STUDIES ON WILDLIFE FORENSICS TO DETECT SPECIES ORIGIN … · i studies on wildlife forensics to detect species origin of sambar (cervus unicolor) meat by k. venkat ragavan b.v.sc

i

STUDIES ON WILDLIFE FORENSICS TO DETECT SPECIES

ORIGIN OF SAMBAR (Cervus unicolor) MEAT

By

K. VENKAT RAGAVAN B.V.Sc.

THESIS SUBMITTED TO THE

ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF

MASTER OF VETERINARY SCIENCE

IN THE FACULTY OF VETERINARY SCIENCE

DEPARTMENT OF VETERINARY PUBLIC HEALTH

COLLEGE OF VETERINARY SCIENCE, TIRUPATI

ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY

HYDERABAD

JUNE, 2003

ii

CERTIFICATE

Mr. K. VENKAT RAGAVAN has satisfactorily prosecuted the course of

research and that the thesis entitled “STUDIES ON WILDLIFE FORENSICS TO

DETECT SPECIES ORIGIN OF SAMBAR (Cervus unicolor) MEAT”

submitted is the result of original research work and is of sufficiently high standard

to warrant its presentation to the examination. I also certify that the thesis or part

thereof has not been previously submitted by him for a degree of any University.

Date : (Dr. S. UMAMAHESWARA RAO)

Major Advisor

Professor & University Head

Department of Veterinary Public Health

College of Veterinary Science

Tirupati – 517 502 (A.P)

iii

CERTIFICATE

This is to certify that the thesis entitled “STUDIES ON WILDLIFE

FORENSICS TO DETECT SPECIES ORIGIN OF SAMBAR (Cervus unicolor)

MEAT” submitted in partial fulfilment of the requirements for the degree of

MASTER OF VETERINARY SCIENCE of the Acharya N.G. Ranga Agricultural

University, Hyderabad, is a record of the bonafide research work carried out by Mr.

K. VENKAT RAGAVAN under my guidance and supervision. The subject of the

thesis has been approved by the Student’s Advisory Committee.

No part of the thesis has been submitted for any other degree or diploma. The

published part has been fully acknowledged. All assistance and help received during

the course of investigation have been duly acknowledged by the author of the thesis.

(Dr. S. UMAMAHESWARA RAO)

Chairman of the Advisory Committee

Thesis approved by the Student’s Advisory Committee

Chairman : (Dr. S. UMAMAHESWARA RAO) ______________

Professor and University Head



Tirupati – 517 502

Member : (Dr. P. MASTHAN REDDY) ______________

Professor and Head

Department of Livestock Products Technology



Member : (Dr. I. SANKARA REDDY) ______________

Associate Professor in Dairy Microbiology

Dairy Technology Programme



iv

LIST OF CONTENTS

CHAPTER

NUMBER

TITLE PAGE

NUMBER

I 1. INTRODUCTION 1

II 2. REVIEW OF LITERATURE 5

2.1 Anatomical and histological methods 5

2.2 Physical, chemical and biochemical methods 6

2.3 Sodium dodecyl sulphate – Poly Acrylamide Gel

Electrophoresis (SDS-PAGE)

6

2.4 Immunochemical tools 8

2.4.1 Double Immuno Diffusion (DID) test 9

2.4.2 Single Radial Immuno Diffusion (SRID) test 14

2.4.3 Immuno Electrophoresis (IE) 15

2.4.4 Counter Immuno Electrophoresis (CIE) 16

2.5 Enzyme Linked Immuno Sorbent Assay (ELISA) 17

2.6 Western blot technique (Electroblot Immuno Assay) 19

2.7 Iso Electric Focusing (IEF) 19

2.8 Other methods 21

2.8.1 DNA probe Technology 21

2.8.2 Polymerase Chain Reaction (PCR) 22

2.8.3 Wildlife Forensics – Micro satellite analysis of

DNA

22

III 3 MATERIALS AND METHODS 24

3.1 Preparation of sambar muscle protein antigens 24

3.1.1 Collection and processing of sambar flesh 24

3.1.1.1 Materials 24

3.1.1.2 Method 25

1. Determination of protein concentration 25

2. Standardization of the method of processing for

antigen preparation

25

v

CHAPTER

NUMBER

TITLE PAGE

NUMBER

3. Processing of sambar muscle 25

3.2 Preparation of fresh and thermostable muscle

protein antigens (TMP) of field samples (cattle,

buffalo, sheep, goat, pig and rabbit)

28

3.3 Production and preparation of hyperimmune sera to

sambar muscle protein antigens

29

3.3.1 Materials 29

3.3.2 Method 29

1. Immunization of rabbits with fresh muscle protein

antigens of sambar

29

2. Immunization of rabbits with thermostable muscle

protein antigens (TMP) of sambar

30

3. Immunization of sheep with fresh muscle antigens

of sambar

31

4. Immunization of sheep with thermostable muscle

protein (TMP) antigen of sambar

31

3.4 Preparation of binary meat mixtures 32

3.5 Immunochemical tools to detect wildlife sambar

meat

33

3.5.1 Double Immuno Diffusion (DID) test 33


3.5.1.2 Method 33

1. Pre-coating of the slides 33

2. Preparation and casting of the gel 34

3. Washing drying and staining of the gels 34

3.5.2 Single Radial Immuno Diffusion (SRID) technique 35


3.5.2.2 Method 35

3.5.3 Counter Immuno Electrophoresis (CIE) 37

vi

CHAPTER

NUMBER

TITLE PAGE

NUMBER


3.5.3.2 Method 37

3.6 Sodium Dodecyl Sulphate Poly Acrylamide Gel

Electrophoresis (SDS – PAGE))

38

3.6.1 Preparation of reagents 38

1. Acrylamide Bis acrylamide stock solution (100ml) 38

2. Resolving gel buffer (1.5m Tris – HCl pH 8.8) 39

3. Stacking gel buffer (0.5m Tris HCl buffer pH 6.8) 39

4. 10% Sodium Dodecyl Sulphate (SDS) 39

5. Sample Buffer 39

6. Tank Buffer 40

7. 10% Ammonium per sulphate 40

8. Staining solution 40

9. Destaining solution 40

3.6.2 Gel preparation 41

3.6.3 Gel preparation procedure 41

3.6.4 Sample preparation 42

3.6.4.1 Fresh antigen 42

3.6.4.2 Heated antigen 42

3.6.5 Sample loading 42

3.6.6 Staining and destaining 44

3.6.7 Molecular weight determination 44

3.7 Electroblot immuno assay (semi–dry) 45

3.7.1 Materials 45

3.7.1.1 Preparation of Buffers 45

1. Anode buffer I, pH 10.4 45

2. Anode buffer II, pH 10.4 45

3. Cathode buffer, pH 9.4 46

4. Tris buffered saline (TBS), pH 7.5 46

vii

CHAPTER

NUMBER

TITLE PAGE

NUMBER

5. TBS – Tween 20 (TBS – T) 46

6. Blocking solution 47

7. Antibody buffer 47

8. HRP labeled goat anti rabbit Ig – G conjugate

1/5000)

47

9. Substrate buffer (0.5 M sodium citrate pH 5.2 for

HRP system)

47

10. Substrate solution for HRP system 47

3.7.2 Method 48

IV 4. RESULTS 51

4.1 Development, preparation and standardization of

immuno chemical reagents for identification of

wildlife sambar meat

51

4.1.1 Standardization of the method of processing for

antigen preparation and preparation of sambar

muscle protein antigens

51

4.1.2 Preparation of sambar fresh muscle protein antigens 52

4.1.3 Preparation of thermostable muscle protein (TMP)

antigen of sambar

52

4.1.4 Production and preparation of hyperimmune sera to

sambar fresh muscle protein antigens

53

4.1.4.1 Hyperimmune sera in rabbits 53

4.1.4.2 Hyperimmune sera in sheep 53


thermostable muscle protein (TMP) antigens of

sambar

53

4.1.5.1 Hyperimmune sera in rabbits 53

4.1.5.2 Hyperimmune sera in sheep 54

viii

CHAPTER

NUMBER

TITLE PAGE

NUMBER

4.2 Preparation of fresh and thermostable muscle

protein (TMP) antigens of domestic species (field

samples)

54

4.3 Standardization of immunochemical methods to

detect wildlife sambar meat (fresh muscle antigen)

57

4.3.1 Standardization with rabbit antisera 57

4.3.1.1 Double Immuno Diffusion (DID) test 57

4.3.1.2 Counter Immuno Electrophoresis (CIE) 57

4.3.2 Standardization with sheep antisera 58



4.3.2.3 Comparison of species of animals for optimum

responsiveness in the antibody production against

sambar fresh muscle antigen

58

4.4 Standardization of immunochemical methods to

detect wildlife sambar meat (thermostable muscle

protein antigen)

63

4.4.1 Standardization with rabbit antisera 63


4.4.1.2 Single Radial Immuno Diffusion (SRID) test 63


4.4.2 Standardization with sheep antisera 64


4.4.2.2 Single Radial Immuno Diffusion (SRID) test 65



responsiveness in the antibody production against

sambar thermostable muscle protein (TMP) antigen

65

ix

CHAPTER

NUMBER

TITLE PAGE

NUMBER

4.5 Determination of protein profile of muscle protein

of sambar and different species of animals and

sambar by SDS – PAGE

67

4.5.1 Comparative analysis of SDS – PAGE of fresh

muscle proteins of sambar and different species of

domestic animals

67

4.5.2 Comparative analysis of SDS – PAGE of TMP

antigens of sambar and different species of animals

69

4.5.3 Comparison of the protein profile of fresh and TMP

antigens of sambar muscle protein

69

4.5.4 Comparison of the protein profile of fresh and TMP

antigen of other domestic animals

70

4.6 Electroblot immunoassay (western blotting) of

cooked meat (TMP antigens) of sambar and other

domestic species

70

4.7 Comparison of SDS – PAGE analysis and western

blotting of TMP antigens of sambar and other

domestic species

72

4.8 Detection of adulterated wildlife (sambar) meat

with the meat of domestic species by Immuno

Diffusion and Immuno Electrophoresis

73

4.8.1 Detection of sambar fresh meat adulterated with the

meat of domestic species

73

4.8.1.1 Detection with hyperimmune sera raised in rabbits 73

4.8.1.1.1 Double Immuno Diffusion (DID) test 73

4.8.1.1.2 Counter Immuno Electrophoresis (CIE) 76

4.8.1.2 Detection with hyperimmune sera raised in sheep 76



x

CHAPTER

NUMBER

TITLE PAGE

NUMBER

4.8.2 Detection of sambar cooked meat (TMP)

adulterated with the meat of domestic species

77

4.8.2.1 Detection with hyperimmune sera raised in rabbits

against sambar TMP antigens

77

4.8.2.1.1 Double Immuno Diffusion 77

4.8.2.1.2 Single Radial Immuno Diffusion (SRID) test 78


4.8.2.2 Detection with hyperimmune sera raised in sheep

against sambar TMP antigens

80


4.8.2.2.2 Single Radial Immuno Diffusion (SRID) test 80

4.8.2.2.3 Counter Immuno Electrophoresis 80

4.9 Detection of sambar meat adulterated with the meat

of domestic animals by electroblot immunoassay

(western blotting)

83

4.10 Detection of sambar meat adulterated with buffalo

meat by Electroblot immunoassay (western blotting)

83

V 5 DISCUSSION 84

VI 6 SUMMARY 101

LITERATURE 108

APPENDICES 118

xi

LIST OF ILLUSTRATIONS

FIGURE

NUMBER

DESCRIPTION

PAGE

NUMBER

1. Schematic flow diagram for preparation of fresh sambar

muscle protein antigens

26

2. Schematic flow diagram for preparation of thermostable

muscle protein (TMP) antigens of sambar

27

3. Arrangement of sheets in semidry blot unit 48

4. Photograph of the DID reactions comparing the

precipitation reaction of sambar fresh muscle antigen

against rabbit anti sambar sera and sheep anti sambar

sera. Peripheral wells contain I rabbit anti sambar sera

and II sheep anti sambar sera while the central wells

contains sambar muscle (fresh) antigen.

55

5. CIE of sambar muscle (Fresh) antigen. 56

6. Photograph showing the negative precipitation reaction

between cooked muscle extracts of sambar and domestic

animals against rabbit anti sambar serum.

59

7. Photograph showing the precipitation reaction of

thermostable muscle protein antigen of sambar against

sheep anti sambar (heated) sera.

60

8. Standardization of SRID test for sambar Thermostable

muscle protein (TMP) antigen perceptible ring diameters

of precipitation reaction was noticed only with 50%

rabbit anti sambar sera incorporated in the gel.

61

9. CIE of Thermostable muscle protein (TMP) antigen of

sambar.

62

10. SDS – PAGE analysis of fresh muscle antigens of

sambar and other domestic animals muscle samples.

66

xii

FIGURE

NUMBER

DESCRIPTION PAGE

NUMBER

11. SDS – PAGE analysis of Thermostable muscle protein

(TMP) antigen of sambar and other domestic muscle

samples.

68

12. Immunoblot analysis of Thermostable muscle protein

(TMP) of antigens of sambar and other domestic muscle

heated samples.

71

13. Photograph showing the precipitation reaction between

rabbit anti sambar (fresh) serum and sambar muscle

(fresh) antigen experimentally adulterated with buffalo

muscle (fresh) extract.

74

14. CIE of sambar muscle (fresh) extract experimentally

adulterated with buffalo muscle (fresh) extract.

75

15. CIE of Thermostable muscle protein (TMP) antigen of

sambar experimentally adulterated with buffalo muscle

(treated) extract.

79

16. Immunoblot analysis of 1% adulterated Thermostable

muscle protein (TMP) of sambar with other domestic

species muscle extracts.

81

17. Immunoblot analysis of adulterated Thermostable muscle

protein (TMP) antigen of sambar with Buffalo muscle

extract.

82

xiii

LIST OF TABLES

TABLE

NUMBER

TITLE PAGE

NUMBER

1 Preparation of binary meat mixtures 32

2 Protein concentration of fresh muscle extracts of

different species

52

3 Comparative studies on protein concentration of fresh

and heated muscle extracts (TMP) of different species

54

xiv

ACKNOWLEDGEMENTS

I avail this unique opportunity with pride to express my whole

hearted indebtedness to my guide Dr. S. Umamaheswara Rao Ph.D.,

Professor and University Head, Department of Veterinary Public

Health, College of Veterinary Science, Tirupati for his inspiring and

able guidance and unfailing support during the course of this research

and throughout my M.V.Sc programme.

I have immense pleasure in expressing my heartfelt gratitude to

the member of the advisory committee, Dr. P. Masthan Reddy,

Professor and Head, Department of Livestock Products Technology,

College of Veterinary Science, Tirupati for rendering his genuine

cooperation and help during the course of this investigation.

I am highly indebted to Dr. I. Sankara Reddy, Associate

Professor in Dairy Microbiology, Dairy Technology Programme,

College of Veterinary Science, Tirupati for acting as a member of the

advisory committee and for his marked advice while going through the

manuscript of the thesis.

My sincere thanks are due to Dr. A.Seshagiri Rao, Associate

Dean , College of Veterinary Science, Tirupati and to Dr.C.R.K

Reddy, then Associate Dean, College of Veterinary Science,

Tirupati for providing necessary facilities to carry out the present

investigation.

xv

My grateful thanks are due to Sri. K. Subba Rao, Principal Chief

Conservator of Forests, Govt. of Andhra Pradesh for giving kind

permission to collect sambar flesh. I am extremely thankful to Sri E.

Narasimhulu, I.F.S Curator, S V Zoological Park Tirupati for giving

necessary permission to collect sambar flesh for my research work.

My sincere thanks are due to Dr. Ramana, Zoo Veterinarian

Sri Venkateswara Zoological Park ,Tirupati without whose willing

co-operation and help, the collection of required sambar flesh for the

current investigation would not have been possible.

With deep sense of gratitude I acknowledge the help and

cooperation received from Dr. D.V.R. Sai Gopal, Ph.D., Department

of Virology, S.V. University, Tirupati for his help, whole hearted

cooperation and for his kind permission to avail the facilities in his

department.

I am highly thankful to Sri M. R. L. Prabhu, Head of the

Department of Biochemistry, College of Veterinary Science,

Tirupati for his encouragement, support and cooperation.

I am extremely thankful to Dr. V.H Rao, Associate Professor

and Head, Department of Animal Physiology, College of Veterinary

Science, Tirupati for kindly providing experimental rabbits for my

research work.

