml of sterile normal saline. Heart blood- sterile normal saline

mixture was dispensed into 10 ml of enrichment transport medium (BHI broth) for

transportation to the Mastitis Research Lab. Department of Clinical Medicine & Surgery,

University of Agriculture, Faisalabad. Immediately after arrival at this Lab., 0.1 ml aliquots

of heart blood –normal saline –transport medium were cultured on casein-sucrose-yeast

(CSY) agar plates containing 5% calf blood and blood agar plates. In addition to aspirated

heart blood samples, swab samples of heart blood were collected by drenching sterile swab

(IMPROSWAB™, Microbiological Transport Swab, IMPROVE®, Guangzhou Improve

Medical Instruments Co., Ltd.) with heart blood (OIE, 2012). The swabs were cultured on

casein-sucrose-yeast (CSY) agar plates and blood agar plates.

3.1.2 Isolation and biochemical and molecular confirmation of P. multocida

The following features were considered for identification of P. multocida. It forms

smooth grayish glistening translucent colonies of nearly 1 mm diameter on blood agar after

24 hours incubation at 37°C. It did not grow on MacConkey‟s agar. On Gram staining, the

organism appeared as Gram –ve, short, ovoid bipolar staining coccoid with pleomorphism

(OIE, 2012). Further confirmation of the organism and type (i.e., group B) was done by

biochemical reactions (Butt et al., 2003; Ashraf et al., 2011; Ekundayo et al.,2008) and by

using PCR primers (Townsend et al., 1998; OIE, 2012).

30

3.1.3 Synthetic oligonucleotide primers

Table 3.1: The oligonucleotide primer pair used for PCR based confirmation of

Pasteurella multocida (Townsend et al., 1998; OIE, 2012)

PCR amplification using the aforementioned primers was carried out as described by

Townsend et al. (1998). Primers (KTT72 and KTS P61) were got synthesized from Gene

Link, USA. These primers specifically produce products of approximately 620 bp from HS-

causing type B isolates of P. multocida.

3.1.4 PCR Reaction

Suspect colonies were sub-cultured and subjected to biochemical testing (Butt et al.,

2003; Ashraf et al., 2011). After confirmation, the colony was streaked onto sheep blood

agar plate. Following overnight incubation at 37 °C, a single colony was picked to run the

PCR reaction. PCR was carried out in a 50µl reaction volume using Techne Touchgene

Gradient PCR Thermal Cycler (Bibby Scientific Limited, UK). Single bacterial colony was

added to 48µl of PCR reaction mixture consisting of: 35µl DD-water; 5µl, 10XPCR buffer;

5µl MgCl2 (25mM), 0.2µl TaqDNA polymerase (5U/µl), 1µl deoxynucleotide triphosphate

(dNTP) mix (10mM each), 1µl Forward primer (25 p moles/µl), 1 µl Reverse primer (25 p

moles/µl). Cycling profile used for PCR reaction was as under: The program used for

diagnosis of P. multocida with primer pair KTT72/ KTS P61: 94 °C for 5 min (1 cycle); 95

°C for 1 min., 55 °C for 1 min, 72 °C for 1 min (30 cycles); 72 °C for10 min (1 cycle). PCR

reaction product were subjected to gel electrophoresis (1.5 agarose gel, 85 volts for 20

minutes), stained by Ethidium bromide and visualized by gel doc system (Bio-Rad, USA).

3.1.5 Pathogenicity (virulence) of 3 candidate vaccinal isolates of P. multocida

LD50 (virulence) of candidate vaccinal P. multocida isolates (n= 3) was determined in

six weeks old Swiss albino mice following the procedure described by Butt et al. (2003). One

Primer Sequence Product Size Primer Specificity

KTT72 5‟-GGCTCGTTTGGATTAGTAAG-3‟

618 bp

HS-causing type-B-specific

KTS P61 5‟-ATCCGCTAACACACTCTC-3‟

31

tenth of an ml (i.e. 0.1 ml) of each tenfold dilution (10-1

to 10-10

in sterile normal saline) of 18

hours old cultures grown in 1% nutrient broth (Merck™

, Darmstadt, Germany) was

inoculated intraperitoneally in mice using 10 mice for each dilution. A set of another 10

mice, for each dilution was kept as control and injected with sterile normal saline. Mortality

and time of death associated with each dilution was recorded to calculate LD50. Blood smears

prepared from the cardiac blood of dead mice were stained with Giemsa‟s stain. In addition,

the cardiac blood samples of dead mice were cultured on blood agar and CYS agar plates for

reisolation of P. multocida (Naz et al., 2012).

3.2 Sources of milk samples, isolation of candidate vaccinal S. aureus

and Str. agalactiae isolates and their partial characterization

Milk samples from 37 quarters of cows (n=10) and buffaloes (n=9) managed at the

Livestock Experimental Station, University of Agriculture, Faisalabad, Ayub Agriculture

Research Institute, Faisalabad, Raja Asghar dairy farm, Mehar Anayat dairy farm, Raja wala

and Bilal Rashid dairy farm, and suffering from peracute/acute clinical mastitis (Radostitset

al., 2007; Schalm et al., 1971) were sampled aseptically following the procedures described

by Hogan et al. (1999). Immediately after collection, milk samples were placed on crushed

ice in a thermos chest (Coleman®; the Coleman Company Ltd, USA.) and transported to the

Mastitis Research Lab. Department of Clinical Medicine and Surgery, University of

Agriculture, Faisalabad. On arrival at this lab, using a platinum-rhodium inoculation loop,

0.01 ml (i.e. one hundredth of a milliliter) of each milk sample was inoculated onto blood

agar (Oxoid Ltd., Basingstoke, Hampshire, England) plate containing 5% defibrinated ovine

blood and MacConkey‟s agar plate (Hogan et al., 1999; National Mastitis Council, Inc. USA,

1987; Claxton and Ryan, 1993). Four milk samples were cultured on each agar plate of 100

mm diameter. After inoculation of milk samples, blood agar and MacConkey‟s agar plates

were incubated aerobically at 37ºC for 48 hours, followed by examination of the growth (if

present) by Gram staining, catalase reaction and other relevant tests (Appendix II and

Appendix III). β hemolytic, Gram positive, catalase positive coccal isolates that grew on

blood agar plates were presumptively identified as staphylococci and subjected to tube

coagulase test. (Hogan et al., 1999; National Mastitis Council, Inc. USA, 1987). Briefly, a

loop-full of growth of each isolate presumptively identified as a Staphylococcus was

32

inoculated into 0.5 ml of rabbit plasma diluted 1:1 with sterile normal saline (Appendix IV)

and incubated at 37oC for 24 hours. Staphylococcal isolates with positive tube coagulase test

at 4 hours were provisionally identified as S. aureus, whereas staphylococcal isolates

negative in tube coagulase test till 24 hours were categorized as coagulase negative

staphylococci (National Mastitis Council, Inc. USA, 1987; National Mastitis Council, Inc.

USA, 1990). Four selected S. aureus isolates that displayed wide hemolytic zones on blood

agar plates (candidate vaccinal isolates) were biotyped using a commercially available

Staphylococcus kit (API® STAPH; bioMerieux, France). Latex Slide Agglutination test was

performed using Staphytect Plus® kit (Oxoid Ltd, Basingstoke, UK) for the determination of

clumping factor, protein A and certain polysaccharides found only in S. aureus (Rashid,

2011; Raza, 2012).

Gram positive, catalase-negative, esculin-negative, CAMP- positive (Appendix VI),

and β-hemolytic cocci (i.e. clear hemolysis) were considered as isolates of Streptococcus

agalactiae (Altwegg and Bockemühl, 1998). Blood agar with 0.1% esculine and 0.01%

ferric citrate was used for testing the isolates for CAMP reaction (National Mastitis Council,

Inc. USA, 1990). Three isolates of Streptococcus agalactiae were biotyped using a

commercially available kit of genus Streptococcus (API® 20-STREP kit bioMerieux,

France). Isolates other than those of Staphylococcus and Streptococcus that grew on blood

agar plates were not further processed.

3.2.1 Molecular confirmation of S. aureus

3.2.1.1 Detection of nuc gene by polymerase chain reaction (PCR)

The vaccinal isolate of S. aureus was confirmed by PCR by targeting ‘nuc’ a

chromosomal gene encoding extracellular thermo-stable nuclease activity (a specific target

for S. aureus; Brakstad et al., 1992), using a commercially available Multiplex PCR kit

(BactReadyTM

Multiplex PCR system; Genescript, USA). Sequence of forward and reverse

primer used and size of PCR product have been mentioned in Table 3.2. All primers were

purchased from GeneLinkTM

(USA) through a local vendor (The Worldwide Scientific,

Lahore, Pakistan).

33

Table 3.2: Sequence of nuc gene primers used for confirmation of S. aureus and PCR

product size

Targeted

genes

Sequence Product

Size

Reference

nuc forward

nuc reverse

Nuc 5‟- GCG ATT GAT GGT GAT ACG GTT-3‟

Nuc 5‟-AGC CAA GCC TTG ACG AAC TAA AGC-3

279bp Brakstad et al.

(1992)

3.2.1.2 Preparation of genomic DNA from S. aureus

For PCR, genomic DNA was prepared using extraction buffer (BR-A buffer)

available with BactReady Multiplex PCR system (Genescript, USA). Briefly, single, isolated,

18-hour old colony from blood agar plate was picked and suspended in 25 µl of DNase free

water and 1µl of cell suspension was mixed with 20 µl of BR-A/extraction buffer. The lysate

was spun down by short centrifugation (2000 rpm for 1 minute) and the supernatant

containing gDNA was used directly for amplification.

3.2.1.3 PCR amplification

The reaction was performed using BactReadyTM

Multiplex PCR system following the

manufacturer instructions. The reaction mixture (20 μl) was prepared in thin walled with flat

cape, DNase-RNase free 0.2 ml thermo-tubes (Thermo-scientific, UK) with 1 μl of template

DNA. Amplifications were performed using a micro-processed controlled SwiftTM Maxi

Thermal Cycler Block (ESCO Technologies Inc. France) under the following conditions:

activation of ScriptTM DNA polymerase at 94 ºC for 15 minutes, followed by 35 cycles of

denaturation (95ºC for 1 min), annealing (55ºC for 1 min), extension (72ºC for 1 min), and a

final extension step at 72ºC for 3 minutes. The composition of reaction mixture is given in

the Table 3.3. The amplicons were analyzed by agrose gel electrophoresis using a horizontal

mini agarose gel electrophoresis system (ENDUROTM Labnet International Inc.,

Woodbridge, New Jersey, USA). A mixture of undiluted PCR products (5 μl) and 5X loading

dye (1 μl; Fermentas Thermo Fischer Scientific Inc., UK) was loaded to 1.2% agrose gel

(multipurpose agarose, low EEO, multipurpose, Fischer Scientific Ltd, Loughborough, UK)

containing ethidium bromide (0.5 μg./ml; Fischer Scientific Ltd, Loughborough, UK) for

DNA staining. The gels were run in 1X TAE buffer (50X TAE Buffer, Fischer Scientific Ltd,

Loughborough, UK) at 70 volts (80mA) for 1 hour. The amplicons were visualized on a

34

trans-illuminator (Vilbert Lourmart, Cedex France) and saved using gel doc system (Bio-

Rad, USA). The size of the products was measured using a ready-to- use 100 bp molecular

marker (O gene-Ruler 100bp DNA ladder, Fermentas, Thermo-scientific, UK).

Table 3.3: Composition of PCR reaction mixture used for amplification of nuc (S.

aureus specific) gene

Reagents Volume (μl) Final concentration

PCR grade (DNase free) water 7

Forward primer 1 200nM

Reverse primer 1 200nM

DNA solution 1

PCR Premix 10

Total volume 20

3.3 Detection of biofilm production trait of S. aureus

3.3.1 Qualitative determination by tube method (TM)

Tube method as described by Christensen et al. (1982) and Mathur et al. (2006) was

followed for qualitative determination of biofilm. The isolates were grown in tubes

containing Tryptone Soya Broth (TSB; Oxoid, UK) with 1% glucose (TSBglu). After

inoculation, tubes were incubated at 37 oC for 24 hours, decanted carefully, washed with

phosphate buffered saline (PBS, pH 7.3), dried and stained with safranin for 8 minutes.

Excess stain was removed by washing twice with de-ionized water. The production of visible

film at walls and bottom of the tubes after staining with safranin was interpreted as positive

result for biofilm production. The ring formation along the walls of the tubes was interpreted

as a negative result for biofilm production trait. The intensity of biofilm production was

subjectively assessed on a score of 0 to 4, depending upon the retention of safranin color at

the walls and bottom of the tubes after washing with deionized water.

3.3.2 Qualitative determination by Congo red agar method (CRA)

Biofilm production trait of 4 selected candidate vaccinal S. aureus isolates (Table 3.4)

was also determined by the Congo red agar method (Freeman et al., 1989; Mathur et al.,

2006). Fresh growth of each test isolate on blood agar plate was inoculated onto 4 separate

35

Congo red agar (CRA) plates and incubated for 24 hours at 37oC. Interpretation of biofilm

production by this method was as follows (Mathur et al., 2006).

Positive result: Appearance of black colored colonies having dry crystalline

consistency.

Weak biofilm production: Pink colonies with no dry crystalline consistency (although

occasionally darkening at the center).

Intermediate biofilm production: Darkening of the colonies with the absence of a dry

crystalline colonial morphology.

Table. 3.4: Composition and preparation of Congo red agar medium (Mathur et al.,

2006)

Ingredients Quantity (g/l)

Brain heart infusion broth (BHI, Oxoid, UK) 37 g

Congo red stain (BDH Chemical Ltd, Poole, England) 0.8 g

Sucrose 50 g

Agar technical (Oxoid, UK) 10 g

Congo red stain was dissolved in distilled water, followed by sterilization at 121oC

for 15 minutes. Mixture of brain heart infusion broth (BHI), sucrose and agar technical was

sterilized in a separate container at 1210C for 15 minutes and cooled to approximately 55

0C

before mixing it with autoclaved Congo red stain. The final mixture was then quickly

dispensed into sterilized Petri plates of 100 mm diameter.

3.3.3 Quantitative determination of biofilm production by spectrophotometric method

Quantitative evaluation of biofilm production trait of 4 candidate vaccinal

staphylococcal isolates was performed spectrophotometerically on flat bottom 96 well

ELISA plate (Greiner® Germany) by the method of Mathur et al. (2006) modified by Rashid

(2011). Each well of the plate was filled with 5 μl culture broth (i.e. Tryptone Soya Broth

with 1% glucose inoculated with the test isolate) and 95 μl of fresh Tryptone Soya Broth

(TSB; Oxoid, UK) with 1% glucose (TSBglu). As negative controls, 3 wells were filled with

100 μl of TSBglu and 2 wells with 100 μl of de-ionized water to check the nonspecific

binding of the culture medium. Plates were incubated for 18 hours at 37oC. Following

incubation, the contents of the plates were discarded by sharply striking the inverted ELISA

36

plate against the top of a disposal tray. Using a multichannel micro-dispenser, each well was

washed 4 times with 0.3 ml of phosphate buffered saline (PBS; pH 7.2). Biofilms formed by

adherent „sessile‟ organisms in plates were fixed with sodium acetate (2%) and stained for 10

minutes with crystal violet (0.1% w/v). Excess stain was removed by thorough washing with

de-ionized water and plates were left for drying. It was noticed that adherent staphylococcal

cells formed biofilm on all sides of wells and were stained uniformly with crystal violet.

Optical density (OD) of wells of ELISA plate was determined by ELISA auto reader (BioTek

Instruments, Highland Park, USA) at 570 nm (OD 570 nm) wavelength. These OD values

were considered as an index of adherence of bacteria to the surface and reflected the intensity

of biofilm production. Experiment was performed in duplicate and repeated two times. The

average was calculated both from cultured wells and negative control wells. In order to

recompense for background absorbance, OD readings from sterile culture medium (Tryptone

Soya Broth), fixative (sodium acetate 2%) and dye (crystal violet 0.1% w/v) were averaged

and subtracted from all individual test values. The mean OD value obtained from media

control well was subtracted from all the test OD values (Mathur et al., 2006; Rashid, 2011).

3.3.4 Pathogenicity of 4 candidate vaccinal isolates of S. aureus

Pathogenicity of candidate vaccinal isolates of S. aureus (n= 4) was determined in

rabbits by subcutaneous inoculation of 0.2 ml of bacterial suspensions (1x1010

cells / ml)

grown in nutrient broth. Suspension of each isolate was inoculated separately in 5 rabbits.

The morbidity/mortality was observed for up to 48 hours post inoculation (Shakoor et al.,

2006; Ahmad, 2009; Hirsch and Strauss, 1964).

3.3.5 Pathogenicity of 2 candidate vaccinal isolates of Str. agalactiae

Pathogenicity of candidate vaccinal isolates of Str. agalactiae (n= 2) was determined in

rabbits by subcutaneous inoculation of 0.2 ml of bacterial suspensions (1x109

cells / ml)

grown in nutrient broth. Suspension of each isolate was inoculated separately in 5 rabbits.

The morbidity/mortality was observed for up to 48 hours post inoculation (Shakoor et al.,

2006; Ahmad, 2009; Hirsch and Strauss, 1964). Dead rabbits were subjected to necropsy

examination.

37

3.4 Immunogenicity testing of vaccinal isolates of P. multocida, S. aureus

and Str. agalactiae

Immunogenic response of vaccinal isolates was determined in rabbits. To this end, 30

adult male rabbits were randomly divided into 3 equal groups (RaA, RaB and RaC) of 10

animals each. Rabbits in each of the 3 groups were respectively inoculated with 0.2 ml

formalin killed suspensions (1×106 per ml) of P. multocida (n=3), S. aureus (n=4) and Str.

agalactiae (n=2) subcutaneously twice at an interval of 7 days (Athar, 2007). Serum

antibody titers were determined by in-house indirect ELISA. Briefly, each of the vaccinal

isolates was grown on nutrient-whey broth, followed by inactivation with formalin (0.1%).

The growths were washed, sonicated separately (Sajjad-ur-Rehman et al., 2004) and the

supernatants were estimated for total protein contents by using Bradford reagent. ELISA

plates were coated with bicarbonate buffer (Sigma, USA) containing 15 µg protein

(antigen)/ml and blocked with bovine serum albumin (0.1%). Antibodies were detected by

protein G-HRP conjugate (LSI Vet, France). TMB (3, 3‟, 5, 5‟-Tetramethylbenzidine) was

finally used as substrate to detect the conjugation and reaction was stopped with sulphuric

acid (1M H2SO4). Plates were read at 450 nm wave length for optical density.

3.5 Antimicrobial susceptibility testing of candidate bacterial isolates

(Antibiogram)

The susceptibility of 9 bacterial isolates (P. multocida, n= 3; S. aureus, n=4 and Str.

agalactiae, n=2) to 11 different antibiotics was tested by disc diffusion method on Mueller –

Hinton medium (Oxoid Ltd., Basingstoke, Hampshire, England), according to the guidelines

of Clinical Laboratory Standards Institute (CLSI, 2012). For Streptococci, Mueller –Hinton

medium was enriched with 5% ovine blood. The plates were incubated at 37°C for 24 hours

and inhibition zones were measured with the antibacterial zone gauge. Staphylococcus

aureus ATCC (American Type Culture Collections- Rockville, Maryland USA) 25923 was

used as a sensitive quality control.

3.6 Selection of vaccinal P. multocida isolate

From a pool of three candidate vaccinal P. multocida isolates, the isolate No. 1 with a

strong pathogenicity (LD50=10-7.75

) and immunogenicity was selected as a vaccinal isolate for

the production of Montanide® adjuvanted combined HS-Mastitis vaccine.

