Evaluating Diagnostic Tests for Bovine Tuberculosis in Tanzania

HARRY STEELE BODGER MEMORIAL SCHOLARSHIP 2008 REPORT Evaluating Diagnostic Tests for Bovine Tuberculosis in Tanzania Morogoro and Serengeti, Tanzania 1 st September to 4 th October 2008 Kathryn Allan Background In Tanzania, Mycobacterium bovis which causes tuberculosis (TB) in cattle is wide spread. It has the broadest host range of all bacteria within the Mycobacterium tuberculosis complex group and frequently infects other mammalian hosts including humans and a range of wildlife species. The impact of M. bovis infection in cattle seems to vary between geographical areas. M. bovis infection has been reported to cause high morbidity and mortality in South African wildlife (Keet et al. 1996, 2000; de Lisle et al. 2002) but infections have been reported in Tanzanian wildlife with no obvious signs of clinical disease; M. bovis was isolated from greater than 10 per cent of carcases of apparently healthy wildebeest with no gross lesions on post-mortem examination (Cleaveland et al. 2005). There are a number of questions still to be answered about the apparent variability of pathogenicity caused by the infection in wildlife and livestock. Three main areas for further research have been highlighted: 1. The relationship between infections in livestock, wildlife and human populations 2. The role of different populations in disease maintenance and transmission 3. The factors determining pathogenicity in various populations. Within some populations, for example, in cattle in the UK (Sales et al. 2001), there is known to be a degree of M. bovis genetic diversity and this is also believed to occur across sub-saharan Africa. As yet, little genotyping data or information about the relationship between strain type and host pathogenicity is available for this region. One hypothesis is that the genetics of the causal agent may play a role in disease pathogenicity and transmissibility. It has also been suggested that within strains of M. bovis, there may be subgroups, each with a different and specific host preference (Smith et al. 2006). Determining the effects and impacts of each strain type will have important implications for long term disease control. 1

Previous investigations of the epidemiology of bovine tuberculosis in Africa have been hampered by the poor performance of tests in the field and there is an urgent need for robust, reliable and practical methods of TB diagnosis. Currently, the intradermal tuberculin test (skin test) is the routine test for bovine TB in the UK and other European countries. However, the use of this test in sub-saharan Africa has traditionally been limited by tuberculin availability, the need for repeat visits and the lack of suitable handling facilities. An additional problem with the skin test is the widespread cross-reactivity with environmental mycobacteria which can limit its use. Therefore, the use of more sensitive and specific tests such as the gamma-interferon immunoassay has been proposed as an alternative to increase the probability of accurately detecting infected animals. Thus far, its use in developing countries has been limited by the requirement for specialized facilities, the need for rapid transportation of samples to laboratories and the high costs involved. In Tanzania, there are several protected areas where close interactions between humans, domestic animals and wildlife create high risk situations for the transmission of TB between populations. One such area is the Serengeti ecosystem in Northern Tanzania which extends to the Masai Mara in south western Kenya. Although the use of protected land by local people is restricted, the areas are not fenced and wildlife is free to move through human settlements. Human-wildlife conflicts arise in the border areas where local people still live in a traditional manner; for example, crop damage by elephants is a real concern for small scale farmers. Serengeti National Park Sokoine University of Agriculture, Morogoro Figure 1: Map of Tanzania showing Serengeti National Park and Morogoro (Source: Tanzania, no. 3667, rev. 5 January 2005; permission granted) Recent data from Tanzania including the Serengeti area indicates that M. bovis is an important cause extrapulmonary TB in humans in addition to being a threat to livestock and wildlife health (Mfinanga et 2