I am very much thankful to Dr. J. Rama Prasad, Professor and

Head, Department of Animal Nutrition, College of Veterinary

Science, Tirupati for his whole hearted cooperation and for kindly

providing experimental sheep for my research.

xvi

I am extremely thankful to Dr. P.K. Sriraman Ph.D., Head

(Rtd), Department of Pathology, College of Veterinary Science,

Hyderabad for his invaluable help and for taking excellent photographs

of my research work.

I Wholeheartedly express my grateful thanks to Sree N. Bhaskara

Reddy, M.Lib.Science, Librarian, ANGRAU Regional Library,

Tirupati for his help and cooperation.

I sincerely thank Dr. A. Jagdeesh Babu, M.V.Sc., and Dr. T.

Srinivasa Rao, M.V.Sc., Assistant Professors, Department of

Veterinary Public Health College of Veterinary Science, Tirupati for

their affectionate encouragement, help and cooperation.

I express my heartfelt gratitude to Mr. Venkata Prasanna,

Research Scholar, Department of Virology, S.V. University, Tirupati

for his technical help, immense support and encouragement and I

thankfully acknowledge the invaluable help and affection extended by

my colleagues and friends, Tamilmani, Rajarajan, Manigandan,

Anandaraja, Sarvana Kumar, Siva, Kumareshan, Upendra.

I have no words to express my heartful thanks to my father Sri.

M. Kandasamy, mother Smt. K. Baby Rani and sister Miss.

Premalatha and Mrs. R. Jayachitra for their inspiration, moral

support, everlasting affection and encouragement during the course of

my study.

I express my sincere thanks to Mr. V. Chandra Sekhar for his

excellent typing of the thesis.

(K. VENKAT RAGAVAN)

xvii

Name of the author : K. VENKAT RAGAVAN

Title of the thesis : “STUDIES ON WILDLIFE FORENSICS

TO DETECT SPECIES ORIGIN OF

SAMBAR (Cervus unicolor) MEAT”

Degree to which it is

submitted : MASTER OF VETERINARY SCIENCE

Faculty : VETERINARY SCIENCE

Department : VETERINARY PUBLIC HEALTH

Major Advisor : Dr. S. UMAMAHESWARA RAO BVSc., MSc(Vety.)., Ph.D.

Professor and University Head




University : Acharya N.G. Ranga Agricultural

University

Year of Submission : June, 2003

ABSTRACT

Attempts were made to develop suitable wildlife forensic tools

with immunologic basis to detect and identify sambar (Cervus unicolor)

meat in the experimentally adulterated meat samples of domestic animal

species viz., buffalo, cattle, sheep, goat, pig and rabbit both fresh and

cooked (thermostable muscle protein (TMP)) meat.

xviii

PBS, pH 7.3 was found to be a suitable diluent for preparation of

antigens both from fresh and cooked meat of sambar, buffalo, cattle,

sheep, goat, pig and rabbit.

Two kinds of experimental animals viz. rabbit and sheep were

immunized with sambar muscle protein antigen both fresh and cooked

(TMP) to study their ability to develop specific antibody. The rabbit, a

distinct phylogenetic animal was found to be more suitable when

compared to sheep which is a phylogenetically related species as

evidenced by the immunocompetence of the sera to recognize and react

with TMP antigens leading to the formation of precipitation reaction.

The sambar flesh, the source of antigen for the current

investigation was collected immediately from the naturally dead sambar

(either by natural death or by accidental death without any infectious

etiology) from Sri Venkateswara Zoological Park, Tirupati.

The conventional immunochemical methods like DID and CIE

tests were used to detect wildlife sambar meat, both fresh and cooked

(TMP) muscle antigens, were standardized using hyperimmune sera

xix

raised in both, rabbit and sheep. The results of precipitation reaction

were quick within 6 hours of incubation for fresh muscle antigens with

rabbit and sheep antisera, while the precipitation reaction was delayed

by 72 hours for TMP antigens with rabbit antisera only but the reaction

was absent with sheep antisera.

The protein profile of sambar fresh and TMP antigens was studied

by subjecting to SDS – PAGE and a comparison was made with those of

other domestic animal species. The electrophoretogram revealed that

sambar fresh muscle protein was resolved into seven protein bands with

molecular weights of 60kd, 52kd, 44kd, 42kd, 30kd, 22kd and 12kd,

while the buffalo muscle protein profile resolved into five protein bands

with molecular weights of 60kd, 52kd, 44kd, 42kd and 22kd. The cattle

meat profile was resolved into three protein bands: 40kd, 30kd and 12kd,

while the sheep meat protein profile resolved into six bands: 60kd, 52kd,

44kd, 22kd and that of goat meat protein profile was resolved into five

protein bands: 60kd, 52kd, 44kd, 42kd and 12kd.

The electrophoretogram of TMP antigens of sambar and other

species of domestic animals like buffalo, sheep, goat and pig except

cattle showed a partially denatured two protein complex with diffused

xx

migration pattern (44kd and 42kd). The two low molecular weight

protein bands 22kd and 12kd were present only in sambar probably

species specific.

The electroblot immuno assay (western blot) technique could

identify serologically related TMP antigens of 44kd and 42kd of sambar,

buffalo, sheep, goat and pig. Another serologically related protein 40kd

was noticed only in cattle although not appreciated very well in sambar.

Two serologically identified proteins with molecular weight of 22kd and

12kd were present only in sambar, possibly species specific.

The tests like DID and CIE were carried out using rabbit or sheep

antiserum to detect adulteration of sambar fresh meat from binary

mixtures of fresh meat of buffalo, cattle, sheep, goat, rabbit and pig.

These tests could detect the fresh meat adulteration up to 1% level while

detection of sambar cooked meat (TMP) adulterated with cooked meats

(TMP) of domestic animals could be up to a level of 10% only with

rabbit antisera. The sheep antisera against TMP antigen of sambar did

not detect the adulteration.

xxi

Western blotting could identify cooked (TMP) sambar meat

adulterated with the cooked (TMP) meats of buffalo, cattle, sheep, goat

and pig as low as up to a level of 1%. The experimental adulteration of

cooked (TMP) sambar meat with cooked buffalo meat when subjected to

western blotting, the detection was as low as 0.1% but not at 0.05%

level.

xxii

LIST OF ABBREVIATIONS

ADS - Ammonium per Sulphate

AGIEF - Agarose Gel Iso Electric Focussing

B – C - Buffalo muscle extract – Cooked

B – F - Buffalo muscle extract – fresh

BE - Boiling resistant Ethanol

C – C - Cattle muscle extract – Cooked

C – F - Cattle muscle extract – fresh

CIE - Counter Immuno Electrophoresis

COE - Cross Over Electrophoresis

DAB - Di amino benzidine

DID - Double Immuno Diffusion

DNA - De Oxyribo Nucleic Acid

ELISA - Enzyme Linked Immuno Sorbent Assay

F – F - Fowl muscle extract – fresh

FCA - Freund’s Complete Adjuvant

G – C - Goat muscle extract – Cooked

G – F - Goat muscle extract – fresh

HA - Haem agglutination

HCl - Hydrochloric Acid

HRP - Horse Radish Peroxidase

IE - Immuno Electrophoresis

IEF - Iso Electric Focussing

Kg - Kilogram

xxiii

MW - Molecular Weight

P – C - Pig muscle extract – Cooked

P – F - Pig muscle extract – fresh

PBS - Phosphate Buffer Saline

PCR - Polymerase Chain Reaction

PVDF - Poly Vinylidine Diflouride

R – C - Rabbit muscle extract – Cooked

S – C - Sheep muscle extract – Cooked

S – F - Sheep muscle extract – fresh

SDS – PAGE - Sodium Dodecyl sulphate – Poly Acrylamide

Gel Electrophoresis

SRID - Single Radial Immuno Diffusion

TBS - Tris Buffered Saline

TBS - Tris Buffered Saline – Tween

TMP - Thermostable muscle protein

µl - Microlitre

µg - Microgram

V/V - Volume / Volume

xxiv

CHAPTER I

1. INTRODUCTION

Preservation of wildlife ecosystem and protection of wildlife

fauna and flora are imperative and uncompromised edicts to maintain

natural ecological balance.

International trade in wildlife and its products is worth around $17

billion a year (Agrawal, 2000). Unfortunately illegal trade in wildlife,

probably the world’s second largest illegitimate business, is on its

increase. In order to check this menace, a convention of international

trade in endangered species (CITES) of world flora and fauna was

brought into existence by International union for conservation of nature

and natural resources (IUCN). The government of India had also enacted

the wildlife (protection) Act 1972 to provide protection for the

endangered and threatened species and their hunting is prohibited.

However, the opening up of international market for meat foods

and also increased desire of man to taste new culinary dishes prepared

with wildlife meat may lead to fraudulent substitution of meat from

protected species of wildlife. The meat of such animal species at times

1

xxv

poached or often misrepresented for flesh of cattle, buffalo, sheep, goat

or pig.

Under such circumstances speciation of meats and meat products

is mandatory and it is therefore essential to develop an appropriate

methodology to detect the species origin of such meats in order to

contest in a court of law.

Determining the species of meat origin is an integral part of food

regulatory control with respect to economic fraudulence. Besides this,

correct species identification is important to the consumer for reasons of

specific food allergies or religious dietary restrictions.

In order to combat these problems, species identification of meats

acquired great importance and is a challenging task in food hygiene,

food quality control programmes, medico legal cases and veterinary

forensic medicine. Authentic detection of species origin of meat

however remains a nagging problem for food regulatory agencies at

national and global level.

In spite of decades research carried out in this field, the

problem still remains unsolved in the absence of a single reliable fool

xxvi

proof technique to detect such unscrupulous mixing. The potential of

immunoassay as a method of choice for replacing less specific and more

costly methods has been recently appreciated.

Sambar, black buck and other deer species are prohibited from

hunting in Andhra Pradesh as per wildlife (protection) Act, 1972 and

other reserve forest regulations. Sambar are frequent victims targeted by

poachers for their meat and hide. The meat of such animal species is

often misrepresented for flesh of cow, buffalo, sheep or goat leading to

medico legal cases (Umamaheswararao, 2003).

With scanty reported literature on wildlife forensics, a study was

under taken to develop a methodology for the detection of sambar meat

experimentally adulterated with the meat of other domestic animal

species using immunochemical techniques with the following objectives.

1. To study the efficacy of using fresh and thermostable muscle protein

(TMP) antigen of sambar to develop species specific antibodies in

rabbits and sheep in order to identify the fresh as well as cooked

sambar (Cervus unicolor) meat.

xxvii

2. To evaluate the suitability of immunochemical techniques like

double immuno diffusion (DID) test and counter immuno

electrophoresis (CIE) test etc., in identifying fresh as well as cooked

sambar muscle.

3. To apply the so developed wildlife immunochemical forensic tool to

detect sambar meat when adulterated with the meat of other domestic

animals.

xxviii

CHAPTER II

2. REVIEW OF LITERATURE

The identification of species origin of meat is not only imperative

in a given case but also the detection of level of adulteration is equally

essential for appropriate regulatory control of marketing of such meats.

Methods employed generally to detect the origin of meats are

anatomical and histological; physical, chemical and biochemical;

electrophoretic; immunochemical; enzyme linked immunosorbent assay

(ELISA); Immunoblotting and Isoelectric focusing techniques. Hence a

brief survey of the literature pertaining to these techniques would enable

the investigator to choose the most expedient procedure bearing in mind

the circumstances expected: whether the test should cover domestic

species, wild species or closely related species.

2.1 ANATOMICAL AND HISTOLOGICAL METHODS :

The species identification of meats based on anatomical and

histological methods such as length of carcass, ribs on the thorax and the

size of the muscle fibers is not always useful in the detection of

adulteration and lacks authenticity especially in forensic cases. 5

xxix

2.2 PHYSICAL, CHEMICAL AND BIOCHEMICAL METHODS:

Colour, consistency and texture of meats were among the physical

characteristics used for differentiation of meats which are highly variable

within same species (Ginsberg, 1948 and Krishna, 1998).

According to Sherikar et al (1988) chemical tests for species

identification of meats such as estimation of body fats, refractive index,

Iodine number, carotene content , fatty acid composition, linoleic acid

content, ratio of histidine dipeptides, glycogen content, myoglobin

content, muscle enzymes and muscle nitrogen fractions are highly

variable and cannot be used with accuracy to rely upon.

2.3 SODIUM DODECYL SULPHATE - POLY ACRYLAMIDE

GEL ELECTROPHORESIS (SDS-PAGE) :

SDS-PAGE electrophoresis was successfully applied by several

workers to study the band pattern of sarcoplasmic proteins and

myofibrillar proteins (Scopes and Penny, 1971 and Rattire and

Regenstien, 1977).

Ramadas (1972) reported species specific muscle protein band

patterns of bullock, buffalo, dog, pig and poultry while in case of sheep

and goat the band patterns were identical but different from other

xxx

species. Prasad (1974) using SDS – gel electrophoresis, established a

relationship of muscle protein band pattern of different species between

different age and sex groups.

Lawrie et al (1977) while studying the identification of meats in

fresh and processed foods by starch gel electrophoresis and PAGE,

indicated water insoluble myofibrillar and water soluble sarcoplasmic

muscle protein patterns for quantitative identification of meat proteins.

Babiker et al (1981) used a modified linear gradient poly

acrylamide gel electrophoresis technique and identified cooked horse

meat (1200C) from cooked beef on the basis of myofibrilar protein

pattern.

Yamamoto et al (1982) advocated the use of SDS – PAGE for

the detection of various non meat extenders in meat products.

Govindarajulu (1982) differentiated beef from mutton on the basis

of species specific electrophoretic pattern on starch gel electrophoresis.

Anneli Skrokki and Osmo Harmi (1994) quantitatively

differentiated beef, pork, venison, reindeer and mutton and detected

adulterated minced beef with pork by employing modified PAGE.

xxxi

Nath et al (1999) by employing SDS – PAGE made it possible to

identify the meats of sheep, goat, cattle, buffalo, pig, chicken based on

total number of protein bands, their relative position and concentration,

ionic charges together with differences in RF values.

2.4 IMMUNOCHEMICAL TOOLS :

The remarkable specificity of serological reactions was amply

demonstrated by Landsteiner (1945). Various immunochemical methods

adopted by different workers gave promising results in the identification

of species origin of meats.

Fresh meat antigens can be successfully used to produce species

specific antibodies for the identification of various meats. But the

identification of cooked meats through serological means will be

difficult as the muscle protein may get denatured during heating. The

main disadvantages of immunochemical techniques were cross reactions

among related species and failure to identify the origin of cooked meats

(Krishnamurthy, 1998).

2.4.1 Double Immuno Diffusion test (DID) :

xxxii

The application of double immuno diffusion (DID) or Agar gel

precipitation test (AGPT) was first carried out by Pike and Sulkin (1957)

and they could detect the adulteration of beef with horse meat.

Fugate and Shelton (1971) employed immuno diffusion technique

for the detection of mixed muscle samples.

Ramadas (1972) observed that agar gel immuno diffusion (AGID)

technique was a reliable technique for differentiating meats of closely

related species.

Poli et al (1977) differentiated animal proteins in cooked sausages

employing agar gel diffusion method.

Pandey and Pathak (1974) could not distinguish the cow meat

from buffalo meat by agar gel diffusion test.

Beljaare and Oleman (1977) compared PAGE and DID test for

detection of non meat protein in meat products and concluded that DID

was superior over PAGE.

Flego and Bor gheese (1979) made comparison of DID with that

of cross over electrophoresis (COE) method and concluded that COE

xxxiii

was 26-45 times more sensitive than DID in the detection of pork, beef,

horse and chicken meat.

Sherikar et al. (1979) concluded that inspite of cross reactions,

DID is useful in differentiating meats of various species viz, cattle,

buffalo, sheep, goat, pig and poultry.

Hayden (1979) used myoglobin of ovine, porcine and equine

muscle as antigens for antibody production in rabbits and successfully

detected the presence of meat of these species.

Hayden (1980) could successfully employ agar gel immuno

diffusion and could quantitate the pig, horse and rabbit flesh in ground

beef using troponin and myoglobin as test proteins.

Hayden (1981) could detect mammalian adrenal antigens in beef

sausages at 5% level of adulteration while the avian kidney – adrenal

antigens were detected at 10% level of adulteration by means of agar gel

immunodiffusion technique.

Nath (1986) experimented on the use of cross immunization in

phylogenetically related species using freeze dried muscle extractives as

xxxiv

antigen and successfully detected the species origin of fresh meats by

using DID.

Sherikar et al. (1987) used extracts of raw and partially heated

(700C) muscles of buffalo, cattle, sheep, goat, pig and poultry as antigen

and raised anti rabbit antibody to identify and differentiate their raw,

heated (700C) and boiled (100

0C) meats. They observed cross reactions

between related species but anti chicken sera and antisera to heated pig

muscle extracts gave species specific reaction. Boiled (1000C) meat

extracts did not elicit any antibody production and did not react with

these antisera. Antibody to raw meats stored at -200C for 9 years were

found continue to yield similar results as that of fresh antiserum.

