EVALUATING DIAGNOSTIC TESTS TO ASSESS THE EPIDEMIOLOGICAL STATUS OF BOVINE TUBERCULOSIS. FARIS DELIL YESUF I.D.No. DPV (VEP)

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1 EVALUATING DIAGNOSTIC TESTS TO ASSESS THE EPIDEMIOLOGICAL STATUS OF BOVINE TUBERCULOSIS FARIS DELIL YESUF I.D.No. DPV (VEP) DEPARTMENT OF VETERINARY EPIDEMIOLOGY & PREVENTIVE MEDICINE MADRAS VETERINARY COLLEGE CHENNAI TAMIL NADU VETERINARY AND ANIMAL SCIENCES UNIVERSITY 2012

2 EVALUATING DIAGNOSTIC TESTS TO ASSESS THE EPIDEMIOLOGICAL STATUS OF BOVINE TUBERCULOSIS FARIS DELIL YESUF I.D.No. DPV (VEP) Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy In VETERINARY EPIDEMIOLOGY AND PREVENTIVE MEDICINE to the Tamil Nadu Veterinary and Animal Sciences University DEPARTMENT OF VETERINARY EPIDEMIOLOGY & PREVENTIVE MEDICINE MADRAS VETERINARY COLLEGE CHENNAI TAMIL NADU VETERINARY AND ANIMAL SCIENCES UNIVERSITY 2012

3 Dedicated To My late Father & Family

4 TAMIL NADU VETERINARY AND ANIMAL SCIENCES UNIVERSITY DEPARTMENT OF VETERINARY EPIDEMIOLOGY & PREVENTIVE MEDICINE MADRAS VETERINARY COLLEGE CHENNAI CERTIFICATE This is to certify that the thesis entitled " EVALUATING DIAGNOSTIC TESTS TO ASSESS THE EPIDEMIOLOGICAL STATUS OF BOVINE TUBERCULOSIS " submitted in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy in the discipline of Veterinary Epidemiology and Preventive Medicine to the Tamil Nadu Veterinary and Animal Sciences University, Chennai , is a record of bonafide research work carried out by FARIS DELIL YESUF under my guidance and that no part of the thesis has been submitted for the award of any other degree, diploma, fellowship or other similar titles or prizes and that the work has not been published in part or full in any scientific or popular journal or magazine. Date: Place: Chennai (Dr. P.I. GANESAN) CHAIRMAN RECOMMENDED Date:... Place: APPROVED EXTERNAL EXAMINER Chairman : (Dr. P.I. GANESAN) Members : 1. (Dr. K. KUMANAN) 2. (Dr. B. SAMUEL MASILAMONI RONALD) 3. (Dr. M. VIJAYA BHARATHI)

5 CURRICULUM VITAE Name : FARIS DELIL YESUF Date of Birth : Major Field of specialization : Veterinary Epidemiology and Preventive Medicine Minor Field of specialization : Animal Biotechnology Educational Status : Completed M.V.Sc. in 2007 Addis Ababa University, FVM Debre Zeit, Ethiopia Enrolled for Ph. D. in 2009 Awards / Fellowships : Nil Conferences/Seminar/Workshop/ Symposium Participated : Eleven Publications : Research article : four Marital Status : Married Permanent Address : Wollo University, School of Veterinary Medicine Assistant Professor P. O. Box 1145 Dessie, Ethiopia Phone (line): Mobile: farisyesuf12@gmail.com Membership in Professional society : 1. Ethiopian Veterinary Association

6 Acknowledgements

7 ACKNOWLEDGEMENTS First of all, I would like to offer a deep sense of gratitude and indebtedness to Dr. P.I. Ganesan, Chairman Advisory Committee, Professor and Head, Department of Veterinary Epidemiology and Preventive Medicine, MVC, and Chairman, Advisory Committee, for his intellectual guidance, constant encouragement, critical suggestions, and willingness to listen my thought during this research work. I should use this opportunity to express my thanks to Dr. M. Sekar, former Chairman Advisory Committee, Professor, Department of Veterinary Public Health and Epidemiology, MVC, for smooth interaction and cordial guidance. I wish to express grateful and cordial thanks to Dr. K. Kumanan, Member of Advisory Committee, Director of Research, TANUVAS, for successful guidance and overall involvement in this work. I am honoured to express my deepest gratitude to Dr. B. Samuel Masilamoni Ronald, Member of Advisory Committee, Associate Professor, Bacterial Vaccine Research Centre, TANUVAS, for an end to end tangible involvement during the entire period. I am thankful to Dr. M. Vijaya Barathi, Member of Advisory Committee, Assistant Professor, Department of Veterinary Epidemiology and Preventive Medicine, MVC, for involvement and constant encouragement. I would like to express my deep sense of gratitude to Dr. K. S. Venkataraman, Professor (Rtd.), Department of Veterinary Epidemiology and Preventive Medicine, MVC, for invaluable suggestions, constant encouragement and overall fatherly approach. I do not have words strong enough to express my feelings. I appreciate your determination to listen others opinion. I would like to thank Dr. L. Gunaseelan, Professor and Head, Department