al. 2005, Kazwala et al. 2006). The presence of a wildlife reservoir in this area may be particularly important in maintaining infection in cattle. The Sokoine University of Agriculture (SUA) in Morogoro, central Tanzania is a regional centre for excellence which offers a range of agriculture-based courses ranging from horticulture and forestry to animal health to over 2000 students each year. It is the only university to offer veterinary training in Tanzania and between 20 and 30 students graduate each year who will go on to work mostly in government positions. SUA is actively involved in a number of projects and international collaborations promoting animal health and welfare throughout Tanzania. Professor Rudovick Kazwala, a recognised expert in bovine tuberculosis, is based at the faculty of veterinary medicine at SUA and works closely with the Universities of Edinburgh and Glasgow. One ongoing project is a BBSRC-funded collaboration between SUA, Swiss Tropical Institute, Centre for Tropical Veterinary Medicine, University of Figure 2: The faculty of Veterinary Medicine at SUA Edinburgh and Veterinary Laboratories Agency (UK). Dr. Tiziana Lembo (previously of the University of Edinburgh, now at the University of Glasgow) has been working with the Professor Kazwala and his laboratory team to investigate the epidemiology of M. bovis in Tanzania. The project aims to assess the performance and practicality of different diagnostic tests in field conditions, characterise the diversity of M. bovis strains in different host species to determine host preferences, phylogeographical relationships and associated health impacts of each strain type. The team is due to host a regional training course funded by the Wellcome Trust to establish a network for bovine TB in East Africa. Linking with these existing bovine TB projects in north-western Tanzania and Morogoro, my visit had three main objectives; 1) To obtain field training in current techniques specific to livestock systems and Mycobacteria and to understand constraints and limitations of sample collection, diagnosis and disease surveillance in the field. Study sites The existing bovine TB projects are based in two main areas; firstly around Morogoro district with close proximity to SUA for rapid handling of samples, and secondly; around the Serengeti National Park (SNP) in Northern Tanzania. The Serengeti ecosystem presents a rare opportunity to study interactions and disease transmission between livestock, wildlife and people. The SNP is home to large numbers of African wildlife and is particularly famous for the annual migration of wildebeest between the SNP and the Masai Mara in Kenya. In the areas surrounding the SNP, the indigenous people live in traditional settlements and homesteads made from local resources such as wood, mud and cow dung (Figure 3). 3

Figure 3: Traditional village and houses in Mugumu district, northern Tanzania Most families own livestock; zebu cattle predominate in these areas and are considered to be a sign of wealth. Goats and sheep are also common and there are close interactions between farmers and their livestock. Young calves and lambs may be kept inside the house at night while adult cattle rest in a boma in the centre of a small cluster of houses. Cattle are grazed in a nomadic fashion in many areas and frequently encounter wild ruminants as they move around the borders of the park. The way of life in these villages creates many opportunities for transmission of disease between wild and domestic ruminants and also between livestock and farming families due to their close contact. Bovine Tuberculosis Diagnostic Testing Post mortem sampling Post mortem (PM) examination and sampling was performed on cattle from slaughterhouses in Northern Tanzania. Cattle were purchased for slaughter and confined for three days for ante-mortem inspection, blood sampling and testing by Bovigam assay and skin test. Following slaughter, carcases were inspected for TB lesions in collaboration with trained meat inspectors and samples were collected to be tested for the presence of Mycobacterium species. Diagnostic Testing Samples were collected from cattle in the Morogoro region of Tanzania, known to be infected with bovine TB from previous skin tests and PM findings, and from a teaching dairy herd of Ayrshire cattle belonging to SUA. 4

Figures 4&5: Cattle gathered for sampling with SUA students watching data recording closely! Figure 6: Cow after inoculation with bovine and avian antigen testing in the rest of the country were not encountered by us. The procedure for the skin test can be found in appendix 1. Intradermal tuberculin testing (skin test) The intradermal tuberculin test (skin test) was performed on 20 Ayrshire cattle ranging in age from six months to seven years. The herd also included a borrowed bull at this time. In Tanzania, it is still common practice to share bulls especially those of improved genetic breeding potential. These bulls may run with a number of different herds throughout the year thus acting as potential source of disease transmission. In addition to the skin test, jugular venepuncture was performed to collect samples for gamma interferon immunoassays. SUA is fortunate to have good handling facilities and stocks men so many of the logistical difficulties facing routine 5