Bansal and Mandokhot (1988) used DID, Counter

immunoelectrophoresis (CIE), Immunoelectrophoresis (IE) and micro

passive haemagglutination (HA) test to detect beef, sheep, goat, pig and

chicken meats and proved that DID and CIE were equally sensitive and

specific and concluded that DID was more suitable because of its

simplicity and reliability.

xxxv

Sherikar et al. (1988) identified species of origin in cooked (700C)

meat mixtures of closely related species by agar gel immuno diffusion

technique using specific antisera to adrenal heat stable antigens.

Radhakrishna et al. (1988) reported immunogenic nature of

thermostable muscle protein (TMP) of goat, sheep, ox and buffalo and

the sera raised against these proteins were made monospecific by cross

absorption technique and thereby they could identify the respective TMP

as well as the extractives from raw beef and from those canned spiced

mutton curry.

Karkare et al. (1988) studied species origin of meat in

commercially processed canned meat products viz, beef, kheema, corned

beef, mutton kheema, mutton chunk curry and pork mince. Species

specific antisera was raised in rabbits to heat stable antigens of adrenal

gland of cattle, buffalo, sheep, goat and pig and conducted DID and CIE

by using saline extracts and ethanol precipitated proteins of the canned

meat products as antigens. Their results revealed that rabbit anti adrenal

sera could identify the species origin of meats in canned products while

other components in the canned meat products viz, additives, condiments

etc., did not interfere in the test reactions.

xxxvi

Suryarao (1990) conducted experiments in identifying the species

origin of meats using testicular antigens of sheep, goat, cattle and buffalo

employing serological tests including DID, IE, CIE and rocket immuno

electrophoresis technique. He could detect adulterated meat as low as at

1% level in fresh meats and at 10-20% level in cooked meat mixtures.

Reddy et al. (1990) made the antisera raised against adrenal

boiling resistant and ethanol precipitable (BE) antigens of cattle and

sheep species specific by immuno absorption and detected cooked meat

at 1% level using DID.

Saisekhar and Reddy (1995) developed monospecific antisera to

troponin of cattle and buffalo and detected fresh beef at 10% level and

fresh buffalo meat at 1% level but these sera could not detect cooked

meats.

Reddy and Reddy (1995) could detect either fresh or cooked

meats as low as at 10% level in cooked state and at 20% level in fresh

condition from a mixture of beef or cara beef or mutton using DID

technique.

xxxvii

Sasidhar (1995) developed species specific sera against native as

well as heated porcine testicular extract antigens and detected cooked

pork up to a level of 2% and fresh pork up to 0.5% level with DID and

CIE techniques.

Sridharlingareddy (1996) concluded that, of the tests employed

DID and CIE were superior to detect adulteration in fresh meats of

cattle, buffalo and pig using brain extract antigens of respective species.

2.4.2 Single Radial Immuno diffusion (SRID) test :

Suryarao (1990) conducted single radial immuno diffusion (SRID)

to find out fresh buffalo meat antigen and concluded that SRID can be

used to determine the level of adulteration.

2.4.3 Immuno Electrophoresis (IE) :

Graber and Williams (1953) were the pioneers of this technique

and they could find out the use of immunoelectrophoresis (IE) for

analyzing the quality and purity of antibody rich serum fractions of

immunized animals.

xxxviii

Durand and Schneider (1962) used IE for species identification of

cattle, buffalo, horse, sheep, goat, donkey and camel by observing

precipitation lines and their number and position.

Ramadas and Misra (1981) employed IE for species

differentiation of bullock, buffalo, horse, goat, sheep, pig and chicken

according to the number and position of precipitation arcs by using alum

precipitated muscle extracts of the respective species as antigens.

Reddy (1986) successfully used IE and detected species specific

antibodies for identification of cooked beef and mutton. His studies

further revealed that two precipitation lines found to be common for all

the antigens tested viz., muscle extract antigens of cattle, buffalo, sheep

and goat.

2.4.4 Counter Immuno Electrophoresis (CIE) :

The diffusion of antigen and antibody is facilitated by the

application of low voltage current and by using this technique,

Schweiger et al. (1982) and Brehmer (1983) identified species origin of

meat in various mixed meat products.

xxxix

Nath (1986) used freeze dried muscle extracts as antigen for

immunization in phylogenetically related species and employed DID and

CIE for species identification and found that CIE was more sensitive

than DID.

Allsup (1987) concluded that CIE was more rapid and highly

sensitive in species identification of fresh and frozen meats and offals

using commercially produced bovine, porcine and equine antisera.

Karkare et al. (1988) employed DID and CIE using adrenal

boiling resistant and ethanol precipitable (BE) antigens for species

identification of cattle, buffalo, sheep, goat and pig and concluded that

CIE was most sensitive.

Lajon et al. (1989) detected added poultry meat in charcuterie

products by employing DID, CIE, Lourell IES and ELISA. CIE and

Lourell IES methods detected up to a level of 15% added poultry meat

while ELISA could detect up to 5% fresh meats. None of these

techniques could detect poultry meat in cooked samples.

Srinivasarao (1996) employed DID, CIE and IE tests and

concluded that DID and CIE were more sensitive than IE in the detection

xl

of adulteration using native brain extracts of sheep, goat and rabbit as

antigen.

Using CIE, species of origin in cooked meat mixtures of closely

related species have been identified by Sherikar et al. (1988), Reddy et

al. (1990), Suryarao (1990), Nath et al. (1999) and Sivakumar et al.

(1999).

2.5 ENZYME LINKED IMMUNO SORBENT ASSAY ( ELISA) :

Kang’ethe et al. (1984) and Manz (1985) employed ELISA for the

detection of adulteration in cooked meats both qualitatively and

quantitatively using heat treated beef and pork muscle containing antigen

component precipitated in the alpha 2 (α 2) globulin region.

Both competitive and indirect ELISA procedures were capable of

detecting as low as 5% pork or mutton in beef (Dincer et al., 1987).

Kang’ ethe and Gathuma (1987) used thermostable muscle antigen

and developed monospecific antisera in goats and detected adulteration

of cooked, sterilized and autoclaved meat and meat products. Kang’ethe

and Lindquist (1987) using ELISA could detect beef in pork sausages at

10% and 5% level but not at 1% level.

xli

Sawaya et al. (1990) using antisera in sheep raised against

autoclaved porcine muscle extract could detect pork at 1% level mixed

in beef and mutton at 0.5% level.

Sherikar et al. (1993) compared various immunological

techniques such as DID, CIE and ELISA in meat speciation using

thermostable species specific anti adrenal sera and found that ELISA

could detect adulteration as low as at the 1%level.

Gacheru et al. (1994) described a sandwich ELISA for speciation

of cooked meat samples of cattle, buffalo, camel, pig, horse and bush pig

and detected the adulterants at 1% level.

Mandokhot and Kotwal (1997) advocated the use of thermostable

antigens in the ELISA test to get promising results in the identification

of cooked meats.

2.6 WESTERN BLOT TECHNIQUE (ELECTROBLOT IMMUNO

ASSAY) :

By Western Blot technique, Chen and Hsieh (2001) characterized

a porcine specific thermostable muscle protein from cooked pork

successfully and the same was identified as troponin.

xlii

2.7 ISO ELECTRIC FOCUSING (IEF) :

Sinclair and Slattery (1982) reported that differentiation of buffalo

meat from beef as well as sheep, horse, kangaroo and pig was possible

by employing isoelectric focusing (IEF) but the differentiation of closely

related species such as sheep and goat or horse and donkey was not

possible by this method.

Isoelectric focusing was a sensitive and suitable technique for

identification of fresh meats according to Abraham and Leela Nayar

(1983).

Abraham (1987) found that agarose isoelectric focusing technique

was found to be effective for species identification of raw and cooked

meats. Species specific differences existed in the protein band profiles of

beef, cara beef, chevon and mutton on agarose gel isoelectric focusing

(AGIEF) using urea extracts of raw meats and this technique could be

used for rapid and reliable species identification in the raw meats.

Sherikar et al. (1988) successfully demonstrated that buffalo,

cattle, sheep, goat, pig and poultry meats could be differentiated by

using poly acrylamide gel isoelectric focusing. The bands of the raw and

xliii

heated (700C) meats behaved characteristically and distributed in a

particular pH range specific for respective species. However, the bands

of boiled and autoclaved meats were diffused and could not be

differentiated.

Mc Cormick et al. (1992) employed IEF technique in the

detection of meat from game and domestic species. Banding patterns in

meat from prong horn, mule, deer, white tailed deer, sheep, moose, pork

bison, elk, caribon, reindeer, beef and goat could be characterized with

IEF combined with specific enzyme staining for creatine kinase.

However the inability to separate some games species from domestic

species was the limitation of the application of this technique.

2.8 OTHER METHODS :

2.8.1 DNA probe Technology :

DNA assay technique was applied to the identification of meat

species specific sequence of DNA in the meat (Chikuni et al., 1990;

Wintero et al., 1990; Ebbehoj and Thomson, 1991 a & b). However the

probes prepared from genomic DNA showed cross reactions between

cattle, buffalo and sheep (Chikuni et al., 1990).

xliv

Ebbehoj and Thomson (1991a) concluded that the DNA

hybridization technique applying 32p labeled probes were useful for

quantitative determination in the quality control of heat treated meat

product.

Wintero et al. (1990) compared DNA hybridization with other

techniques and could detect raw pork in beef at 0.4% by CIE, 1% by

DID, 0.5% by IEF and DNA hybridization.

2.8.2 Polymerase Chain Reaction (PCR) :

Chikuni et al. (1994) by employing polymerase chain reaction

(PCR) technique showed 92% of homology between sheep and goat and

they were differentiated by APa1 digestion of the PCR products because

sheep had I A APa1 site and goat had no site in the PCR products.

Rasool et al. (1995) developed a simple method for the

identification of raw meats of various species based on repetitive DNA

sequence. They also further showed that raw meat derived from the

family bovidae can be identified using restriction enzyme analysis.

xlv

Matsunaga et al. (1999) could successfully employ PCR to

identify species origin of meats from cattle, pig, chicken, sheep, goat and

horse.

2.8.3 Wildlife Forensics – Micro satellite analysis of DNA :

Micro satellite DNA markers have been determined for population

samples of moose and white tailed deer and the genotyping of individual

samples have been automated using DNA sequence and gene scan and

genotype software, and these innovations facilitated rapid analysis of

evidence in criminal cases (Irv Kornfield 1999).

Coffin et al. (1999) developed micro satellite loci for a number of

big game species including pronghorn.

Wilson and White (1999) have developed a protocol using highly

repetitive satellite DNA markers to identify and quantify the amount of

game tissue present in a processed meat sample. They have used p32

radio actively labeled genomic DNA of the game species being detected

in hybridized to southern blotted DNA digested with species diagnostic

restriction enzymes which revealed species specific banding patterns

without cross homology to other game or domestic species.

xlvi

CHAPTER III

3. MATERIALS AND METHODS

All the glassware used in this work were of Corning (India) make.

The chemicals used to prepare different buffers and reagents were of

either Analar (BDH) or E-Merck. The fine chemicals used were of

Sigma make. Quartz distilled water (pH 6.8 to 6.9) was used throughout

this work to prepare buffers and reagents.

3.1 PREPARATION OF SAMBAR MUSCLE PROTEIN

ANTIGENS :

3.1.1 Collection and processing of sambar flesh :

3.1.1.1 Materials :

xlvii

1. PBS, pH 7.3 (Appendix I)

2. 0.15M saline

3. Sambar (Cervus unicolor) flesh

Four to five kilograms of flesh without any subcutaneous fat was

collected from the wildlife Sambar (Cervus unicolor) maintained at Sri

Venkateswara Zoological Park, Tirupati. The flesh was collected

immediately after the death of the animal (either natural death or death

due to accidents but without history of any infectious etiology) without

any subcutaneous fat, fascia and connective tissue. The samples were

transported to the laboratory immediately and washed with PBS and

stored at –200C in aliquots of 1 kg each till used.

3.1.1.2 Method :

1. Determination of protein concentration :

Protein concentration of samples was estimated as per the standard

method described by Lowry et al (1951).

2. Standardization of the method of processing for antigen

preparation :

In order to extract muscle proteins, two diluents viz., 0.15M saline

and Phosphate buffer saline (PBS) pH 7.3 were tried and standardization

was done depending upon the protein concentration in the solute as per

the method of Lowry et al (1951).

24

xlviii

3. Processing of sambar muscle :

The muscle was washed with PBS, pH 7.3 thrice, minced with

sterilized forceps and homogenized and the fresh muscle protein

antigens were prepared as illustrated in the flow diagram (Fig.1) while

the method for thermostable protein (TMP) antigens is depicted in

(fig.2).

Fig.1 SCHEMATIC FLOW DIAGRAM FOR PREPARATION OF

FRESH SAMBAR MUSCLE PROTEIN ANTIGENS

100 gm of muscle +200 ml of PBS, pH 7.3

Homogenate

Centrifugation@

, 5000 rpm/30 mts

Residue discarded Supernatant

xlix

Preserved with thiomersal

(1:10,000) and stored at -200C

__________________________________________________________

@ HITACHI – HIMAC CR 21 F

Fig. 2. SCHEMATIC FLOW DIAGRAM FOR PREPARATION OF

THERMOSTABLE MUSCLE PROTEIN (TMP) ANTIGENS OF

SAMBAR 50 gm of muscle + 100 ml of PBS, pH 7.3

Homogenate

Boiling water / 1hr

Centrifuged at 3000 rpm@

/ 15 min

Supernatant

Autoclaved at 15 lb psi and centrifuged at

15000 rpm @

/ 30 min at 40C

Supernatant

3 vol of ethyl alcohol / 18 hrs at room

temperature

Centrifuged at 5000 rpm @

/ 15 min at

40C

l

Precipitate

30 ml of PBS, pH 7.3, centrifuged at

2000 rpm@

/ 30 min at 40C

Supernatant (TMP)

__________________________________________________________ @ HITACHI – HIMAC CR 21 F

The supernatant contained thermostable muscle protein (TMP)

antigen and was stored at -200C in 1 ml aliquots and preserved with

thiomersal (1 in 10,000) in screw capped tubes.

3.2 PREPARATION OF FRESH AND THERMOSTABLE

MUSCLE PROTEIN ANTIGENS (TMP) OF FIELD

SAMPLES (CATTLE, BUFFALO, SHEEP, GOAT, PIG

AND RABBIT) :

Muscle antigens were prepared from field samples comprising

species of cattle, buffalo, sheep, goat, pig and chicken from the slaughter

houses of Tirupati municipality and other local slaughter houses. The

preparation of respective muscle protein antigens was carried out as

already described in 3.1. These antigens were catalogued and identified

as cattle muscle extract – fresh (C-F), cattle muscle extract – cooked (C-

C), buffalo muscle extract – fresh (B-F), buffalo muscle extract – cooked

(B – C), sheep muscle extract – fresh (S-F), sheep muscle extract –

cooked (S-C), goat muscle extract – fresh (G-F), goat muscle extract –

li

cooked (G – C), pig muscle extract – fresh (P-F), pig muscle extract –

cooked (P-C), rabbit muscle extract – fresh (R-F), rabbit muscle extract

cooked (R – C).

3.3 PRODUCTION AND PREPARATION OF HYPERIMMUNE

SERA TO SAMBAR MUSCLE PROTEIN ANTIGENS :

Hyperimmune sera to both fresh and heated antigens of sambar

muscle were prepared in different species of animals.

3.3.1 Materials :

1. Rabbits

2. Sheep

3. Freund’s Complete Adjuvant (FCA) *

4. Sambar fresh muscle antigen

5. Sambar thermostable muscle protein (TMP) antigen

6. Thiomersal (1 : 10,000)

3.3.2 Method :

1. Immunization of rabbits with fresh muscle protein antigens of

sambar :

Three healthy rabbits weighing about 1.2 kg were used for

immunization. The method followed was that of Williams and Chase

(1967) with slight modification. One initial intramuscular injection in

lii

divided doses was given at multiple sites with 1 ml of (1:1) mixture of

__________________________________________________________

*Sigma chemical company, St Louis, USA.

sambar muscle protein antigen with a protein concentration of 1.16

mg/ml. A similar injection was repeated 2 weeks later. Blood was

collected seven days after the last injection. If antibody titre of serum is

not satisfactory another injection was given and the animal was bled

after giving rest for one week. The respective sera were pooled and

preserved at –20 0C after adding thiomersal to a final concentration of 1

in 10,000.