8 of Veterinary Public Health and Epidemiology, MVC, for valuable suggestions and constructive criticism. I am very much thankful to Dr. K. Sathyabama, Assistant Professor, Department of Veterinary Epidemiology and Preventive Medicine, MVC, for constant encouragement and overall support. I extend my heartfelt thanks and appreciation to Dr. N.R. Senthil for extensive help throughout my study period. His smooth and friendly interaction made my stay in India pleasant. I like to thank Dr. S. Balakrishnan and Dr. M. Ashok Kumar, Assistant Professors, Department of Veterinary Public Health and Epidemiology, MVC; and Dr. P. Kannan for their valuable suggestions and help in all the time when I was in need. I would like to extend my appreciation to Director of Clinic, MVC, Chennai; Professor and Head of PGRIAS, Kattuppakam; and Professor and Head of URF, Madvaram for cordial cooperation during sample collection. It is time to express and give credit to my senior and juniors in my department: Dr. O.R. Vinodhkumar, Dr. Saleem Dhar, Dr Keneisezo Kuotsu, Dr. Gowri Yale, Dr. V.Bhanu Rekha, Dr. S. Shankar, Dr. R. Mohanasundari, Dr. S. Bhavya, Dr. M. Krishnaveni and Dr. Vinita Arulmozhi for their enlightening friendship during this study time. I wish to express my sincere thanks to my friend Dr. Tsegalem Abera, Department of Veterinary Microbiology, MVC, for constant encouragement and day to day companion during my stay in India. I am extremely grateful to Mr. C. Janakachakravarthy, laboratory technician, Department of Veterinary Epidemiology and Preventive Medicine, MVC, for tremendous assistance throughout the study period.

9 It is time to give credit to Departments of Animal Biotechnology, Bio- Informatics and Library Sciences for free access to resources they have during the entire study period. I would like to extend my thank to Wollo University, Dessie, Ethiopia and Ministry of Education, The Federal Democratic Republic of Ethiopia, for this opportunity and financial support. Words are not strong enough to express my appreciation to Diplomats and staffs of Ethiopian Embassy, New Delhi, India for coordinating the entire study process with financial and moral support.. I would like to thank Tamil Nadu Veterinary and Animal Sciences University and ICAR, India for the opportunity provided. My heartfelt thanks, love and appreciation to: my mother Kedja Habib, you are the inception for all what I am right now; to my sisters and brothers for your care and affection since my childhood. My beloved spouse, Eyerusalem Mengesha, I appreciate your strength to take the responsibility of our family during this time. My elder son, Elabem, I know you have been in need of me but you let me finish the journey uninterrupted. Was that because you know what I was for? Miss you all always! Overall I am ever grateful to the almighty who blessed me abundantly with good health and strength to complete this work. Faris Delil Yesuf

10 Abstract

11 ABSTRACT Title Name of the student I.D.NO. Degree for which thesis is submitted Chairman College University : EVALUATING DIAGNOSTIC TESTS TO ASSESS THE EPIDEMIOLOGICAL STATUS OF BOVINE TUBERCULOSIS : FARIS DELIL YESUF : DPV (VEP) : Ph.D., in Veterinary Epidemiology and Preventive Medicine : Dr. P.I. GANESAN, Ph.D., Professor and Head Department of Veterinary Epidemiology and Preventive Medicine Madras Veterinary College, Chennai : Madras Veterinary College, Chennai-7 : Tamil Nadu Veterinary and Animal Sciences University, Chennai-51 Year : 2012 Evaluation and assessment of the epidemiological status of bovine tuberculosis (btb) using different diagnostic techniques have been carried out in organized, unorganized dairy farms and slaughter house materials in urban and suburban areas. In organized farms btb diagnostic tests including Single Intradermal Comparative Cervical Tuberculin (SICCT) test, Gamma Interferon (IFN-γ) assay, Enzyme Linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction

12 (PCR) were employed. From these organized farms, 195 animals were screened by SICCT test of which 7(4.1%) were reactors. Skin test reactivity has been compared among the two management systems adopted by organized farms including those practicing semi-grazing and zero- grazing. Higher percentage of animals from semi-grazing type of management i.e.7 out of 114 (6.1%) were found reactors to SICCT test compared with zero-grazing 1 out of 81 (1.2 %). A total of 117 animals from the same organized farms that were screened by SICCT test were also assessed by IFN-γ assay for btb and 13 animals (11.1 %) were positive. The prevalence figure among the two management systems using IFN-γ assay compared. As that of SICCT test, more number of animals, 9 out of 36 (25%) from semi-grazing type of management were positive compared with zero-grazing which was 4 out of 81 animals (4.9%). Comparison of SICCT test and IFN-γ assay indicated 5 (4.3%) animals to be positive by both tests and 101 (86.3%) negative by both tests with 90.6% concordance. The two tests showed moderate agreement (kappa=0.44). Comparison of SICCT test and ELISA displayed 3 (2.6%) animals to be positive by both tests and 105 animals (89.7%) negative by both tests with 92.3% concordance. The two tests showed moderate agreement (kappa= 0.36). With regard to ELISA, from the same organized farms that were screened by SICCT test and IFN-γ assay, 117 parallel sera were assessed for btb antibody using commercial kit. Of 117 animals, 8 (6.8 %) were seropositive. Comparison of ELISA and IFN-γ assay revealed that 2 animals (1.7%) to be positive by both tests and 99 animals (84.6) negative by both tests with 86.3% concordance. The two tests showed slight agreement (kappa=0.13). The overall btb prevalence in organized farms by using these three diagnostic tests: SICCT test, IFN-γ assay and ELISA in the current investigation