Table 1: Results of the skin test on Ayrshire cattle Animal ID Age AV0 AV3 Bov0 Bov3 Result* Interpretation of ITT BULL Unknown 10 13 7 13 3 Inconclusive (Borrowed) 39 7y5m 7 8 7 9 1 Inconclusive 76 5y2m 6 9 5 10 2 Inconclusive 100 4y3m 8 9 6 9 2 Inconclusive 122 3y4m 6 6 6 7 1 Inconclusive 138 2y7m 6 9 7 8-2 Negative 141 2y6m 9 9 7 7 0 Negative 142 2y6m 8 14 7 20 7 Positive 152 2y1m 10 10 7 9 2 Inconclusive 901 1y 6 6 4 4 0 Negative 902 1y9m 8 9 6 9 2 Inconclusive 906 1y3m 6 8 6 10 2 Inconclusive 908 1y4m 6 10 5 10 1 Inconclusive 909 1y2m 5 8 5 8 0 Negative 910 1y2m 4 7 4 8 1 Inconclusive 912 1y1m 4 5 3 3-1 Negative 915 11m 6 8 5 5-2 Negative 917 9m 5 6 3 3-1 Negative 919 6m 4 6 3 5 0 Negative 920 6m 5 8 3 5-1 Negative *Result = (Bov3-Bov0)-(Av3-Av0) Interpretation of the Intradermal Tuberculin skin test X = (Bov3-Bov0)-(Av3-Av0) X 4 = positive 1 X < 4 = Inconclusive 1 > X negative Of the 20 cattle tested, one positive reactor was identified which had clinical signs of respiratory disease (5%), there were 10 inconclusive test results (50%) and 9 negative tests results (45%). Analysis of test results by age group category showed all animals below one year of age to test negative using the skin test. All animals above 36 months of age gave inconclusive test results (see figure 7 below). 6

Figure 7: Chart showing test results per age group Current limitations of sampling, diagnosis and disease surveillance in the field Experiencing field research conditions gave me a greater appreciation of the challenges to be overcome to implement effective control programmes. Good communication and community liaison is vital to encourage local participation and to ensure availability of cattle for diagnostic tests and follow up visits. The use of the intradermal tuberculin skin test is limited in the field by the need for repeat visits and handling facilities in the field in addition to the potential for cross reactivity with environmental mycobacteria. Gathering cattle together can be quite a challenge in these areas as many graze far from the homestead and there is an obvious lack of adequate handling facilities at most of the local villages. The exception was in one village near Mugumu on the North West edge of SNP. Here, a local initiative had constructed the only cattle handling facilities in the district which consisted of a simple but effective wooden cattle race and crush. This facility had considerably improved management of cattle and was shared by all farmers in the vicinity. Such facilities, in addition to aiding in routine management procedures, can facilitate implementation of large scale disease control programmes such as diagnostic testing and vaccination campaigns. Most villages were not so fortunate however, and the lack of handling facilities was a great hindrance in managing herds of cattle in a safe and efficient manner. 7

2) Given the limitations of current techniques for in vivo detection, to assess the performance and practicality of an improved diagnostic test, the Bovigam, so to expand its use to larger scale studies in the Serengeti region and elsewhere in Tanzania. Gamma interferon immunoassay (Bovigam Immunoassay) Samples collected for validation of the Bovigam from herds of cattle in Morogoro and around SNP known to be infected with bovine TB on the basis of skin tests and PM findings. Bovigam is a gamma interferon immunoassay which is used to conduct an in vitro blood based assay of cell-mediated immunity to M. bovis PPD tuberculin to detect bovine tuberculosis infection. Tuberculin PPD is presented to lymphocytes in whole blood culture. Production of gamma interferon from cells is then detected using a monoclonal antibody sandwich ELISA. Lymphocytes of animals which are free from M. bovis infection will not be stimulated to produce gamma interferon by M. bovis PPD tuberculin. Figure 8: Blood sampling cattle for gamma interferon The test used as a confirmatory test in cattle in the UK for assay positive or inconclusive reactors within three to 30 days after application of the skin tests. The immunoassay is more sensitive that the skin test and can detect earlier infections. The Bovigam immunoassay was used to run the gamma interferon assay. A summary of the procedure is given below (full details are available in appendix 2): 1. Collect samples by jugular venepuncture into heparinised vacutainers. DO NOT REFRIDGERATE. 2. Culture within 30 hours of collection. Aliquot blood into three vials into which one of three substances is added; a. Phosphate buffered saline (negative control) b. Bovine PPD antigen c. Avian PPD antigen 3. Incubate samples at 37 C for 16-24 hours (whole blood). 4. Centrifuge sample and harvest plasma. Plasma can either be tested immediately or stored at minus 35 C for up to 7 days or minus 20 C for several months. 5. Each sample is assayed in duplicate and controls in triplicate. 6. Microplates coated with monoclonal antibody to gamma interferon are supplied for the assay. All reagents (except conjugate) must be brought to room temperature before use. 7. Controls; positive Bovine gamma interferon, negative phosphate buffered saline 8. For assay procedure; see appendix 2. 9. Plates are read using a microplate reader with both 450nm filter and 620-650 nm filters. 8