2. Immunization of Rabbits with thermostable muscle protein

antigens (TMP) of sambar :

Three healthy rabbits weighing about 1.2 kg were used for

immunization. The method followed was that of Williams and Chase

(1961) with slight modification. One initial intramuscular injection in

divided doses was given at multiple sites with 1 ml of (1:1) mixture of

thermostable muscle protein antigen of sambar with a protein

concentration 0.88 mg/ml. Such similar injections were repeated at

fortnight intervals and a total of five injections were made with a total

protein concentration of 1.1 mg every time. After resting the animals for

liii

one week after last injection, the animal was bled, serum was separated

and after adding with thiomersal to a final concentration of 1 in 10,000

and preserved at – 200C.

3. Immunization of sheep with fresh muscle antigens of sambar :

A healthy adult sheep (ram) aged 3 years was immunized against

fresh sambar muscle protein, a similar procedure as described above

with a total protein concentration of 4.64 mg each time. A total of two

intramuscular injections were given. After resting the animals for one

week after last injection, the animal was bled, serum was separated and

preserved at –200C after adding thiomersal to a final concentration of 1

in 10,000.

4. Immunization of sheep with thermostable muscle protein (TMP)

antigen of sambar :

A healthy adult sheep (ram) aged about 3 years was immunized

against TMP antigen of sambar, a similar procedure as described above

with a total protein concentration of 3.52 mg / injection. A total of three

intramuscular injections were given. After resting the animals for one

week after last injection, the animal was bled, serum was separated and

liv

preserved at – 200C after adding thiomersal to a final concentration of 1

in 10,000.

3.4 PREPARATION OF BINARY MEAT MIXTURES :

Twenty percent muscle extracts either fresh or autoclaved muscles

of cattle, buffalo, goat, sheep pig and rabbit were prepared and binary

meat mixtures of one species meat with another was achieved by mixing

20% extract solutions in different proportions, so as to arrive at different

percentages of mixing as illustrated below.

Table 1. Preparation of binary meat mixtures

Sambar muscle

extract (ml)

Cattle muscle extract

(ml)

Percentage of

adulteration (%)

0.5 0.5 50

0.25 0.75 25

0.10 0.90 10

0.05 0.95 5.0

0.025 0.975 2.5

0.01 0.990 1.0

0.005 0.995 0.5

0.001 0.999 0.1

0.0005 0.9995 0.05

lv

Experimental adulteration was carried out similarly with muscle

extracts of buffalo, sheep, goat, pork and rabbit.

3.5 IMMUNOCHEMICAL TOOLS TO DETECT WILDLIFE

SAMBAR MEAT :

3.5.1 Double immuno diffusion (DID) test :

Double Immuno diffusion (DID) test employing agarose was

carried out to detect the presence of precipitating antibody to the sambar

muscle protein antigen, fresh or cooked, as per the method described by

Ouchterlony (1958).

3.5.1.1 Materials :

1. Glass slides, 75mm x 75mm

2. Eppendorf automatic pipette *

3. 0.7% Agarose@

4. Barbital buffer pH 8.6 (Appendix I)

5. 2% saline and physiological saline(Appendix I)

6. staining solutions (Appendix II)

(a) Amido Black stain

(b) Coomassie Brilliant blue stain

7. Destaining solutions (Appendix II)

3.5.1.2 Method :

1. Pre-coating of the slides :

Ten ml of 0.2% agarose solution was prepared while hot and glass

slides were smeared and dried.

lvi

_________________________________________________________

* Eppendorf, West Germany

@ Type I agarose, Sigma chemical company, St Louis, USA

2. Preparation and casting of the gel :

The method followed was that of Parulekar (1982). Four ml of the

hot dissolved agarose in buffer was spread over the pre-coated slide to

get uniform thickness of 1.3 mm and allowed to solidify. The gel was

allowed to harden by transferring the slides to a refrigerator in a humid

chamber. Then a five well pattern was made with a 3 mm distance

between the edges of adjacent well with a diameter of 3mm. Each well

was charged with antigen and antibody separately as the case may be

with Eppendorf automatic micropipette. Then the slides incubated at 40C

for 18 to 24 hr. After diffusion was complete, the precipitation patterns

were read directly. Some slides were washed, dried and stained.

3. Washing drying and staining of the gels :

After completion of precipitation reaction and before staining, the

unprecipitated proteins present in the gel were removed by washing in

2% saline followed by physiological saline and distilled water over the

day. For drying, the gel was covered with a Whatman no.1 filter paper

lvii

moistened with sterilized distilled water and left overnight in the

incubator at 370C.

The dried gel on the slides was stained with Amido black or

Coomassie Brilliant blue for 5 to 10 minutes followed by destaining with

destaining solution for about 15 minutes. Then rinsed in tap water and

dried.

3.5.2 Single Radial Immuno Diffusion (SRID) technique :

Single radial Immuno diffusion (SRID) technique developed by

Mancini et al (1965) with slight modifications was used to quantify

sambar thermostable muscle protein (TMP) antigens.

3.5.2.1 Materials :

1. Phosphate buffer saline, pH 7.3

2. Barital buffer pH 8.6

3. Agarose 0.7%@

4. Specific antiserum

3.5.2.2 Method :

Agarose dissolved in buffer (1.4%) was kept at 560C in a water

bath. Hyperimmune serum raised against sambar thermostable muscle

protein (TMP) antigens diluted in the buffer was added to equal volume

of agarose solution kept in the water bath to a final concentration

lviii

__________________________________________________________

@ Type I agarose, Sigma chemical company, St Louis, USA

of 5%. Similarly antiserum in buffer was also added to agarose to a final

concentration of 5%. These mixtures in appropriate quantities were over

layered on precoated glass slides so as to obtain a gel thickness 1.3 mm.

After solidification, wells of 3 mm diameter were punched out at a

distance of 12 mm between the wells.

The wells were charged with several dilutions of sambar muscle

protein (TMP antigen) and incubated at 40C in humid chamber for 24 to

48 hours.

The formation of antigen antibody precipitate rings around the

wells were observed and their diameters were measured. Some slides

were washed and stained by the method described already in 3.5.1.2.

Duplicate determinations were made for every sample on different

slides. A standard curve was constructed by plotting precipitate ring

diameters against the corresponding log of protein concentrations of the

antigen. This curve a linear one was used for calculation of sambar

muscle proteins (TMP) in the test sample.

lix

3.5.3 Counter Immuno Electrophoresis (CIE) :

Procedure of Jad (1982) was followed.

3.5.3.1 Materials :

1. Electrophoresis apparatus and power pack.#

2. Glass slides, 75mm x 75mm

3. Whatman No. 1 filter paper wicks

4. Agarose@

5. Barbital buffer pH 8.6

6. Hyper immune sera

7. Antigen

3.5.3.2 Method :

Agarose was prepared in barbital buffer, pH 8.6. Each

microscopic slide was layered with 2.5ml of agarose and after setting,

two wells with a diameter of 3mm were punched at a distance of 2mm

from each well. Ten µl of antigen was filled in the cathodal well and the

test serum in the anodal well with a micropipette.

The electrophoresis chambers were filled with 60 ml of barbital

buffer, pH 8.6. Four microscope glass slides were placed in the

_____________________________________________________

# Systronics Electrophoresis Supply 610

@ Type 1 agarose, Sigma chemical company, St Louis, USA

lx

electrophoresis chamber and contacts were made by means of Whatman

no.1 filter wicks. Electrophoresis was carried out for 5 minutes with a

constant current supply of 15 ma per slide. The slides were observed for

precipitation bands.

The electrophoresed slides were washed, dried and stained as

described earlier (3.5.1.2).

3.6 SODIUM DODECYL SULPHATE POLY ACRYLAMIDE

GEL ELECTROPHORESIS (SDS - PAGE) :

SDS – PAGE was performed by using the procedure of Laemmli (1970).

3.6.1 Preparation of reagents :

Stock solutions

1. Acrylamide Bis acrylamide stock solution (100 ml) :

Acrylamide – 30.0g

N’N1 methylene bis acrylamide – 0.8g

The above two chemicals were dissolved in 80 ml of distilled

water and made up to 100 ml of distilled water. Filtered the solution and

stored at 40C in an amber coloured bottle.

2. Resolving gel buffer (1.5m Tris – HCl pH 8.8) :

Tris base – 18.15 g

lxi

Tris base was dissolved in 80 ml of distilled water and pH was

adjusted to 8.8 with 0.1 N HCl and made up to 100 ml with distilled

water.

3. Stacking gel buffer (0.5m Tris HCl buffer pH 6.8) :

Tris base – 6.0 g

It was dissolved in 80 ml of distilled water and pH was adjusted to

6.8 with 0.1 N HCl and made up to 100 ml with distilled water.

4. 10% Sodium dodecyl sulphate (SDS) :

SDS – 10 g

Dissolved in 10 ml of distilled water and stored at room

temperature.

5. Sample Buffer :

0.5 M Tris – HCl buffer (6.8 pH) – 1.0 ml

Glycerol - 0.8 ml

10% SDS - 1.06 ml

2-mercaptoethanol - 0.4 ml

0.05% Bromophenol blue - 0.2 ml

Distilled water - 4.0 ml

The above solutions were mixed thoroughly and stored at room

temperature.

lxii

6. Tank Buffer :

Tris Base - 450 mg

Glycine - 2.16 g

SDS - 150 mg

Distilled water - 150 ml

7. 10% Ammonium per sulphate :

Ammonium per sulphate - 100 mg

Dissolved in 1 ml of distilled water and prepared just before use.

8. Staining solution :

Coomassie brilliant blue (R – 250) - 100 mg

Methanol - 50 ml

Glacial acetic acid - 7 ml

Dissolved and mixed the above chemicals thoroughly and made

up to 100 ml with distilled water.

9. Destaining solution :

Methanol - 40 ml


The above solutions were mixed and made up to 200 ml with

distilled water.

3.6.2 Gel preparation :

lxiii

Stock solution Resolving gel (12%) Stacking gel (4%)

30% Acrylamide 4.0 ml 1.3 ml

Resolving gel Buffer 5.0 ml -

Stacking gel buffer - 2.5 ml

10% SDS 0.2 ml 0.1 ml

10% APS (Ammonium

per sulphate

0.1 ml 0.05 ml

Distilled water 6.7 ml 6.04 ml

TEMED 20 µl 10 µl

3.6.3 Gel preparation procedure :

The vertical slab gel@

unit in the casting mould with 1 mm spacer

was used. Resolving gel solutions was prepared as given under

preparation of reagents. The solution was poured into the sandwich to a

level of 2 cm from the top. Distilled water was gently added along the

wall of the sandwich to form uniform gel surface after polymerization.

After polymerization the water on the resolving gel was poured off.

Stacking gel was prepared and overlayed on the resolving gel. The comb

was inserted into the sandwich of stacking gel. The gel was allowed to

polymerize for 30 min.

__________________________________________________________

@ Broviga Electrophoresis Apparatus, Chennai.

3.6.4 Sample preparation :

3.6.4.1 Fresh antigen :

lxiv

The sample was prepared by taking 50 µl of sambar fresh antigen

C-F, B-F, S-F, G-F and 50 µl of sample buffer mixed with each sample

and prepared.

3.6.4.2 Heated antigen :

The sample was prepared by taking 50 µl of thermostable muscle

protein (TMP) antigen of sambar, C-C, B-C, S-C, G-C, P-C and 50 µl of

sample buffer mixed with each sample and prepared.

3.6.5 Sample Loading :

Comb was slowly removed from the gel after polymerization from

the sandwich. The lower and upper chambers were filled with electrode

buffer. Fresh antigens were prepared and loaded in the wells as

mentioned below.

Lane: 1 Protein marker* (Phosphorylase B 97.4 kd, Bovine

serum albumin 68 kd, Ovalbumin 43 kd, Carbonic

unhydrase 29 kd, Soyabean Trypsin inhibitor 20 kd

Lysozyme 14.3kd).

__________________________________________________________

* GENEI, Bangalore

Lane: 2 Fresh muscle antigen of sambar

Lane: 3 Buffalo muscle extract – fresh (B-F)

Lane: 4 Cattle muscle extract – fresh (C-F)

Lane: 5 Sheep muscle extract – fresh (S-F)

lxv

Lane: 6 Goat muscle extract – fresh (G-F).

Another gel was prepared as mentioned above and the heated

muscle extracts (antigen) were prepared and loaded in the wells as

mentioned below.

Lane: 1 Protein marker (Phosphorylase B 97.4 kd, Bovine

serum albumin 68kd, Ovalbumin 43kd, Carbonic

unhydrase 29kd, Soyabean Trypsin inhibitor 20kd

Lysozyme 14.3kd).

Lane: 2 Thermostable muscle protein (TMP) antigen of

sambar.

Lane: 3 Buffalo muscle extract – Cooked (B-C)

Lane: 4 Cattle muscle extract – Cooked (C-C)

Lane: 5 Sheep muscle extract – Cooked (S-C)

Lane: 6 Goat muscle extract – Cooked (G-C)

Lane: 7 Pig muscle extract – Cooked (P-C)

The electrophoresis unit was connected to the power pack and

voltage was initially adjusted to 60 V and when the samples entered in

the resolving gel the current was adjusted to 100V. This was

disconnected when the moving dye touched the bottom edge of the gel.

3.6.6 Staining and Destaining :

The gel was carefully dismantled after completion of run and the

gel was immersed in a glass tray containing staining solution for 2 hours.

lxvi

Later the gel was destained in destaining solution until the back ground

of the gel became clear.

3.6.7 Molecular weight determination :

The marker proteins used in electrophoresis were Phosphorylase B

97.4 kd, Bovine serum albumin 68 kd, Ovalbumin 43 kd, Carbonic

unhydrase 29 kd, Soyabean Trypsin inhibitor 20 kd, Lysozyme 14.3 kd

used as the marker protein standards (GENEI)

The distance migrated by the meat proteins and marker proteins

were recorded and a standard curve was plotted by taking distance

migration on x-axis and molecular weights on y-axis. The molecular

weight of the meat proteins were calculated from the standard curve.

3.7 ELECTROBLOT IMMUNO ASSAY (SEMI – DRY) :

Electroblot immuno assay was performed according to the

procedure of Walker (1986)

3.7.1 Materials :

3.7.1.1 Preparation of Buffers :

lxvii

1. Anode buffer – I – pH 10.4

0.3 M Tris - 3.63 g


Methanol - 20 ml

Tris was dissolved in 50 ml of distilled water and pH was adjusted

to 10.4 with 0.1 N HCl. The solution was made up to 80 ml with distilled

water and to this 20 ml of methanol was added.

2. Anode buffer II, pH 10.4

0.0125 M Tris - 0.3 g


Methanol (20%) - 20 ml

Tris was dissolved in 50 ml of distilled water and pH was adjusted

to 10.4 with 0.1 N HCl. The solution was made up to 80 ml with distilled

water and to this 20 ml of methanol was added.

3. Cathode buffer, pH 9.4

0.02 M Tris - 0.3 g

0.04 M Glycine - 0.3 g


Methanol (20%) - 20 ml

lxviii

Tris and glycine were dissolved in 50 ml of distilled water made

up to 80 ml with distilled water and to this 20 ml of methanol was added.

4. Tris buffered saline (TBS), pH 7.5

0.02 M Tris - 2.42 g

0.15 M Nacl - 29.24 g

The above chemicals were dissolved in 900 ml of distilled water

and pH was adjusted to 7.5 with 0.1 N HCl. The solution was made up to

1000 ml with distilled water.

5. TBS – Tween 20 (TBS – T)

TBS - 1000 ml

Tween 20 - 0.5 ml

Mixed well and stored at 40C

6. Blocking solution

Non fat dried milk powder - 5 g

TBS - 100 ml

Mixed well by stirring on a magnetic stirrer for 10 min and stored

at 40C.

7. Antibody buffer

Non fat dried milk powder - 5 g

TBS – T - 100 ML

Mixed well by stirring on a magnetic stirrer for 10 min and stored

at 40C.

8. HRP labeled goat anti rabbit Ig – G conjugate (1/5000)

lxix

Conjugate - 4µl

Antibody buffer - 20 ml

9. Substrate buffer (0.5 M sodium citrate pH 5.2 for HRP system)

Tris sodium citrate - 735 mg

The above chemical was dissolved in 30 ml of distilled water and

pH was adjusted to 5.2 with 0.1N HCl and made up to 50 ml with

distilled water.

10. Substrate solution for HRP system

3, 31 diamino benzidine (DAB) - 6 mg

DAB was dissolved in 9 ml of substrate buffer and 1 ml of 0.3%

cobaltous chloride and 10 ml of 30% H2O2 were added and mixed well.

It was used immediately.