13 with interpretation of positive if an animal is positive by any of these tests was 15.9%. In unorganized farms, ELISA and PCR using milk and nasal swabs were employed during the current investigation. Out of 193 sera, 101 were bovine samples that attended MVC Teaching Hospital and 92 from small holder dairy farms in Salem district, 11 (10.9%) and 3 (3.3%) animals were seropositive, respectively. Similarly, 207 milk samples, 167 from MVC Teaching Hospital outpatient unit and 42 from small holder dairy farms from Salem district that were assessed for MTC by IS6110 PCR. In this study, 39 (23.6%) from MVC and 3 (7.1%) from Salem district were shedding organisms in milk. On the other hand, out of 158 nasal swabs collected from MVC out-patient unit and screened for the same MTC, 8 (5.1%) were excreting MTC organisms in nasal secretions. A total of 388 carcasses were inspected for visible lesions (VL) in-situ and tissue samples from lungs, bronchial lymph nodes, spleen and heart with or without VL were also collected for further analysis. Of all carcasses and/or organs inspected, VLs were observed in 22 (5.7%) organs. Tissue impression smears from organs with or without VLs were stained using Ziehl-Neelsen stain and acidfast bacilli were observed in 23 (5.9%). The same tissue samples were further analyzed by PCR using IS6110 primer. Of 388, PCR detected 43 tissue samples (11.1%) positive for MTC. The presence of visible lesions and detection of AFB in tissues impression smears using ZN staining technique has been compared and a statistically highly significant difference (P<0.01) at 99% level of significance observed. Attempt has been made to culture MTC using the 43 PCR positive tissue samples and additional 6 PCR negative tissue samples by Lowenstein Jensen (LJ) media. Of 49 tissue samples, colonies characteristic of MTC were detected in 13 (26.5%) and further confirmed by ZN staining technique and IS6110 PCR. However, no growth observed in none of the 6 PCR negative tissue samples.

14 Out of 211 nasal swabs collected from the above same animals and screened using IS6110 PCR, 9 nasal swabs (4.3 %) were positive. A total of 56 slaughter house sera samples collected from the above same animals were assessed by ELISA and 12 sera (21.4%) were positive. Overall, 1024 cattle and buffaloes both from organized farms unorganized farms and slaughter house were screened using either of the aforementioned diagnostic tests of which 137 animals or their carcasses were positive by at least one of the tests used in current investigation. The prevalence of btb by considering an animal as positive if it is found to be positive by at least one diagnostic test was 13.4 %. The overall prevalence of btb by using these three tests in organized farms in the current assessment is 15.9% which is lower than the previous reports of the same farms. However, compared with other countries that adopt appropriate preventive and control measures, this figure is too high and could be taken as alarm to institute appropriate preventive and control measures including, entry level screening for newly purchased animals; routine practice of regular btb screening in the farm; test and disposal and/or segregation for positive and doubtful animals, respectively; retesting of doubtful animals after 2 months using tuberculin test or shorter using IFN-γ assay; separate pasture and farm worker allotment for test positive and negative animals until disposed; regular screening of the environmental specially soil and use of appropriate decontaminants to clear soil contamination; and adoption of regular screening techniques for milk to reduce cow-calf transmission and public health hazard. Key words: Bovine tuberculosis; Single Intradermal Comparative Cervical Tuberculin test; Gamma Interferon test; Enzyme-Linked Immunosorbent Assay; Polymerase Chain Reaction; Ziehl Neelsen staining, Visible lesions.

15 List of Contents

16 LIST OF CONTENTS S.No Title Page No. LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF ABBREVIATIONS 1 INTRODUCTION 1 2 REVIEW OF LITERATURE HISTORICAL BACKGROUND ETIOLOGY EPIDEMIOLOGY Occurrence Source of Infections Methods of Transmission Inhalation Oral route Vertical and pseudo-vertical transmissions Auto-contamination Host range Risk Factors Age Gender Breed Body condition Immune status Genetic resistance and susceptibility Herd-Level Risk Factors Access to human excretion 12

17 S.No Title Page No Herd size Type of cattle industry Management PATHOGENESIS IMMUNITY DISTRIBUTION OF BOVINE TUBERCULOSIS Global Scenario Indian Scenario Tamil Nadu DIAGNOSIS Acid Fast Staining Tuberculin tests Principle of tuberculin test and its drawback Types of tuberculin tests Single Intradermal Tuberculin (SIT) test Single Intradermal Comparative Cervical tuberculin (SICCT) test Double Intradermal Comparative Cervical test Sensitivity and specificity of tuberculin test Other limitations of tuberculin test Short thermal test Stormont test The gamma-interferon assay Polymerase Chain Reaction (PCR) PCR using tissue samples Limitations of PCR Slaughter house post-mortem survey 33

18 S.No Title Page No ELISA Cultural identification Economic losses due to Bovine tuberculosis Zoonotic importance Control and Eradication Test and segregation Disinfectants for mycobacteria 39 3 MATERIALS AND METHODS MATERIALS Study area Study animals Sample collection Serum Plasma Milk Nasal swab Tissues Materials required for SICCT Test Materials required for IFN-γ Assay Materials required for ELISA Materials required for Ziehl-Neelsen Staining Materials required for PCR DNA extraction kits Other diagnostics required for PCR Material required to prepare 50x TAE Preparation of Ethidium bromide Material required for culture Preparation of Antibiotic solution 46

19 S.No Title Page No. 3.2 METHODS Single Intradermal Comparative Cervical Tuberculin Test Gamma Interferon (IFN-γ) assay test procedure ELISA Ziehl-Neelsen staining Polymerase Chain Reaction (PCR) DNA Extraction Tissue samples DNA extraction DNA extraction from milk and nasal swabs Agarose Gel Electrophoresis Culture Farm management and epidemiological information Data management and statistical analysis 56 4 RESULTS DIAGNOSIS OF btb IN ORGANIZED FARMS Single Intradermal Comparative Cervical Tuberculin Test Comparison of SICCT test results from different sources Comparison of different breeds reaction to SICCT test for Age-wise btb prevalence using SICCT test Gamma Interferon (IFN-γ) Assay Comparison of IFN-γ assay results from different sources Breed-wise prevalence of btb by using IFN-γ assay Age-wise prevalence of btb using IFN-γ assay Enzyme Linked Immunosorbent Assay (ELISA) Comparison of btb seroprevalence from different sources Breed-wise btb seroprevalence using ELISA 68