Figure 9: Laboratory work (left) with loaded Bovigam (right) Interpretation of Bovigam results: To interpret the results of the Bovigam immunoassay, the mean for each sample is calculated (nil/bovine/avian PPD OD) and compared to mean OD values for each animal. Table 2: Parameters for validation of Bovigam Positive (indicates M. Bovis infection) Bov.PPD.OD nil.ag.od 0.1 AND Bov.PPD.OD Av.PPD.OD 0.1 Positive bovine IFNγ OD > 0.700 (positive must not deviate by more than 30% from mean OD)* Negative Bov.PPD.OD nil.ag.od < 0.1 AND Bov.PPD.OD Av.PPD.OD < 0.1 Negative bovine IFNγ OD < 0.130 (maximum variation of ± 0.04)* *If these conditions are not met then the test results are invalid and should be repeated. Table 3: Results from Bovigam immunoassay in 29 cattle from the Morogoro District Sample no. Cow ID Bov.OD-nil.OD Bov.OD-Av.OD Test result 1 12 0.588 0.532 Positive 2 16 0.843 0.834 Positive 3 19 0.065 0.039 Negative 9

4 44 0.338 0.349 Positive 5 50 0.014 0.381 Inconclusive 6 52 0.065 0.242 Inconclusive 7 53 1.422 1.133 Positive 8 60 0.085 0.063 Negative 9 62 1.397 1.084 Positive 10 67 0.265 0.173 Positive 11 76 0.98 0.926 Positive 12 82 0.168 0.16 Positive 13 89 0.185 0.193 Positive 14 91 0.065 0.032 Negative 15 98 0.391 0.329 Positive 16 99 0.345 0.298 Positive 17 104 0.204 0.112 Positive 18 105 0.304 0.312 Positive 19 106 0.003 0.026 Negative 20 108 0.286 0.255 Positive 21 120 0.39 0.207 Positive 22 122 0.016 0.005 Negative 23 130 0.811 0.726 Positive 24 142 0.076 0.017 Negative 25 144 0.021 0.002 Negative 26 906 0.018 0.022 Negative 27 909 0.043 0.012 Negative 28 ARSU 0.781 0.77 Positive 29 A-BULL 0.104 0.1 Positive Limitations: 1. Incorrect techniques 2. Using other anticoagulants other than heparin 3. Excess levels of circulating IFNγ 4. Immunosuppresion 5. Contaminated reagents Other potential problems for widespread use of Bovigam test in Tanzania for detection of bovine TB are; Samples must be transported to the laboratory for stimulation within 30 hours of collection and must be kept at 22 C ± 5 C Refrigeration and appropriate storage of reagents can be difficult due to unreliable power supplies which compromise the institutions ability to maintain suitable refrigeration temperatures. There are also environmental factors to consider as well. For example, room 10