38

3.7 Selection of vaccinal S. aureus isolate

The selection was based primarily on the level of biofilm production, pathogenicity

and immunogenicity of the candidate isolates. A beta hemolytic S. aureus isolate number 1

with the strongest biofilm production ability in tube method and in spectrophotometric

method was selected as a vaccinal isolate for preparation of montanide® adjuvanted

combined hemorrhagic septicemia-mastitis vaccine (Table 3.5). The selected isolate showed

highest values in tube method and spectrophotometric method but intermediate in CRA. The

selection was dependent on tube method and spectrophotometric methods because CRA was

an additional test. This isolate was strongly pathogenic and immunogenic.

Table 3.5: Selection of vaccinal isolate by biofilm detection methods and other traits

Isolate I.D. Spectrophoto

metric OD

Tube

method

score

Biofilm

production

degree on

Congo red

agar (CRA)

Pattern of

hemolysis

on blood

agar

Pathog

enicity

Immuno

genicity

1* 0.31 4 Strong Beta 100 1.23

2 0.19 2 Intermediate Beta 80 1.11



* Isolate was selected on the basis of tube method score, spectrophotometric method score

and pattern and intensity of hemolysis. The current designation of this isolate is Mastitis

Research Lab is 2 as it originated from an acutely mastitic cross bred cow managed at the

Livestock Experimental Station, University of Agriculture, Faisalabad.

3.8 Selection of vaccinal Str. agalactiae isolate

From a pool of three candidate vaccinal Str. agalactiae isolates, the isolate (SA-100)

with strong pathogenicity and immunogenicity and displaying API® 20 STREP biochemical

profile of 3462414 was selected as a vaccinal isolate for the production of Montanide®

adjuvanted combined HS-Mastitis vaccine. This isolate was recovered from a cow suffering

from periodic flare ups of clinical mastitis. The cow belonged to Livestock Experimental

Station, University of Agriculture, Faisalabad.

39

3.9 Preparation of Montanide® adjuvanted combined HS-Mastitis

vaccine

3.9.1 Preparation of antigen of P. multocida

Procedure adopted by Nuclear Institute of Agriculture and Biology (NIAB)

Faisalabad (OIE, 2012) for production of NIAB HS vaccine was used. A single colony of P.

multocida was picked from nutrient agar plate, inoculated into 50 ml of CSY broth and

incubated for 24 hours at 37 °C. One liter of CSY broth was inoculated with 12.5 ml of P.

multocida pure culture, and incubated at 37°C for 36 hours with constant agitation. The

bacterial sediment (from one liter broth) was harvested by centrifugation at 7000 rpm for 20

minutes. The bacterial sediment was dissolved in 50 ml normal saline and the bacterial

suspension inactivated by addition of 0.4 % of 37 % formaldehyde. The inactivated bacterial

suspension was placed at 37 °C for 24 hours. The sterility of this suspension was checked by

inoculation onto blood agar, nutrient agar, MacConkey‟s agar and citrate agar plates at 37°C.

The inactivated bacterial sediment was harvested by centrifugation and supernatant

discarded. Dilution factor was determined to attain a transmittance of 20% with the help of

spectrophotometer at 540 nm. Normal saline was added to whole bacterial sediment to attain

20% transmittance. The resulting final solution was designated as prepared antigen and was

used for vaccine preparation.

3.9.2 Preparation of antigens of S. aureus and Str. agalactiae

The selected vaccinal isolates of S. aureus and Str. agalactiae were grown

independently in modified nutrient broth (nutrient broth supplemented with 10% sterile

bubaline whey) at 37 ºC for 48 hours on an orbital shaker set at 60 rpm under aerobic

conditions. The suspensions were confirmed for culture purity by Gram‟s staining technique

and inoculation onto blood agar and MacConkey‟s agar plates. Pure cultures of S. aureus and

Str. agalactiae in broth were inactivated by addition of formalin (0.4% V/V) and then

centrifuged at 6000xg for 1 hour at 4°C and resuspended in PBS (pH 7.2). Bacterial

concentrations of each of the vaccinal organisms were adjusted to 1× 109

cells/ml

spectrophotometrically (Hirsch and Strauss, 1964). For incorporation of crude extract of S.

aureus and Str. agalactiae, supernatant fluid collected from 48-hours old broth cultures of

respective isolate were autoclaved at 121 °C for 20 minutes. The autoclaved supernatant was

added to the vaccine preparation at the concentration of 5 mg of dry weight per dose (Athar

40

et al., 2007).

3.9.3 Preparation of combined HS-Mastitis vaccine

Montanide® adjuvanted bacterin-toxoid was prepared by adding Montanide® ISA

201 VG (Seppic, France) in 50/50 (W/W) ratio to a suspension of plain bacterins of S.

aureus, Str. agalactiae, P. multocida and crude extracts of S. aureus and Str. agalactiae. The

beaker containing oil phase was fitted to a homogenizer and stirred at 200 rpm. The antigens

in aqueous phase were then added slowly over a period of 30 seconds. Stirring was increased

to 2000 rpm for 10 minutes. The 5 ml of Montanide® adjuvanted bacterin- toxoid finally

contained 5x 109

cells each of S. aureus, Str. agalactiae and P. multocida, toxin extracts of S.

aureus, Str. agalactiae, formalin, thimerosal, sodium azide and Montanide® ISA 201 VG to

make a total volume of 5 ml (Athar, 2007; Yousaf et al., 2009; Giraudo et al., 1997).

3.10 Evaluation of quality of vaccine

3.10.1 Sterility

The prepared vaccine was cultured on blood agar and MacConkey‟s agar plates for

checking its sterility. The inoculated plates were incubated at 37 °C for 48 hours and

observed for any growth.

3.10.2 Safety and side effects

Four cows and buffaloes were vaccinated with double the recommended dose of

vaccine (10 ml) and monitored for 14 days for local and systemic reactions if any. In

addition, 5 mice were inoculated intramuscularly with 0.2 ml of the vaccine and observed for

5 days for occurrence of any untoward local and systemic reaction (OIE, 2012).

3.10.3 Challenge- protection assay (Vaccination- challenge study)

Fifty mice divided randomly into 5 groups (M-1 thru M-5) were immunized

intraperitoneally with 0.1 ml doses of the vaccine administered at 15 day interval. The mice

were then challenged with inocula 30 days after administration of the second dose of vaccine.

The procedure for preparation and administration of inocula is as under:

One ml inoculum containing 109 CFU ml

-1 of each of the vaccinal isolates (S. aureus, Str.

agalactiae and P. multocida) grown in modified nutrient broth for 24 hours at 37 oC,

centrifuged at 6,000×g for 30 minutes at 4°C, and resuspended in peptone solution (0.5% w/v

of peptone and 0.5% NaCl), was inoculated intraperitoneally in vaccinated and control mice.

41

The numbers of dead mice within 3 days of the challenge were recorded and compared to

calculate the percent survival rate (Giraudo et al., 1997; Athar, 2007)

3.10.4 Stability

The physical stability of vaccine was evaluated by storing vaccine vials at 4 °C and

7° C for 6 months and parameters (pH, sedimentation, color, appearance, syringibility) were

recorded at monthly intervals (OIE, 2012; Athar, 2007)

Phase II: Field Evaluation of Montanide® Adjuvanted Combined HS-

Mastitis Vaccine in Target Dairy Species

3.11 Animals, their management and treatment-based grouping

A total of 70 mastitis free lactating animals (buffaloes n=45; cows n=25), 50 lactating

cows and buffaloes microbiologically positive for S. aureus/Str. agalactiae (buffaloes n=25,

cow n=25) and dairy calves aged up to 1 year (buffalo calves n=70; cow calves n=50) were

utilized for the field evaluation of combined HS-mastitis vaccine. The animals were selected

from those maintained at government institutes (Livestock Experimental Station, University

of Agriculture, Faisalabad, Ayub Agriculture Research Institute, Faisalabad, etc.,) and two

private dairy farms (Raja dairy farm, Samundri road, Faisalabad and Mehar Anayat dairy

farm, Raja wala, Faisalabad). Non vaccination status against HS during the last 6 months and

low or no P. multocida antibody titers (ELISA- based) were another criterion for selecting

animals for field trial. The grouping of these animals, their experimental treatments, and

HS/mastitis status at the time of initiation of trial is depicted in Table 3.6. General principles

for the design of clinical trials with special reference to mastitis and HS described by

Thorburn (1990) and OIE (2012) were followed.

42

Table 3.6: Grouping of animals, their experimental treatments, and HS/mastitis status

at the time of initiation of field trial of combined HS- mastitis vaccine

Groups Experimental

treatment

Dairy species and

number (n) of

animals

HS-mastitis status at the time of

initiation of trial

A Vaccination twice

at 21 day interval

Buffalo (n=30)

Cow (n=10)

Milk cultu re –ve for S. aureus and

Str agalactiae, CMT/ SFMT –ve;

no history of vaccination against

HS during the last six months, no

or low antibody titers against P.

multocida

B (control) No vaccination Buffalo (n=15)

Cow (n=15)

Milk culture –ve for S. aureus and

Str. agalactiae, CMT/ SFMT –ve;

no history of vaccination against

HS during the last six months, no

or low antibody titers against P.

multocida

C (Sub

clinically

mastitic

buffaloes and

cows)

Vaccination twice

at 21 days

interval

Buffalo (n=15)

Cow (n=15)

Milk culture +ve for S. aureus/Str.

agalactiae, CMT/ SFMT +ve in

one or more quarter but no clinical

signs of mastitis

D (Sub

clinically

mastitic

buffaloes and

cows)

Infected

control

No vaccination Buffalo (n=10)

Cow (n=10)

Milk culture +ve for S. aureus/Str.

agalactiae, CMT/ SFMT +ve in

one or more quarter but no clinical

signs of mastitis

E

Vaccination twice

at 21 days

interval

Buffalo calves aged

up to 1 year (n=50)

Cow calves aged up

to 1 year (n=25)

No history of vaccination against

HS during the last six months

together with no or very low

antibody titer against P. multocida

F (Control)

No

vaccination

Buffalo calves aged

up to 1 year (n=20)

Cow calves aged up

to 1 year (n=25)

No history of vaccination against

HS during the last six months

together with no or very low

antibody titer against P. multocida

IMI=Intrammamry infection; SFMT=Surf field mastitis test; CMT=California mastitis test

43

3.12 Evaluation parameters

3.12.1 Hemorrhagic septicemia

3.12.1.1 ELISA- based antibody titers against P. multocida

ELISA-based antibody titers against P. multocida were determined from 20 % of

randomly selected vaccinated and control animals at day 0, 60, 120, and 180 post

vaccination. Serum antibody titers were determined by in-house indirect ELISA, as described

in the preceding section 3.4.

3.12.1.2 Incidence of HS

Incidence of HS in vaccinated and control animals over a period of 180 days if any was

recorded. The diagnosis of HS was based on typical signs (De Alwis, 1992).

3.12.1.3 Local and systemic reactions

Local and systemic reactions in vaccinated animals (OIE, 2012) were monitored.

3.13 Mastitis

3.13.1 ELISA-based antibody titers against S. aureus and Str. agalactiae

ELISA-based antibody titers against S. aureus, and Str. agalactiae were determined

from at least 20 % of randomly selected vaccinated and control animals at day 0, 60, 120, and

180 post vaccination (Qureshi and Saxena, 2014). Serum antibody titers were determined by

in-house indirect ELISA, as described in the preceding section 3.4.

3.13.2 Incidence and prevalence of S. aureus and Str. agalactiae mastitis

Incidence and prevalence of S. aureus and Str. agalactiae mastitis over a period of

180 days were determined by microbiological examination of aseptically collected quarter

foremilk samples as described by Hogan et al. (I999).

3.13.3 Mastitis screening tests based prevalence and incidence of mastitis

Surf Field Mastitis test (Muhammad et al., 2010) and California Mastitis Test

(Schalm et al., 1971) based prevalence and incidence of mastitis was determined at day 60,

120, and 180 post vaccination.

3.13.4 Severity of mastitis in vaccinated and control animals

Severity of clinical mastitis in vaccinated and control groups was determined as per

Athar (2007).

44

3.13.5 Isolation and identification of pathogens from clinically mastitic animals

Clinically mastitic quarters of vaccinated and unvaccinated animals were subjected to

microbiological examination of aseptically collected milk samples to determine the nature of

mastitis causing pathogens (Hogan et al., I999).

3.13.6 Milk somatic cell count

Somatic cell count was monitored in vaccinated and control groups at day 60, 120,

and 180 post vaccination (Singh and Ludri, 2000; Shailja and Singh, 2002; Athar et al., 2007

and Rashid, 2011).

3.13.7 Effect of vaccination on milk yield

Mean milk yields (liters per 24 hours) were recorded on monthly basis for six months post

vaccination. Similar observations were made in unvaccinated groups (Nickerson, 1994)

3.13.8 Vaccine efficacy

Proportionate reduction in S. aureus, Str. agalactiae mastitis and HS attack rates

(AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts were calculated

from the relative risk (RR) of these diseases among the vaccinated group using the following

formulas (Weinberg and Szilagyi, 2010).

ARU - ARV

Efficacy = -------------------------- × 100

ARU

Efficacy= (1-RR) × 100

3.14 Statistical Analysis

The data collected were subjected to statistical analysis using ANOVA and other

appropriate design software package (Anonymous, 2004). Mammary quarters were

considered as unit of concern for prevalence, incidence and somatic cell count. For serum

and milk whey antibody titers buffaloes and cows were the unit of concern. Significance was

declared as P≤0.05 (Steel et al., 1997). Cost benefit analysis was performed by partial

budgeting technique/spread sheet cost- benefit analysis (Miller, 1986).

45

CHAPTER 4

RESULTS

PHASE-I

4.1 Pasteurella multocida isolates recovered from peripheral blood,

femur, and heart blood of HS affected animals

Microbiological examination of peripheral blood, femur, and heart blood of HS

affected animals on CSY and blood agar plates yielded growth of only three isolates of P.

multocida. No P. multocida isolate was recovered from peripheral blood and swab samples

of heart blood. Culturing of heart blood and femur bone marrow samples respectively yielded

growth of two and one isolate of P. multocida (Table 4.1).

Table 4.1: Pasteurella multocida isolates recovered from peripheral blood, femur, and

heart blood of HS affected animals

Nature of samples and animal species No. of samples

cultured on CYS*

and blood agar

plates

No. of P. multocida

isolates that grew on

CYS*/blood agar plates

Peripheral blood of HS affected buffalo

calves

5 -

Peripheral blood of HS affected adult

buffaloes

2 -

Femur bone marrow samples collected

from adult buffaloes that had died of HS

during the post 48-72 hours

4 1

Heart blood samples collected from

buffaloes calves killed at the terminal

stage of HS

4 2

Heart blood swab samples collected

from buffalo calves killed at the terminal

stage of HS

4 -

*Casein-sucrose-yeast agar

4.2 Cultural and biochemical characteristics of suspected isolates of P.

multocida Suspected isolates of P. multocida formed smooth grayish, glistening translucent

colonies of nearly 1 mm diameter on blood agar after 24 hours incubation at 37°C (Plate 1).

No growth was recorded on MacConkey‟s agar on subculturing. On Gram staining, the

46

organism appeared as Gram –ve, short, ovoid bipolar coccoid, with some degree of

pleomorphism. Growth on CYS agar was smooth, yellowish, mucoid and larger in size than

that on blood agar (Plate 2).

Plate 1. Growth of a P. multocida Plate 2. Growth of a P.multocida on a

Casein- on a blood agar plate sucrose-yeast agar plate

4.2.1 Biochemical reactions of P. multocida isolates

All three P. multocida isolates were positive for indol, ODC, glucose, fructose, and

catalase, negative for H2S production, lactose, D-dulcitol and maltose. Variable results were

recorded for D-sorbitol and arabinose (Table 4.2).

Table 4.2: Biochemical profiles of P. multocida isolates (n=3) recovered from femur

bone marrow and heart blood samples

Biochemical test No. of P. multocida isolates showing + ve or –ve

reaction

Indol All three isolates showed a +ve reaction

ODC* All three isolates showed a +ve reaction

H2S on TSI All three isolates showed a -ve reaction

D-sorbitol Two isolates showed +ve reaction and one isolate

showed -ve reaction

D-dulcitol All three isolates showed a -ve reaction

Arabinose

One isolate showed +ve reaction and two isolates

showed-ve reaction

Glucose All three isolates showed a +ve reaction

Fructose All three isolates showed a +ve reaction

Lactose All three isolates showed a -ve reaction

Catalase All three isolates showed a +ve reaction

Maltose All three isolates showed a -ve reaction *ODC = Ornithine decarboxylase activity.

47

4.2.2 PCR based confirmation of P. multocida isolates

These isolates of P. multocida were confirmed by amplification of P. multocida

specific gene using KTSP61/KTT7 set of primers. These primers yielded a product of 618

bp on electrophoresis which confirmed these isolates as P. multocida type B.

4.2.3 Antimicrobial susceptibility testing of 3 candidate vaccinal P. multocida isolates

All 3 isolates of P. multocida were sensitive to enrofloxacin, amoxicillin, ceftiofur

sodium and oxacillin and resistant to tetracycline, fusidic acid, and penicillin. All three

isolates displayed an intermediate sensitivity to vancomycine. A variable degree of

susceptibility was displayed to lincomycine, septran and gentamicin (Table 4.3).

Table 4.3: Antibiotic susceptibility profiles of 3 candidate vaccinal isolates of P.

multocida against 11 antibiotics/antimicrobials

Designation of P.

multocida isolate

Antibiotics /antimicrobials and their susceptibility profiles

Linco

Enro

Amox

Tetra

Vanco

Sept

Gen

Fus.acid

Pen

Ceft

Ox

P. multocida

isolate No.1

R S S R I R S R R S S

P. multocida

isolate No. 2

S S S R I I I R R S S

P. multocida

isolate No. 3

S S S R I I S R R S S

Linco= Lincomycin; Enro= Enrofloxacin; Amox= Amoxicillin; Tetra= Tetracycline; Van=

Vancomycine; Sept= Septran (Sulfamethoxazole+Trimethoprim; Gen=Gentamicin;

Fus.acid= Fusidic acid; Pen=Penicillin G; Ceft= Ceftiofur sodium; Ox=Oxacillin; S=

Sensitive; I= Intermediate; R= Resistant

4.2.4 Pathogenicity of the 3 candidate vaccinal isolates of P. multocida in mice

Table 4.4 depicts the mouse LD50 of 3 candidate vaccinal isolates of P. multocida. As

can be seen in this table, isolate No.1 (vaccinal isolate) was more virulent than isolates no.2

and 3, the latter two isolates were equal in terms of virulence in mouse. Necropsy

examination of all mouse dying in 24 hours revealed clear septicemic lesions consisting of

necrotic foci on the dorsal surface of liver and hemorrhages in lungs, trachea and spleen.

Giemsa‟s stained smears prepared from the cardiac blood of dead mice revealed the presence

of cocco bacilli. Culturing of heart blood samples on blood agar and CYS agar revealed the

presence of colonies of P. multocida with characteristics as described in section 4.2 above.

48

Table 4.4: Pathogenicity of the 3 candidate vaccinal isolates of P. multocida in mice

Designation of P. multocida isolate LD50

P. multocida isolate No.1* 10-7.75

P. multocida isolate No. 2 10-7.50

P. multocida isolate No.3 10-7.50

*vaccinal isolate

4.3 Staphylococcus aureus isolates recovered from peracute/acute

clinically mastitic quarters (n=37) of dairy cattle and buffaloes

Microbiological examination of 37 clinically mastitic (peracute/acute) quarters of

cows (n=10) and buffaloes (n=9) on blood agar and MacConkey‟s agar plates yielded growth

of 10 suspect S. aureus isolates. These isolates grew only on blood agar and there was no

growth on MacConkey‟s agar plates. Four of these isolates showed wide β-hemolysis and

were subjected to further characterization in terms of biotyping, biofilm determination, Latex

agglutination, antibiogram, PCR, pathogenicity and immunogenicity.