20 S.No Title Page No Age-wise btb seroprevalence using ELISA PCR using nasal swabs and milk DIAGNOSIS OF btb IN UNORGANIZED FARMS Enzyme Linked Immunosorbent Assay (ELISA) Breed-wise seroprevalence of btb using ELISA Milk PCR Nasal swabs DIAGNOSIS OF btb IN POST-MORTEM SAMPLES Tissue samples Tuberculous lesions visibility Ziehl-Neelsen staining Comparison of different tissue and presence of AFB Polymerase Chain Reaction (PCR) for tissue Comparison of presence of gross lesions and tissue PCR Comparison of different tissue in relation with IS6110 PCR Culture Nasal swab PCR Enzyme Linked Immunosorbent Assay (ELISA) COMPARISON OF DIFFERENT btb DIAGNOSTIC TESTS Comparison of SICCT test and IFN-γ assay Comparison of SICCT test and ELISA Comparison of IFN-γ assay and ELISA Comparison of PCR for milk and nasal swabs Comparison of Ziehl-Neelsen staining and tissue PCR Farm management and epidemiological information 86 5 DISCUSSION DIAGNOSIS OF btb IN ORGANIZED FARMS 87

21 S.No Title Page No Single Intradermal Comparative Cervical Tuberculin Test Comparison of SICCT test results from different sources Comparison of different breeds reaction to SICCT test for btb Age-wise btb prevalence using SICCT test Gamma Interferon (IFN-γ) Assay Comparison of IFN-γ assay results from different sources Breed-wise prevalence of btb by using IFN- γ test Age-wise prevalence of btb using IFN-γ assay Enzyme Linked Immunosorbent Assay (ELISA) Age-wise btb seroprevalence using ELISA DIAGNOSIS OF btb IN UNORGANIZED FARMS Enzyme Linked Immunosorbent Assay (ELISA) Milk PCR Nasal swabs DIAGNOSIS OF btb IN POST-MORTEM SAMPLES Tuberculous lesions visibility Ziehl-Neelsen staining Polymerase Chain Reaction (PCR) Culture COMPARISON OF DIFFERENT btb DIAGNOSTIC TESTS Comparison of SICCT test with IFN-γ assay Comparison of SICCT test and ELISA Comparison of ZN staining and tissue PCR Farm management and epidemiological information SUMMARY REFERENECES 107

22 List of Tables

23 LIST OF TABLES Table No. Title Page No. 1 Layout for dispensing blood and antigens into 24-well 48 2 Single Intradermal Comparative Cervical Tuberculin test result for btb in two organized dairy farms with different management systems in and around Chennai 3 Comparison of Single Intradermal Comparative Cervical Tuberculin test result in different breeds from organized farms in and around Chennai 4 Age-wise prevalence of btb using Single Intradermal Comparative Cervical Tuberculin test result 5 Prevalence of btb in organized farms managed in two different 63 management systems by using IFN-γ assay 6 Breed-wise prevalence of btb using IFN- γ assay 65 7 Age-wise prevalence of btb using IFN-γ assay 67 8 Seroprevalence of btb in organized farms using ELISA 68 9 Breed-wise seroprevalence of btb in organized farms using 68 ELISA 10 Age-wise seroprevalence of btb in organized farms using ELISA Seroprevalence of btb in unorganized farms Breed-wise seroprevalence of btb in unorganized farms Detection of MTC DNA using IS6110 PCR in milk from 73 unorganized farms 14 Comparison between visibility of lesions and presence of AFB 76

24 15 Percentage prevalence of AFB in different tissues, Perambur 79 slaughter house Chennai 16 Comparison of post-mortem lesions and PCR Detection of MTC DNA in different organs by using IS6110 PCR 80

25 List of Figures

26 LIST OF FIGURES Figure No. Title Page No. 1 Comparison of SICCT test results in various breeds of cattle and 62 buffaloes from organized farms in and around Chennai 2 Breed-wise prevalence of btb by using IFN-γ assay from large 66 ruminants in and around Chennai

27 List of Plates

28 LIST OF PLATES Plate No. Title Page No. 1 A typical SICCT test reactor -Jersey cross cow 61 2 A typical SICCT test reactor buffalo 61 3 Gamma Interferon (BOVIGAM) Enzyme-linked Immunosorbent Assay plate Enzyme-linked Immunosorbent Assay plate IS6110 milk PCR gel electrophoresis picture 74 6 Miliary tuberculosis on mesentery 77 7 Miliary tuberculosis on heart 77 8 Lungs with granulomatous lesions 78 9 Ziehl Neelsen acid fast stained tissue impression smear (1000x magnification) IS6110 tissue PCR gel electrophoresis picture Characteristic colonies of Mycobacterium spp. on LJ media slant 12 Ziehl-Neelsen stained culture smear with characteristic AFB (1000x magnification) 83 83

29 List of Abbreviation

30 LIST OF ABBREVIATIONS AFB BC BCG bp btb Acid-fast bacilli Before Christ Bacillus Calmette Gurein Base pair Bovine tuberculosis ºC Degree Celsius cfu CMI CD CFU CFP-10 DTH DNA EDTA ELISA ESAT-6 gm hrs IFN-γ IS IU LN LAC-OP colony forming unit Cell mediated immunity Cluster of Differentiation Colony forming unit Culture filtrate protein-10 Delayed type hypersensitivity Deoxy ribonucleic acid Ethylene diamine tetraacetic acid Enzyme Linked Immunosorbent Assay Early Secreted Antigenic Target-6 Gram Hours Interferon Gamma Insertion sequence International Unit Lymph node Large Animal Clinic out-patient

31 MVC ml mm mg mm nm ND OD OIE OP OTF PBS PCR PPD rpm Madras Veterinary College Millilitre Millimetre Milligram Millimolar Nanometer Non Descript Optical Density Office Internationale des Epizooties Out-patient Officially tuberculosis free Phosphate buffer saline Polymerase Chain Reaction Purified protein derivative Revolutions per minute SIT SICCT TAE TB TE UV μl Single intradermal test Single Intradermal Comparative Cervical Tuberculin Tris acetate EDTA Tuberculosis Tris EDTA Ultra violet Microlitre