temperature in Tanzania is often above 30 C and it can be difficult to maintain suitable conditions for running tests within the lab as well as storage of reagents. Problems encountered while running the Bovigam test predominantly involved validation of the test results in comparison with the controls. On a number of occasions, the optical densities of the control samples did not meet the required criteria for test validation. Critical evaluation of the test procedure in the lab identified a number of potential causes of this failure; ensuring suitable operating and storage environments was a key issue. In addition, the potential for contamination and deactivation of reagents required reagents to be renewed on a regular basis which increases the cost of running this test long term. The specific requirement for maintaining suitable laboratory conditions for the Bovigam immunoassay questions the robustness of this test in the field. 3) To obtain training in techniques for Mycobacterium culturing and typing on post-mortem samples collected during abattoir surveys. Culture and Genus Typing PCR of Mycobacterium species (RD4 PCR) Laboratory culture of tissues from carcases was performed and positive cultures were then sub-cultured to allow identification of Mycobacterial characteristics for species classification and to differentiate between atypical mycobacteria and mycobacteria of the M. tuberculosis complex. RD4 PCR can be used to distinguish between complex (M. bovis and M. tuberculosis) and non-complex Mycobacterium species. The PCR procedure can be found in appendix 3. Complex mycobacteria species are indicated by the presence of a band at 372 base pairs in addition to the 1030 base pair genus product (see figure 11). Complex species can be further investigated using spoligotyping to identify the specific strain of mycobacterium isolated (Sinclair et al 1995). This information may then be used to indicate the source of infection with the intention to create a map of strains across East Africa. So far, all strains of M. bovis that have been identified are consistent with Tanzanian strains of the bacteria. 11

Figure 10: Routine tests used for TB diagnosis in the laboratory at SUA from samples collected from cattle in the slaughterhouse and at post mortem examination Samples from lungs and thoracic lymph nodes are collected Culture for 6-8 weeks Ziehl Nielsen staining Negative (end) Acid fast bacteria (positive) Mycobacterium genus PCR Mycobacterium tuberculosis Complex RD4 PCR M. bovis (+) (-) M. TB Non complex STOP Table 4: Results of RD4 PCR as shown in Figure 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 LADDER NC C C I NC C C C C C NC C C C LADDER 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 LADDER C C C C C neg C c1 c2 c3 c4 LADDER Controls: c1 = Mycobacterium tuberculosis c2 = Water (negative control) c3 = Mycobacterium bovis c4 = Mycobacterium avium Results: C = Mycobacterium complex (M. bovis/m. tuberculosis) I = Mycobacterium intracellulare NC = Non complex STOP 12

Figure 11: Results of RD4 PCR Conclusions of Field and Laboratory Work My experience in Tanzania was incredibly valuable in many ways. I gained first hand experience of field and developing country research and have a greater understanding of many of the challenges facing attempts at disease control within Africa. I thoroughly enjoyed my time working with SUA in the field and the laboratory. I was fortunate to spend some time around Serengeti National Park to appreciate the reality of the challenges facing researchers attempting disease control. I was privileged to be welcomed into the homes of local people to gain first hand experience of the typical living conditions and lifestyles of the local people and the challenges facing disease control in these areas. I was also able witness part of the annual migration between SNP and Masai Mara in Kenya. The incredible numbers of wildebeest and zebra passing through livestock grazing areas and close to settlements was an excellent example of the magnitude of wildlife interactions with local farmers. The limitations of the diagnostic tests were apparent during my time in the field and also in the laboratory. Working conditions are challenging for the use of standard field tests and more sensitive laboratory tests (e.g. gamma interferon immunoassay) as these require the samples to remain under precise storage conditions and to be processed quickly. There are many remote areas it would be impossible at present to use the more sensitive tests due to long travelling distances to laboratory facilities and poor transport networks. Some practical difficulties with the Bovigam test were encountered in the lab. The trouble-shooting discussions resulting from these problems gave me a deeper understanding of the specific requirements of the test as well as its delicate nature. Experiencing water shortages and power cuts first hand emphasised how challenging it can be to store reagents and samples suitably, even in some of the best facilities in the country! These problems have since been solved and the SUA TB diagnostics team has continued to assess the larger scale viability of using this test in Tanzania 13