4.3.1 Confirmation and characterization of 4 candidate vaccinal S. aureus isolates

Staphylococcus aureus isolates (n=4) that grew on blood agar were, β- hemolytic,

catalase positive, Gram positive cocci and were thus presumptively identified as members of

the genus Staphylococcus. On performing tube coagulase test, all of these 4 isolates tested

positive for coagulase production at 4 hours. Seven-digit numerical profiles of these 4

isolates with API® STAPH (bioMerieux, France) test strips are given in Table 4.5.

Table 4.5: Seven-digit numerical biotype (biochemical) profiles of 4 candidate vaccinal

S. aureus isolates determined by using API® STAPH

S. No. Candidate vaccinal S.

aureus isolate No.

Seven-digit

numerical

profile

Identification level

1 1* 6736153 Good identification (S. aureus id= 90.3%)

2 2 6736053 Low discrimination

3 3 6336151 Good identification

4 4 6716152 Low discrimination

* Vaccinal isolate

49

Plate 3 depicts the biochemical reactions of one of the 4 candidate vaccinal S. aureus isolates

Plate 3. Seven-digit numerical biotype profile of a candidate vaccinal S. aureus isolates

determined by API® STAPH.

Latex agglutination test using Staphytect plus® kit was positive for all of the four candidate

vaccinal S. aureus isolates (Plate 4)

Plate 4. Latex agglutination test positive and negative reactions using Staphytect plus® kit

50

4.3.2 Antimicrobial susceptibility of candidate vaccinal S. aureus isolates

All 4 isolates of S .aureus were sensitive to lincomycin, enrofloxacin, amoxicillin,

ceftiofur sodium and oxacillin and resistant to tetracycline. A variable degree of

susceptibility was displayed to vancomycine, septran, gentamicin, fusidic acid and penicillin,

(Table 4.6).

Table 4.6: Antibiotic susceptibility profiles of 4 candidate vaccinal isolates of S. aureus

against 11 antibiotics/antimicrobials

Designation of

S. aureus isolate


Linco

Enro

Amox

Tetra

Vanco

Sept

Gen

Fus.acid

Pen

Ceft

Ox

S. aureus isolate

No.1*

S S S R I I I R R S S

S. aureus isolate

No.2

S S S R S S S S S S S

S. aureus isolate

No.3

S S S R I I S R R S S

S. aureus

isolate No.4 S S S R S S S S S S S




Sensitive; I= Intermediate; R= Resistant, *vaccinal isolate

4.3.3 Pathogenicity of the 4 candidate vaccinal isolates of S. aureus in rabbits

Table 4.7 depicts the results of pathogenicity testing of 4 candidate vaccinal isolates

of S. aureus in rabbits. As can be seen in this table, 100, 80, 80 and 60% rabbits died within

18 hours of inoculation of isolates No. 1, 2, 3 and 4, respectively. Necropsy examination of

dead rabbits revealed septicemia along with congested lungs and heart and petechiation on

serosal surface of intestine, with straw colored fluid in abdominal cavity. Stained smears of

blood and straw colored fluid revealed S. aureus under microscope. Each isolate was also

isolated from straw colored fluid in the abdominal cavity.

51

Table 4.7: Pathogenicity of the 4 candidate vaccinal isolates of S. aureus in rabbits

Designation of S.

aureus isolate

Percent mortality of rabbits within 18 hours of inoculation of 0.2 ml

of bacterial suspension containing 1x1010

cells / ml of respective

isolate.

S. aureus isolate No.1* 100

S. aureus isolate No.2 80



*vaccinal isolate

4.3.4 Biofilm production by candidate vaccinal S. aureus isolates (n=4)

4.3.4.1 Tube method

Table 4.8 depicts tube method-based scores of biofilm production by 4 candidate

vaccinal S. aureus isolates. As can be seen in this table, S. aureus isolate number 2, 3 and, 4

showed a moderate level of biofilm production whereas isolate number 1 showed strong

biofilm production.

Table 4.8: Tube method-based scores of biofilm production by candidate vaccinal S.

aureus isolates (n=4)

S. No. Candidate vaccinal S. aureus isolate No. Biofilm production score*

1 1** 4 (strong )

2 2 2 (moderate)

3 3 2 (moderate)

4 4 2 (moderate)

**Vaccinal isolate *As per Christensen et al. (1982).

4.3.4.2 Spectrophotometric method

Table 4.9 depicts the result of spectrophotometric based estimates of biofilm

production traits of 4 candidate vaccinal isolates of S. aureus. All 4 isolates produced

biofilms, however highest OD (0.31nm) was noted in isolate # 1, whilst OD values ranged

between 0.17- 0.22. Thus the isolate #1 was considered as highest producer of biofilm in

spectrophotometric method.

52

Table 4.9: Spectrophotometric method based optical densities (OD values) for biofilm

production of 4 candidate vaccinal S. aureus isolates

S. No. Candidate vaccinal S. aureus isolate No. OD values*

1 1** 0.31

2 2 0.19

3 3 0.17

4 4 0.22

*As per Mathur et al. (2006) **vaccinal isolate. The ODs of control wells (distal water)

were between 0.01-0.03

4.3.4.3 Congo red agar method

Table 4.10 depicts degrees of biofilm production trait of 4 candidate vaccinal S. aureus

isolates on Congo red agar (CRA). As can be seen in this table, S. aureus isolate number 2, 3,

and 4 showed intermediate degree of biofilm production whereas isolate number 1 showed

strong degree of biofilm production on Congo red agar.

Table 4.10: Degrees of biofilm production trait of 4 candidate vaccinal S. aureus

isolates on Congo red agar (CRA)

S. No. Candidate vaccinal S. aureus isolate No. Biofilm production degree on

Congo red agar (CRA)*

1 1** Strong

2 2 Intermediate

3 3 Intermediate

4 4 Intermediate

**Vaccinal isolate * As per Freeman et al. (1989) and Mathur et al. (2006)

4.3.4.4 PCR based confirmation of S. aureus isolates

All 4 candidate vaccinal isolates were confirmed as S. aureus as they displayed

extracellular thermostable nuclease (nuc) activity in PCR by yielding a 279 bp product.

53

4.4 Streptococcus agalactiae isolates recovered from peracute/acute

clinical mastitis from dairy cattle and buffaloes.

Microbiological examination of 37 clinically mastitic (peracute/acute) quarters of cows

(n=10) and buffaloes (n=9) on blood agar yielded growth of 2 suspect Str. agalactiae

isolates.

4.4.1 Confirmation and characterization of candidate vaccinal Str. agalactiae isolate

On blood agar, suspect Str. agalactiae isolates (n=2) displayed β-hemolysis. Microscopic

examination of Gram stained smears of these isolates revealed Gram positive cocci. On

catalase test, these isolates were negative. CAMP test reaction on blood agar with 0.1%

esculine and 0.01% ferric citrate was positive for both the isolates.

Plate 5: Growth of a Str. agalactiae on a blood agar plate

4.4.2 Seven-digit numerical biotype (biochemical) characteristics of candidate vaccinal

isolates

Biotypes 2 candidate vaccinal Str. agalactiae isolates in terms of seven-digit

numerical profiles determined by using a commercially available kit API® 20 STREP,

bioMérieux, France) have been depicted in Table 4.11.

54

Table 4.11: Seven-digit numerical (biochemical) profiles of 2 candidate vaccinal Str.

agalactiae isolates determined by API® 20 STREP (bioMerieux, France)

S.

No.

Candidate vaccinal Str.

agalactiae isolate No.

Seven-digit

numerical profile

Identification level

1 1* 3462414 Very Good identification

(Str. agalactiae % id= 99.9)

2 2 3462415 Very Good identification

(Str. agalactiae % id= 99.9)

* Vaccinal isolate

Plate 6: Seven-digit numerical biotype profile of a candidate vaccinal Str.

agalactiae isolates determined by API® 20 STREP

4.4.3 Antimicrobial susceptibility of candidate vaccinal Str. agalactiae isolates

All 2 isolates of Str. agalactiae were sensitive to lincomycin, enrofloxacin,

amoxicillin, vancomycine, septran, gentamicin, fusidic acid, Penicillin, ceftiofur sodium, and

oxacillin. A variable degree of susceptibility was displayed to, tetracycline (Table 4.12).

55

Table 4.12: Antibiotic susceptibility profiles of 2 candidate vaccinal isolates of Str.

agalactiae against 11 antibiotics/antimicrobials

Designation

of Str.

agalactiae

isolate


Linco

Enro

Amox

Tetra

Vanco

Sept

Gen

Fus.acid

Pen

Ceft

Ox

Str.

agalactiae

isolate No.1

S S S S S S S S S S S

Str. agalactiae

isolate No. 2 S S S R S S S S S S S




Sensitive; I= Intermediate; R= Resistant.

4.4.4 Pathogenicity of the 2 candidate vaccinal isolates of Str. agalactiae in rabbits

Results of pathogenicity testing of 2 candidate vaccinal isolates of Str. agalactiae in

rabbits have been presented in Table 4.13. As can be seen in this table, 80, and 60 % rabbits

died within 18 hours of inoculation of isolate no 1, and 2, respectively. Necropsy

examination of dead rabbits revealed septicemia along with congested lungs, kidneys and

heart and petechiation on serosal surface of intestine and straw colored fluid in abdominal

cavity. Stained smears of straw colored abdominal fluid revealed Str. agalactiae under

microscope. Each isolate was also isolated from straw colored fluid in the abdominal cavity.

Table 4.13: Pathogenicity of the 2 candidate vaccinal isolates of Str. agalactiae in rabbits

Designation of

Str. agalactiae

isolate

Percent mortality of rabbits within 18 hours of inoculation of 0.2 ml of

bacterial suspension containing 1x1010

cells / ml of respective isolate.

Str. agalactiae

isolate No.1*

80

Str. agalactiae

isolate No.2

60

*vaccinal isolate

4.4.5 Immunogenicity of the 3 candidate vaccinal isolates of P. multocida in rabbits

Indirect ELISA-based serum antibody titers in rabbits to 3 candidate vaccinal isolates

of P. multocida, have been presented in table 4.14. As is evident from this table, among these

three isolates, P. multocida isolate No.1 elicited antibody titers higher than those induced by

56

isolate No.2 and 3 on all the three post inoculation sampling days (i.e.

day 7, 14, and 21).

Table 4.14: Indirect ELISA-based serum antibody titers in rabbits to 3 candidate

vaccinal isolates of P. multocida

Designation of P. multocida isolate D-0 D-7 D-14 D-21

P. multocida isolate No.1* 0.26 0.96 1.56 1.9

P. multocida isolate No.2 0.20 0.8 1.2 1.4

P. multocida isolate No. 3 0.1 0.86 1.3 1.8

*vaccinal isolate

4.4.6 Immunogenicity of the 4 candidate vaccinal isolates of S. aureus in rabbits

Table 4.15 depicts indirect ELISA-based serum antibody titers in rabbits to 4

candidate vaccinal isolates of S. aureus. As is evident from this table, amonge these three

isolates, S. aureus No.1 elicited antibody titers higher than those induced by isolate No.2, 3

and 4 on all the three post inoculation sampling days (i.e. day 7, 14 and 21).


vaccinal isolates of S. aureus

Designation of S. aureus isolate D-0 D-7 D-14

D-21

S. aureus isolate No.1* 0.19 0.6 1.3 1.23

S. aureus isolate No.2 0.18 0.5 1.2 1.11



*vaccinal isolate

4.4.7 Immunogenicity of the 2 candidate vaccinal isolates of Str. agalactiae in rabbits

Indirect ELISA-based serum antibody titers in rabbits to 2 candidate vaccinal isolates

of Str. agalactiae, have been presented in table 4.16. As is evident from this table, among

these three isolates, Str. agalactiae isolate No.1 elicited antibody titers higher than those

induced by isolate No.2 on all the three post inoculation sampling days (i.e.

day 7, 14, and 21).

57


vaccinal isolates of Str. agalactiae

Designation of Str. agalactiae isolate D-0 D-7 D-14 D-21

Str. agalactiae isolate No.1* 0.113 0.878 1.13 1.12

Str. agalactiae isolate No.2 0.19 0.6 1.3 1.1

*vaccinal isolate

4.5 Final selection of vaccinal isolates from amongst the candidate

vaccinal isolates of P. multocida, S. aureus and Str. agalactiae for

vaccine production

The most pathogenic (table 4.4) and most immunogenic (table 4.14) P. multocida was

selected for vaccine production. From amongst the 4 candidate S. aureus isolates, the

strongest biofilm producing (table 4.8) the most pathogenic (table 4.7) and most

immunogenic (table 4.15) S. aureus isolate was selected for vaccine production. Similarly,

the more pathogenic (table 4.13) and more immunogenic (table 4.16) of the 2 candidate Str.

agalactiae isolates was selected for vaccine production.

Table 4.17: Composition of Montanide® adjuvanted combine HS-mastitis vaccine.

Ingredients Each 5m ml dose contained

P. multocida 5 × 109 cells

Staphylococcus aureus ( 6736153) 5 × 109 cells

Streptococcus agalactiae (3462414) 5 × 109

cells

Crude toxin extract 1.5 ml

Montanide® ISA 201 VG 2.5 ml

Formalin <0.02 ml

Thimeosal 50 µg=0.00005 g

Sodium azide 50 µg=0.00005 g

PBS Q. S.

58

Plate 7: Montanide® adjuvanted combined hemorrhagic septicemia-mastitis vaccine

Table 4.18: Indirect ELISA-based serum antibody titers in rabbits to vaccinal isolates of P.

multocida, S. aureus and Str.agalactiae

Vaccinal Isolates D-0 D-7 D-14 D-21

P. multocida isolate No.1* 0.26 0.96 1.56 1.9

S. aureus isolate No.1* 0.19 0.6 1.3 1.23

Str. agalactiae isolate No.1* 0.113 0.878 1.13 1.12

* Vaccinal isolates

59

Fig. 4.1 Indirect ELISA-based serum antibody titers in rabbits to vaccinal isolates

4.6 Evaluation of quality of vaccine

4.6.1 Sterility

The vaccine prepared was cultured on blood agar and MacConkey‟s agar plates

showed not any growth in 48 hours ,which indicated the sterility of the vaccine under trial.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

D-0 D-7 D-14 D-21

Seru

m a

nti

bo

dy

tite

rs in

rab

bit

s

Days

S. aureus

Str.agalctiae

P.multocida

60

4.6.2 Stability

4.6.2.1 Effect of temperature and duration of storage on the physical stability of

Montanide® adjuvanted combined hemorrhagic septicemia-mastitis

vaccine.

The physical stability of vaccine were evaluated by storing vaccine vials at 4°C and

37°C for 6 months and parameters (pH, sedimentation, color, appearance, syringibility) were

recorded at monthly intervals.

Table 4.19: Effect of temperature and duration of storage on the physical stability of

Montanide® adjuvanted combined hemorrhagic septicemia-mastitis

vaccine

Temperature Days in Storage

7 90 180

4°C Good Good Satisfactory



Good = Milky white suspension without any sedimentation; Satisfactory = Milky with a

small portion of water phase temporarily separated which upon shaking readily becomes

suspension

4.6.3 Safety and side effects

4.6.3.1 Comparative safety of Montanide® adjuvanted combined hemorrhagic

septicemia-mastitis vaccine in cattle and buffaloes.

Subcutaneously administration of vaccine was associated with a varying degree of

mild to moderate swelling at injection site in cows and buffaloes that subsided within 3-7

days while intramuscular administration caused either no or a very mild swelling at the

injection site that subsided within 48 hours. No systemic effects were noted.

61

Table 4.20: Comparative safety of Montanide® adjuvanted combined hemorrhagic

septicemia-mastitis vaccine in cattle and buffaloes

Groups

of cows

and

buffaloes

Vaccine Number

of

animals

Dose and

Route

Mortality Severity of

swelling at

injection

site

Cows

Montanide®

Adjuvanted Combined

Hemorrhagic

Septicemia-Mastitis

Vaccine

4

10 ml SC Nil Moderate

10 ml IM Nil Mild

Buffaloes

Montanide®

Adjuvanted Combined

Hemorrhagic

Septicemia-Mastitis

Vaccine

4 10 ml SC Nil Mild

10 ml IM Nil Nil

Control Placebo 4 No

treatment

Nil Nil

Nil Nil

SC=Subcutaneously; IM=intramuscularly; Mild: swelling at the injection site, which subside

within 48 hours; Moderate: swelling at the injection site, which subside within 3-7 days

Table 4.21: Comparative safety of Montanide® adjuvanted combined hemorrhagic

septicemia-mastitis vaccine in mice

Groups

of mice

Vaccine dose and route of

inoculation

Mice no. Mortality Morbidity

/patterns

M1 Montanide® Adjuvanted Combined

Hemorrhagic Septicemia-Mastitis

Vaccine

1 Nil Nil

2 Lethargy

3 Nil

4 Nil

5 Nil

M2 Placebo(control) 1 Nil Nil

2 Nil

3 Nil

4 Nil

5 Nil

None of the mice died following intramuscularly administration of 0.2 ml of the vaccine.

Only one mice had shown lethargy during first hours of after injection.

62

4.6.4 Challenge- protection assay (Vaccination- challenge study)

4.6.4.1 Vaccination challenge protection assay of Montanide® adjuvanted

combined hemorrhagic septicemia-mastitis vaccine in mice

Fifty mice divided randomly into 5 groups (M-1 thru M-2) were immunized

intraperitoneally with two 0.1 ml doses of the vaccine administered at 15-day interval. The

mice were then be challenged with inocula 30 days after administration of the second dose of

vaccine. The inocula were prepared and administered as under.

One ml containing 109 CFU ml

-1 of each of the vaccinal isolates (S. aureus, Str.

agalactiae and P. multocida) grown in modified nutrient broth for 24 hour at

37oC,centrifuged at 6,000×g for 30 minutes at 4°C, and resuspended in peptone solution

(0.5% w/v of peptone and 0.5% NaCl) were inoculated intraperitoneally in vaccinated and

control mice. The number of dead mice within 3 days of the challenge were recorded and

compared to calculate the per cent survival rate.

Table 4.22: Vaccination challenge protection assay of Montanide® adjuvanted

combined hemorrhagic septicemia-mastitis vaccine in mice

Group of

mice

Vaccine use for

immunization

Vaccine ,

inoculum*dose

and route

No. of mice Safety

(Survival

rate %) Total

number of

mice used

No. of

dead mice

within 3

days

M1

Montanide®

Adjuvanted

Combined

Hemorrhagic

Septicemia-

Mastitis Vaccine

0.1 ml IP twice at

15 days interval;

challenged with 1

ml of live

inoculum IP 30

days of post –

booster

administration

25 0 100

M2 Unvaccinated

control 25 23 20

*109

CFU ml-1

each of vaccinal isolate of P. multocida, S. aureus, and Str. agalactiae

63

None of the mice in group M-1 (vaccination twice before challenging) died within 3

days of the challenging IP(Intra peritoneal) with I ml of live inoculum containing 109

CFU

ml-1

of each of the vaccinal isolates whereas only two mouse survived from the control

group (which also died later on day 4). This translated into green signal for all the

experimental Montanide® Adjuvanted Combined Hemorrhagic Septicemia-Mastitis Vaccine

to be used safely in the future trials whereas death of the 23 mice in the unvaccinated control

group (within three days) proved its lethality.