Fig. 3. Arrangement of sheets in semidry blot unit

(-)

Cathode plate

Cathode buffer filter papers

Slab gel

PDVF membrane

Anode II buffer filter papers

Anode I buffer filter papers

Anode plate

(+)

lxx

3.7.2 Method :

50 µl of TMP antigen of sambar, C – C, B – C, S – C, G – C, P –

C and 50 µl of sample buffer (1: V/V) mixed and electrophorosed by 12%

SDS – PAGE described earlier. Electroblotting was carried out by using

semi dry blot apparatus#.

__________________________________________________________

# Millipore Semidry Blot Apparatus.

The anode buffers I, II and cathode buffer were prepared and

taken separately in petriplates. The unstained gel was soaked in cathode

buffer and shaken for 20 minutes at room temperature. Seven sheets of

Whatman no.3 filter papers were cut in the size of the gel and soaked

two sheets in anode buffer I, two sheets in anode buffer – II and three

sheets in cathode buffer. The poly vinylidine diflouride (PVDF)

membrane was cut in the gel size and soaked in 100% methanol for 15

seconds and they soaked in deionized water for 15 seconds. Membrane

was equilibrated in anode buffer II for 5 minutes and the sheets were

arranged in semidry blot unit according to the fig.3 and the unit was

connected to power pack and current was adjusted to 54 mA per 1 hour.

lxxi

After the electrophoretic transfer of proteins, the membrane was

incubated on a shaker with blocking solution for 2h at room temperature.

The membrane was incubated for 1 hour in 1/40 dilution of homologous

antiserum (antibody) buffer. After 3 washings each 5 min in TBS – T,

the membrane was incubated for 1 hour in 1/1000 dilution of horse radish

peroxidase labeled goat anti rabbit antibodies in antibody buffer. The

membrane was washed thrice by 5 minutes interval in TBS – T and

transferred to a substrate solution. Colour development was recorded

visually and the reaction was stopped by washing the membrane in

distilled water. The membrane was dried and photograph was taken and

kept under dark condition.

lxxii

CHAPTER IV

4. RESULTS

4.1 DEVELOPMENT, PREPARATION AND STANDARDI-

ZATION OF IMMUNOCHEMICAL REAGENTS FOR

IDENTIFICATION OF WILDLIFE SAMBAR MEAT :

4.1.1 Standardization of the method of processing for antigen

preparation and preparation of sambar muscle protein

antigens :

As the soluble proteins of muscle are the potent antigens with

appropriate epitopes to stimulate the specific antibody production, the

solubility of sambar muscle proteins in two different solvents was tested

and standardized depending upon the total concentration of protein. The

same methodology was used in standardizing the appropriate buffer

lxxiii

system while extracting the muscle proteins of other domestic animals as

well. The comparative results of protein estimation of muscle proteins of

various domestic animals and sambar are presented in the table 2.

Table 2. Protein concentration of fresh muscle extracts of different

species

Species

Protein concentration

PBS pH 7.3

(mg / ml)

0.15 M NaCl

(mg / ml)

Sambar 1.16 0.56

Cattle 0.52 0.36

Buffalo 0.48 0.40

Sheep 0.42 0.36

Goat 0.48 0.44

Pig 0.36 0.32

Rabbit 0.40 0.36

4.1.2 Preparation of sambar fresh muscle protein antigens:

The sambar muscle was processed as illustrated in the fig.1 and

the fresh muscle protein antigens were prepared accordingly. The total

protein present was found to be 1.16 mg/ml of the fresh muscle antigen.

4.1.3 Preparation of thermostable muscle protein (TMP) antigen of

sambar :

51

lxxiv

The sambar muscle was processed and TMP antigens were

prepared as illustrated in the fig.2. The total protein present was found to

be 0.88 mg/ml of the TMP antigens.

4.1.4 Production and preparation of hyperimmune sera to sambar

fresh muscle protein antigens :

4.1.4.1 Hyperimmune sera in rabbits :

Satisfactory titre of antibody could be observed after giving three

injections of fresh sambar muscle antigen with a total protein

concentration of 3.48 mg / rabbit.

4.1.4.2 Hyperimmune sera in sheep:

Two injections of sambar fresh muscle antigen at fortnight interval

were sufficient to produce required titre of antibody in the serum of

sheep with a total protein concentration of 9.28 mg of antigen.


thermostable muscle protein (TMP) antigens of sambar :

4.1.5.1 Hyperimmune sera in rabbits :

lxxv

Satisfactory titre of antibody could be observed after giving five

injections of TMP antigen of sambar with a total protein concentration of

4.4 mg/ rabbit.

4.1.5.2 Hyperimmune sera in sheep :

Three injections of TMP antigen of sambar with total protein

concentration of 10.56 mg were given to produce required titre of

antibody in the serum of sheep.

4.2 PREPARATION OF FRESH AND THERMOSTABLE

MUSCLE PROTEIN (TMP) ANTIGENS OF DOMESTIC

ANIMAL SPECIES (FIELD SAMPLES):

The field samples (cattle, buffalo, sheep, goat, pig and rabbit)

were processed, fresh and thermostable antigens were prepared and

protein concentration was estimated. The results of protein estimation of

muscle extracts (antigen) of field samples are presented in the table 3.

Table 3. Comparative studies on protein concentration of fresh and

cooked muscle extracts (TMP) of different species

Species

Protein concentration

Fresh (mg / ml) TMP (mg / ml)

Sambar 1.16 0.88

Cattle 0.52 0.34

Buffalo 0.48 0.32

Sheep 0.42 0.24

Goat 0.48 0.36

lxxvi

Pig 0.36 0.28

Rabbit 0.40 0.28

Fig. 4 Photograph of the DID reactions comparing the precipitation

reaction of sambar fresh muscle antigen against rabbit anti sambar sera

and sheep anti sambar sera. Peripheral wells contain I rabbit anti sambar

sera and II sheep anti sambar sera while the central wells contains

sambar muscle (fresh) antigen.

lxxvii

Fig. 5 CIE of sambar muscle (fresh) antigen.

Left wells - Sambar muscle (fresh) antigen

Right wells - 1, 2 – Rabbit anti sambar sera

3, 4 – Sheep anti sambar sera

lxxviii

4.3 STANDARDIZATION OF IMMUNOCHEMICAL METHODS

TO DETECT WILDLIFE SAMBAR MEAT (FRESH

MUSCLE ANTIGEN ) :

4.3.1 Standardization with rabbit antisera :

4.3.1.1 Double Immunodiffusion (DID) test :

Rabbit antisera raised against fresh antigen of sambar muscle

showed lines of identity when analyzed in Ouchterlony DID test within

six hours after incubation at 40C and this optimum titre could be

achieved after giving three injections with a total protein concentration

of 3.48 mg/rabbit.

The precipitation reaction between sambar fresh muscle extract

(antigen) and the hyperimmune sera is presented in the fig. 4.

4.3.1.2 Counter Immuno Electrophoresis (CIE):

The counter immuno electrophoresis (CIE) with sambar fresh

antigens revealed one sharp precipitation line between antigen and

antibody well and the same is presented in the fig. 5.

4.3.2 Standardization with sheep antisera :

lxxix

4.3.2.1 Double Immunodiffusion (DID) test:

Sheep antisera raised against fresh antigens of sambar meat

showed lines of identity when analyzed in Ouchterlony DID test within

six hours after incubation at 40C and this optimum titre could be

achieved after giving three injections with a total protein concentration

of 9.28 mg/sheep.

The precipitation reaction between sambar fresh muscle extract

(antigen) and the hyperimmuno sera is presented in the fig. 4.


The CIE with sambar fresh antigen revealed one sharp

precipitation line between antigen and antibody well and the same is

shown in the fig.5.


responsiveness in the antibody production against sambar

fresh muscle antigen :

The species of animals chosen for the production of antibody

against sambar fresh muscle antigen viz., rabbit and sheep apparently

responded similarly in the yield of satisfactory titre of antibody so

produced and hence the antisera produced in these two species of

animals were utilized in various serological tests conducted.

lxxx

Fig. 6. Photograph showing the negative precipitation reaction between

cooked muscle extracts of sambar and domestic animals against rabbit

anti sambar serum.

Central well - Sheep anti sambar serum

Peripheral wells - 1. Sambar muscle extract – TMP

2. Cattle muscle extract – Cooked

3. Buffalo muscle extract – Cooked

4. Sheep muscle extract – Cooked

5. Goat muscle extract – Cooked

6. Pig muscle extract – Cooked

lxxxi

Fig. 7. Photograph showing the precipitation reaction of thermostable

muscle protein (TMP) antigen of sambar against rabbit anti sambar sera.

Central well - Thermostable muscle protein antigen of

sambar.

Peripheral wells - Rabbit anti sambar sera against TMP

antigen of sambar.

lxxxii

Fig.8. Standardization of SRID test for sambar TMP antigen.

Perceptible ring diameters of precipitation reaction were noticed only

with 50% rabbit anti sambar sera incorporated in the gel.

lxxxiii

Fig. 9. CIE of Thermostable muscle protein (TMP) antigen of Sambar

Left wells - TMP antigen of Sambar

Right wells - Rabbit antisambar sera

lxxxiv

4.4 STANDARDIZATION OF IMMUNOCHEMICAL METHODS

TO DETECT WILDLIFE SAMBAR MEAT

(THERMOSTABLE MUSCLE PROTEIN ANTIGEN) :

4.4.1 Standardization with rabbit antisera :

4.4.1.1 Double Immunodiffusion (DID) test:

Rabbit antisera raised against TMP antigens of sambar meat

showed perceptible lines of identity when analysed in DID test only after

72 hours when incubated at 40C and the same is presented in the fig. 7.

4.4.1.2 Single Radial Immunodiffusion (SRID) test :

Two buffer systems phosphate buffer saline pH 7.3 with ionic

strength of 0.01 and sodium barbital buffer pH 8.6 with ionic strength of

0.05 were tested to standardize the suitable buffer test system for single

radial immunodiffusion (SRID) test. The hyperimmune sera raised

against sambar fresh antigen in rabbits was tested. The ring diameters

were clear and sharp in the case of PBS than that of sodium barbital

buffer and hence the former test system was followed. The anti sambar

sera raised in rabbits reacted well and showed clear precipitin rings and

hence anti sambar sera raised in rabbits was used throughout the test.

lxxxv

As the test results would largely depend upon the antibody content

in the agarose gel, a series of dilutions of 5%, 10%, 25% and 50% of

rabbit antisera were carried out with known protein concentration of

fresh sambar meat to select the optimal dilutions which produced a clear

precipitation reaction as shown in the fig. 8.

The estimations were made with five dilutions of fresh

sambar meat and the average precipitation ring diameters so obtained

were used to construct a linear standard curve by plotting the ring

diameters against the log of protein concentration of fresh sambar meat.


The CIE with sambar TMP antigens revealed one sharp

precipitation line between antigen and antibody well and the same is

shown in the fig. 9.

4.4.2 Standardization with sheep antisera :

4.4.2.1 Double immunodiffusion (DID) test :

Sheep antisera raised against TMP antigens of sambar meat did

not show any precipitation reaction when analysed in DID test even after

72 hours of incubation.

lxxxvi

4.4.2.2 Single Radial Immunodiffusion (SRID) test :

Perceptible immuno diffusion could not be noticed around the

antigen wells even after incubating at 40C for 96 hr.


The CIE with sambar TMP antigens revealed no precipitation

reaction between antigen and antibody well.


responsiveness in the antibody production against sambar

thermostable muscle protein (TMP) antigen :

The TMP antigens of sambar did stimulate the antibody

production in rabbits but could not in sheep as evidenced by the

precipitation reaction observed in a variety of serological tests viz. DID,

SRID and CIE.

lxxxvii

Fig. 10. SDS – PAGE analysis of fresh muscle antigens of sambar and

other domestic animal muscle samples

Lane: 1 Protein marker (Phosphorylase B 97.4 kd, Bovine

serum albumin 68 kd, Ovalbumin 43 kd, Carbonic

unhydrase 29 kd, Soyabean Trypsin inhibitor 20 kd,

Lysozyme 14.3kd)

Lane: 2 Fresh muscle antigen of sambar

Lane: 3 Buffalo muscle extract – fresh (B-F)

Lane: 4 Cattle muscle extract – fresh (C-F)

Lane: 5 Sheep muscle extract – fresh (S-F)

Lane: 6 Goat muscle extract – fresh (G-F).

4.5 DETERMINATION OF PROTEIN PROFILE OF MUSCLE

PROTEIN OF SAMBAR AND DIFFERENT SPECIES OF

ANIMALS BY SDS – PAGE :

lxxxviii

The muscle proteins of different species of animals viz. buffalo,

cattle, sheep, goat, pig and sambar were subjected to SDS–PAGE to

assess their comparative molecular weights of protein profile so

delineated.

4.5.1 Comparative analysis of SDS – PAGE of fresh muscle proteins

of sambar and different species of domestic animals :

The electrophoretic separation of migrated proteins of sambar

muscle protein depending upon their molecular weight was resolved into

seven protein bands with molecular weights of 60 kd, 52 kd, 44 kd, 42

kd, 30 kd, 22 kd and 12 kd while the buffalo muscle protein profile

resolved into five protein bands with molecular weights of 60 kd, 52 kd,

44 kd, 42 kd and 22 kd. The cattle meat profile was resolved into three

protein bands with molecular weights of 40 kd, 30 kd and 12 kd while

the sheep meat protein profile resolved into six protein bands with

molecular weights of 60 kd, 52 kd, 44 kd, 42 kd and 22 kd and that of

goat meat protein profile was resolved into five protein bands 60 kd, 52

kd, 44 kd, 42 kd and 12 kd as shown in the fig. 10.

lxxxix

Fig. 11. SDS – PAGE analysis of thermostable muscle protein (TMP)

antigen of sambar and other domestic animal muscle samples.

Lane: 1 Protein marker (Phosphorylase B 97.4kd, Bovine serum

albumine 68kd, Ovalbumin 43kd, Carbonic unhydrase

29kd, Soyabean Trypsin inhibitor 20kd, Lysozyme 14.3 kd)

Lane:2 Thermostable muscle protein (TMP) antigen of sambar.

Lane: 3 Buffalo muscle extract –Cooked (B-C)

Lane: 4 Cattle muscle extract –Cooked(C-C)

Lane: 5 Sheep muscle extract –Cooked(S-C)

Lane: 6 Goat muscle extract –Cooked (G-C)

Lane: 7 Pig muscle extract –Cooked (P-C)

4.5.2 Comparative analysis of SDS – PAGE of TMP antigens of

sambar and different species of animals :

xc

The electrophoretic migration pattern of the protein bands of TMP

antigens of sambar was resolved into three bands, one major and two

minor. The major protein band comprising the proteins with molecular

weights of 44kd and 42kd was diffused in its migration probably due to

partial denaturation. The minor protein bands were 22kd and 12kd. A

similar pattern of diffused migration of two protein complex was

observed in the protein profile of the muscle proteins of other species

viz. buffalo, sheep, goat and pig except in cattle but conspicuously

absent were the two protein bands with molecular weights of 22 kd and

12 kd which were present in sambar only as shown in the fig. 11.

4.5.3 Comparison of the protein profile of fresh and TMP antigens

of sambar muscle protein :

The sambar fresh muscle protein profile when subjected to SDS –

PAGE resolved into seven bands and the same when subjected to SDS –

PAGE after cooking (TMP) resolved into only three protein bands one

major and two minor. The major protein band, a diffused one, possibly

reflects the denatured two protein complex viz. 44 kd and 42 kd while

the minor protein bands viz 22 kd and 12 kd were heat stable and the

xci

migration observed was similar in both the electrophoretic pattern of

analysis fresh muscle as well as heated (TMP) muscle protein.

4.5.4 Comparison of the protein profile of fresh and TMP antigen of

other domestic species :

The protein profile of fresh meat of buffalo, sheep and goat which

showed a similar electrophoretic pattern of migration of four protein

bands with 60 kd, 52 kd, 44 kd and 42 kd. However after subjecting to

heat treatment, three protein bands were noticed one major (comprising

44kd and 42kd of molecular weight) but merged into a single band

probably representing partial denaturation as evidenced by the diffused

pattern (fig. 10 and 11) and two minor protein bands with molecular

weights of 22kd and 12kd.

4.6 ELECTROBLOT IMMUNOASSAY (WESTERN BLOTTING)

OF COOKED MEAT (TMP ANTIGENS) OF SAMBAR AND

OTHER DOMESTIC SPECIES :

The electrophoretic migration of the cooked meat protein (TMP)

of sambar, buffalo, cattle, sheep, goat and pig followed by immunoassay

analysis were identified by using polyclonal antibodies raised in rabbits.

xcii

Fig. 12. Immunoblot analysis of thermostable muscle protein (TMP) of

antigens of sambar and other domestic animal muscle samples.