32 μg Microgram % % WHO w/v χ 2 ZN World Health Organization Weight by volume Chi-square Ziehl-Neelsen

33 Introduction

34 CHAPTER I INTRODUCTION Bovine tuberculosis (btb) is a chronic, infectious and contagious disease of livestock, wildlife and humans caused by infection with Mycobacterium bovis. It is a disease which poses a major economic problem and a significant public health risk in several countries in the world (Pollock et al., 2005). The public health concern is particularly significant in developing countries due to scarce preventive and/or control measures (Etter et al., 2006). Infection of cattle with M. bovis is usually chronic and can remain subclinical for long periods. Infected cattle can become infectious long before they exhibit any obvious clinical signs or lesions typical of tuberculosis (TB) detectable even with most careful veterinary examination. Even if present, the clinical signs of TB in cattle are not pathognomonic. As a result, effective antemortem surveillance for bovine tuberculosis (btb) must primarily rely on the detection of infected cattle at an early stage by the use of sensitive immunodiagnostic tests (De la Rua-Domenech et al., 2006). Bovine TB has almost been eradicated in developed countries after the implementation of preventive and control measures such as test and culling of infected animals or pasteurization of milk but foci of infection is still maintained due to a number of wildlife maintenance hosts including badgers in UK (Corner, 2006), Eurasian wild boar in Spain (Jaroso et al., 2010), white-tailed deer and mule deer in USA (Böhm et al., 2007), African buffalo and wildebeest in southern Africa (Renwick et al., 2006) brushtail possum in New Zealand (Buddle et al., 2000) and many more.

35 Since btb remains a worldwide problem, it is imperative to intensify control and preventive measures aimed at its eradication. As the real incidence of M. bovis on human health is still unknown, it is essential to advance the eradication of btb worldwide by means of adequate programmes, especially in developing countries. Even if the risk to human health is low in most developed countries, the HIV pandemic raises concern about its impact on the transmission of M. bovis to and between humans (Grange, 2001). Individuals with HIV/AIDS infection are the highest risk groups (Ayele et al., 2004). It is estimated that M. bovis might infected over 50 million cattle worldwide with resulting economic losses of approximately $3 billion (Hewinson, 2001). Traditional test-and-slaughter policies based on skin testing with tuberculin have not been fully successful so that additional diagnostic tests are required (Pollock et al., 2005). The success of btb eradication depends upon the removal of infected animal before it becomes source of infection for other animals and contaminates their environment. None of the tests currently available for the diagnosis of btb allow a perfectly accurate determination of the infection status of cattle. Some diagnostic techniques have poor sensitivity resulting in higher false negative animals and some have low specificity leading to more number of false positive animals, resulting in culling of btb free animals. Culling btb free animals is not affordable in most developing countries due to associated economic loss to the farmer or government if compensation plan is implemented. The false negative result on the other hand results in the maintenance of infection. In general, the key measure of diagnostic test accuracy is the relationship between sensitivity and specificity, which determines the false-positive and false-negative proportions. Therefore, this work has been done to identify effective diagnostic techniques through the simultaneous use of two or more screening methods for bovine tuberculosis investigation in different farms under different management conditions with the following specific objectives:

36 i. To detect the status of bovine tuberculosis in organized and unorganized dairy units in some parts of Tamil Nadu by employing different diagnostic tests. ii. To identify management factors contributing to the occurrence of bovine tuberculosis in organized farms. iii. To evaluate btb diagnostic tests viz Polymerase Chain Reaction (PCR), Interferon gamma EIA and Enzyme-Linked Immunosorbent Assay (ELISA).

37 Review of Literature

38 CHAPTER II REVIEW OF LITERATURE 2.1. HISTORICAL BACKGROUND According to archaeological evidences, tuberculosis (TB) in humans dates back at least to 4000 or 5000 B.C. and it has been one of the worst enemies of mankind; considered by some to be the greatest killer in history. Robert Koch in 1882, reporting his discovery of the causative agent of TB in man, claimed that the most dreaded infectious disease like bubonic plague rank far behind TB if the number of victims which a disease claims is the measure of its significance. Statistic shows that one-seventh of all human beings die of TB (Selgelid, 2008). It is a disease of antiquity that has been found in the mummies of ancient Egypt. It was described by Hippocrates and Aristotle in the fifth century B.C. as phthisis, which was translated into English as consumption (Hart et al., 1996). The turning point in the history of tuberculosis occurred on March 24th 1882 where Robert Koch had observed and cultured the bacillus responsible for tuberculosis. Koch termed them Tuberkelbacillen (bacilli of tuberculosis). In 1898, Theobald Smith observed certain differences between tubercle bacilli of bovine and human origin named these two variants of the tubercle bacillus ETIOLOGY Bovine tuberculosis is caused by Mycobacterium bovis, which is a slow growing non-chromogenic acid-fast bacillus with exceptionally wide mammalian host range but cattle are considered to be the primary hosts (Ayele et al., 2004; Thoen, 2010). O Reilly and Daborn (1995) stated that M. tuberculosis and M. bovis are pathogenic for cattle; however tuberculosis in cattle in most countries is invariably caused by M. bovis (Cousins et al., 1991). M. bovis is a member of Mycobacterium tuberculosis complex that also includes M. tuberculosis, M. africanum, M. microti, M. caprae, M. canetti M.