4.6.5 ELISA- based antibody titers generated by Montanide® adjuvanted combined

HS-mastitis vaccine against P. multocida, S. aureus and Str. agalactiae in cows

and buffaloes

Prevaccination serum IgG antibody titers (optical density values; OD values) against

P. multocida, S. aureus and Strep agalactiae were not significantly different in vaccinated

and unvaccinated groups (Table 4.23). The highest anti-staphylococcal IgG values (OD

2.56) were recorded in non-mastitic group at day 60 that remained significantly higher

throughout the observation period (180 days) than prevaccination titer (day zero).

The result of present study revealed that the administration of Montanide®

adjuvanted combined HS-mastitis vaccine produced higher antibody titers against P.

multocida, S. aureus and Str. agalactiae in vaccinated group (A and C) compared to un-

vaccinated groups( B and D) as shown in table 4.23 and Fig. 4.2. The antibody titers were

higher in non-diseased vaccinated (group A) animals compared to diseased vaccinated

animals ( group C) at 0, 60, 120 and 180 days of post -vaccination as evident in Table 4.23.

It was observed that the antibody titers against P. multocida, S. aureus and Str.

agalactiae augmented at 60 and 120 days of vaccine administration in non-diseased

vaccinated animals (Table 4.23) and decreased at 180 days of post-vaccination (Table 4.23).

Data was statistically designed by 2 factor CRD and comparison of means was done by using

Tukey‟s test at 5% level of significance.

64

Table 4.23: ELISA-based antibody titers generated by Montanide® adjuvanted

combined HS-mastitis vaccine against P. multocida, S. aureus and Str.

agalactiae in cows and buffaloes

Type of

antibody

determination

Sampling

day

Group A Group B Group C Group D

Anti- P.

multocida

0 0.47±0.006b 0.35±0.005d 0.59±0.004a 0.41±0.007c

60 2.55±0.027a 0.31±0.011d 1.63±0.006b 0.36±0.006c

120 2.13±0.013a 0.31±0.015c 0.74±0.005b 0.29±0.006d

180 1.31±0.009a 0.20±0.005d 0.70±0.005b 0.30±0.004c

Anti-S. aureus

0 0.38±0.007b 0.36±0.005c 1.42±0.010a 0.31±0.007d

60 2.56±0.015a 0.41±0.006c 1.73±0.006b 0.39±0.007d

120 2.14±0.011a 0.32±0.007c 0.84±0.007b 0.21±0.006d

180 1.32±0.008a 0.30±0.007c 0.80±0.006b 0.30±0.006c

Anti-Str.

agalactiae

0 0.37±0.007b 0.27±0.005c 0.51±0.004a 0.21±0.006d

60 1.86±0.009a 0.32±0.006b 1.87±0.008a 0.29±0.007c

120 1.88±0.005a 0.49±0.006c 1.40±0.006b 0.19±0.005d

180 0.56±0.005b 0.45±0.005c 0.80±0.005a 0.31±0.006d

Means followed by different letters in a row or in a column are statistically significant

(P˂0.05) with type of antibody titers determinant.

A= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis vaccinated

cows and buffaloes (Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve);

B= un-vaccinated cows and buffaloes (Milk culture –ve for S. aureus and Str. agalactiae,

CMT/ SFMT –ve); C= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis

vaccinated cows and buffaloes (Milk culture +ve for S. aureus and Str. agalactiae, CMT/

SFMT +ve); D= un-vaccinated cows and buffaloes (Milk culture +ve for S. aureus and Str.

agalactiae, CMT/ SFMT +ve)

65

Fig. 4.2: ELISA-based antibody titers generated by Montanide® adjuvanted

combined HS-mastitis vaccine against P. multocida, S. aureus and Str.

agalactiae in cows and buffaloes

A= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis vaccinated

cows and buffaloes (Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve);

B= un-vaccinated cows and buffaloes (Milk culture –ve for S. aureus and Str. agalactiae,

CMT/ SFMT –ve); C= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis




4.7 Incidence of HS

4.7.1 Number of new HS cases in vaccinated and control cows, buffaloes and calves

during 180 days post- vaccination period

Table 4.24 and Fig.4.3 depict the number of new HS cases in vaccinated and control

cows, buffaloes and calves during 180 days post- vaccination period. As it can be seen in this

0 60 120 180 0 60 120 180 0 60 120 180

anti-P.multocida anti-staph anti-strep

OD

450n

m


66

table, none of the 10 vaccinated cows developed HS over the entire observation period of 180

days. Only 1 of the 30 vaccinated buffaloes (3.33 %) developed HS during this period. In the

control group (un-vaccinated), 1 cow (6.66 %) and 2 buffaloes (13.33 %) developed HS

during this period. None of the 25 cow calves vaccinated with Montanide® adjuvanted

combined HS-mastitis vaccine developed HS during the post vaccination observation period

of 6 months. One of the 50 buffalo calves (2 %) vaccinated with this vaccine developed HS

during this period. One of 25 un-vaccinated cow calves (4%) and 3 of 20 buffalo calves

(15%) developed clinical HS over the observation period of 6 months. In as much as very

few animals (vaccinated or un-vaccinated) developed HS during the observation period of 6

months, incidence data was not subjected to any statistical analysis. Pasteurella multocida

could be isolated from only 1 buffalo calf (group F) that succumbed to peracute HS. Isolation

of P. multocida from other cases were not successful.

Table 4.24: Number of new HS cases in vaccinated and control cows, buffaloes and

calves during 180 days post- vaccination period

Groups of buffaloes,cows and calves

Number of new HS cases in vaccinated and

control cows, buffaloes and calves during 180

days post- vaccination period

Day

0-59

Day

60-119

Day

120-180

Cumulative

incidence over 180

days period

A (vaccinated) cows n=10 0 0 0 0

buffaloes n=30 0 0 1 1

B (un-vaccinated) cows n=15 0 0 1 1


C (vaccinated)

cows n=15 0 0 0 0


D (un-vaccinated) cows n=10 0 0 0 0


E ( vaccinated )

cow calves n=25 0 0 0 0

buffalo calves

n=50

0 0 1 1

F( un-vaccinated)

cow calves n=25 0 0 1 1

buffalo calves

n=20

0 1 2 3

67

A= Montanide® adjuvanted combined HS-mastitis vaccinated cows and buffaloes

(Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve; no history of HS

vaccination during the last 180 days); B= un-vaccinated cows and buffaloes (Milk culture –

ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve; no history of HS vaccination during

the last 180 days); C= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis



agalactiae, CMT/ SFMT +ve); E= vaccinated cow and buffalo calves; no history of HS

vaccination during the last 180 days F= un-vaccinated cow and buffalo calves; no history of

HS vaccination during the last 180 days.

Fig. 4.3: Number of new HS cases in vaccinated and control cows, buffaloes and

calves during 180 days post- vaccination period

A= Montanide® adjuvanted combined HS-mastitis vaccinated cows and buffaloes

(Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve; no history of HS

vaccination during the last 180 days); B= un-vaccinated cows and buffaloes (Milk culture –

ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve; no history of HS vaccination during

the last 180 days); E= vaccinated cow and buffalo calves; no history of HS vaccination

0

0.5

1

1.5

2

2.5

3

3.5

A B C D E F

Nu

mb

er o

f n

ew H

S

Day Day Day Cumulative incidence over 180 days period

68

during the last 180 days F= un-vaccinated cow and buffalo calves; no history of HS

vaccination during the last 180 days.

4.8 Incidence and prevalence of S. aureus and Str. agalactiae mastitis

4.8.1 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on quarter-

based incidence of Str. agalactiae and S. aureus IMIs in cows and buffaloes

initially culture negative and CMT/ SFMT negative

Tabel 4.25 and Fig. 4.4 depict the effect of Montanide® adjuvanted combined HS-

mastitis vaccine on quarter-based incidence of Str. agalactiae and S. aureus IMIs in cows

and buffaloes initially culture negative and CMT/ SFMT negative. As can be seen in these

depictions, of the 10 vaccinated cows, only 1 quarter each of two cows developed new Str.

agalactiae and S. aureus intramammary infection (IMI) over the 6 months period. None of

the 30 vaccinated buffaloes developed new IMI with Str. agalactiae whereas 1 quarter

developed new S. aureus IMI. Of the 15 un-vaccinated cows, new IMI with Str. agalactiae

was detected in one quarter whereas 5 quarters of 4 un-vaccinated cows developed new IMI

with S. aureus over a period of 180 days. New IMIs with Str. agalactiae and S. aureus were

detected in 1 and 3 quarters respectively of 15 un-vaccinated buffaloes. Amalgamating the

new Str. agalactiae and S. aureus IMIs in vaccinated cows and buffaloes and comparing the

corresponding figure with that of un-vaccinated cows and buffaloes would indicate that the

quarter–based incidence of these mastitis 2 pathogens over the 6 months observation period

was 3.3 folds higher in un-vaccinated cows and buffaloes.

69

Table 4.25: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on

quarter-based incidence of Str. agalactiae and S. aureus IMIs in cows and

buffaloes initially culture negative and CMT/ SFMT negative

Groups of cows and buffaloes Quarter based cumulative incidence over 180

days post vaccination period

A (vaccinated)

Cows n=10 Str. agalactiae 1

2 S. aureus 1

Buffaloes n=30 Str. agalactiae 0

1 S. aureus 1

B (un-

vaccinated)


6 S. aureus 5


4 S. aureus 3

IMI=Intramammary infection; SFMT=Surf field mastitis test; CMT=California mastitis test

A= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis vaccinated cows and

buffaloes (Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve) B= un-

vaccinated cows and buffaloes (Milk culture –ve for S. aureus and Str. agalactiae, CMT/

SFMT –ve)

Fig 4.4: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on

quarter-based incidence of Str. agalactiae and S. aureus IMIs in cows and

buffaloes initially culture negative and CMT/ SFMT negative



buffaloes (Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve) B= un-


SFMT –ve)

0

1

2

3

4

5

6

Str

. ag

ala

ctiae

S. au

reus

Str

. ag

ala

ctiae

S. au

reus

Str

. ag

ala

ctiae

S. au

reus

Str

. ag

ala

ctiae

S. au

reus

Cows n=10 Buffaloes n=30 Cows n=15 Buffaloes n=15

A (vaccinated) B (un-vaccinated)

Ov

era

ll n

ew

In

fecti

on

s (

incid

en

ce)

70


based incidence of Str. agalactiae and S. aureus IMIs in cows and buffaloes initially

culture positive and CMT/ SFMT positive

Effect of Montanide® adjuvanted combined HS-mastitis vaccine on quarter- based

incidence of Str. agalactiae and S. aureus IMIs in cows and buffaloes initially culture

positive and CMT/ SFMT positive have been depicted in table 4.26 and Fig. 4.5. As is

evident from the data given in this table and figure, the cumulative incidence of Str.

agalactiae plus S. aureus in vaccinated cows and buffaloes (1 new quarter infected by each

of these pathogens) was 6 magnitude lower than the incidence of these pathogens (12 new

quarters infected by these pathogens) over a six month post vaccination period.


quarter- based incidence of Str. agalactiae and S. aureus IMIs in cows and

buffaloes initially culture positive and CMT/ SFMT positive

Groups of cows and buffaloes

Quarter-based cumulative incidence over 180

days post vaccination period

C

(vaccinated-

diseased)


1 S. aureus 1


1 S. aureus 1

D (un-

vaccinated-

diseased)


7 S. aureus 5


5 S. aureus 3


C= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis vaccinated cows and

buffaloes (Milk culture +ve for S. aureus and Str. agalactiae, CMT/ SFMT +ve) D= un-


SFMT +ve)

71

Fig: 4.5: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on

quarter- based incidence of Str. agalactiae and S. aureus IMIs in cows and

buffaloes initially culture positive and CMT/ SFMT positive





SFMT +ve)

0

1

2

3

4

5

6S

tr. ag

ala

ctiae

S. au

reus

Str

. ag

ala

ctiae

S. au

reus

Str

. ag

ala

ctiae

S. au

reus

Str

. ag

ala

ctiae

S. au

reus


C (vaccinated- diseased) D (un-vaccinated-diseased)

Ov

era

ll n

ew

In

fecti

on

s (

incid

en

ce)

72


based time points prevalence of Str. agalactiae and S. aureus IMIs in cows and

buffaloes initially culture positive and CMT/SFMT positive

Table 4.27 and figure 4.6 show the effect of Montanide® adjuvanted combined HS-

mastitis vaccine on quarter- based time points prevalence of Str. agalactiae and S. aureus

IMIs in cows and buffaloes initially culture positive and CMT/SFMT positive. As can be

seen in these depictions, at day 0, the number of Str. agalactiae and S. aureus infected

quarters of vaccinated –diseased group (group C) was numerically comparable to Str.

agalactiae and S. aureus infected quarters of un-vaccinated –diseased group (i.e. group D).

At day 60, day 120 and day 180 post vaccination, the number of Str. agalactiae and S.

aureus infected quarters in vaccinated –diseased group (group C) was lower than the number

of quarters infected by these two pathogens at day 0. In other words, vaccination reduced the

prevalence of Str. agalactiae and S. aureus IMIs over the entire observation period of 180

days. Towards the end of study period (i.e. day 180), the prevalence of Str. agalactiae and S.

aureus IMIs was slightly higher than the prevalence of these pathogens at day 60 and day

120. In the un-vaccinated- diseased group (group D), the number of Str. agalactiae and S.

aureus infected quarters increased over the observation period of 180 days. Thus the

prevalence of these pathogens at day 60, day 120, and day 180 was higher than their

prevalence at day 0.

73


quarter- based time points prevalence of Str. agalactiae and S. aureus

IMIs in cows and buffaloes initially culture positive and CMT/ SFMT

positive

Groups of cows and

buffaloes

Number of quarters detected

culturally positive for Str.

agalactiae and S. aureus at

different days post vaccination

Day 0 Day 60 Day 120 Day 180

C

(Vaccinated)

Cows

n=15

Str. agalactiae 9 (15%) 4(6.7%) 3(5%) 4(6.7%)

S. aureus 17(28.3%) 10(16.7%) 10(16.7%) 11(18.3%)

Buffaloes

n=15

Str. agalactiae 6(10%) 2(3.3%) 2(3.3%) 3(5%)

S. aureus 12(20%) 6(10%) 6(10%) 7(11.7%)

D (un-

vaccinated)

Cows

n=10

Str. agalactiae 5(12.5%) 7(17.5%) 7(17.5%) 8(20%)

S. aureus 12(30%) 17(42.5%) 16(40%) 17(42.5%)

Buffaloes

n=10

Str. agalactiae 3(7.5%) 5(12.5%) 5(12.5%) 4(10%)

S. aureus 8(20%) 11(27.5%) 11(27.5%) 10(25%)


C= Montanide® adjuvantead combined hemorrhagic septicemia-mastitis vaccinated cows

and buffaloes (Milk culture +ve for Str. agalactiae and S. aureus, CMT/ SFMT +ve); D= un-

vaccinated cows and buffaloes (Milk culture +ve for Str. agalactiae and S. aureus, CMT/

SFMT +ve)

74

Fig 4.6: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on

quarter- based time points prevalence of Str. agalactiae and S. aureus

IMIs in cows and buffaloes initially culture positive and CMT/ SFMT

positive


C= Montanide® adjuvantead combined hemorrhagic septicemia-mastitis vaccinated cows

and buffaloes (Milk culture +ve for Str. agalactiae and S. aureus, CMT/ SFMT +ve); D= un-


SFMT +ve)

0

2

4

6

8

10

12

14

16

18S

tr. ag

ala

ctiae

S. au

reus

Str

. ag

ala

ctiae

S. au

reus

Str

. ag

ala

ctiae

S. au

reus

Str

. ag

ala

ctiae

S. au

reus


C (Vaccinated) D (un-vaccinated)

Ov

era

ll t

ime p

oin

ts p

rev

ale

nce


75

4.9 Mastitis screening tests based prevalence and incidence of mastitis

4.9.1 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on CMT and

SFMT based quarter incidence of mastitis in cows and buffaloes initially culture

negative for S. agalactiae and S. aureus and CMT/ SFMT negative

Table 4.28 and Figure 4.7 depict the quarter-based incidence rates of new subclinical

and clinical mastitis cases in vaccinated and control cows, and buffaloes (initially culture

negative for S. agalactiae and S. aureus and CMT/SFMT negative) during 180 days post-

vaccination period. As it can be seen in these depictions, 4 of 40 quarters (10%) of

vaccinated cows developed mastitis over the entire observation period of 180 days. Only 2 of

the 120 quarters (1.6 %) of vaccinated buffaloes developed mastitis during this period. In the

control group (un-vaccinated), 8 (13.33%) quarters of 15 cow and 6 (10%) quarters of 15

buffaloes developed mastitis during this period. In as much as a fairly few quarters of

vaccinated or unvaccinated developed subclinical or clinical mastitis during the observation

period of 6 months, incidence data were not subjected to any statistical analysis.

Table 4.28: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on CMT and

SFMT-based quarter incidence of mastitis in cows and buffaloes initially culture



Number of new quarters reacting

positive in CMT and SFMT over

180 days post –vaccination period

A ( vaccinated –culture –ve for Str.

agalactiae and S. aureus and

CMT/ SFMT –ve )

Cows n=10 4 (10 % )

Buffaloes

n=30 2 (1.6 % )

B (un- vaccinated –culture –ve for

Str. agalactiae and S. aureus and

CMT/ SFMT –ve)

Cows n=15 8 (13.33 % )

Buffaloes

n=15 6 (10 % )

SFMT=Surf field mastitis test; CMT= California mastitis test


buffaloes (Milk culture –ve for Str. agalactiae and S. aureus, CMT/SFMT –ve) B= un-

vaccinated cows and buffaloes (Milk culture –ve for Str. agalactiae and S. aureus, CMT

/SFMT –ve)

76

Fig: 4.7: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on CMT and

SFMT-based quarter incidence of mastitis in cows and buffaloes initially culture


SFMT=Surf field mastitis test; CMT= California mastitis test


buffaloes (Milk culture –ve for Str. agalactiae and S. aureus, CMT/SFMT –ve) B= un-

vaccinated cows and buffaloes (Milk culture –ve for Str. agalactiae and S. aureus, CMT

/SFMT –ve)

0

1

2

3

4

5

6

7

8

9


A ( vaccinated -culture -ve for Str. agalactiaeand S. aureus and CMT/ SFMT -ve )

B (un- vaccinated -culture -ve for Str.agalactiae and S. aureus and CMT/ SFMT -ve)

CM

T/S

FMT

-bas

ed

qu

arte

r in

cid

en

ce

77

4.9.2 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on

CMT/SFMT- based quarter time points prevalence of mastitis in cows and

buffaloes initially negative for Str. agalactiae and S. aureus plus CMT/SFMT

negative

Table 4.29 and Figure 4.8 show the effect of Montanide® adjuvanted combined HS-

mastitis vaccine on CMT/SFMT- based quarter time points prevalence of mastitis in cows

and buffaloes initially negative for Str. agalactiae and S. aureus plus CMT/SFMT negative.

As can be seen in these depictions, at day 0, the numbers of CMT/SFMT positive quarters of

vaccinated cows and buffaloes (group A) were broadly comparable to the corresponding

numbers in control (un-vaccinated; group B) cows and buffaloes. In the vaccinated cows, an

increase of 2.5 % was noted at day 60 and day 120. At day 180 a further increase of 7.5 % in

the CMT/SFMT quarter prevalence of mastitis was recorded. In vaccinated buffaloes,

CMT/SFMT quarter prevalence of mastitis did not change much; quarter prevalence at day 0,

day 160, day 120, and day 180 being 4.16, 3.33, 4.16, and 5%, respectively. In the case of

un-vaccinated cows (control), increases of 11.67, 13.34 and 13.34 % were registered at day

60, day 120 and day 180, respectively. Un-vaccinated buffaloes witnessed a sharp increase of

8.33% in CMT/SFMT- based quarter prevalence of mastitis at day 60 whereas at day 120 and

180 a steady small further increases of CMT/SFMT-based quarter prevalence of mastitis was

observed.