Lane: 1 Thermostable muscle protein (TMP) antigen of sambar.

Lane: 2 Buffalo muscle extract – Cooked (B-C)

Lane: 3 Cattle muscle extract – Cooked (C-C)

Lane: 4 Sheep muscle extract – Cooked (S-C)

Lane: 5 Goat muscle extract – Cooked (G-C)

Lane: 6 Pig muscle extract – Cooked (P-C)

The western blot analysis identified the serologically related TMP

antigens of sambar along with other species viz. 44kd and 42kd of

xciii

sambar, buffalo, sheep, goat and pig. Although 40kd protein band of

sambar could not be appreciated very well, it was identified as one of the

serologically related protein that was present in cattle.

The serologically identified proteins with molecular weight of

22kd and 12kd were present only in sambar.

4.7 COMPARISON OF SDS – PAGE ANALYSIS AND WESTERN

BLOTTING OF TMP ANTIGENS OF SAMBAR AND

OTHER DOMESTIC SPECIES :

A diffused two protein complex band comprising 44kd and 42kd

was observed in sambar, buffalo, sheep, goat and pig in SDS – PAGE

electrophoresis, and this was serologically identified common to all of

these animals by Western blotting.

The serologically identifiable protein specific to only sambar were

22kd and 12kd as seen in the western blotting (fig. 12).

4.8 DETECTION OF ADULTERATED WILDLIFE

(SAMBAR) MEAT WITH THE MEAT OF DOMESTIC

SPECIES BY IMMUNODIFFUSION AND IMMUNO

ELECTROPHORESIS :

xciv

4.8.1 Detection of sambar fresh meat adulterated with the meat

of domestic species :

4.8.1.1 Detection with hyperimmune sera raised in rabbits :

4.8.1.1.1 Double Immunodiffusion (DID) test :

Detection of experimental adulteration of sambar muscle extract

with that of cattle, buffalo, sheep, goat, pig and rabbit was done by the

standardized serological tests.

The presence of sambar meat could be established up to 1% level

of adulteration as evidenced by perceptible lines of identity in the DID

test when the anti sambar (fresh) sera raised in rabbits was allowed to

react with a series of muscle extracts of each species such as cattle,

buffalo, sheep, goat, pig and rabbit as illustrated in the table. 1. The

precipitation reaction, however could not be observed at 0.5% level.

xcv

Fig. 13. Photograph showing the precipitation reaction between rabbit

anti sambar (fresh) serum and sambar muscle (fresh) antigen

experimentally adulterated with buffalo muscle (fresh) extract.

Central well - Rabbit anti sambar serum (fresh)

Peripheral wells. 1 – 25%

2 – 10%

3 – 5%

4 – 2.5%

5 – 1%

6 – 0.5% of Sambar (fresh) muscle extract

xcvi

Fig. 14. CIE of sambar muscle (fresh) extract experimentally adulterated

with buffalo muscle (fresh) extract.

Left wells - 1 – 10%

2 – 5%

3 – 2.5%

4 – 1%

5 – 0.5 % of Sambar (fresh ) muscle extract

Right wells - Rabbit anti sambar sera.

4.8.1.1.2 Counter immuno electrophoresis (CIE):

xcvii

The results of CIE using different levels of adulteration are

furnished in the fig. 14. The antibody in the anti sambar hyper immune

sera raised in rabbits could react with the sambar meat as low as at 1%

level when adulterated with the meats of other species such as cattle,

buffalo, sheep, goat, pig and rabbits , within 5 minutes resulting in the

formation of a sharp line . Depending upon the presence of percentage of

sambar meat in the adulterated meats , the precipitation line found to be

thick to that of feeble and thin. However the sambar meat could not be

detected at 0.5% level of adulteration.

4.8.1.2 Detection with hyperimmune sera raised in sheep :

4.8.1.2.1 Double Immunodiffusion (DID) test :

The presence of sambar meat could be established up to 1%

level of adulteration as evidenced by perceptible lines of identity in the

DID test when the anti sambar (fresh) sera raised in rabbits was allowed

to react with a series of muscle extracts of each species such as cattle,

buffalo, sheep, goat, pig and rabbit as illustrated in the table.1. The

precipitation reaction, however could not be observed at 0.5% level.

4.8.1.2.2 Counter immuno electrophoresis (CIE):

xcviii

The antibody in the anti sambar hyper immune sera raised in

rabbits could react with the sambar meat as low as at 1% level when

adulterated with the meats of other species such as cattle, buffalo, sheep,

goat, pig and rabbit within 5 minutes. Depending upon the presence of

percentage of sambar meat in the adulterated meats, the precipitation line

found to be thick to that of feeble and thin. However the sambar meat

could not be detect at 0.5% level of adulteration.

4.8.2 Detection of sambar cooked meat (TMP) adulterated with the

meat of domestic species :

4.8.2.1 Detection with hyperimmune sera raised in rabbits against

sambar TMP antigens:

4.8.2.1.1 Double Immunodiffusion :

When the anti sambar hyper immune sera raised in rabbits was

allowed to react with TMP antigens of sambar meat, lines of identify

could be noticed only after 72 hours of incubation at 40C.

However the same precipitation reaction could not be noticed with

the adulterated meat of sambar with the meat of domestic animals such

as cattle, buffalo, sheep, goat, pig and rabbit. Therefore the hyper

immune sera as well as the adulterated meat mixtures were concentrated

to two folds by means of using poly ethylene glycol 600 and then the test

was run. The so concentrated hyper immune serum when allowed to

xcix

react with the concentrated adulterated meat mixtures could result in the

perceptible precipitation lines and lead to the detection of adulteration up

to 10% level but not at 5% level.

4.8.2.1.2 Single Radial Immunodiffusion (SRID) test :

The precipitation reaction around the antigen well could be

observed with the whole sambar muscle extract when the agarose gel

incorporated with 50% of the hyper immune sera. But none of the

sambar meat adulterated with meat of domestic species such as cattle,

buffalo, sheep, goat, pig and rabbit showed any precipitation reaction.

4.8.2.1.3 Counter Immuno Electrophoresis (CIE):

When the hyperimmune sera and the adulterated meat mixtures

were electrophorosed, appropriate precipitation reaction could be seen in

the form of a sharp precipitin line between antigen and antibody well

(fig.15), however depending upon the level of presence of percentage of

sambar meat in the adulterated meat mixtures, the precipitation reaction

found was formation of thick line to that of thin and faint. Thus the

adulteration could be detected up to a level of 10% but not at 5% level.

c

Fig. 15. CIE of thermostable muscle protein (TMP) antigen of sambar

experimentally adulterated with cooked buffalo muscle extract (TMP).

Left wells - 1 – 25%

2 – 10%

3 – 5%

4 – 2.5%

5 – 1% of TMP of sambar

Right wells - Rabbit anti sambar sera (heated)

4.8.2.2 Detection with hyperimmune sera raised in sheep against

sambar TMP antigens:

ci

4.8.2.2.1 Double Immuno Diffusion (DID) test :

The hyper immune sera raised in sheep when allowed to react with

TMP antigens of experimentally adulterated sambar meat with the meat

of domestic species, no perceptible reaction could be noticed even at

50% level even after incubating at 96 hours. The DID test conducted

with concentrated hyper immune sera and the adulterated meat mixtures

did not result in any perceptible precipitation reactions.

4.8.2.2.2 Single Radial Immuno Diffusion (SRID) test :

The anti sambar hyper immune sera raised in sheep against

sambar TMP antigens could not react even with concentrated sambar

TMP antigens and as such it could not detect any of the sambar meat

adulterated with the meat of other domestic species such as cattle,

buffalo, sheep, goat, pig and rabbit.

4.8.2.2.3 Counter Immuno Electrophoresis :

The hyper immune sera raised in sheep against sambar TMP

antigens when electrophoresed along with sambar TMP antigen in the

CIE test did not result in perceptible precipitation line and as such none

of the adulterated meat mixtures could be identified with the hyper

immune sera raised in sheep.

cii

Fig. 16. Immunoblot analysis of 1% adulterated thermostable muscle

protein (TMP) of sambar with other domestic species muscle extracts

(heated).

Lane 1 Thermostable muscle protein antigen of sambar

Lane 2 1% of sambar adulterated with cattle muscle extract

Lane 3 1% of sambar adulterated with buffalo muscle extract

Lane 4 1% of sambar adulterated with sheep muscle extract

Lane 5 1% of sambar adulterated with goat muscle extract

Lane 6 1% of sambar adulterated with pig muscle extract

ciii

Fig. 17 Immunoblot analysis of adulterated thermostable muscle protein

(TMP) antigen of sambar with cooked buffalo muscle extract.

Lane: 1 Thermostable muscle protein (TMP) antigen of sambar.

Lane: 2 0.5% TMP of sambar adulterated with buffalo muscle extract

Lane: 3 1% TMP of sambar adulterated with buffalo muscle extract




4.9 DETECTION OF SAMBAR MEAT ADULTERATED

WITH THE MEAT OF DOMESTIC ANIMALS BY

civ

ELECTROBLOT IMMUNOASSAY (WESTERN

BLOTTING) :

By western blot analysis, the adulteration of sambar cooked meat

(TMP) at 1% level with the meat of buffalo, sheep, goat and pig was

detected with distinct migration of protein with molecular weights of

44kd, 42kd, 22kd and 12kd.

4.10 DETECTION OF SAMBAR MEAT ADULTERATED

WITH BUFFALO MEAT BY ELECTROBLOT

IMMUNOASSAY (WESTERN BLOTTING)

The cooked buffalo meat (TMP) containing experimentally added

sambar cooked meat (TMP) at 1%, 0.5%, 0.25%, 0.1% and 0.05% when

subjected to western blotting with polyclonal serum raised in rabbits

against TMP antigen of sambar showed that this immunoassay could

detect sambar meat up to as low as 0.1% level but not at 0.05% level

as shown in the fig. 17.

CHAPTER V

5. DISCUSSION

cv

Certain species of wildlife like sambar and black buck are

protected species in Andhra Pradesh as they are declared as endangered

species and as such they are prohibited from hunting as per reserve

forest regulations. Now a days there is increased litigation over

misrepresentation of flesh of wildlife for flesh of domestic species like

cow, buffalo, sheep or goat (Umamaheswararao, 2003). Under such

circumstances development and standardization of wildlife forensic tools

are essential as authentic testing would be mandatory and essential in

order to detect the species origin of such meats and also to contest in a

court of law. This formed the back ground for this current study.

Till recent past, several studies were made on identification of

species origin of meats, involving various domestic species of food

animals (Reddy 1986, Giridharreddy 1994, Srinivasarao 1996,

Sridharlingareddy 1996, and Krishnamurthy 1998), however wildlife

species left untouched may be due to under reporting of poaching. In the

absence of adequate reported literature on wildlife forensics in India, any

investigator would have to fall back on immunological studies as the

case with other workers who investigated the meat speciation of

domestic animals. Therefore immunochemical determination of species

origin of meats of wildlife was attempted with the hypothesis that the

84

cvi

test antigens could be 1) obtainable in a reasonably pure form, 2)

sufficiently antigenic and foreign to the host animal, 3) thermostable to

withstand the heat treatment made during the purification process and 4)

sufficient enough to induce the production of specific antibody.

Besides conventional immunochemical methods for identification

of wildlife species (sambar) origin of meats, the present studies carried

out include certain advanced immunoassays like western blotting

(electro blot immuno assay) to detect the level of experimental

adulteration of sambar meat with the meat of other species of domestic

animals.

Trials were conducted with two diluents PBS, pH 7.3 and 0.15M

NaCl in order to standardize the suitable buffer system in which more

soluble protein goes into the solution without disrupting the antigenic

character of protein or without much loss of protein of test antigens of

the sambar meat system as evidenced by the concentration of proteins so

estimated with each diluent. The estimated protein concentration of meat

extract of different domestic animals including sambar is shown in the

table 2 which reveals that PBS, pH 7.3 is a preferred choice of buffer

system of solvent where the concentration of protein is more with every

cvii

species of the animal tested when compared with the other diluent

0.15M NaCl. Hence PBS pH 7.3 was used throughout the experiments

while extracting the muscle proteins either fresh or heated.

As a first step towards the development of wildlife forensic tools

for identification of sambar meat, the preparation of antigen from fresh

muscle extract (fig.1) and from heated muscle extract, the preparation of

thermostable proteins (fig.2) and the production of respective antibody

with appropriate immunization procedure was standardized.

The sera from the experimental animals (rabbits and sheep),

before immunizing them, were screened for the presence of antibodies

against the muscle protein antigens of sambar, cattle, buffalo, sheep,

goat, pig and rabbit and found to contain none.

Identification of species origin of meats of different food animals

depends on the production of species specific antibody by developing

antibody in phylogenetically related animals or by using species specific

protein fractions which are thermostable present in liver, testicles, brain,

pituitary and muscles as cited by Reddy et al (1990).

cviii

For the production of specific antibody against sambar muscle

antigen, two species of animals, rabbits and sheep were utilized in the

hyperimmunization experiment .Rabbit is chosen for immunization

because it is phylogenetically removed farther from the source of antigen

so that major antigenic differences will be recognized by immune

apparatus (Warnecke and Sarfle,1968; Pinto,1961;Ramadas and

Misra,1981) while sheep phylogenetically closely related to sambar for

generating immune response to the fine antigenic differences that exist

between isoantigens (Bansal and Mandokhot ,1988 ; Nath ,1986 and

Reddy ,1986 ).

However in our experiments, both of these animals responded

similarly in the antibody production to the sambar fresh muscle antigens.

But sheep failed in responding to the production of antibody against

TMP antigens of sambar, while rabbits responded adequately although

required pumping of antigen a few more times.

Possibly the reason for inadequate response of sheep to antibody

production against TMP antigens of sambar could be due to denaturation

of proteins having fine antigenic differences which could have

stimulated antibody production had the antigen been not heated. This

cix

interpretation gains strength further as evidenced by the results of the

serological experiments carried out as described in 4.3.

Therefore, TMP antigen was used to develop species specific

antibody for identification of sambar muscle besides fresh muscle

antigen in order to develop wildlife forensics utilizing the

immunochemical techniques.

Utilizing the hyperimmune sera raised in rabbits and sheep,

different conventional serological tests were tried and standardized for

the detection of sambar meat from a mixture (binary mixture) of

adulterated meats of different domestic species of animals.

The DID test carried out with sambar fresh meat antigen using

homologous antisera raised in rabbits and sheep showed precipitation

reaction with lines of identity (fig.4) when incubated at 40C within six

hours and is in full agreement with the findings of Giridhar reddy, 1996;

Sherikar et al.,1993; and Allsup,1987 who used rabbit antisera to

identify fresh muscle antigens of domestic species. However similar

results could be obtained with the hyperimmune sera raised in rabbits or

sheep when allowed to react with the fresh muscle antigen of buffalo and

cattle, while the precipitin lines were thin and feeble with the fresh

cx

muscle antigens of sheep and goat but not with pig. These results concur

with the findings of Sherikar et al. (1979) and Sherikar et al. (1987) who

noticed cross reactions between meats of heterologous species viz.,

cattle, buffalo, sheep, goat and pig and also with the findings of Pandey

and Pathak (1975), Hayden (1979) and Allsup (1987) who observed

cross reactions between cattle, horse, lamb and pig. Therefore it could be

inferred that sambar can also be considered as phylogenetically related

to other species viz., buffalo, cattle, sheep and goat but not with pig.

The DID test carried out with the hyperimmune sera raised in

rabbits and sheep against TMP antigens of sambar when tested with

homologous antigens and of heterologous species i.e., cattle, buffalo,

sheep, goat and pig resulted in interesting findings contrary to the

findings of the DID test using fresh muscle antigen. The rabbit antisera

reacted only with TMP antigens of homologous species (sambar) but not

with any of the heterologous species, and it could be due to denaturation

of serologically related proteins during heat treatment. These findings

are slightly deviated from the findings of earlier workers who noticed

faint reactions while working with speciation of meat of domestic

animals (Sherikar et al., 1987). However the hyperimmune sera raised in

sheep did not react even with homologous and heterologous species

cxi

antigen as the proteins specific for sambar might have been denatured

and hence could not elicit appropriate antibody production and there is

no reported literature to draw parallels. However, Carpenter (1965) was

of the view that heat caused denaturation of protein resulting in the loss

of antigenic fraction to form an antigen antibody complex leading to the

formation of precipitin line.

Ramdas and Misra (1981) and Sherikar et al. (1979) made

speciation of meats based on the number of precipitation lines using

antiserum raised in rabbits in DID test and immuno electrophoresis (IE).