Analysis of the skin test results by age group shows that all animals below the age of 12 months tested negative. In animals over 36 months there were no negative test results which may be a consequence of exposure to either Mycobacteria tuberculosis complex pathogens or cross-reactivity to environmental mycobacteria. Use of the gamma interferon immunoassay should allow differentiation between the two. While the current tests still have their value in many situations, the logistics of carrying out these out in the field still limit their use on a wider scale. There have been attempts to develop simpler and cheaper techniques for serological detection of infected animals. One such test which has great potential for further development and use is a novel rapid test (RT) which makes use of lateral flow technology to detect TB in a range of species (Chembio diagnostic systems, Inc.). Preliminary studies have shown this to be convenient and accurate and research into field test performance is a priority for future work. My time at SUA coincided with World Rabies Day on 28 th September 2008. The Alliance for Rabies Control (ARC) in collaboration with SUA and the Ifakara Health Institute (IHI) had coordinated a large scale vaccination campaign in the Ulanga district in southern Tanzania. The campaign in this region was initiated by staff at the IHI who, while carrying out field work in the region, had witnessed a rabid dog bite 9 people in one day. The IHI was able to help the people in this region get the essential post exposure treatment which is all too often not administered due to logistical and financial difficulties. Ulanga district had high numbers of deaths from rabies during the previous year which prompted staff from the IHI to contact ARC and to schedule vaccination campaigns in this region similar to those which had already been performed on other areas of Tanzania. To assist with these campaigns, a number of students from SUA were due to join the teams and I was able to accompany them to see a different part of the country and another component to the battle against zoonotic disease in Tanzania. Figure 11: The Rabies vaccination teams in our World Rabies Day T-shirts and myself in action 14

Finally, while in Tanzania, I was offered a four month position with IDEAL (Infectious Disease in East African Livestock project), as project veterinarian. This Wellcome Trust-funded collaboration between ILRI, University of Edinburgh and University of Pretoria aims to evaluate the burden of infectious disease in indigenous cattle in Kenya, and investigate a number of risk factors including co-infections and genetics (www.ideal-project.org). The experience I gained of research in East Africa while working with your scholarship was a key to my appointment and also equipped me with valuable skills to contribute to this project. Acknowledgements I am extremely grateful to the trustees of the Harry Steele Bodger Memorial Fund for making this experience possible and to Helena Cotton for all her help. I would like to thank my supervisors, Dr. Sarah Cleaveland and Dr. Tiziana Lembo for their help, encouragement and practical support to organise my trip and work in Tanzania. I am grateful to Professor Rudovick Kazwala for welcoming me to SUA and to Ally Kitime and Ndaki Lukiko for their teaching and assistance in the laboratory. Many thanks to the following people who I worked with in the field; Jo Halliday, Dr. Katie Hampson, Dr. Heather Ferguson (Ifakara Health Insitute), Cleophas Simon and the rest of the teams, who were a pleasure to work with. 15

Appendix 1: Procedure for the Single Intradermal Comparative Tuberculin Test (Performed under EU regulation 64/432/EEC) 1. Identify the animal by its ear tag number and record identification 2. Shave two sites in the middle third of the neck on one side (the right hand side was used in this test), one above the other, separated by about 12cm. 3. At each site, a fold of skin should be measured using a calliper and the measurement recorded. 4. PPD-bovine and PPD-avian (Central Veterinary Institute, Lelystad, The Netherlands) should be injected (0.1ml) intradermally. The upper site should be used for the avian PPD and the lower site for the bovine PPD). 5. Re-visit after 72 hours 6. Re-measure the same skin fold at each site using callipers and record the measurement. The same operator should make the measurements on both occasions. 7. Standard interpretation is then applied to the results a. If the reaction to bovine PPD is > 4.0 mm greater than to avian PPD the test is considered positive. b. If the reaction to bovine PPD is between 1.0 and 4.0 mm greater than to avian PPD the test is considered inconclusive. c. If the reaction to bovine PPD is < 1.0mm greater that to avian PPD the test is considered negative. 16