78


CMT/SFMT- based quarter time points prevalence of mastitis in cows

and buffaloes initially negative for Str. agalactiae and S. aureus plus

CMT/SFMT negative


Number of quarters reacting positive in

CMT/SFMT at different days post

vaccination


A(vaccinated –

culture –ve for Str.

agalactiae and S.

aureus and CMT/

SFMT –ve )

Cows n=10 3 (7.5%) 4 (10%) 4 (10 %) 6 (15%)

Buffaloes

n=30 5 (4.16% ) 4 (3.33 % ) 5 (4.16 %) 6 (5 %)

B (un- vaccinated –

culture –ve for Str.

agalactiae and S.

aureus and CMT/

SFMT –ve)

Cows

n=15 5 (8.33% ) 12 (20 % ) 13 (21.67%) 13 (21.67 %)

Buffaloes

n=15 3 (5% ) 8 (13.33% ) 9 (15 %) 10 (16.67%)

CMT= California mastitis test; SFMT=Surf field mastitis test


buffaloes (Milk culture -ve for Str. agalactiae and S. aureus CMT /SFMT -ve); B= un-

vaccinated cows and buffaloes (Milk culture -ve for Str. agalactiae and S. aureus, CMT

/SFMT -ve)

79


CMT/SFMT based quarter time points prevalence of mastitis in cows and

buffaloes initially negative for Str. agalactiae and S. aureus plus CMT/SFMT

negative

CMT= California mastitis test; SFMT=Surf field mastitis test 2121




/SFMT -ve)

0

2

4

6

8

10

12

14

Co

ws

n=

10

Buffa

loe

s

n=

30

Co

ws

n=

15

Buffa

loe

s

n=

15

A(vaccinated -culture -ve for Str. agalactiaeand S. aureus and CMT/ SFMT -ve )

B (un- vaccinated -culture -ve for Str.agalactiae and S. aureus and CMT/ SFMT -

ve)

CM

T/S

FM

T-b

ased

qu

art

er

tim

e p

oin

ts p

rev

ale

nce


80

4.9.3 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on CMT and

SFMT based quarter incidence of mastitis in cows and buffaloes initially culture

positive for S. agalactiae and S. aureus and CMT/ SFMT positive

Three additional quarters of 15 vaccinated- disease cows (group C) reacted positive in

CMT/ SFMT over 180 days post vaccination period. The corresponding figure in 15

vaccinated-diseased buffaloes (group C) was 2. In the un-vaccinated-diseased cows (group

D), 8 new quarters developed CMT/ SFMT positivity over 180 days post vaccination period

whereas the corresponding figure for the un-vaccinated –diseased buffaloes (group D) was 6

( table 4.30 , Figure 4.9).

Table 4.30: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on CMT

and SFMT based quarter incidence of mastitis in cows and buffaloes

initially culture positive for S. agalactiae and S. aureus and CMT/ SFMT

positive

SFMT=Surf field mastitis test; CMT=California mastitis test



vaccinated- diseased cows and buffaloes (Milk culture +ve for S. aureus and Str. agalactiae,

CMT/ SFMT +ve)

Groups of cows and buffaloes Quarter-based cumulative incidence (new

cases) over 180 days post vaccination period

C (vaccinated- diseased)

Cows

n=15 3

Buffaloes

n=15 2

D (un-vaccinated-diseased)

Cows

n=10 8

Buffaloes

n=10 6

81

Fig: 4.9: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on CMT

and SFMT based quarter incidence of mastitis in cows and buffaloes

initially culture positive for S. agalactiae and S. aureus and CMT/ SFMT

positive




vaccinated- diseased cows and buffaloes (Milk culture +ve for S. aureus and Str. agalactiae,

CMT/ SFMT +ve)

0

1

2

3

4

5

6

7

8

9


C (vaccinated- diseased) D (un-vaccinated-diseased)

CM

T/SF

MT-

bas

ed q

uar

ter

inci

den

ce

82

4.9.4 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on

CMT/SFMT- based time points quarter prevalence of mastitis in cows and

buffaloes initially positive for Str. agalactiae and S. aureus plus CMT/SFMT

positive

Table 4.31 and Figure 4.10 show the effect of Montanide® adjuvanted combined HS-

mastitis vaccine on CMT/SFMT- based quarter time points prevalence of mastitis in cows

and buffaloes initially positive for Str. agalactiae and S. aureus plus CMT/SFMT positive.

As can be seen in these depictions ,at day 0, the number positive quarters of vaccinated –

diseased group (group C) was numerically comparable to positive quarters of un-vaccinated

–diseased group (i.e. group D). At day 60, day 120 and day 180 post vaccination, the number

of positive quarters in vaccinated –diseased group (group C) was lower than the number of

quarters positive at day 0. In other words, vaccination reduced the prevalence over the entire

observation period of 180 days. Towards the end of study period (i.e day 180), the prevalence

was slightly higher than at day 60 and day 120.

In the un-vaccinated- diseased group (group D), the number of positive quarters increased

over the observation period of 180 days. Thus the prevalence of these pathogens at day 60,

day 120, and day 180 was higher than their prevalence at day 0.

83


CMT/SFMT- based time points quarter prevalence of mastitis in cows

and buffaloes initially positive for Str. agalactiae and S. aureus plus

CMT/SFMT positive


Number of quarters detected positive by

CMT/SFMT at different post vaccination

days


C

(Vaccinated)

Cows n=15 24(40%) 17 (28.33%) 16 (26.67 %) 18 (30%)

Buffaloes n=15 16 (26.67%) 10 (16.67%) 10 (1 6.67%) 12 (20 %)

D (un-

vaccinated)

Cows n=10 17 (42.50 %) 32 (80 %) 31 (77.5 %) 33 (82.5 %)

Buffaloes n=10 11 (27.5 %) 22 (55%) 22 (55%) 20 (50 %)



buffaloes (Milk culture +ve for Str. agalactiae and S. aureus, CMT/ SFMT +ve); D= un-


SFMT +ve)

84


CMT/SFMT- based time points quarter prevalence of mastitis in cows

and buffaloes initially positive for Str. agalactiae and S. aureus plus

CMT/SFMT positive



buffaloes (Milk culture +ve for Str. agalactiae and S. aureus, CMT/ SFMT +ve); D= un-


SFMT +ve)

0

5

10

15

20

25

30

35C

ow

s

n=

15

Buffa

loe

s

n=

15

Co

ws

n=

10

Buffa

loe

s

n=

10

C (Vaccinated) D (un-vaccinated)

CM

T/S

FM

T-b

ased

tim

e p

oin

ts q

uart

er

pre

vale

nce


85

4.10 Severity of mastitis in vaccinated and control animals 4.10.1 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on the

severity of mastitis.

Table 4.32 and Fig. 4.11 depict the effect of Montanide® adjuvanted combined HS-

mastitis vaccine on the severity of mastitis in vaccinated–non-diseased, control-non-diseased,

vaccinated –diseased, control-diseased groups. As can be seen from these depictions, mean±

SEM of the severity scores of group A (vaccinated; CMT/SFMT and culture –ve) were lower

than those of group B (control; CMT/SFMT and culture –ve). Similarly, mean± SEM of the

severity scores of group C (vaccinated; CMT/SFMT and culture +ve) were less than those of

group D (control; CMT/SFMT and culture +ve).

Table 4.32: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on the

severity of mastitis in vaccinated–non-diseased, control-non-diseased,

vaccinated –diseased, control-diseased groups of cows and buffaloes

Groups of cows

and buffaloes

Number of

animals

Mean±SEM* of

the group quarter

mastitis severity

scores**

Distribution of quarters as per

their mastitis severity scores

Group

A=Vaccinated

(CMT/ SFMT and

Culture –ve)

cows n=10 0.125±0.0639

3 quarters with score =1

1 quarter with score=2

buffaloes n=30 0.025±0.0186

1 quarters with score=1

1 quarter with score =2

Group B = Control

(CMT/ SFMT and

Culture -ve)

Cows n=15 0.20±0.0744




buffaloes n=15 0.12±0.0481



Group C =

Vaccinated CMT/

SFMT and Culture

+ve)

Cows n=15 0.05±0.0402



buffaloes n=15 0.033±0.0234


Group D = Control

(CMT/ SFMT and

Culture +ve)

Cows n=10 0.20±0.0859



buffaloes n=10 0.15±0.0706



* aggregate of the quarter severity scores divided by the total of quarters in the respective

groups, ** As per Roberson (2003)


86




/SFMT -ve) C= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis


SFMT +ve) D= un-vaccinated- diseased cows and buffaloes (Milk culture +ve for S. aureus

and Str. agalactiae, CMT/ SFMT +ve)

Fig. 4.11: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on the

severity of mastitis in vaccinated–non-diseased, control-non-diseased,

vaccinated –diseased, control-diseased groups of cows and buffaloes





/SFMT -ve) C= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis


SFMT +ve) D= un-vaccinated- diseased cows and buffaloes (Milk culture +ve for S. aureus

and Str. agalactiae, CMT/ SFMT +ve)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

cow

s n

=10

bu

ffalo

es n

=3

0

Co

ws n

=15

bu

ffalo

es n

=1

5

Co

ws n

=15

bu

ffalo

es n

=1

5

Co

ws n

=10

bu

ffalo

es n

=1

0

Group A=Vaccinated(CMT/ SFMT and

Culture -ve)

Group B = Control(CMT/ SFMT and

Culture -ve)

Group C = VaccinatedCMT/ SFMT and

Culture +ve)

Group D = Control(CMT/ SFMT and

Culture +ve) c

Mean

±S

EM

87

4.11 Milk somatic cell count

4.11.1 Effect of Montanide® adjuvanted combined HS-mastitis vaccine on the

quarter milk somatic cell count (× 105

ml of milk) in vaccinated–non-

diseased, control-non-diseased, vaccinated –diseased and control-diseased

groups of cows

A non-significant decrease in somatic cell count was noted at day 60, and day 120 in

vaccinated-non-diseased cows (group A). At day 180 post vaccination, somatic cell counts in

this group was non-significantly higher than at day 0, day 60 and day 120. In unvaccinated–

non-diseased cows (group B), a non-significant increase in SCC was recorded at day 60,120

and 180. Vaccination of diseased cows (group C) affected a significant decrease in SCC on

all post vaccination observation time points (day 60, 120 and 180) albeit at day 180, a slight

increase in SCC was noticed. Somatic cell counts in unvaccinated –diseased cows (group D)

differed non-significantly at day 0, 60, 120, and 180 (Table 4.34; Fig.4.12).

A non-significant decrease in somatic cell count was noted at day 60, and day 120 in

vaccinated-non-diseased buffaloes (group A). At day 180 post vaccination, somatic cell

counts in this group was non-significantly higher than at day 0, day 60 and day 120. In

unvaccinated–non-diseased buffaloes (group B), a non-significant increase in SCC was

recorded at day 60, 120 and 180. Vaccination of diseased buffaloes (group C) affected a

highly significant decrease in SCC on all post vaccination observation time points (day 60,

120 and 180) albeit at day 180, a slight increase in SCC was noticed. Somatic cell counts in

unvaccinated –diseased buffaloes (group D) differed non-significantly at day 0, 60, 120, and

180 (Table 4.36; Fig.4.13).

88



ml of milk) in vaccinated–non-diseased, control-

non-diseased, vaccinated –diseased, control-diseased groups of cows

Source of variation Degrees of

freedom

Sum of squares Mean squares F-value

Group

Day

Group x Day

Error

Total

3

3

9

80

95

201.257

7.036

23.537

2.287

234.118

67.0858

2.3454

2.6152

0.0286

2346.31***

82.03***

91.47***

*** = Highly significant (P<0.001)

Table: 4.34: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on the



diseased, control-non-diseased, vaccinated –diseased, control-diseased

groups of cows

Group of cows Days with reference to first vaccination

Mean 0 60 120 180

A 2.14 ± 0.02c 2.09 ± 0.07

c 2.08 ± 0.03

c 2.31 ± 0.04

c 2.15 ± 0.04

C

B 2.16 ± 0.08c 2.23 ± 0.07

c 2.25 ± 0.08

c 2.38 ± 0.09

c 2.25 ± 0.03

C

C 5.77 ± 0.09a 3.13 ± 0.11

b 3.16 ± 0.09

b 3.26 ± 0.07

b 3.83 ± 0.56

B

D 5.73 ± 0.11a 5.78 ± 0.07

a 5.66 ± 0.07

a 5.71 ± 0.11

a 5.72 ± 0.02

A

Mean 3.95 ± 1.03A 3.31 ± 0.85

B 3.29 ± 0.82

B 3.41 ± 0.79

B

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

Small letters represent comparison among interaction means and capital letters are used for

overall mean.

A= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis vaccinated cows

(Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve); B= Un vaccinated

cows (Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve ); C=

Montanide® adjuvanted combined hemorrhagic septicemia-mastitis vaccinated cows (Milk

culture +ve for S. aureus and Str. agalactiae, CMT/ SFMT +ve); D= Un vaccinated cows

(Milk culture +ve for S. aureus and Str. agalactiae, CMT/ SFMT +ve)

89

Fig.4.12: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on the




groups of cows

A= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis vaccinated cows

(Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve); B= un-vaccinated

cows (Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve;); C=

Montanide® adjuvanted combined hemorrhagic septicemia-mastitis vaccinated cows (Milk

culture +ve for S. aureus and Str. agalactiae, CMT/ SFMT +ve); D= un-vaccinated cows

(Milk culture +ve for S. aureus and Str. agalactiae, CMT/ SFMT +ve;)

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

D-0 D-60 D-120 D-180

Milk

so

mat

ic c

ell

cou

nt

(co

ws)


90





groups of buffaloes

Source of variation Degrees of

freedom

Sum of squares Mean squares F-value

Group

Day

Group x Day

Error

Total

3

3

9

80

95

182.326

6.918

21.77

2.661

213.674

60.775

2.306

2.418

0.0333

1827.46***

69.34***

72.73***

*** = Highly significant (P<0.001)





groups of buffaloes

Group of buffaloes Days with reference to first vaccination

Mean 0 60 120 180

A 2.11 ± 0.01c 2.01 ± 0.01

c 2.04 ± 0.01

c 2.16 ± 0.02

c 2.08 ± 0.02

C

B 2.15 ± 0.02c 2.18 ± 0.04

c 2.21 ± 0.06

c 2.23 ± 0.10

c 2.19 ± 0.01

C

C 5.49 ± 0.05a 2.89 ± 0.04

b 2.91 ± 0.08

b 3.17 ± 0.06

b 3.61 ± 0.53

B

D 5.43 ± 0.09a 5.46 ± 0.07

a 5.41 ± 0.05

a 5.66 ± 0.03

a 5.49 ± 0.04

A

Mean 3.79 ± 0.96A 3.13 ± 0.79

C 3.14 ± 0.77

C 3.30 ± 0.81

B

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean.

A= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis vaccinated buffaloes

(Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve); B= Un vaccinated

buffaloes (Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve;); C=

Montanide® adjuvanted combined hemorrhagic septicemia-mastitis vaccinated buffaloes

(Milk culture +ve for S. aureus and Str. agalactiae, CMT/ SFMT +ve); D= Un vaccinated

buffaloes (Milk culture +ve for S. aureus and Str. agalactiae, CMT/ SFMT +ve;)

91

Fig. 4.13: Effect of Montanide® adjuvanted combined HS-mastitis vaccine on the quarter

milk somatic cell count (× 105 ml of milk) in vaccinated–non-diseased, control-

non-diseased, vaccinated –diseased, control-diseased groups of buffaloes


(Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve); B= un vaccinated



(Milk culture +ve for S. aureus and Str agalactiae, CMT/ SFMT +ve);D= un vaccinated

buffaloes (Milk culture +ve for S. aureus and Str. agalactiae, CMT/ SFMT +ve)

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

D-0 D-60 D-120 D-180

Milk

so

mat

ic c

ell

cou

nt

(bu

ffal

oes

) Group A Group B Group C Group D

92

4.12 Effect of vaccination on milk yield

4.12.1 Milk yield (Mean ±SE; L/24 hours) in vaccinated and control cows and buffaloes

at different post-vaccination days

At Day 0, cows of group A (vaccinated-non-diseased cows) and B (unvaccinated-

non-diseased cows) differed non-significantly in terms of milk yield per day (Table 4.37). On

Day 0, cows of group C (vaccinated-diseased) and group D (unvaccinated diseased) yielded

significantly lesser milk per day than those of group A (vaccinated-non-diseased) and B

(unvaccinated-non-diseased). At Day 60 and Day 120, per day milk yield of cows of group A

(vaccinated-non-diseased) was non-significantly (P ˃ 0.05) higher than their milk yileds at

Day 0. At Day, 60 milk yiled of cows of group A (vaccinated-non-diseased) and B

(unvaccinated-non-diseased) differed non-significantly from each other whereas group C

cows (vaccinated diseased) and group D cows (unvaccinated-diseased) produced

significantly lower milk than those of group A (vaccinated-non-diseased). At this day, group

D cows (unvaccinated diseased cows) yielded significantly lower milk than those of group C

cows (vaccinated-diseased). At Day 120, cows of group A (vaccinated-non-diseased) and B

(unvaccinated-non-diseased) differed non-significantly from each other in their yielded.

Whereas group C cows (vaccinated diseased) and group D cows yielded significantly lower

milk quantity than group A cows (vaccinated-non-diseased) and group B cows

(unvaccinated-non-diseased). Group D cows also yielded significantly lower quantity of milk

than group C cows (vaccinated-diseased). At Day 180, cows of group A had significantly

higher yield than those of group B cows (unvaccinated-non-diseased). However, group C

(vaccinated diseased) was significantly different both from group A (vaccinated-non-

diseased) and B (unvaccinated-non-diseased cows). Group D cows (unvaccinated diseased)

had the lowest yield was significantly lower than those of all of the 3 groups. Over all the

highest yielded was recorded in group A cows (vaccinated-non-diseased) followed by group

B cows (unvaccinated-non-diseased), group C cows (vaccinated-diseased). The lowest

overall yielded was recorded in cows of group D (unvaccinated diseased) that was

significantly lower than those of group A, B and C cows. Entire study period of all groups

differed significantly different from one another in their yieled.

93

Buffaloes of Group A (vaccinated-non-diseased) and group B (unvaccinated-non-

diseased) differed non-significantly from each other in terms of milk yield per day at Day 0.

Similarly, buffaloes of group C (vaccinated diseased) and those of group D (unvaccinated

diseased) yielded non-significantly different quantities of milk per day at day 0 (Table 4.37).

However, group C buffaloes (vaccinated-diseased) and group D buffaloes (unvaccinated-

diseased) had significantly lower yield than those of group A (vaccinated-non-diseased) and

group B (unvaccinated-non-diseased). At Day 60, buffaloes of group A (vaccinated-non-

diseased) and B (unvaccinated-non-diseased) had a non-significant difference among

themselves, group C (vaccinated-diseased-buffaloes) and D (unvaccinated-diseased

buffaloes) was significantly differed from group A and B (unvaccinated-non-diseased

buffaloes). Group D (unvaccinated-diseased buffaloes) also had significantly lower values

than group C (vaccinated-diseased buffaloes). At day 120 buffaloes of group A (vaccinated-

non-diseased buffaloes) had highest milk yield values followed by group B (unvaccinated-

non-diseased buffaloes), C and lowest values observed in group D (unvaccinated diseased

buffaloes). All the groups were significantly different from one another. At day 180 buffaloes

group A (vaccinated-non-diseased buffaloes) had a highest values and was significantly

different from all other group. Group B (unvaccinated-non-diseased buffaloes), C

(vaccinated-diseased buffaloes), and D (unvaccinated-diseased buffaloes) had non-

significantly different yield values from each other but significantly lower from group A

(vaccinated-non-diseased). Over all highest values present in group A (vaccinated-non-

diseased) followed by group B (unvaccinated-non-diseased), C (vaccinated diseased

buffaloes) and lowest values was observed in group D (unvaccinated-diseased buffaloes). All

the groups were significantly different from one another.