39 pinnipedii, and M. bovis BCG strain (Collins, 2011). M. bovis closely resembles M. tuberculosis, and precise identification of and distinction between the two can be established by biochemical and molecular biology techniques (Ayele et al., 2004). At the genome level, it shares more than per cent identity with M. tuberculosis (Garnier et al. 2003; Parra et al., 2008). The genome of M. bovis contains around 4000 genes. Since the cell wall is the interface between the bacillus and host; genes encoding cell wall structures show the greatest degree of variation due to selection. Therefore, the greatest degree of sequence variation between the human and bovine bacilli is in genes encoding cell wall and secreted proteins (Hewinson et al., 2006). Mycobacterium spp. in general have thick waxy envelope so that they are extremely resistant to external influences. Despite being obligatory intracellular parasite, the bacilli in pus and morbid discharges remain viable for several weeks. But the bacilli in culture are destroyed by direct sunlight within few hrs. The bacilli remain viable for at least five months in faeces and survive for up to approximately 400 days in running water. Cultures in physiological saline solution and egg-based media have been observed to survive for days and at least six years, respectively (Phillips et al., 2003). 2.3 EPIDEMIOLOGY Occurrence All species and age groups are susceptible to M. bovis but cattle, goats and pigs are most susceptible. Sheep and horse show a high natural resistance. In most developed countries tuberculosis in animals is now rare with occasional severe outbreak occurring in small group of herds. This is the result of rigorous TB control programs in place for many years by strict test and slaughter policy. At present, the presence of the disease is signaled by detection in carcasses at abattoirs (McGavin and Zachary, 2006; Radostits et al., 2010).

40 2.3.2 Source of infection Infected cattle are the main source of infection for other cattle where the causative agent is excreted in the exhaled air, in sputum, faeces (from both intestinal lesions and coughed-up and swallowed sputum from pulmonary lesions), milk, urine, vaginal and uterine discharges, and discharges from open peripheral lymph nodes (Radostits et al., 2010). But generally, infection of the lungs by inhalation is the most common in adult animals, whereas ingestion of infected milk or colostrum is more predominant in young animals (McGavin and Zachary, 2006; Thoen, 2010). The organisms are mostly transmitted by aerosol or droplets of exudates with bacilli when animals or man at advanced stage of TB cough or sneeze. Ingestion of feed and water contaminated with infectious materials including faeces, urine or exudates from diseased animals are other sources of infection (Thoen, 2010). Hutchings and Harris (1997) suggested pasture contamination with the urine, faeces and/or sputum of infectious badgers to be the main route of transmission from badger to cattle. Active latrines are avoided by most cattle until the grass sward length in the rest of the field is reduced. During dry period where grass sward length is much reduced, amount of soil ingested increases maximizing the chances of ingesting M. bovis in the contaminated soil. Transmission from an infectious animal to a susceptible animal depends on proximity, which in turn depends on close contact and ventilation. Herd size is a risk factor for the incidence of btb (Goodchild and Clifton-Hadley, 2001). Wildlife can play an important role in the introduction of tuberculosis caused by M. bovis into cattle herds in some countries. Nevertheless; some of the initial infection, and eventually most of the spread within an infected herd may be due to cattle-to-cattle transmission. Adequate bio-security can reduce the spread of infection between herds and management measures such as adequate

41 ventilation, reduction of group sizes and hygienic measures can reduce transmission within herds (Goodchild and Clifton-Hadley, 2001) Methods of Transmission There are several routes of infection but respiratory and gastrointestinal tracts are primary routes of transmission for M. bovis. Experimental studies involving exposure of animals to M. bovis via different routes including intratracheal, oral, intravenous and intraperitoneal demonstrated that the nature and extent of tuberculous lesions vary with the route of exposure (Palmer et al., 2002) Inhalation Respiratory transmission through the inhalation of contaminated aerosols or fomites is the most common and efficient form of transmission which require low number of organisms as an infective dose (Kaneene, 2008). Menzies and Neill (2000) suggested the probability of a single bacillus within a droplet nucleus to be sufficient to establish infection within the bovine lung. Johnson et al. (2007) investigated the degree of lesion advancement and granuloma distribution in experimentally infected four groups with different doses of M. bovis (1cfu, 10 cfu, 100 cfu and 1000 cfu). In their finding a similar lesion advancement and granuloma distribution between the lowest dose group (1 cfu) and the highest dose group (1000 cfu) have been noticed using different diagnostic tests. Neill et al. (1990, 1992) observed that respiratory route of infection most important source especially in animals in close contact with each other. Similarly, Menzies and Neill (2000) suggested that close contact between animals is a major risk factor for btb infection.

42 Oral route Ingestion of M. bovis is considered to be the second most common route of infection (Pollock and Neill, 2002). However, Phillips et al. (2003) suggested that M. bovis infection is unlikely to be acquired directly from eating grass contaminated by other cattle since cattle do not usually graze where other cattle deposit faeces. Nevertheless, once faeces are disaggregated, mycobacteria may survive longer in the environment under adequate climatic conditions. Generally, mycobacteria are not frequently and regularly excreted in cattle faeces, even by a heavily infected animal reducing chance of faeco-oral infection (Neill et al., 1988) Vertical and pseudo-vertical transmissions Ozyigit et al. (2007) described vertical transmission of M. bovis from an infected dam to her calf through congenital infection in utero. Zanini et al. (1998) suggested ingestion of contaminated colostrums or milk as another means of btb transmission from dam to calf. Transmission through close contact (pseudovertical) between a cow and its calf by grooming has been mentioned as a possible risk factor too (Phillips et al., 2003) Auto-contamination According to Phillips et al. (2003), cattle that become infected through the oral route might further emit contaminated aerosols during regurgitation. The animal might inhale these contaminated aerosols and a subsequent respiratory infection can occur; as few as one bacilli can infect an individual via respiratory route (Neill et al., 1988) Host range M. bovis has an exceptionally wide host range of all known pathogens (O Reilly and Daborn, 1995 and Smith et al., 2006). The disease has been reported in cattle, bison, buffaloes, marsupials, hares, equines, camels, pigs, sheep, goats, deer, antelopes, elephants, cats, dogs, foxes, mink, badgers, ferrets, non-human primates and man (Francis, 1947 and 1958).