Appendix 2: Procedure summary for Bovigam immunoassay Add 50 µl of green diluent to wells Add 50 µl of test and control samples (control last) and mix Incubate for 60 minutes Wash (repeat six times) and drain Add 100 µl of conjugate to wells (diluted to working strength in blue diluent) Incubate for 60 minutes Wash (repeat six times) Prepare enzyme solution Add 100 µl of enzyme to wells Incubate 30 minutes (protect from direct sunlight) Add 50 µl of enzyme stopping solution and mix Read absorbance in five minutes using 450 nm filter and again with a 620-650 nm filter 17

Appendix 3: Procedure summary for genus typing PCR of Mycobacterium species Procedure: 1. Initial denaturation at 95 C for 10 mins 2. Followed by 35 cycles of: Denaturation at 95 C; 1 min annealing primer at 61 C; 30 seconds Extension at 72 C; 2 mins. 3. Final elongation at 72 C; 10 mins 4. Hold at 4 C Electrophoresis performed at 100 volts on Agarose 1.5% gel of 1x TAE buffer and ethidium bromide at 0.3 µg/ml. Result 1. 1030 base pairs all members of the genus of Mycobacteria (i.e. primers Mycgen-F/R) 2. 180 base pairs M. Avium subspecies including M. Paratuberculosis (primers Mycgen F/M YcAv R) plus 1030 base pairs 3. 850 base pairs M. Intracellulare (primers Myc int-f/ Mycgen-R) in addition to 1030 base pairs genus product 4. 372 base pairs M. Tuberculosis complex (with TB-1-F/ TB-1-R) in addition to 1030 base pair genus product. Two bands indicate a specific PCR product and respective species-specific PCR product Positive controls; M. Avium M. Intracellulare M. Bovis M. Tuberculosis 18

Appendix 4: Summary of expenses Flight: London Heathrow Nairobi, Kenya 449.70 Travel and voluntary work Insurance (6 weeks) 70 Medical costs: Vaccination (yellow fever and 77 meningitis ACWY) Malaria prophylaxis 28 Visas 60 Accommodation 194.80 National Park fees 75 Transport (Kenya to Tanzania, and within 116.20 Tanzania) Total (excluding food costs) 1070.70 References: Bovigam data sheet, Prionics AG, Switzerland Cleaveland, S. et al 2005. Tuberculosis in Tanzanian Wildlife. Journal of Wildlife Diseases, 41: 446-453 de Lisle, G.W. et al 2002. Tuberculosis in free-ranging wildlife: detection, diagnosis and management. Revue scientifique et Technique de l Office International de Epizooties, 19:689-701 Kazwala, R. R. 1996. Molecular epidemiology of bovine tuberculosis in Tanzania. PhD Thesis, University of Edinburgh, Scotland Keet, D.F. et al 1996. Tuberculosis in buffaloes (Syncerus caffer) in the Kruger National Park: spread of the disease to other species. The Onderstepoort Journal of Veterinary Research 63: 239-244 Keet, D.F et al 2000. Tuberculosis in free-ranging lions (Panthera leo) in the Kruger National Park. Proceedings of the South African Veterinary Association Biennial Congress, Durban, Kwazulu-Natal, 20-22 September 2000. 10pp. Lembo, T. (2007) Bovine Tuberculosis in Serengeti, Tanzania: genetic factors affecting disease patterns in multiple hosts; Study Protocol Mfinanga, S.G.M et al. 2005. Mycobacterial adenitis: the role of Mycobacterium bovis, non tuberculous mycobacteria, HIV infection and risk factors in Arusha, Tanzania. East Africa Medical Journal, 81: 171-178 Sales, M.P.U. et al 2001. Genetic diversity among Mycobacterium bovis isolates: a preliminary study of strains from animal and human sources. Journal of Clinical microbiology, 39: 4558-4562 19

Sambrook, J. et al 1989. Molecular cloning: A laboratory manual, 2 nd ed. Cold Spring Harbour Press. Sinclair, K. et al 1995 A multiplex PCR for distinguishing M. tuberculosis from M, tuberculosis complex. Molecular and cellular probes, 9: 291-295 Smith, N.H et al. 2006. Bottlenecks and broomsticks: the molecular evolution of Mycobacterium bovis. Nature reviews. Microbiology, 4: 670-681 20