Overall milk yield of vaccinated cows and buffaloes (non-diseased or diseased) was

significantly higher than those of unvaccinated cows and buffaloes (non-diseased or

diseased). In other words, vaccination had a positive effect on the milk yield of both healthy

(free from S. aureus and Str. agalactiae) and diseased (infected with S. aureus and Str.

agalactiae) cows and buffaloes.

94

Table 4.37: Milk yield (Mean ±SE; L/24 hours) of vaccinated and control cows and

buffaloes at different post-vaccination days

Animal

groups

Animals and

their numbers

Milk yield (Mean ±SE; L/24 hours) at

different post-vaccination days

Overall

milk yield

(Mean±SE

L/24 hours) Day-0 Day-60 Day-120 Day-180

A

Cows (n= 10) 7.89± 0.15a 8.17± 0.12

a 7.95±0.17

a 5.45±0.16

bc 7.36±0.55

A

Buffaloes (n=

30) 9.13±0.05

ab 9.71±0.06

a 9.52±0.03

a 6.22±0.10

d 8.64± 0.71

A

B

Cows (n= 15) 7.87± 0.19a 7.72± 0.11

a 7.67±0.20

a 4.49±0.22

d 6.93±0.70

B

Buffaloes (n=

15) 9.10±0.18

ab 9.13±0.09

ab 8.57±0.31

b 5.15±0.36

ef 7.99± 0.82

B

C

Cows (n= 15) 5.21± 0.13c 6.13±0.19

b 6.01± 0.14

b 4.76±0.25

cd 5.53±0.28

C

Buffaloes

(n=15) 6.11± 0.25

d 7.10±0.18

c 6.43±0.28

cd 4.72±0.23

f 6.09±0.43

C

D

Cows (n= 10) 5.18± 0.12c 5.10±0.08

cd 4.95± 0.17cd 3.32± 0.11

e 4.64±0.38

D

Buffaloes

(n=10) 6.17± 0.14

d 5.86± 0.13

de 5.27± 0.04

ef 3.79±0.12

f 5.27±0.45

D

Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).

Small letters represent comparison among interaction means and capital letters are used for

overall mean.


buffaloes (Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve); B= un-


SFMT –ve); C= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis


SFMT +ve);D= un vaccinated cows and buffaloes (Milk culture +ve for S. aureus and Str.


95

Fig.4.14: Milk yield (Mean ±SE; L/24 hours) in vaccinated and control cows and

buffaloes at different post-vaccination days


(Milk culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve); B= un vaccinated



(Milk culture +ve for S. aureus and Stragalactiae, CMT/ SFMT +ve);D= un-vaccinated

buffaloes (Milk culture +ve for S. aureus and Str. agalactiae, CMT/ SFMT +ve)

4.13 Cost- benefit analysis of Montanide® adjuvanted combined

hemorrhagic septicemia-mastitis vaccine

4.13.1 HS related cost -benefit calculations

Table 4.38 depicts the number of HS cases in vaccinated and control cows, buffaloes

and calves during 180 days post- vaccination period and pecuniary losses based on market

prices of affected animals.

0

2

4

6

8

10

12

cows (10) buffaloes(30)

cows (15) buffaloes(15)

cow (25) buffalo (50) cow (20) buffalo (25)

A B C D

Animal groups

Milk

yie

ld (

per

lact

atio

n)

Day Day Day Day

96

Table 4.38: Number of HS cases in vaccinated and control cows, buffaloes and calves

during 180 days post- vaccination period and pecuniary losses based on

market prices of affected animals

Groups of buffaloes, cows and calves

Cumulative

HS incidence

over 180 days

period

Monitory losses (Rs) based on

the prevailing market prices

(buffalo Rs=125000; cow

Rs=100000; buffalo and cow

calf Rs =25000) of HS affected

animals

A (vaccinated) cows n=10 0 0

buffaloes n=30 1 125,000

B (un-vaccinated)

cows n=15 1 100,000


C (vaccinated)

cows n=15 0 0

buffaloes n=15 0 0

D (un-vaccinated) cows n=10 0 0


E ( vaccinated )

cow calves n=25 0 0

buffalo calves n=50 1 250,00

F( un-vaccinated)

cow calves n=25 1 25,000

buffalo calves n=20 3 75,000

A= Montanide® adjuvanted combined HS-mastitis vaccinated cows and buffaloes (Milk

culture –ve for S. aureus and Str. agalactiae, CMT/ SFMT –ve; no history of HS vaccination

during the last 180 days); B= un-vaccinated cows and buffaloes (Milk culture –ve for S.

aureus and Str. agalactiae, CMT/ SFMT –ve; no history of HS vaccination during the last

180 days); C= Montanide® adjuvanted combined hemorrhagic septicemia-mastitis

vaccinated buffaloes (Milk culture +ve for S. aureus and Str. agalactiae, CMT/ SFMT +ve);

D= un vaccinated cows and buffaloes (Milk culture +ve for S. aureus and Str. agalactiae,

CMT/ SFMT +ve); E= vaccinated cow and buffalo calves; no history of HS vaccination

during the last 180 days F= un-vaccinated cow and buffalo calves; no history of HS

vaccination during the last 180 days.

97

Summary

Vaccinated groups

a) Financial losses due to HS associated mortality= Rs. 150,000

b) Cost of two shots of the Montanide® adjuvanted combined HS-mastitis vaccine

( Rs. 60/dose)= Rs. 17,400

Total of a and b = Rs. 167,400

Un-vaccinated groups

a) Financial losses due to HS associated

mortality= Rs. 575,000

Benefits of vaccination (credits) i.e. differential of the debits in un-vaccinated

groups minus debits in vaccinated groups =Rs. 407,600

4.13.2 Mastitis related cost -benefit calculations

4.13.2.1 Mastitis control realted benefits accruing from use of Montanide®


Table 4.39 depicts mastitis control realted benefits accruing from the use of

Montanide® adjuvanted combined hemorrhagic septicemia-mastitis vaccine.

98

Table 4.39: Mastitis control related benefits accruing from use of Montanide®


Groups of cows and

buffaloes

Total

Milk

yield

(L)

Total L

of milk

produced

over 180

days trial

period

Additional milk

(L) produced by

vaccinated

animals vis-à-vis

un-vaccinated

animals over a

180 days period

Cost of 2

shots of

vaccination

@ Rs=60

per dose

Economic

benefit @

RS.70/L of milk

A

(vaccinated)

cows

n=10 13266

59,922 19,638

4,800 1,374,660

buffaloes

n=30 46,656

B (un-

vaccinated)

cows

n=15

18,711 40,284 - 0

buffaloes

n=15

21,573

C (

vaccinated )

cows

n=15

14,931 31,374 13,536 3600 947,520

buffaloes

n=15

16,443

D ( un-

vaccinated)

cows

n=10

8,352 17,838 - 0

buffaloes

n=10

9,486

A=vaccinated cows and buffaloes (CMT/ SFMT and Culture –ve); B=unvaccinated cows and

buffaloes (CMT/ SFMT and Culture –ve); C=vaccinated cows and buffaloes (CMT/ SFMT

and Culture +ve); D=un-vaccinated cows and buffaloes (CMT/ SFMT and Culture +ve)

Summary

Cost of two shots of the Montanide® adjuvanted combined HS-mastitis vaccine

(@ Rs. 60/dose) to 50 cows and buffaloes = Rs. 6000 (debit)

Price of additional milk of 50 cows and buffaloes =Rs 1,658,700 (credit)

Benefits in terms of mastitis control (credit – debit) = Rs 1,652,700

Net financial benefit due to HS control + mastitis control = Rs 2,060,300

As can be seen above, the use of Montanide® adjuvanted combined HS-mastitis vaccine had

a net financial benefit worth Rs 2,060,300

99

4.14 Calculation of vaccine efficacy

4.14.1 Calculation of vaccine efficacy for HS in cows, buffaloes and calves

Vaccine efficacy was measured by calculating the incidence rates (attack rates) of HS

among vaccinated and unvaccinated animals and determine the percentage reduction in the

incident rate of disease among vaccinated animals compared to unvaccinated animals. The

basic formula is

ARU - ARV

Vaccine efficacy = -------------------------- × 100

ARU

Vaccine efficacy can also be expressed in relative risk (RR) which is the ratio of ARV to

ARU

Vaccine Efficacy= (1-RR) × 100

Where VE =vaccine efficacy, ARU = Attack rate in the unvaccinated population,

ARV=Attack rate in the vaccinated population

Table 4.40: Calculation of vaccine efficacy for HS in cows, buffaloes and calves

Number of new HS cases in vaccinated and control cows,

buffaloes and calves during 180 days post-vaccination period

Clinical status Group A, C and E=

Vaccinated

Group B, D and F=Non- vaccinated

Diseased a=2 b=8

Healthy c=143 d=87

ARU = b/d = 8/87 = 0.092 ARV= a/c= 2/143 = 0.014

ARU - ARV

Vaccine efficacy (VE) = ------------------- × 100

ARU

0.092– 0.014

VE= ------------------ × 100 = 84.78%

0.092

100

4.14.2 Calculation of vaccine efficacy for mastitis in cows and buffaloes

Vaccine efficacy was measured by calculating the incidence rates (attack rates) of

mastitis among vaccinated and unvaccinated animals and by determining the percent

reduction in the incidence rate of disease among vaccinated animals compared to

unvaccinated animals

Table 4.41: Calculation of vaccine efficacy for mastitis in cows and buffaloes

Quarter based cumulative incidence over 180 days post

vaccination period

Clinical status Group A and C= Vaccinated Group B and D=Non- vaccinated

Diseased a=5 b=22

Healthy c=65 d=28

ARU = b/d = 22/28 = 0.79 ARV= a/c= 5/65 = 0.077

ARU – ARV

Vaccine efficacy (VE) = ------------------- × 100

ARU

0.79 – 0.077

VE= ------------------ × 100 = 90.25%

0.79

101

CHAPTER 5

DISCUSSION

Hemorrhagic septicemia (HS) and mastitis are two of the 10 most prevalent and

important diseases of dairy animals in Pakistan (Khan et al., 1991; Khan et al., 1994-1995).

Although, vaccination against HS is practiced in routine, the application of mastitis control

measures is almost totally nonexistent, save a small number of large commercial dairy herds.

A combined HS-mastitis vaccine, therefore holds the promise of simultaneous control of

these two rife dairy animal diseases in Pakistan. The present investigations were carried out

in pursuit of this objective by following the internationally acceptable guidelines/

requirements of a vaccine preparation and testing (EMEA, 2000).

5.1 Preparation of combined HS-mastitis vaccine and its evaluation against HS

An attempt was made to use P. multocida isolates, isolated from the field cases of

clinical HS. In the context of the present study, a calf was defined as any bovine or bubaline

animal aged up to one year (http://en.wikipedia.org/wiki/calf). This is the age group of cattle

and buffaloes most susceptible to HS (Farooq et al., 2011; Khan et al., 2011). The septicemia

in HS occurs at the terminal stage of the disease. Therefore, blood samples taken from sick

animals before death may not always contain P. multocida organisms. This microorganism is

also not consistently present in the nasal secretions of sick animals. A blood sample or swab

collected from the heart is satisfactory if it is taken within a few hours of death. If the animal

has been dead for a long time, a long bone, free of tissue, can be taken. If there is no facility

for postmortem examination, blood can be collected from the jugular vein by incision or

aspiration. Blood samples in any standard transport medium should be dispatched on ice and

well packed to avoid any leakage. Blood smears from affected animals are stained with

Gram, Leishman‟s or methylene blue stains. The organisms appear as Gram-negative,

bipolar-staining short cocco-bacilli. No conclusive diagnosis can be made on the basis of

direct microscopic examinations alone. Blood samples, or swabs eluted into 2–3 ml sterile

physiological saline are cultured. Alternatively, the surface of a long bone is swabbed with

alcohol and split open. The marrow is extracted aseptically and cultured. Direct culture is

102

usually satisfactory only if the material is fresh and free from contaminants or post-mortem

invaders that would otherwise overgrow any Pasteurella present.

The failure to recover P. multocida in overwhelming majority of clinical cases in the

present study is broadly congruent with the findings of previous studies (Butt et al., 2003;

Ashraf et al., 2011; Ekundayo et al., 2008). Many blood samples collected from HS

affected animals failed to yield the etiologic agent on cultural examination because

septicemia develops only in the terminal stages of this disease (Center for Food Security and

Public Health and Institute for International Cooperation in Animal Biologics an OIE

Collaborating Center, 2009). This notion was borne out by the findings of the present study

wherein blood samples of all 5 buffalo calves and 2 adult buffaloes tested negative on

cultural examination. Long bones from the suspected carcasses of HS were found to be

sample of choice for isolation of P. multocida. Isolation of P. multocida has been attempted

from a wide range of organs such as kidney, heart, liver, brain, tonsils, and lymph nodes from

experimentally infected calves (Chowdhury et al., 2014; OIE, 2012; Singh et al., 2007).

From the liver and blood samples, recovery of the causative organisms is poor. Gross

pathological changes might have inactivated the causative agents in these samples (Naz et

al., 2012). In the present study, microbiological examination of 7 blood samples (collected

from five HS affected calves and two HS affected adult buffaloes) on CYS agar did not yield

growth of P. multocida. The negative blood culture results in these cases may be attributed to

the notion that P. multocida appears in blood only at the terminal septicemic stages of the

disease (Carter and De Alwis, 1989). A total of 3 isolates of P. multocida could be recovered

on microbiological examination of peripheral blood, femur, and heart blood of HS affected

animals on CSY and blood agar plates. No P. multocida isolate was recovered from

peripheral blood and swab samples of heart blood. Culturing of heart blood and femur bone

marrow samples respectively yielded growth of two and one isolate of P. multocida.

Suspected isolates of P. multocida formed smooth grayish, glistening translucent colonies of

nearly 1 mm diameter on blood agar after 24 hours incubation at 37°C. No growth was

recorded on MacConkey‟s agar on subculturing. On Gram staining, the organism appeared as

Gram –ve, short, ovoid bipolar staining coccobacilli with some degree of pleomorphism.

Growth on CYS agar was smooth, yellowish, mucoid and larger in size than that on blood

103

agar. All three P. multocida isolates were positive for indol, ODC, glucose, fructose, and

catalase, while negative for H2S production, lactose, D-dulcitol and maltose. Variable results

were recorded for D-sorbitol and arabinose. These isolates of P. multocida were confirmed

by amplification of P. multocida specific gene using KTSP61/KTT7 set of primers, which

yielded a product of 618 bp on electrophoresis that confirmed these isolates as P. multocida

type B. Conditions for HS-causing type-B-specific PCR were similar to those described for

P. multocida-specific PCR (Townsend et al., 1998). The usefulness of these primers has been

reported for identification of serogroup B strains. HS-causing type-B-specific PCR primers

can also be used in a Multiplex PCR with the P. multocida-specific primers, dramatically

decreasing the time required for P. multocida detection and presumptive identification of the

HS-serotype. To date, the HS-causing type-B-specific PCR remains 100% specific for HS-

causing type B serotypes of isolated P. multocida (OIE, 2012). Type B cultures with the

predominant somatic antigen being either type 2 or 5 are identified by the amplification of a

~620 bp fragment with the KTSP61/KTT7 set of primers.

All 3 isolates of P. multocida were sensitive to enrofloxacin, amoxicillin, ceftiofur

sodium and oxacillin and resistant to tetracycline, fusidic acid, and penicillin. All three

isolates displayed an intermediate sensitivity to vancomycine. A variable degree of

susceptibility was displayed to lincomycine, Septran, and gentamicin. Kehrenberg et al.

(2001) has reviewed antibiotic susceptibility of P. multocida that causes HS.

LD 50 of the three candidate vaccinal isolates of P. multocida in the present study are

broadly in agreement with those reported by Butt et al. (2003). These workers reported the

mouse LD50 of 19 field isolates of P.multocida. The LD50 of these isolates ranged from 10-7.75

to 10-7.50

. According to OIE (2012), the type B: 2 strains associated with HS are highly

virulent for mice and rabbits with LD50 ranging from 1and10 viable microrganisms.

A high degree of immunogenicity is one of the cardinal considerations in selecting an

isolate for vaccine production. As per Muneer et al (2005) comparativetly high antibody

titers were observed in animals of all age groups vaccinated with oil adjuvant vaccine

throughout the trial period while the titers in animals vaccinated with alum precipited

vacccine declined after 3 months following vaccination and reached at minimal level 180

104

days post vaccination. According to the workers, results justified the replacement of alum

precipited vaccine with the newly developed oil adjuvant vaccine for the control of HS in

animals. In the present study, Montanide® ISA 201 VG was used as an adjuvant. Vaccines

formulated with Montanides® (Seppic, France) are cheaper and these adjuvants are ready-to-

use mineral oil adjuvants. The use of Montanides® in veterinary vaccines as adjuvants has

shown very promising results. The use of a Montanide® adjuvanted mastitis vaccine

reportedly induced significantly high immune response against α and β toxins of S. aureus

and pseudocapsule in sheep and cattle (Nordhaug et al., 1994a; Tollersurd et al., 2001).

Montanide® adjuvanted vaccine is reported to be less viscous and less irritant than traditional

Freund‟s adjuvanted vaccine. Montanide® ISA 201 VG adjuvanted quadrivalent foot-and-

mouth vaccine has been reported to elicit superior immune response compared to that elicited

by aluminium hydroxide gel. The vaccine resulted in rapid immune response that was

maintained for longer duration (Patil et al., 2002). Owing to these desirable attributes, the

combined HS-mastitis vaccine tested in the present study utilized Montanide as an adjuvant.

The number of new HS cases in vaccinated and control cows, buffaloes and calves

during 180 days post-vaccination period was investigated. None of the 10 vaccinated cows

and 25 cow calves developed HS over the entire observation period of 180 days. Only 1 of

the 30 vaccinated buffaloes (3.33%) developed HS during this period. In the control group

(un-vaccinated), 1 cow (6.66 %) and 2 buffaloes (13.33 %) developed HS during this period.

One of the 50 buffalo calves (2%) vaccinated with this vaccine developed HS during this

period. One of 25 un-vaccinated cow calves (4%) and 3 of 20 buffalo calves (15%)

developed clinical HS over the observation period of 6 months. Pasteurella multocida could

be isolated from only 1 buffalo calf (group F) that succumbed to peracute HS. Isolation of P.

multocida from other cases were not successful. The Montanide® adjuvanted combined

hemorrhagic septicemia-mastitis vaccine thus seems protective for HS over 6 months trial

period. This vaccine had a vaccine efficacy of 84.78% against HS. Nevertheless, in view of a

limited number of test animals utilized in the present study, a much larger clinical trial

involving a much larger number of bovines and bubaline subjects is clearly warranted. In as

much as the use of a combined HS-mastitis vaccine has not been reported heretofore, it is not

possible to compare the results of the present study with those of any previous study.

105

5.2 Evaluation of combined HS-mastitis vaccine against mastitis

With milk production of around 52.6 million tons annually, Pakistan is the fourth largest

milk producing country in the world (Pakistan Economic Survey, 2014-15). Mastitis is a

major production limiting disease of dairy animals all over the world. It not only reduces

milk quantity but also depreciates the milk quality, thus adversely affecting milk production

economics (DeGraves and Fetrow, 1993; Sordillo et al., 1997). On a pattern equivalent to

most other countries, mastitis is the most important health problem of dairy animals in

Pakistan (Cady et al, 1983; Ajmal, 1990; Egenolf, 1990). In Pakistan, about 37 years ago,

losses due to clinical mastitis were estimated at Rs.240 million annually in Punjab province

alone (Chaudhry and Khan, 1978). Staggering economic losses associated with mastitis

warrant a well-planned mastitis control program to curtail losses for profitable dairying.