In the present study also two precipitation lines were observed in the

DID test using rabbit antiserum with sambar fresh muscle antigen while

the DID test showed only one precipitation line with TMP antigens and

was species specific for sambar muscle only without any cross reactions

with TMP antigens of domestic animals.

The TMP antigen which was used to raise antibody in rabbits was

composed of heat stable proteins with molecular weight ranging from

44000 daltons to 12000 daltons. Some of these proteins are myofibrillar

protein fractions corresponding to tropomyosin and troponin fractions as

observed by Radhakrishna et al. (1989) pertaining to mutton and beef.

cxii

The rabbit antisera developed against TMP antigen was species specific

as it did not show cross reactions with other species of animals as

observed by Giridharreddy (1994) in the case of species specific TMP

antigens of pork.

In order to estimate the levels of presence of sambar meat in the

adulterated meats of other species, an attempt was made to standardize

single radial Immuno diffusion (SRID) test. The phosphate buffer saline

(PBS), pH 7.3 with ionic strength of 0.01 found to be the suitable test

system during the course of standardization with sodium barbital buffer,

pH 8.6 with ionic strength of 0.05. The anti sambar sera raised in rabbits

reacted well and showed clear precipitin rings around the antigen well

with 50% antiserum in agarose after incubating at 40C for 72 hours. The

SRID test conducted with sheep antiserum did not yield any visible

precipitin rings as in the case of DID test.

Although DID test is a reliable technique for differentiating meats

of different species (Ramadas 1972, Beljaar and Oleman 1977, Sherikar

et al., 1979, Hayden 1980, Nath 1986, and Bansal and Mandokhot

1988), it is cumbersome, time consuming and costly. Hence counter

immuno electrophoresis (CIE) was tried and standardized.

cxiii

The sambar fresh muscle antigen along with hyperimmune sera

raised in rabbit or sheep charged in the cathode well and anode well

respectively, when electrophoresed resulted in a clear sharp precipitin

line between antigen and antibody well (Fig 5 ). When the hyperimmune

serum raised in rabbits or sheep allowed to react with fresh muscle

antigens of cattle, buffalo, sheep and goat and pig reacted similarly in

the CIE test resulting in the formation of a sharp precipitin line which is

in agreement with the findings of Gridharreddy (1994); Allsup (1987)

and Sherikar et al. (1993) who obtained similar results while working

with the speciation of meats of domestic animals. However, there was no

visible precipitation reaction with that of pig in both the tests.

The TMP antigens of sambar when subjected to CIE test utilizing

sheep antisera raised against sambar TMP antigens, did not yield any

precipitation reaction unlike rabbit antiserum which had given a faint

precipitin line after running for 5 minutes followed by incubation at 40C

for 24 hours which resulted in increased sharpness of the precipitin line.

These results are in agreement with the findings of Giridharreddy,

(1994) who could observe similar results with porcine TMP antigens and

cxiv

Sherikar et al. (1993), using TMP antigens of cattle, buffalo, sheep, goat

and pig with homologous serum.

To delineate the sambar fresh muscle proteins with different

molecular weights, SDS – PAGE studies were carried out to examine

whether SDS – PAGE can be recommended as a standard procedure for

detection of adulteration. Comparative studies were also carried out with

muscle proteins of other species viz., cattle, buffalo, sheep and goat.

Upon comparison, the protein bands with similar molecular

weights viz 60 kd, 52 kd, 44 kd, and 42 kd were more or less similar in

sambar, buffalo, sheep and goat. Strangely cattle muscle protein profile

appears to be somewhat different in that these proteins are conspicuously

absent while it contained one protein band with 40 kd.

The protein band with a molecular weight of 30 kd was present

only in sambar and cattle, while it was absent in all other species.

The protein band with a molecular weight of 22 kd could be seen

in sambar, buffalo and sheep while it was absent in cattle and goat.

cxv

But a common low molecular protein band with a molecular

weight of 12kd was noticed in all the species of animals except in

buffalo.

Although work on wildlife is scanty, a similar type of work

involving meat samples of venison, beef and pork, when electrophoresed

showed species specific separation patterns (Anneli Skrokki, 1994).

Furthermore, our results are also in agreement with those of Ramadas

(1972), Prasad (1974), Govindarajulu (1982) and Nath et al. (1999) who

established a relationship of species specific muscle protein band

involving different species.However further work on SDS – PAGE is

required to identify the species specific proteins of sambar.

The electrophoretogram of TMP antigens of sambar with different

species of animals revealed a diffused protein band comprising protein

bands with molecular weights of 44kd and 42kd were seen in all the

species of animals including sambar except cattle, probably the diffused

pattern was due to partial denaturation. The specific protein bands

conspicuous only to sambar were low molecular weight protein bands of

22kd and 12kd, which were totally absent in any of the species of

animals when subjected to the SDS – PAGE as shown in the fig. 11.

cxvi

These results further prove that TMP antigens were species specific in

that they can be relied upon while detecting adulteration (Babiker et al.,

1981).

Identification of specific immunological proteins which are of

diagnostic significance can be made accurately by latest and advanced

technique of western blotting which gives a concrete evidence. This

latest technique was applied in the present study to identify the specific

immunological proteins which are species specific to sambar. This

technique involved separation of TMP antigens of sambar in gels

followed by electrophoretic transfer of the separated polypeptides to

nitrocellulose membrane for probing specific proteins by antisera raised

in rabbits.

The sambar TMP antigens when subjected to western blotting

resulted in the serologically identified TMP antigens of sambar i.e 44kd

and 42kd along with other species, buffalo, sheep, goat and pig but not

with cattle. Further the serologically identified proteins with molecular

weight of 22kd and 12kd were confined only to sambar. Although the

diffused pattern of the migration of the protein complex comprising

44kd and 42kd in sambar was serologically identified with other species

cxvii

of animals and the migration of this protein complex is diffused in all the

species possibly due to partial denaturation and as such the proteins with

22kd and 12kd molecular weight were probably species specific to

sambar and the same can be of diagnostic value in forensic studies. Our

findings are in agreement with those of Chen and Hsieh (2001) who

adopted western blotting technique and identified a thermostable 24kd

protein as species specific to pig.

The experimentally adulterated fresh meat of domestic animals

with fresh sambar meat (binary mixtures) when subjected to DID test

with rabbit or sheep antiserum, showed precipitation lines up to 1% level

of adulteration but not at 0.5% level and these results are in agreement

with those of Suryarao (1990) who used testicular antigens to detect

fresh meat of buffalo meat adulterated up to 10% level.

The DID test when used to detect adulterated cooked meats of

domestic animals with sambar could identify up to a level of 10% of

adulteration which is in agreement with those of Giridharreddy (1994)

who could detect adulteration of cooked pork up to a level of 20% and

Sherikar et al. (1993) who detected adulteration of cooked meats up to a

level of 5 -10%.

cxviii

Unsuccessful attempts were made to standardize single radial

immunodiffusion (SRID) test in order to estimate the levels of

adulteration. Although 50% of hyperimmune sera raised in rabbit was

required to be incorporated in agarose, it could not detect any of the

adulterated cooked meats.

Counter immunoelectrophoresis (CIE) was successfully

standardized as it is supposed to be sophisticated, reliable (without false

positive or false negative results) quick, rapid and economical.

The CIE test could detect the adulteration of sambar fresh meat as

low as up to 1% level with rabbit or sheep antisera which is in agreement

with those of Suryarao (1990).

The homologous hyperimmune serum raised in sheep could not

detect sambar meat in the adulterated cooked meats in CIE test similar

to that of DID test. The incompetence of the antibody to detect

adulteration could be due to failure of denatured proteins to stimulate

antibody production probably because of close phylogenetic relationship

with that of sambar.

cxix

The TMP antigens of sambar elicited adequate immune response

in rabbits which are phylogenetically distinct and the hyperimmune sera

so raised could detect adulteration up to a level of 10% but not at 5%

level and these results are in agreement with those of Giridhar reddy

(1994) and Suryarao (1990) who could detect adulteration up to a level

of 20% while Sherikar et al. (1993) detected up to a level of 5-10% of

adulteration.

Successful attempts were made to detect the presence of cooked

meat of sambar (TMP) in the experimentally adulterated cooked (TMP)

meats of domestic animals by using western blotting technique. Cooked

meats of phylogenetically related animals like buffalo, cattle, sheep and

goat and that of pig were experimentally adulterated at 1% level and

subjected to western blotting which could detect sambar cooked meat up

to 1% level in all the cooked meats of animal species tested. These

results could not be compared as the literature is lacking in this direction.

Western blotting studies were continued to identify the presence

of sambar cooked meat and its cut off level by selecting the cooked meat

of a domestic animal which is phylogenetically closely related and in

this case buffalo cooked (TMP) meat was experimentally adulterated

cxx

with TMP antigens of sambar with varying percentage. The polyclonal

antisera could detect serologically related proteins of sambar in these

binary mixtures as low as up to 0.1% level but not at 0.05% level as

shown in the fig. 17.

The binary mixtures of fresh meats adulterated with sambar fresh

meat antigen when subjected to immunological tests, the adulteration

could be detected at 1% level by DID and CIE, while DID and CIE tests

could detect the level of adulteration up to 10% of TMP antigens of

sambar. Western blotting analysis could detect the level of adulteration

of TMP antigens of sambar as low as 1% level when adulterated with the

meat of domestic animals like buffalo, cattle, sheep, goat and pig. The

western blotting could detect adulteration of cooked buffalo meat with

cooked sambar (TMP) meat as low as 0.1% level indicating the

reliability, sensitivity and specificity of the test.

However, continued studies are required in this direction

involving other wildlife also in order to standardize this technique as

dependable and fool proof tool in wildlife forensics.

cxxi

CHAPTER VI

6. SUMMARY

Experimental investigations were carried out to develop

appropriate wildlife forensic tools to detect species origin of sambar

(Cervus unicolor) meat for the first time.

Preparation of muscle protein antigens of sambar and other

species of domestic animals both fresh and thermostable proteins (TMP)

was standardized after conducting experimental trials with two diluents

PBS, pH 7.3 and 0.15 M NaCl. Most of the soluble proteins of the

muscle extracts which are potent antigens and necessary in forensic

work could be extracted more with PBS, pH 7.3 compared to 0.15 M

NaCl as evidenced by the estimated concentration of the protein.

Therefore PBS, pH 7.3 was selected and used in the experiments.

Suitability of experimental animals to develop specific antibody

against sambar muscle protein antigen was examined using rabbit, a

cxxii

phylogenetically distinct animal from the source of the antigen to have

major antigenic differences recognized by the immune apparatus while

sheep, a phylogenetically closely related to sambar to induce immune

response to the antigenic differences that exist between isoantigens.

Although both of these animals immunologically responded adequately

to the fresh muscle antigen of sambar, rabbit was found to be an ideal

choice for adequate induction of immune response to the thermostable

muscle proteins (TMP) when compared to sheep, probably because of

the immune apparatus in sheep failed to recognize the fine differences

between the isoantigens. Therefore, rabbit antisera against TMP antigens

of sambar was used throughout the present investigation.

Sambar fresh muscle antigens were prepared from the sambar

flesh collected immediately from the naturally dead sambar (either by

natural death or by accidental death without any infectious etiology)

from Sri Venkateswara Zoological Park, Tirupati.

Sambar fresh muscle antigens were prepared in PBS, pH 7.3,

while thermostable muscle protein (TMP) antigen was the boiling

resistant and ethanol precipitate of cooked meat extract.

cxxiii

Hyperimmune sera was raised to both sambar fresh muscle

antigens and TMP antigens in rabbits and sheep. Rabbit, a

phylogenetically distinct animal responded adequately to both fresh and

TMP antigen while sheep, a phylogenetically related species responded

to fresh antigens but not to TMP antigens.

The immunochemical methods like DID test and CIE test to detect

wildlife sambar fresh muscle antigens were standardized using

hyperimmune sera raised in both rabbit and sheep.

The DID test revealed lines of identity with sambar fresh muscle

antigen when allowed to react with homologous antiserum of rabbit and

two precipitation lines were noticed. The precipitation reaction with

sheep antisera showed lines of identity with only one precipitation band.

Lines of identity were also observed with the fresh muscle antigens of

buffalo, cattle, sheep, goat and rabbit but not with pig amply indicating

that these species except pig are closely related to sambar. When the

DID test was conducted with TMP antigens of sambar using

hyperimmune sera raised in rabbits, lines of identity were noticed after

prolonged incubation at 40C for 72 hours, but not with sheep antisera.

cxxiv

In order to improve upon the sensitivity, specificity and rapidity,

CIE test was standardized using both fresh muscle antigens and TMP

antigens of sambar.

The CIE test did give a sharp precipitation line between antigen

and antibody well with both the type of antigens when allowed to react

with respective sera raised in rabbits. The gel was electrophorosed for 5

min at 40C. The CIE test could detect the fresh antigens of species of

different domestic animals such as cattle, buffalo, sheep, goat, rabbit but

not pig, while such cross reactions were not noticed when tested against

TMP antigens of domestic animal species. The sheep antisera raised

against TMP antigens of sambar showed no cross reactions with any of

the phylogenetically related species of domestic animals and not even

with homologous antigen.

In order to detect the percentage level of sambar meat in the

experimentally adulterated meats of domestic animals, SRID test was

standardized using rabbit antisera. The SRID test could be standardized

with 50% of the hyperimmune sera incorporated in the agarose gel.

However SRID test could not detect the presence of sambar meat in any

cxxv

of the binary mixtures and as such SRID test was found to be unsuitable

as an immunochemical tool in wildlife forensics.

SDS – PAGE studies were also conducted to study the protein

profile of sambar fresh and TMP antigens and a comparison was made

with those of other domestic animal species. The electrophoretogram

revealed that sambar fresh muscle protein was resolved into seven

protein bands with molecular weights of 60kd, 52kd, 44kd, 42kd, 30kd,

22kd and 12kd while the buffalo muscle protein profile resolved into

five protein bands with molecular weights of 60kd, 52kd, 44kd, 42kd

and 22kd. The cattle meat profile was resolved into three protein bands:

40kd, 30kd and 12kd while the sheep meat protein profile resolved into

six bands: 60kd, 52kd, 44kd, 22kd and that of goat meat protein profile

was resolved into five protein bands: 60kd, 52kd, 44kd, 42kd and 12kd.

The electrophoretogram of TMP antigens of sambar and other

species of domestic animals (buffalo, sheep, goat and pig except cattle)

showed a partially denatured two protein complex with diffused

migration pattern (44kd and 42kd). Two protein bands with low

molecular weights 22kd and 12kd were present only in sambar but not in

other animals possibly species specific.

cxxvi

The electroblot immuno assay (western blot) identified

serologically related TMP antigens, 44kd and 42kd of sambar, buffalo,

sheep, goat and pig. Lone serologically related protein 40kd was noticed

in cattle although not appreciated very well in sambar. The serologically

identified proteins with molecular weight of 22kd and 12kd were present

only in sambar meat, possibly species specific.

The conventional immunochemical tests like DID and CIE were

carried out using rabbit or sheep antiserum to detect adulteration of

sambar fresh meat from binary mixtures of fresh meat of buffalo, cattle,

sheep, goat, rabbit and pig. These tests could detect the adulteration up

to 1% level, while detection in adulterated cooked meats of domestic

animals could be up to a level of 10% only with hyperimmune sera

raised in rabbits against TMP antigens. The sera raised in sheep against

TMP antigen of sambar could not detect the adulteration.

Western blotting did identify cooked (TMP) sambar meat

adulterated with the cooked meats of buffalo, cattle, sheep, goat and pig

as low as up to a level of 1%. The experimental adulteration of cooked

(TMP) sambar meat with cooked (TMP) buffalo meat, a

phylogenetically closely related domestic animal, detected as low as up

cxxvii

to a level of 0.1% but not at 0.05% level. Thus western blotting proved

to be a promising reliable, specific and sensitive technique in wildlife

forensics.

cxxviii

LITERATURE CITED

Abraham J 1987 Isoelectric focusing as a method for species

differentiation of raw and cooked meats. Ph.D. thesis, Andhra

Pradesh Agricultural University, Hyderabad.

Abraham J and Leela Nayar 1983 Effect of storage of meat in frozen

condition on the sarcoplasmic protein and patterns in agarose

isoelectric focusing. Kerala Journal of Veterinary Science 14: 76-

80.

Agrawal K C 2000 Wildlife of India conservation and management, pp

78. Nidhi publishers, India.

Allsup T N 1987 A comparison of the agar gel immunodiffusion (AGID)

and counter immunoelectrophoresis (CIE) tests for species

identification of imported red meat and offal. Meat Science 20:

119-128.

Anneli Skrokki and Osmo Hormi 1994 Composition of minced meat.

part A: Methods. Meat science 38: 497 – 501.