43 Cattle are the most important host from an anthropocentric point of view (Grange and Collins, 1987). Wilesmith and Clifton-Hadley (1994) and (Gay et al., 2000) reported M. bovis infection in cats and dogs respectively. Goats are very susceptible to an infection with M. bovis, but outbreaks of caprine M. bovis TB are only reported occasionally; they could nevertheless interfere with btb eradication programmes as already observed in cases reported in Spain and western Wales (Seva et al., 2002; Crawshaw et al., 2009). Hence, the risk of contaminating btb-free cattle herds by introducing infected goats should be considered. Ovine disease is rare and usually associated with cattle cases. However, individual cases of M. bovis tuberculosis in sheep has been reported (Malone et al., 2003; Houlihan et al., 2008). Horses are susceptible to infection with M. bovis and could be a potential source if close contact between horses and cattle is common (Monreal et al., 2001). Many species including pig, ferret, feral cat, hare, hedgehog, baboon and lion are spill-over hosts (Buddle et al., 2000) in which infection is not selfmaintaining; whereas opossum, badger, African buffalo and deer act as maintenance hosts (Buddle et al., 2000). The prevalence of the disease, the capacity of excretion, as well as the ethology and ecology of wild animals influence their role as a reservoir for M. bovis. However, demarcation between these two categories of hosts is not clearly established, as the result of dynamic nature of the relationship between maintenance and spill over hosts (Delahay et al., 2002).

44 2.4 Risk Factors Factors like trade, direct contact of btb-free animals with animals from infected herd, mixing of herds from different countries and with infected wild animals, especially in border areas, represent continuous possibilities to reintroduce btb into free herds (Schiller et al., 2011) Age Age is reported to be one of the main individual risk factors. The duration of exposure increases with age so that older animals are more likely to have been frequently exposed than younger ones (Humblet et al., 2009). Griffin et al. (1996) observed that calves were less likely to be reactors to tuberculin test than older in a cross-sectional study carried out in Ireland. Aswathanarayanan et al. (1998) reported greater percentage of reactors in the older age groups. Stamp (1944) observed adults, growers and young stock being susceptible to btb in descending order Gender A cross-sectional study conducted in Tanzania revealed that male cattle were significantly more affected by btb than female animals. Male cattle are kept longer in the herd than females since they are mostly used for tillage purpose. Due to this particular longevity, it is more probable that they stay in contact with infected cattle from other affected herds and in turn get infected; this would imply that between-herd contact is a major source of btb transmission (Kazwala et al., 2001). On the other hand, a cross-sectional study carried out in Uganda from 2006 to 2007 revealed significantly more females positive to the skin test than males which could be due to the relative long stay of female animals for dairy purpose compared with male that are reared as beef cattle (Inangolet et al., 2008).

45 2.4.3 Breed Studies performed in Africa identified the animals breed as a risk factor for a positive skin test. Omer et al. (2001) suggested that imported breeds may be less resistant to btb compared to indigenous breeds, in a cross-sectional study carried out in Eritrea. Similarly, Elias et al. (2008) reported higher btb infection rate in imported dairy cattle in Ethiopia. Resistance to bovine tuberculosis in general has been considered to be confined to zebu breeds (Bos indicus) (O Reilly and Daborn, 1995; Radostits et al. 2010). Thakur et al. (2010) reported marked differences in relation to btb susceptibility among different breeds (pure breeds and their crosses) Body condition In cross-sectional study in Zambia by Cook et al. (1996) a low body condition score (BCS) had been found to be linked to an increased risk of a positive skin reactivity. Similarly, Kazwala et al. (2001) suggested that skin test reactors might have a poor BCS as a consequence of an advanced stage of btb associated with the long-lasting pathological process Immune status Menzies and Neill (2000) suggested the risk of becoming infected with M. bovis increases with immunosuppression similar to diseases, the risk of but yet it has to be scientifically demonstrated. De la Rua-Domenech et al. (2006) revised that the susceptibility to M. bovis may be enhanced in cattle infected with immunosuppressive viruses such as bovine viral diarrhoea Genetic resistance and susceptibility Genetic mechanisms of non-specific immunity including bronchial mucus, efficiency of the mucociliary clearance, active non-specific macrophages in the lungs (and their lysosomal enzymes) as well as their destructive efficiency could eliminate a low-dose M. bovis challenge (Phillips et al., 2002).

46 2.4.7 Herd-Level Risk Factors Access to human excretion Ayele et al. (2004) sited man with clinical genitourinary tuberculosis; at the excreting stage could contaminate cattle in place where people often urinate on pastures. Corner et al. (1990) reported human cases of tuberculosis among people in contact with a herd as a risk factor for finding a skin test reactor. Cattle contamination by a human excreting M. bovis was also reported in Switzerland in 1998 (Fritsche et al., 2004) Herd size Studies carried out in several parts of the world identified herd size as one of the major btb herd-level risk factors (Cleaveland et al., 2007; Oloya et al., 2007). De la Rua-Domenech et al. (2006) suggested that due to the low specificity nature of skin test, increase in herd size raises the probability of false positive reactors. Monaghan et al. (1994) suggested if more animals are skin tested the higher the probability to have a reactor. Ameni et al. (2006) observed the severity of btb being significantly greater in cattle kept indoors at a higher population density than in cattle kept on pasture Type of cattle industry Animals raised outdoors and kept alive long enough to express lesions are more susceptible to develop clinical signs (Humblet et al., 2009). Barlow (1997) reported dairy cows experience more production stress and gathering of cattle during milking increases the risk of btb transmission.