Dairy farmers in developed countries like USA, Canada and Australia are practicing mastitis

control measures, viz. dry cow therapy, post-milking teat dipping, and culling chronically

mastitic cows since long. Due to small herd size, non-progressive attitude of dairy farmers

and lack of any system of milk quality premium, standard mastitis control measures are

difficult to be adopted in Pakistan. Antibiotic treatment has its own demerits including

emergence of antibiotic resistance and drug residues in milk, thus rendering mastitis control

through antibiotics as an unacceptable approach due to public health hazards. In the absence

of any mastitis control program, high antibiotic resistance and poor therapeutic response,

vaccination against some of the most prevalent mastitis pathogens seems a logical choice to

curtail losses associated with mastitis. In recent studies (Athar et al., 2007; Rashid, 2011),

efforts were made to investigate the role of vaccination as an alternative mastitis control

strategy for farms lacking standard mastitis control program. Although, both live attenuated

and dextran sulphate adjuvanted vaccines evaluated in these studies were found valuable in

the control of mastitis in buffaloes, the limitation of these vaccines including need of a cold

chain for live attenuated vaccine, high production cost of dextran sulphate vaccine (Rs. 500

for 2 shots of dextran sulphate adjuvanted vaccine) and short duration immunological

protection were obvious. There is thus a need not only to curtail the cost of mastitis vaccine

but also to prolong its duration of effectiveness. The principal objective of present study

included the evaluation of Montanide® adjuvanted S. aureus plus Str. agalactiae bacterin-

106

toxoid in an effort to obtain desirable and durable immune response at an economical cost.

From the data generated in the present study, the Montanide® adjuvanted combined HS-

mastitis incorporating S. aureus and Str. agalactiae (the two most prevalent mastitis

pathogens in Pakistan as reviewed by Shakoor, 2006) displayed both preventive and curative

qualities. The preventive and curative attributes of this vaccine (Tables 4.25 to 4.31) are in

agreement with the findings of previous studies (Chishti et al., 1992; Nickerson et al., 1993;

Nordhaug et al., 1994a; Calzolari et al., 1997; Giraudo et al., 1997; Qureshi et al., 2000;

Shakoor, 2006; Shakoor et al., 2006; Athar, 2007; Ashfaq et al., 2014). In addition,

mean±SEM of the group quarter mastitis severity scores (Table 3.32) were lower in

vaccinated groups (group A cows =0.125±0.0639, group A buffaloes =0.025±0.0186; group

C cows =0.05±0.0402, group C buffaloes =0.033±0.0234) than in un-vaccinated groups

(group B cows=0.20±0.0186, group B buffaloes 0.12±0.0481; group D cows=0.20±0.0859,

group D buffaloes= 0.15±0.0706). In this respect, the results of the present study are in

conformity with the results reported by Qureshi et al. (2000), Shakoor (2006), Shakoor et al.

(2006), and Athar (2007). Contrarily, however the results of the present study are discordant

with the results reported by Hoedemaker et al. (2001). These workers determined the effect

of an aluminium hydroxide adjuvanted autogenous S. aureus bacterin on the prevalence of S.

aureus, SCC, and clinical mastitis in a dairy herd beset with infections with this organism.

Animal and quarter based prevalence of S. aureus IMIs did not differ between cows treated

with autogenous vaccine or those treated with a placebo. Although, the combined HS-

mastitis vaccine tested in the present study displayed both preventive and curative effect on

mastitis, its use can not be recommended as a substitute for standard mastitis control

measures such as antiseptic teat dipping, dry cow therapy and culling of chronically infected

animals (Bozhkova et al., 1985). Nonetheless, it can be recommended where a „do nothing‟

policy of mastitis control is in vogue. The increase in milk yield of animals treated with

combined HS-mastitis vaccine in the present study is consistent with the findings of Watson

(1987), Leitner et al. (2003) and Athar (2007). In the present study, concentrations of P.

multocida, S. aureus and Str. agalactiae were adjusted to 1×1010

cells/ml in the montanide

adjuvanted HS-mastitis vaccine. Athar (2007) while evaluating the inactivated polyvalent

vaccines for the control of mastitis in dairy buffaloes reported that a concentration of 1010

107

cells/ml of each of three vaccinal isolates (S. aureus, Str. agalactiae, and E.coli) was more

antigenic as compared to 108

and 1012

cells/ml.

The vaccine developed and tested in the present study harbored antigens not only of P.

multocida (the etiologic agent of a very prevalent disease of calves i.e HS) but also those of

S. aureus and Str. agalactiae (the common etiologic agents of heifer mastitis). Its use in

female calves might have exerted a beneficial role in the control of heifer mastitis. The need

to control mastitis in heifers has since long been recognized (Trinidad et al., 1990a; Trinidad

et al., 1990b; Nickerson et al., 1995; Oliver et al., 2003; Oliver et al., 2003, De Vliegher,

2004) and is increasingly coming into focus in dairy farming.

Mondanide® adjuvanted combined HS-mastitis vaccine evaluated in the present

study was not 100% protective for hemorrhagic septicemia (Table 4.24). This vaccination

failure is consistent with findings of some previous studies. Field observations of veterinary

officers in 9 districts of Pakistani Punjab have indicated the occurrence of field outbreaks of

HS despite vaccination with alum- precipitated P. multocida bacterin prepared in public

sector (Sheik et al., 1996). In order to obtain maximum protection from HS vaccine, it is

recommended that the vaccines be prepared from P. multocida strains circulating in the

regions of intended use. FAO recommends a live avirulent HS vaccine prepared from a P.

multocida serotype B: 3 of fallow deer origin for use in Southeast Asia (Carter, 2005).

Despite vaccination and improved management, occurrence of HS outbreaks is a regular

feature each year, especially in endemic areas of the country (Afzal and Muneer, 1990).

Pakistani workers (Shahzad et al., 2013) reported an outbreak of HS in buffalo calves that

had been vaccinated with alum precipitated HS vaccine two months earlier. Arif et al. (2013)

recently documented that subunit or whole bacterin based P. multocida vaccines were less

immunogenic than an anti-idiotypic vaccine prepared against P. multocida B: 2. Implication

of P. multocida as a mastitis pathogen has also been reported (OIE ,2012)

Isolation, cultural, biochemical characteristics of vaccinal isolates of S. aureus and Str.

agalactiae

Isolation, cultural and biochemical characterization was conducted following the

procedures as describe by brown et al. (1981), National Mastitis Council USA (1990), Athar

108

(2007) and Cengiz et al (2015). The procedures for isolation of S. aureus and Str. agalactiae

are inline with those of Gonzalez et al. (1989). Nikerson (1992), Chaudhry and Azam (1995)

and Fazal-ur-Rehman (1995). Staphylococcus aureus isolates (n=4) that grew on blood agar

were β- hemolytic, catalase positive, Gram positive cocci, 7-digit numerical profile with

api® Staph (bioMerieux, France) test strip = 6736153 and, Latex agglutination test

(Staphytect plus® kit) = positive and were thus presumptively identified as members of the

genus Staphylococcus. On performing tube coagulase test, all of these 4 isolates tested

positive for coagulase production at 4 hours. Staphytect plus® kit (Oxoid, UK) test was used

in the present study for additional confirmation of S. aureus based on the biochemical

differences between S. aureus and other coagulase positive staphylococci (Cohen, 1987).

Several components of S. aureus possess not only antigenic properties (ensuing antibodies

production) but also act as mitogens (i.e. they stimulate cell division) towards B-cells and T-

cells. Protein A is a good B-cell mitogen and techoic acid (a cell wall component) is both a

B-cell and T-cell mitogen. Clumping factor C is one of the surface proteins (adhesions)

which endows adherence properties to S. aureus thus enabling this organism to adhere to

mammary gland tissue (Sears, 2002). In the present study, presence of these cardinal

virulence factors of S. aureus was detected by the Staphytect plus® kit (Oxoid, UK).

Streptococcus agalactiae isolates (n=2) displayed β-hemolysis. Microscopic

examination of Gram stained smears of these isolates revealed Gram positive cocci and

showed api® 20 Strep (3462414). On catalase test, these isolates were negative. CAMP test

reaction on blood agar with 0.1% esculine and 0.01% ferric citrate was positive for both the

isolates.

All 4 isolates of S. aureus and 2 isolates of Str. agalactiae were sensitive to

lincomycin, enrofloxacin, amoxicillin, ceftiofur sodium and oxacillin, while resistant to

tetracycline. Turkish workers (Cengiz et al., 2015) determined the antibiotic resistance of 61

Staphylococcal isolates recovered from subclinically mastitic cows to oxacillin,

erythromycin, tetracycline, nitrocefin, and cefoxitin. Of these isolates (S. aureus, n=34 and

coagulase negative staphylococci, n=27), 26, 29, and 8 displayed resistance to erythromycin,

tetracycline, and oxacillin, respectively.This indicates indiscriminate use of certain

109

antibiotics would have impired resistance in S. ureus and Str. agalactiae and would easily

lead to therapeutic failure therefore antibiotic susceptibility profile is highly warranted.

Biofilm production by candidate vaccinal S. aureus isolates (n=4)

The term biofilm refers to a structured community of bacterial cells enclosed in a self-

produced polymeric matrix and adherent to an inert or living surface (Costerton et al., 1999).

The ability of an organism to produce biofilm (also known as slime production) is

significantly associated with its capability to produce diverse illnesses (Tenover et al., 1998).

Biofilm producing bacteria are known to cause numerous human diseases like cystic fibrosis,

native valve encarditis, otitis media, periodontitis and chronic prostatitis (Rodney, 2002).

Several studies have provided the evidence that S. aureus can form biofilms in the

udder of cows suffering from mastitis. Biofilm production endows upon the bacteria an

ability to impair the action of both the host immune system and antimicrobial agents and

allow the persistence of the infection (Melchior et al., 2006a; Melchior et al., 2009).

Recurrent infections are often attributable to biofilm production by bacteria. Biofilm

formation is accompanied by significant genetic and sub-sequent physiological changes in

the microorganisms resulting, inter alia, in a loss of sensitivity to virtually all classes of

antibiotics (Melchior et al., 2006a). Hensen (2000) reported that in bovine primary mammary

epithelial cell assay, sensitivity for neomycin, neomycin and penicillin, neomycin and

tetracycline, neomycin and bacitracin and tetracycline with bacitracin was higher for non-

adherent S. aureus than for adherent staphylococci. An exopolysaccharide matrix is

constituent of biofilm. Owing to this trait, biofilm resist the access of antibiotics to bacteria

(Stewart, 1996). The exopopolymers forming the biofilm shield the bacteria against

components of the host immune system. Extra-cellular slime (slime is the old term for

biofilm) produced by S. epidermidis protected cells from the phagocytic activity of

macrophages and against opsonins as well as reactive oxygen species in polymorphonuclear

leukocytes. Slime production by S. aureus strains was negatively associated with bactericidal

activity of neutrophils (Melchior et al., 2006a).

In the present study, three methods (tube method, spectrophotometric method and

Congo red agar method) were used for determination of biofilm production by 4

110

staphylococcal isolates. The tube method-based scores of biofilm production by 4 candidate

vaccinal S. aureus isolates. S. aureus isolate number 2, 3 and, 4 showed a moderate level of

biofilm production, whereas isolate number 1 showed strong biofilm production. All 4

isolates produced biofilms, however highest OD (0.31nm) was noted in isolate No. 1, whilst

OD values ranged between 0.17- 0.22. Thus the isolate No.1 was considered as highest

producer of biofilm in spectrophotometric method. On Congo red agar (CRA) S. aureus

isolate number 2, 3 and 4 showed intermediate degree of biofilm production whereas isolate

number 1 showed strong degree of biofilm production.

Turkish workers (Turkyilmaz and Eskiizmirliler, 2006) investigated the production of

slime factor in 180 staphylococcal isolates (90 coagulase positive staphylococcus; CoPS and

90 coagulase negative staphylococcus; CoNS) isolated from various animal specimens

(bovine mastitis, dogs with otitis externa and chickens with various infections) with Congo

red agar (CRA), microplate (MP) and standard tube (ST) tests and the results were compared

to each other. The rate of production of slime factor in all of the staphylococcal species

investigated with CRA, MP and ST tests was 61.1, 55.5 and 50.5%, respectively. Of a total

of 60 CoPS and CoNS isolates associated with mastitis, 40 (66.66%) were positive for slime

factor in CRA method. Frequencies of slime factor positive and slime factor negative isolates

of intramammary origin detected by two other methods (MP and ST) were not stated by these

investigators. According to Keskin et al. (2003), CoNS strains isolated from bovine mastitis

milk samples and chicken lesions were more slime productive and adherent as compared to

the strains isolated from healthy animals. Different methods were used for determination of

slime production. Citak et al. (2003) used CRA method to investigate slime production by

CoNS (32.7%) isolated from raw milk samples. They reported that slime production was an

important virulence factor for identifying pathogenic staphylococci. Mathur et al. (2006)

determined the frequencies and scores of biofilm production by 152 non-repetitive, human

clinical isolates of staphylococcus species isolated from blood, infected devices and skin

surface samples received from different tertiary care centers in India. Of the 152

staphylococcus species, 88 (57.8%) displayed a biofilm positive phenotype under the

optimized conditions in the tissue culture plate method and strains were further classified as

high 22 (14.47%) and moderate 60 (39.4%), while in 70 (46.0%) isolates weak or no biofilm

111

was detected. Though the tube method correlated well with the tissue culture plate test for 18

(11.8%) strongly biofilm producing strains, weak producers were difficult to discriminate

from biofilm negative isolates. Screening on Congo red agar did not correlate well with

either of the two methods for detecting biofilm formation in staphylococci. The presence of

the ICA (inter cellular adhesion, one of the components of the genetic make-up of a

particular strain related to biofilm formation in vitro) locus among S. aureus mastitis isolates

was recently investigated by Vasudevan et al. (2003). All 35 isolates tested possessed the

ICA locus, but not all isolates were positive with Congo red agar or the biofilm assay test.

This indicates that there is a high prevalence of the ICA genes among S. aureus mastitis

isolates. These investigators raised the question whether or not the observation that not all

strains produce biofilms ex vivo is related to specific culture conditions (biofilm growth is

stimulated by hypoxic conditions, but culturing in most cases occurs under oxic conditions).

Additional work in this area is clearly warranted. As indicated by the findings of present

study as well as previous studies (Turkyilmaz and Eskiizmirliler, 2006; Mathur et al., 2006;

Keskin et al., 2003, Melchior et al., 2009), the frequencies of biofilm production by different

isolates in a set of isolates is often a function of the methods used to determine biofilm

production trait.

Evaluation of sterility and safety of the Montanide® adjuvanted combined

HS-mastitis Vaccine

Evaluation of sterility and safety along with efficacy, simplicity of use, compatibility

with immune prophylaxis and affordability are the main components of quality of assurance

of the test vaccine (Soulebot et al., 1997). An attempts was made to follow all the standard

procedures as recommended by ISO (1994), FAO (1998) and OIE (2012), for confirming

sterility and safety of the experimental vaccines in laboratory animals. Albino mice, rabbits,

cattle and buffaloes were used for these preliminary studies and it was found that this vaccine

was safe to inject and free of demonstrable side-effects when given at the standard dose rates

(0.2ml IM for mice and 5 ml for cattle and buffaloes). However, this vaccine produced

sluggish movement in one mice, whereas rabbit showed mild local swelling. Similarly, cattle

and buffaloes exhibited moderate swelling when 5 ml of the vaccine was injected SC. As for

as vaccination–challenge studies were conducted in mice, vaccinated mice showed

112

significantly higher survival percentage as compared to those of control groups. The findings

of the present study are in agreement with those of Giraudo et al. (1997) and Athar (2007).

Effect of HS-mastitis combined vaccine on somatic cell count

The somatic cell count is an indicator of inflammation and milk quality (Sharif et al.,

2007). These cells are secreted during the normal course of lactation and are influenced by a

variety of factors like season, breed, environment and infection. The somatic cell count in the

normal buffalo milk varies from 0.5 × 105 to 3.75 × 10

5 with a mean of 1.4 × 10

5 cells per ml

(Silva and Silva, 1994). In cows, SCC in normal milk is always less than 1 × 105 cells per ml.

The possible reason may be compositional changes of the milk, environmental stress and

quarter udder capacity of the buffalo. In the present study values described by Silva and Silva

(1994) of somatic cell count in normal buffalo milk were used to differentiate normal and

mastitic milk. A non-significant decrease in somatic cell count was noted at days 60, day 120

in vaccinated-non-diseased cows and buffaloes. At day 180 post vaccination, somatic cell

count in this group was non-significantly higher than at days 0, 60 and 120. In unvaccinated–

non-diseased cows and buffaloes, a non-significant increase in SCC was recorded at days 60,

120 and 180. Vaccination of diseased cows and buffaloes caused a significant decrease in

SCC on all post vaccination observation time points (days 60, 120 and 180) albeit at day 180,

a slight increase in SCC was noticed. Somatic cell counts in unvaccinated –diseased cows

and buffaloes differed non-significantly at days 0, 60, 120, and 180. These somatic cell count

at 28 days post-treatment are consistent with that of Owens et al. (1988) and Owens et al.

(1995).

Cost-benefit analysis of Montanide® adjuvanted combined HS-mastitis

vaccine

The use of Montanide® adjuvanted combined HS-mastitis vaccine had a net financial benefit

worth Rs 2,060,300. Profitability can be more visible if more lactation length of the animal is

taken into account. Fabre et al. (2004) proved in a study that treatment could be profitable

during the first 6 months of lactation with expected cure rate of 70%. Its value would more

limited with a lower cure rate. In the calculation of cost-benefit ratio only the monitory value

of additional milk produced is consider. These calculations in this regard overlook the benefit

113

of vaccination in terms of milk quality (e.g. decrease somatic cell count). The results of

the present study are in line with the findings of Amorena et al. (1994) and

Shakoor (2006) that the vaccinated animals had a lower somatic cell count and

higher milk production in relation to the control group, suggesting that the

economic benefit of the vaccine may be 13 times higher than the cost.

Calculation of vaccine efficacy

Vaccine efficacy was measured by calculating the incidence rates (attack rates) of

disease among vaccinated and unvaccinated animals and determine the percentage reduction

in the incident rate of disease among vaccinated animals compared to un- vaccinated animals

(Weinberg and Szilagyi, 2010). Vaccine efficacy by using of Montanide® adjuvanted

combined HS-mastitis vaccine for HS and mastitis is 84.78%.and 90.25% respectively.

Similar result were in a separate study conducted in 3 commercial dairy herds in New

Zealand (Pankey et al., 1983)

In conclusion, the combined HS-mastitis vaccine developed and tested in the present study

indicated a preventative efficacy against HS and preventative as well as a curative efficacy

against mastitis. However, in view of preliminary nature of the present study as well as in

view of a fairly small number of animals utilized, a much broader study involving a much

larger number of bovine and bubaline animals is clearly warranted before a definitive

recommendation can be made for its field use.

Future outlook and recommendations

1. In view of a preliminary nature of the study, external validity (trials to be conducted

at other institutions as well as in the field) of the present study needs to be

established.

2. The role of combined HS-mastitis vaccine in conjunction with other mastitis control

measures (e.g. teat dipping, dry cow therapy, segregation etc.) needs to be

determined.

3. The role of combined HS-mastitis vaccine in the control of HS and heifer mastitis

need to be determined.