Babiker S A, Glover P A and Lawrie R A 1981 Improved determination

of horse meat in heated food stuffs. Meat Science 5: 473.

Bansal A K and Mandokhot U V 1988 Studies on differentiation of beef

from meat of other species of animals. I. Comparative specificity

and sensitivity of serum raised in buffalo calves against cattle

antigens. Journal of food science and technology 25 : 146-151.

Beljaare P R and Oleman W J 1977 Collaborative study on the detection

of caseinate and soy protein isolate in meat products. Annales de

la Nutrition et de l’Alimentation 31:603.

Brehmer H 1983 Positive results for soya protein meat products

detection in routine examination. Fleischwirtschaft 63: 1771.

108

cxxix

Carpenter P L 1965 Immunology and serology. 2nd

edition, pp 56. W B

Saunder’s company, London.

Chen F C and Hsieh Y H P 2001 Separation and characterization of

species specific thermostable muscle protein from cooked pork.

Journal of Food Science Vol. 66 No.5 799-803.

Chikuni K Ozutsumi K, Koishikawa T, and Koto S 1990 Species

identification of cooked meat by DNA hybridization assay. Meat

Science 27: 119.

Chikuni K Tabata T Kosugiyama M and Monma M 1994 Polymerase

chain reaction assay for detection of sheep and goat meats. Meat

Science 37: 337 – 345.

Coffin J.W., Corey S. Davis, Deedra Hawk, and curtis stroback, 1999 “A

DNA typing system for pronghorn Antelope and its use in a

poaching investigation from Wyoming” Northwest Association of

forensic scientists, Abstracts, Fall 1999.

Dincer B, Spearow J.L Cassens R.G and Greaser M.L 1987 The effects

of curing and cooking on the detection of species origin of meat

products by competitive and indirect ELISA techniques Meat

Science 20: 253 – 265.

Durand M Schneider R 1962 Immuno electrophoresis of meat juice and

its application to the detection of fraud. Arch. Inst. Pasteur Junic

39: 153 (Veterinary bulletin 33: 2188, 1963).

Ebbehoj K F and Thomsen P D 1991a Species identification of heated

meat products by DNA hybridization. Meat Science 30: 221.

Ebbehoj K F and Thomsen P D 1991b Differentiation of closely related

species by DNA hybridization. Meat Science 30: 221.

Flego R and Borghese R 1979 Analysis of meat production: critical

comparison of double immunodiffusion and COE methods for

minimum antigen detection. Bullettina Provinciali 5: 701-711.

cxxx

Fugate H G and Shelton P R 1971 Biochemical techniques-

Immunodiffusion technique for the identification of animal

species. Journal of AOAC 54: 1152.

Gacheru D N Kang’ethe E K Kaburia H F A and Njeruh F M 1994

Sandwich enzyme immunoassay for speciation of cooked meat

and for detecting trace amounts of adulterants in phylogenetically

related species. Agricultural and forestry Journal 59: 205 – 212.

Ginsberg A 1948 The differentiation of meats by precipitation test.

Veterinary Record 60: 683 – 685.

Govindarajulu M, Ranganatham M and Ramamurthy R 1982 Studies on

the differentiation of beef from mutton by starch gel

electrophoresis. Cherion 11: 6.

Graber, P and Williams C A 1953 Immunoelectrophoretic Analysis. In

Application to Human Biological fluids(eds Graber and Burtin)

Elsevier Publication Co. Amsterdam, London, New York.

Giridharreddy M 1994 Immunochemical detection of pork using

thermostable muscle protein antigens. M.V.Sc Thesis submitted to

the Acharya N.G. Ranga Agricultural University, Hyderabad.

Hayden A R 1979 Immunochemical detection of ovine, porcine and

equine flesh in beef products with antisera to species myoglobin.

Journal of Food Science 44: 494.

Hayden A R 1980 Immunochemical detection of species origin of meat

products. Ph.D Thesis, University of Wisconsin, Madison, W I.

Cited from Hayden A R 1981.

Hayden A R 1981 Use of antisera to heat stable antigens of adrenals for

species identification in thoroughly cooked beef sausages. Journal

of Food Science 46: 11810 – 11813.

Irv Kornfield 1999 Establishment of microsatellite databases for forensic

casework in the northwest united states. Northwest Association of

forensic scientists abstracts fall 1999.

cxxxi

Jad C Y 1982 Counter Immunoelectrophoresis. pp 66 – 107 In Gel

Immunodiffusion techniques in research and laboratory Medicine

(eds S S Kelkar and P M Khare) Popular Prakashan Private

Limited, Bombay.

Kang’ethe E K and Gathuma J M 1987 Species identification of

autoclaved meat samples using anti sera to thermostable muscle

antigens in an enzyme immunology. Meat Science 19: 265 – 270.

Kang’ethe E K and Lindquist K J 1987 Detection beef and pork in fresh

and heat treated meat products using enzyme immunology.

Journal of Food Science and Technology 24: 289 – 293.

Kang’ethe E K Lindquist and Gathuma J M 1984 In Biochemical

identification of meat species (Paterson RLS (ed.) Elsevier

Applied Science Publishers.pp129. cited from Kang’etha and

Gathuma 1987.

Karkare U D Sherikar A T Khot J B and Jaya rao B M 1988 Meat

speciation in commercial canned meats using species antisera to

thermostable antigens of adrenal glands. Indian journal of Meat

Science & Technology Vol 1(2).

Krishnamurthy G 1998 Identification of cooked meats using

thermostable brain antigens M.V.Sc Thesis Acharya N.G. Ranga

Agricultural University. Rajendranagar, Hyderabad.

Lammeli U K 1970 Cleavage of structural proteins during the assembly

of the head of Bacteriophage T4. Nature 227: 680 – 685.

Lajon A, Pauteau M L Demeulemester C Martin J L Pinson M and

Durand P 1989 Identification of added poultry meat in charcuterie

products. Viandes-et-produits-carnes 10: 203-206.

Landsteiner K 1945 Specificity of serological reactions. Dover

publications New York. Cited from Sherikar et al (1987).

Lawrie R A 1977 Meat identification in fresh and processed foods. Food

Technology Australia 20: 347 (FSTA 10, 4s, 505, 1978).

cxxxii

Lowry O H, Rosenbrough N J Farr A L and Randall R J 1951 Protein

measurement with Folin-phenol reagent Journal of Biological

Chemistry 193: 265 – 275.

Mancini G, Carbonara A O and Heremans J F 1965 Single radial

Immunodiffusion. PP 111 – 118. I Gel Immunodiffusion

techniques in research and Laboratory medicine (eds S S Kellar

and P M Khave 1982). Popular Prakshan Private Limited,

Bombay.

Mandokhot U V and Kotwal S K 1997 Enzyme linked immuno sorbant

assay in detection of species origin of meats – A critical appraisal.


Manz J 1985 Detection of heat denatured muscle proteins by means of

ELISA. Determining beef and pork muscle proteins.

Fleiswirtschaft 65: 497 – 499.

Matsunaga T Chikuni K Tanabe R Muroya S Shibata K Yamda J and

Shimura Y 1999 A quick and simple method for the identification

of meat species and meat products by PCR assay. Meat Science

51: 143-148.

Mc Cormick R J, Collins D A, Field R A and Moore T D 1992

Identification of meat from game and domestic species. Journal of

Food Science 57: 516 – 517.

Nath D R 1986 Comparative studies on electrophoresis and

immunodiffusion technique for identification of fresh buffalo and

sheep meat. Ph.D., Thesis, Haryana Agricultural University,

Hisar.

Nath D R Chandir N K and Mandokhot U V 1999 Counter current

Immuno Electrophoresis Technique for Meat Specification and

detection of adulteration in meats. Souvenir cum proceedings of

National Seminar on processing of Meat, Poultry and by-products

for value addition.pp 231.

Nath D R Chandirmani N K and Mandokhot U V 1999 SDS-PAGE for

the different meat species. Proceedings of the National Seminar on

cxxxiii

Processing of Meat, Poultry and by-products for value addition.

pp 219.

Ouchterlony O 1958 Double diffusion in two dimensions PP 19. 16 – 10.

20. In Hand Book of experimental Immunology (ed D M Weir

1978) Volume I. Black well Scientific publications, Oxford.

Pandey R and Pathak R C 1975 Serological relationship of

taxonomically closely related domestic ruminants. Indian Journal

of Experimental Biology 13: 371.

Parulekar S D 1982 Agar gel micro Ouchtorlony radial double diffusion

(AGD). PP 32 – 65. In gel immuno diffusion techniques in

Research and Laboratory Medicine (eds S S Kelkar and P M

Khare). Popular Prakashan Private Limited, Bombay.

Pike R M and Sulkin S E 1957 The agar diffusion precipitation test as a

means of recording evidence of meat adulteration. Journal of

laboratory clinical Medicine 49: 657. Cited from Swart and Wilks

1982.

Pinto F C 1961 Serological identification of ox, buffalo, goat and deer

flesh. British Veterinary Journal 117 : 540.

Poli G, Point A, Balasari A and Cantom C 1977 Ind Aliment 16: 87 cited

from Swart and Wilks 1982.

Prasad V S S 1974 Studies on esterase and other proteins of raw meat of

different species of animals in relation to age and sex M.V.Sc

Thesis, G B Pant University of Agriculture and Technology,

Pantnagar, Uttar Pradesh, India.

Radhakrishna K Vijaya Rao D, Sharma T R 1989 Characterization of the

major component in thermostable muscle proteins. Journal of

Food Science and Technology 26:32.

Radhakrishna K, Vijaya Rao D and Sharma T R 1988 studies on the

electrophoretic and immunodiffusion methods in the

differentiation of mutton and beef subjected to severe thermal

cxxxiv

processing. Journal of Food Science and Technology 25: 280 –

284.

Ramadas P 1972 Physico – chemical and immunological differentiation

of meats of different species of animals. M.V.Sc. Thesis, G B Pant

University of Agriculture and Technology, Pant nagar, Uttar

Pradesh, India.

Ramadas P and Misra D S 1981 Immunoelectrophoretic identification of

meats of bullock, goat, sheep, pig, chicken. Indian Veterinary

Journal 58: 978 – 983.

Rasool T J, Appa Rao K B C, Bhat P P, Rawat A K and Bhat P N 1995

Simple method for differentiation of raw meat from various meat

animals. Potential technique for detection of meat adulteration

Journal of Animal Science 65: 684 – 687.

Rattire H W and Regenstein J M 1977 Action of crude papain on actin

and myosin heavy chains isolated from chicken breast muscle.

Journal of Food Science 42 : 1159.

Reddy P M 1986 Studies on the identification of cooked meats of cattle,

sheep / goat Ph.D. Thesis Haryana Agricultural University, Hisar.

Reddy P M and Reddy G M 1995 Immunochemical detection of pork

using thermostable muscle antigens. Journal of Food Science and

Technology 32: 221 – 223.

Reddy P M and Mandokhot U V and Chandirmani N K 1990 A

comparative study on heated adrenal and immunoglobin G –

antigen for identification of cooked meats of cattle and sheep.


Saisekhar R and Reddy P M 1995 Use of troponin for species

identification of cattle and buffalo meats. Journal of Food Science

32: 68-70.

Sasidhar S 1995 Detection of pork by immunochemical techniques

M.V.Sc Thesis, Andhra Pradesh Agricultural University,

Hyderabad.

cxxxv

Saway W N, Maneesh M S, EL – Rayes, Hussain A and Dashi B 1990

Detection of pork in processed meat by enzyme linked immuno

sorbant assay using anti swine antisera. Journal of Food Science

55: 293.

Schweiger A, Hanning K, Gunther H O and Bandner S 1982

Immunological detection of meat from turkey. In Recent

developments on food analysis. Food Science and Technology

Abstracts 15 : 6S 806, 1983.

Scopes R K and Penny I F 1971 Sub unit sizes of muscle protein as

determined by sodium dodecyl sulphate gel electrophoresis,

Biochemi. Bipphys, Acta, 236:409.

Sherikar A T, Ajinkya S M Khot J B, and Vaidya A M 1979

Differentiation of meats by gel diffusion technique. Indian Journal

of Animal Science 49 : 350.

Sherikar A T Khot J B Jayarao B M and Pillai S R 1987 Species

differentiation in raw and heat treated meats using serological

methods (Agar gel precipitation test). Journal of Food Science and

Technology 24:293.

Sherikar A T, Karkare U D, Khot J B, Jayarao B M and Bhilogaonkar N

Y 1993 Studies on thermostable antigens production of species

specific anti – adrenal sera and comparison of immunological

techniques in meat speciation. Meat Science 33: 121 – 136.

Sherikar A T, Khot J B, Jayarao B M and Pillai S R 1988 Use of species

specific antisera to adrenal heat stable antigens for the

identification of raw and cooked meats by agar gel diffusion and

counter immunoelectrophoretic techniques. Journal of the Science

of Food and Agriculture 44: 63.

Sinclair A J and Slattery W J 1982 Identification of meat according to

species by isoelectric focusing. Australian Veterinary Journal 58:

79.

cxxxvi

Sivakumar K Shanmugam A M and Ramaswami A M 1999 Souvenir

Cum proceedings. National seminar on processing of meat,

poultry and Byproducts for value addition.

Sridharlingareddy V 1996 Use of Brain antigens for species

identification of beef, cara beef and pork, M.V.Sc. Thesis Andhra

Pradesh Agricultural University, Hyderabad.

Srinivasarao Z 1996 Immunochemical detection of mutton, chevon and

rabbit using brain antigens. M.V.Sc. thesis, Andhra Pradesh

Agricultural University, Hyderabad.

Suryarao K 1990 Identification of species origin of cooked meats using

antisera to testicular antigens Ph.D. Thesis Andhra Pradesh

Agricultural University, Hyderabad.

Umamaheswararao S 2003: Personal Communication.

Walker, J.M. 1986 Protein (Western) blotting with immuno detection. In

experiments in Molecular Biology, R.J Slater (ed), PP. 171 – 178.

Humana Press, Clifton, New Jersey U.SA.

Warnecke M O and Sarfle R L 1968 Serological identification of

proteins. Mode of injection and protein extracts for antibody

production Journal of Food Science 33: 131.

Williams C A and Chase M W 1967 Methods in Immunology and

Immunochemistry. PP 246. Volume I and Volume II. Academic

Press, New York and London.

Wilson P J and White N B 1999 “Identification and quantification of

game tissue in processed meat products”. Northwest Association

of Forensic Scientists, Abstracts, Fall 1999.

Wintero A K, Thomsen P D and Davies W 1990 A comparison of DNA

–hybridization, immuno diffusion, counter immuno

electrophoresis and isoelectric focusing for detecting the

admixture of pork to beef. Meat Science 27: 75 – 85.

cxxxvii

Yamamato K, Kobayashi N and Samazima K 1982 Detection of

Soyabean protein in meat products and SDS gel electrophoresis.

Journal of college of Dairying Natural Science 94: 25.

cxxxviii

APPENDICES

Appendix – I

Buffers and reagents

Phosphate buffered saline (PBS), pH 7.3 (Cruickshank, 1965)

NaCl - 8.0 g

K2HPO4 - 1.21 g

KH2PO4 - 0.34 g

Distilled water - 1 Litre

Sodium phosphate buffer saline

Solution A: 0.2 M Monobasic sodium phosphate (27.8 g / Lt NSS)

Solution B: 0.2 M Dibasic sodium phosphate (53.65 g / Lt NSS)

51 ml of solution A was mixed with 49 ml of solution B and pH

adjusted to 6.8 and was finally diluted to a total of 200 ml with NSS.

Sodium barbital buffer

Sodium barbital - 10.31 g

Barbituric acid - 1.84 g

Sodium acetate - 6.80 g

Distilled water to make - 1 Litre

Ionic strength - 0.1

pH - 8.6

The entire solution was diluted to 4 litre with the distilled water.

118

cxxxix

Physiological sodium chloride, ionic strength 0.15, pH 7.4 (Crowle,

1973)

Sodium chloride - 0.88 g


2% Sodium chloride

Sodium chloride - 2 g


Appendix – II

Staining solutions

(a) Amido black stain:

Amido black - 1.5 g

Methanol - 90 ml



(b) Coomassie brilliant blue stain:

Coomassie brilliant blue - 5 g

Ethanol - 450 ml



Destaining solutions

(a) For amido black stain:

cxl

Methanol - 45 ml

Acetic acid - 5 ml


(b) For coomassie brilliant blue stain:

Ethanol - 450 ml



120

Documents

STUDIES ON WILDLIFE FORENSICS TO DETECT SPECIES ORIGIN … · i studies on wildlife forensics to detect species origin of sambar (cervus unicolor) meat by k. venkat ragavan b.v.sc