47 Management Humblet et al. (2009) claimed management system defines cattle-to cattle contact, contact between cattle and contaminated environmental sources and wildlife. However, large-scale studies required to identify which management practices are risk factors in order to adopt preventive and control measures. Cosivi et al. (1998) suggested highest btb incidence in intensive dairy production system. The use of hired or shared bulls also increase the risk of btbintroduction in a herd as suggested by a retrospective matched case-control study carried out in btb-infected farms in Michigan (Kaneene et al., 2002). A cross-sectional study carried out in Eritrea in 2001 suggested that farm size was a risk factor for btb but no information was provided regarding what farm size was considered as being at risk (Omer et al., 2001). Elias et al. (2008) reported that poor housing and management could be involved in the reduction of an animal s resistance to btb. Griffin et al. (1993) indicated that cattle may be at risk if slurry is spread within the two months preceding grazing. In same report, a self-feed silage system was more stressful for animals and thus increased susceptibility to btb. Studies conducted in the UK suggested badgers as a potential source of contamination for silage with urine, faeces or sputum containing M. bovis (Reilly and Courtenay, 2007). Kaneene et al. (2002) identified providing hay on the ground than in feeders, and providing loose hay, rather than in bales, were associated with an increased risk of btb in a retrospective matched case-control study carried out on btb-infected farms in 2002 in Michigan.

48 Studies carried out in the UK, Michigan, Italy and Tanzania suggested that arrival of an infected animal in a btb free herd is one of the major risk factors for introducing btb (Gopal et al., 2006; Kaneene et al., 2002; Marangon et al., 1998; Shirima et al., 2003). A Canada herds tested in outbreak situations was at greater risk if they had either purchased animals from positive farms or requested testing because of possible contact with positive (Munroe et al., 1999). In a study analyzing cattle movements in Great Britain between 1985 and 2003, movement of animals was shown to be a critical risk factor (Gilbert et al., 2005). Gopal et al. (2006) demonstrated animal movements to be greatly responsible for most outbreaks reported in North East England between 2002 and 2004 by molecular typing. Retrospective matched case-control study conducted in Michigan cattle farms in 2001 indicated sharing of pastures or facilities as a risk factor for the transmission of btb when animals come in contact with another herd (Kaneene et al., 2002). Humblet et al. (2009) suggested that the probability of detecting lesions increases with high culling rate in countries where surveillance programmes based on meat inspection are effective. 2.5 PATHOGENESIS Pathogenicity of mycobacteria is a multifactorial phenomenon requiring the participation and cumulative effects of several components. Inhaled bacilli reaching the alveoli are phagocytosed by pulmonary alveolar macrophages and then respiratory infection starts. If alveolar macrophages are successful in destroying the bacteria, infection is obviated. The organism mostly multiply

49 intracellularly, kill the macrophage, and initiate infection from which bacilli spread via airways within the lungs and eventually via the lymph vessels to trachea-bronchial and mediastinal lymph nodes (Pollock et al., 2006; McGavin and Zachary,2006 ). Ingestion of the tubercle bacilli by phagocytes into phagosomes or intracytoplasmic vacuoles protects the organism from bactericidal components in serum. Lysosomes fuse to the phagosomes to form phagolysosomes. Virulent tubercle bacilli elude the microbicidal activities of the hydrolytic enzymes released by the macrophages. The bacilli multiply and destroy the phagocytes. Other phagocytes ingest the increasing number of tubercle bacilli. A small cluster of cells referred as granuloma develops. Cellular responses result in the accumulation of large numbers of phagocytes and finally result in the formation of a tubercle (Laneelle and Daffe, 1991; Palmer and Waters, 2006; Pollock et al., 2006). If the infection is not contained within primary complex, lungs and regional lymph nodes, bacilli disseminate via the lymph vessels to distant organs and other lymph nodes by the migration of infected macrophages. When the inflammatory process containing the mycobacteria erodes the walls of blood vessels and causes vasculitis, haematogenous dissemination occurs. If bacterial dissemination is sudden and massive, numerous small foci of infection develop in many tissues and organs referred to as miliary tuberculosis (McGavin and Zachary, 2006). Tubercles are often found in bronchial, mediastinal and portal lymph nodes. Lungs, liver, spleen and superficial surfaces of body cavities are also affected (Thoen et al., 2009). Differences in the pathogenicity among various species have not been explained (Thoen and Himes, 1986). The challenge dose to establish tuberculosis in calves should be computed on body weight basis, but the big unknown is virulence of the strain of M. bovis employed (Pollock and Neill, 2002).

50 cattle. Dean et al. (2005) reported that 1 cfu is sufficient to induce a disease in 2.6 IMMUNITY The dominant immune response to mycobacterial infections in cattle is cellular rather than humoral in nature (Ritacco et al., 1991; Vordermeier et al., 2004; Welsh et al., 2005). However, mycobacterial infections also induce antibody responses. Generally, specific antibody responses are mostly detected late in disease and correlate with severe progression of disease (Plackett et al., 1989). The response of host to the tubercle bacillus is broad and complex, involving all aspects of the immune system (Flynn and Chan, 2001). M bovis as other member of MTC has evolved to avoid immune clearance and induce chronic lesions (North and Jung, 2004). Following experimental challenge or natural infection with M. bovis, robust DTH and IFN-γ responses are elicited. Thus, a mainstay for TB diagnosis is based on evaluation of these cell-mediated responses either by skin test (Monaghan et al., 1994) or IFN-γ-based assays (Wood and Jones, 2001). Infection with M. bovis stimulates a complex array of cellular immune responses, a dominant component of which is a type 1 CD4+ T-cell response and the tuberculin test used to diagnose bovine TB is an in vivo assay of this response (Morrison et al., 2000). 2.7 DISTRIBUTION OF BOVINE TUBERCULOSIS Global Scenario In Europe, btb is endemic in many countries and constitutes a significant economic burden to the agricultural industries (Schiller et al., 2011) The overall prevalence of btb is currently increasing in the EU. A rise in cattle herd btb positivity had been seen from 0.48% in 2006 to.053% in 2007