4. Larger field trials are needed in particular under field conditions.

114

5. Addition of other bacterial or viral pathogens (e.g Corynebacterium species, E.coli,

leptospira, FMD virus) in the „combo‟ vaccine tested in the present study may be

contemplated.

115

CHAPTER 6

SUMMARY

As per field surveys of major dairy animal diseases in Pakistan, hemorrhagic septicemia (HS)

and mastitis are the two most rife, endemic and economically important diseases of buffalo

and cattle. Hemorrhagic septicemia (HS) is an infectious, highly fatal bacterial disease

caused by Pasteurella multocida serotype B: 2 or Robert's type 1. Vaccination using different

types of vaccines (e.g. formalin-killed alum precipitated bacterin, oil adjuvanted vaccines) is

the single most important control measure against HS in Pakistan and in other Asian,

African, South European and Middle East countries where this disease is endemic. The

vaccines are quite effective when used as per the recommendations of manufacturers and

extension agencies. Alum precipitated bacterin is still the most commonly used HS vaccine

in Pakistan but it gives immunity for a short duration i.e. 3 months and its protective efficacy

is about 60% . Montanide® adjuvanted vaccines are considered to be superior to vaccines

containg other adjuvants. Mastitis is another common dairy disease which although not fatal,

causes colossal economic losses to our resource-poor dairy farmers and milk processing

industry. Research conducted over the past four decades has shown that S. aureus and Str.

agalactiae are the most prevalent mastitis pathogens in dairy animals in Pakistan. Whereas,

vaccination against HS is a fairly well established practice among Pakistan dairy farmers,

adoption of a mastitis control program is virtually nonexistent. Control of this mastitis is

imperative for economically viable dairy farming as well as to produce milk which conforms

to the standards of WTO Accord.

A combined HS-mastitis vaccine may potentially curtail the staggering economic

losses sustained by farmers due to these two diseases. Against this backdrop, the present

study was designed to develop and evaluated a Montanide® adjuvanted combined HS-

mastitis vaccine in laboratory settings and under field conditions. In phase 1 (preparation and

evaluation in laboratory settings), field isolates of P. multocida (n=3), S. aureus (n=4) and

Str. agalactiae (n=2) were scrutinized for their cultural and biochemical charateristics,

antibiograms, pathogenicity/virulence, and immunogenicity. The identity of P .multocida

116

serotype B and S. aureus was confirmed through PCR. The most pathogenic (LD50=10-7.75

; in

mice) and most immunogenic (in rabbits) P. multocida was selected for vaccine production.

From amongst the 4 candidate S. aureus isolates, the strongest biofilm producing (OD value

in spectrophotometric method =0.31) isolate with the highest pathogenicity (percent

mortality in rabbits=100) and immunogenicity (in rabbits) was selected for vaccine

production. Similarly, the more pathogenic (percent mortality in rabbits= 80) and more

immunogenic (in rabbits) of the 2 candidate Str. agalactiae isolates was selected for vaccine

production. Sterile bubaline whey was added @ 10% to nutrient broth for culturing the three

vaccinal organisms with a view to enhance expression of pseudocapsule of S. aureus and to

provide growth factors to Str. agalactiae for vaccine production. A 5ml dose of Montanide®

adjuvanted combine HS-mastitis vaccine contained 5×109 cells each of P. multocida, S.

aureus and Str. agalactiae along with their toxoids, 50μg thimerosal, 50μg sodium azide,

0.02 ml formalin and 2.5 ml Montanide® ISA 201. Evaluation of safety and side effects of

the test vaccine in cows (n=4) and buffaloes (n=4) with its double doses (10ml,

subcutaneously/ intramuscularly) indicated that the vaccine was safe and free of any

clinically significant side effect. The vaccine was found sterile and safe in rabbits. A

challenge-protection assay conducted in mice indicated 100% survival of challenged mice

that had been vaccinated twice 30 days prior to challenge. The vaccine was physically stable

in terms of pH, sedimentation, color, appearance, and syringibility for 6 months observation

period at 37°C.

In Phase II (field evaluation), the combined vaccine was evaluated in cows, buffaloes

and calves. To this end, a total of 70 S. aureus and Str. agalactiae free lactating animals

(buffaloes n=45; cows n=25 divided into vaccinal group A and control group B), 50

lactating cows and buffaloes microbiologically positive for S. aureus/Str. agalactiae

(buffaloes n=25, cow n=25 divided into vaccinal group C and control group D) and dairy

calves (buffalo calves n=70; cow calves n=50 divided into vaccinal group E and control

group F) aged up to 1 year were treated with 2 doses of combined HS-mastitis vaccine at 21

day interval and evaluated for 6 months in terms of ELISA based antibody titers against P.

multocida, S. aureus and Str. agalactiae, incidence of HS, local and systemic reactions,

incidence and prevalence of S. aureus and Str. agalactiae mastitis, mastitis screening tests

117

(Surf field mastitis test and CMT) reaction, severity of mastitis, milk somatic cell count,

effect of vaccination on milk yield, cost benefit analysis and vaccine efficacy.

Administration of Montanide® adjuvanted combined HS-mastitis vaccine produced

significantly (P˂0.05) higher ELISA-based antibody titers against P. multocida, S. aureus

and Str. agalactiae in vaccinated groups (A and C) than in un-vaccinated groups (B and D).

In general, the antibody titers were higher in non-diseased vaccinated (group A) animals

compared to diseased-vaccinated animals (group C) at 0, 60, 120 and 180 days post–

vaccination. It was observed that the antibody titers against P. multocida, S. aureus and Str.

agalactiae augmented at day 60 and 120 of vaccine administration in non-diseased

vaccinated animals and decreased at day 180 of post-vaccination. None of the 10 vaccinated

cows developed HS over the entire observation period of 180 days. Only 1 of the 30

vaccinated buffaloes (3.33%; group A) developed HS during this period. In the control group

(un-vaccinated-non diseased i.e group B), 1 cow (6.66%) and 2 buffaloes (13.33%)

developed HS during this period. None of the 25 cow calves vaccinated with Montanide®

adjuvanted combined HS-mastitis vaccine (group E) developed HS during the post

vaccination observation period of 6 months. One of the 50 buffalo calves (2%) vaccinated

with this vaccine (group E) developed HS during this period. One of 25 un-vaccinated cow

calves (4%; group F) and 3 of 20 buffalo calves (15%; group F) developed clinical HS over

the observation period of 6 months. Pasteurella multocida could be isolated from only 1

buffalo calf (group F) that succumbed to peracute HS. Isolation of P. multocida from other

cases was not successful.

Subcutaneously administration of vaccine was associated with a varying degree of

mild to moderate swelling at injection site in cows and buffaloes that subsided within 3-7

days while intramuscular administration caused either no or a very mild swelling at the

injection site that subsided within 48 hours. No systemic effects were noted (Table 4.20 and

4.21).

Determination of effect of Montanide® adjuvanted combined HS-mastitis vaccine on

quarter-based incidence of S. aureus and Str. agalactiae IMIs in cows and buffaloes

indicated that the quarter–based incidence of mastitis due to these 2 pathogens over the 6

months observation period was 3.3 folds higher in un-vaccinated cows and buffaloes.

118

Mastitis screening tests (Surf field mastitis test and CMT) based incidence of mastitis

in cows and buffaloes initially culture negative for S. aureus and Str. agalactiae indicated

that 4 of 40 quarters (10%) of vaccinated cows developed mastitis over the entire observation

period of 180 days. Only 2 of the 120 quarters (1.6 %) of vaccinated buffaloes developed

mastitis during this period. In the control group (un-vaccinated), 8 (13.33%) quarters of 15

cow and 6 (10%) quarters of 15 buffaloes developed mastitis during this period (Table 4.28).

Analysis of this parameter in cows and buffaloes initially positive for S. aureus and

Str. agalactiae indicated that 3 additional quarters of 15 vaccinated-disease cows (group C)

reacted positive in CMT/ SFMT over 180 days post vaccination period. The corresponding

figure in 15 vaccinated-diseased buffaloes (group C) was 2. In the un-vaccinated-diseased

cows (group D), 8 new quarters developed CMT/ SFMT positivity over 180 days post

vaccination period whereas the corresponding figure for the un-vaccinated –diseased

buffaloes (group D) was 6 ( table 4.30 , Figure 4.9).

The vaccine reduced the prevalence of S. aureus and Str. agalactiae IMIs over the

entire observation period of 180 days. Towards the end of study period (i.e. day 180), the

prevalence of Str. agalactiae and S. aureus IMIs was slightly higher than the prevalence of

these pathogens on day 60 and day 120. In the un-vaccinated- diseased group (group D), the

prevalence of these pathogens on days 60, 120 and 180 was higher than that on at day 0.

Mean± SEM of the mastitis severity score of group A (vaccinated; CMT/SFMT and

culture –ve for S. aureus/Str.agalactiae) was lower than those of group B (control;

CMT/SFMT and culture –ve for S. aureus/Str. agalactiae). Similarly, mean severity scores of

group C (vaccinated; CMT/SFMT and culture +ve for S. aureus/Str. agalactiae) were lower

(Table 4.32) than those of group D (control; CMT/SFMT and culture +ve for S. aureus/Str.

agalactiae).

Cumulative mean somatic cell counts in vaccinated groups (group A and C) were

significantly lower (P˂0.05) than those recorded in respective unvaccinated control groups

(group B and D). The vaccinated groups of both the species differed non-significantly

(P≥0.05) from each other.

119

When compared on the basis of overall milk production for a 6 months study period,

significantly higher (P<0.05) milk yield was recorded in vaccinated cows and buffaloes

(group A and C) as compared to respective unvaccinated control groups ( group B and D).

The vaccinated groups of both species revealed a non-significant difference from each other

with respect to milk yield. Cost-benefit analysis of vaccine indicated a net financial benefit

worth Rs 2,060,300.

The vaccine tested had efficacies of 84.78%.and 90.25% against HS and S.

aureus/Str. agalactiae associated mastitis.

In sum, Montanide® adjuvanted combined HS-mastitis vaccine tested in the present

study demonstrated a preventative role against HS and both preventative and curative role

against S. aureus and Str. agalactiae associated mastitis. In view of the preliminary nature of

the study, establishment of external validity as well as additional work involving much larger

number of cows, buffaloes and calves is clearly warranted.

120

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APPENDICES

Appendix 1: Composition of casein-sucrose-yeast (CSY) agar and

method of its preparation

Composition of Casein-Sucrose-Yeast (CSY) agar

Sr# Ingredients Ingredients (g/l)

1 Casein hydrolysate 3g

2 Sucrose 3g

3 Yeast extract 5g

4 Sodium chloride 5g

5 Anhydrous dipotassium hydrogen orthophosphate 3g

7 Distilled water 1 liter

8 Agar 15g

Method of preparation

The above given ingredients were dissolved in water. The pH was adjusted to 7.3–7.4, after

which 1.5% agar was added. The medium was autoclaved at 1 bar for 15 minutes. After

cooling to 45–50°C, 5% calf blood (antibody-free P. multocida) was added. Finally the

medium was dispensed into sterile petri plate. (0IE, 2012)

Appendix II: Preparation of blood agar medium and collection of

defibrinated blood

Blood from sheep was collected by adopting the procedure described by National Mastitis

Council Inc. (1990). That was

i. Juglar vein was cleared by clipping the hairs over the skin, swabbed with alcohol

followed by tincture iodine.

ii. Blood from the vein was collected in sterile disposable 50 ml syringe fitted with 18

gauge needle by adopting all aseptic precautions.

141

iii. Needle from syringe was removed after blood collection and poured gently into

sterilized flask of 250ml with glass beads previously sterilized by dry heat at 27oC for

40 minutes in hot air oven.

iv. As the blood pour into the flask, it was gently rotated.

v. To defibrinate the blood, flask was vigorously shaken for 10 minute after recapping.

Appendix III: Preparation of culture media and composition

A. Blood agar plates

Blood agar base 40g

Distilled water 1000 ml

Blood agar base manufactured by Difco Laboratories, Detroit, Michigan, USA was used in

this study. In order to tropicalize the medium 4 additional gram of agar technical was added.

Ingredient per litter

Ingredients Ingredients (g/l)

Beef heart infusion from 500.00 g

Bacto tryptose 10.00 g

Sodium chloride 5.00 g

Bacto agar 15.00 g

Additional agar 4.00 g

i. Following the instructions described by National Mastitis Council Inc. (1987)

medium was dissolved, sterilized by autoclaving and cooled to about 500C before

adding 5 percent defibrinated blood

ii. Medium poured into the plates, cooled and incubated at 370C overnight to check the

sterility. In order to preserve the moisture in the plates they stored in the refrigerator

in polythene bags. All plates were used within the period of two weeks.

B. Staph-110 agar plates

Staph-110 agar powder 40g

Distilled water 1000ml

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i. Staph-110 agar was manufactured by Oxoid Ltd, Basingstoke; Hampshire England

was used in this study. In order to tropicalize the medium 4 additional gram of agar

technical was added.

Ingredients (g/ liter)

Ingredients g/ liter

Yeast extract 2.5

Tryptone 10.0

Lactose 2.0

Mannitol 10.0

Sodium chloride 75.0

Di-potassium hydrogen phosphate 5.0

Gelatin 30.0

Agar 15.0

Additional agar 4.0

ii. Following the instructions described by National Mastitis Council Inc. (1987)

medium was dissolved, sterilized by autoclaving and cooled to about 50oC.

iii. Medium poured into the plates, cooled and incubated at 37oC overnight to check the



C.MacConkey Agar Plates

MacConkey Agar Powder 52g

Distilled water 1000ml

i. MacConkey agar was manufactured by Oxoid Ltd, Basingstoke, Hampshire England

was used in this study. In order to tropicalize the medium 4 additional gram of agar

technical was added.

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Peptone 20.0

Lactose 10.0

Bile salts 5.0

Sodium chloride 5.0

Neutral red 0.075

Agar 12.0

Additional agar 4.0

ii. Following the instructions described by National Mastitis Council Inc. (1987)

medium was dissolved, sterilized by autoclaving and cooled to about 50oC.

iii. Medium poured into the plates, cooled and incubated at 37oC overnight to check the



D. Nutrient Broth

i) Nutrient broth powder was manufactured by Merck, Germany.

Nutrient broth powder 8 grams



Peptone from meat 5

Meat extracts 3

ii) Following the instructions described by National Mastitis Council Inc., (1987) nutrient

broth was dissolved in distilled water, then sterilized by autoclave and cooled to 500C.

E. Brain Heart Infusion Broth (BHI)

Brain heart infusion broth powder 37g


i) Brain heart infusion broth powder was manufactured by Oxoid Ltd, Basingstoke,

Hampshire, England.

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Ingredient (g/liter)

Calf brain infusion solids 12.5

Beef heart infusion solids 5

Protease peptone 10

Glucose 2

Sodium chloride 5

Disodium phosphate 2.5

ii) Following the instructions described by National Mastitis Council Inc., (1987) broth

was dissolved in distilled water, then sterilized by autoclave and cooled to 500C.

F. Tryptone Soya Broth

Tryptone soya broth powder 30g


i) Tryptone soybroth powder was manufactured by Oxoid Ltd, Basingstoke, Hampshire,

England.

Ingredient (g/liter)

Pancreatic digest of casein 17

Pancreatic digest of soy bean meal 3

Glucose 2.5

Sodium chloride 5

Dibasic potassium phosphate 2.5

ii) Following the instructions described by National Mastitis Council Inc., (1987)

Tryptone soya broth was dissolved in distilled water, then sterilized by autoclave and

cooled to 500C.

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Appendix IV: Collection, processing and preparation of rabbit plasma

for tube coagulase test to differentiate coagulase positive

and coagulase negative Staphylococci

A tube coagulase test was used to differentiate coagulase positive and coagulase

negative Staphylococci. Tube coagulase positive test at 4 hours confirmed the isolates as

those of S. aureus.

Material required

Rabbit plasma (diluted 1:1 with normal saline). Fresh overnight culture of Gram

positive, catalase positive coccus. Glass tubes

Collection of rabbit plasma

A rabbit of approximately 1.1 kg body weight yielded about 30-38ml of blood. Five percent

solution of sodium EDTA was used as an anticoagulant for the collection of plasma from

rabbit blood.

Preparation of rabbit before slaughtering

The rabbit was restrained in lateral recumbency. The neck of the rabbit was swabbed

with a cotton swab moistened with normal saline to remove the dirt and broken hair attaching

to the body of the animal.

Preparation of EDTA solution

Five percent EDTA solution was prepared at a concentration of 1 g in 20 ml distilled

water. One and a half ml of this solution was gradually added to the blood that gushed from

the neck of the rabbit on slaughtering.

Collection of blood & preparation of plasma

Rabbit was slaughtered with a sharp knife and blood was collected in a beaker placed

under the neck of rabbit. EDTA solution was poured along the blade of the knife for proper

mixing of it with the blood while shaking the underlying beaker gently. The blood from the

beaker was poured into centrifugation tubes which were than centrifuge at 2000-2500rpm for

25 minutes. The plasma was then aspirated with the help of sterilized glass pipette. The

plasma thus separated was diluted 1:1 with sterilized normal saline. Aliquots of plasma were

dispensed into sterile eppendorf tubes which were then stored at -20oC.

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Test Procedure

1. Take rabbit plasma

2. Make 1:1 dilution of the plasma

3. Then 24 hours fresh growth of the isolate was inoculated

4. Tube was placed in incubator at 37oC for 4 hours

5. The plasma was changed into a coagulum after 4 hours in case of positive test and

there was no change in case of negative result.

Appendix V: Preparation of sterile milk whey (Watson and Watson, 1989)

To prepare one liter whey by rennin precipitation.

1) Rennin suspension was prepared by dissolving 0.5mg of rennin in 27ml of saline.

2) The suspension was then added to 1.5 liter defatted bovine milk and kept at 37ºC for

30 minutes

3) The samples were then centrifuged at 10,000Xg for 20 minutes.

4) Whey thus obtained was sterilized by passing first through 0.45 μm membrane filters

and then through 0.22 μm filter.

Appendix VI: CAMP (Christie, Atkins and Munch-Peterson)-Esculine test

(National Mastitis Council, Inc. USA, 1987)

Inoculate a CAMP-esculine plate by streaking a culture of beta-hemolysis producing

S. aureus in a straight line across the center. Picked suspect Streptococcal colonies and

streaked perpendicular to and within 2 to 3 mm of the S. aureus streak. Ten isolates may be

tested per plates, with five on a side incubate the plate inverted for 18-24 hours at 37ºC.

Interpretation: CAMP reaction –complete clearing in a half –circle fashion (arrow –head) in

the partial lysis zone of the S. aureus. Esculine hydrolysis –browning around organism

growth

Appendix VII: Somatic cell count

(Singh and Ludri, 2002; Shailja and Sing, 2002; Athar, 2007; Tanveer, 2009).

Somatic cell count was performed using the technique as modified by Athar (2007) from

Schalm et al. (1971)

1) 10 μl of milk samples were spread on slides having a marked area of 10 mm x 10 mm

using a micropipette, dried at temperature 30-40oC in an oven.

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2) The slides were dipped in xylene for 1-2 minutes to remove fat globules and dried

subsequently

3) The slides were then stained with Newman Lamperts stain for 15 minutes, took out

and dried at room temperature.

4) The dried slides were rinsed with tap water to remove excess of stain from the smear

and dried again at room temperature

5) The poorly stained smears were further stained with blue aliquot of Diff Quick Stain

(Difco Labs., Michigan, USA) for 10-15 seconds rinsed with tap water and dried.

6) Counting of cells at 100X in 50 fields which were multiplied by microscopic factor

(MF) to get the number of cells per ml of milk

MF =d x b

20 x 100

d = diameter of the microscopic field

b = number of microscopic fields

The number was then multiplied by 1000 to calculate number of somatic cells/ml of milk.

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