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1 Centre for Tropical Veterinary Medicine, University of Edinburgh, Royal (Dick) School of Clinical Studies, Easter Bush, Roslin, Midlothian, UK EH25 9RG;
2 Tanzania National Parks, PO Box 3134, Arusha, Tanzania;
3 Dept. of Veterinary Medicine and Public Health, Sokoine University of Agriculture, PO Box 3021, Morogoro, Tanzania;
4 Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, South Africa;
5 Ministry of Water and Livestock Development, PO Box 322, Musoma, Tanzania;
6 Animal Health Division, CSL Limited, 45 Poplar Road, Parkville 3052, Australia;
7 Department of Ecology, Evolution & Behavior, University of Minnesota, 1987 Upper Buford Circle, St. Paul, Minnesota 55108
8 Corresponding author (email: sarah.cleaveland{at}ed.ac.uk)
ABSTRACT:
Bovine tuberculosis, caused by Mycobacterium bovis, is a pathogen of growing concern in free-ranging wildlife in Africa, but little is known about the disease in Tanzanian wildlife. Here, we report the infection status of Mycobacterium bovis in a range of wildlife species sampled from protected areas in northern Tanzania. M. bovis was isolated from 11.1% (2/18) migratory wildebeest (Connochaetes taurinus) and 11.1% (1/9) topi (Damaliscus lunatus) sampled systematically in 2000 during a meat cropping program in the Serengeti ecosystem, and from one wildebeest and one lesser kudu (Tragelaphus imberbis) killed by sport hunters adjacent to Tarangire National Park. A tuberculosis antibody enzyme immunoassay (EIA) was used to screen serum samples collected from 184 Serengeti lions (Panthera leo) and 19 lions from Ngorongoro Crater sampled between 1985 and 2000. Samples from 212 ungulates collected throughout the protected area network between 1998 and 2001 also were tested by EIA. Serological assays detected antibodies to M. bovis in 4% of Serengeti lions; one positive lion was sampled in 1984. Antibodies were detected in one of 17 (6%) buffalo (Syncerus caffer) in Tarangire and one of 41 (2%) wildebeest in the Serengeti. This study confirms for the first time the presence of bovine tuberculosis in wildlife of northern Tanzania, but further investigation is required to assess the impact on wildlife populations and the role of different wildlife species in maintenance and transmission.
Key words: Bovine tuberculosis, Mycobacterium bovis, Serengeti, Tanzania, wildlife.
Mycobacterium bovis infects a wide range of species and is increasingly recognized as an important pathogen of free-ranging African wildlife (Keet et al., 2000a; de Vos et al., 2001; Keet et al., 2001; de Lisle et al., 2002; Michel, 2002). Wildlife tuberculosis is a potential source of infection for domestic livestock and humans (Cleaveland et al., 2002; Michel, 2002), and the disease poses a threat to valuable and endangered wildlife species. For example, M. bovis in the Kruger National Park, South Africa, has resulted in mortality and morbidity in buffalo (Syncerus caffer; Keet et al., 1996), lion (Panthera leo; Keet et al., 2000b), and cheetah (Acinonyx jubatus; de Lisle et al., 2002).
Although M. bovis has been reported for many years in Tanzania (Markham, 1952; Chillaud, 1995), recent studies have focused on infection in cattle (Kazwala, 1996) and very little is known about the extent or impact of bovine tuberculosis (bTB) in wildlife. In the Serengeti National Park, bTB was first recorded in wildlife with acid-fast bacilli detected in granulomatous lesions of giraffe (Giraffa camelopardalis) and eland (Taurotragus oryx) (Roelke-Parker and Parker, 1994). However, culture and molecular characterization of Mycobacteria species were not possible until the establishment of containment laboratories and molecular diagnostic facilities at the Sokoine University of Agriculture (SUA) in 1997. Here, we report results on the bTB infection status of different wildlife species in Tanzania, using results from culture and serologic testing of lions and wild ungulates. The identification of other Mycobacteria species also is reported.
For culture and polymerase chain reaction (PCR) analysis, samples were obtained from a range of sources, including: 1) samples collected opportunistically from carcasses with tuberculous lesions detected at post-mortem examination in the Serengeti National Park between 1999 and 2001 (33°50' to 36°E, 1°30' to 3°7'S) (Fig. 1
); 2) pooled samples of lung and retropharyngeal, mediastinal, mesenteric, and prescapular lymph nodes from wildebeest (Connochaetes taurinus) and topi (Damaliscus lunatus) killed during local meat cropping programs in the Ikorongo–Grumeti Game Reserve, adjacent to the Serengeti National Park, in 2000 (33°57' to 34°52'E, 1°50' to 2°13'S) (Fig. 1); and 3) samples of lung and retropharyngeal, mediastinal, mesenteric, and prescapular lymph nodes showing gross visible lesions collected from animals killed by sport hunters in areas adjacent to Tarangire National Park and in Monduli and Longido hunting areas in northern Tanzania between 1999 and 2001 (Fig. 1). Samples from all wildebeest and topi sampled at Ikorongo–Grumeti Game Reseve were cultured irrespective of whether visible lesions were observed. Lesions in hunter-killed animals were detected by game skinners who had been trained in meat inspection by SUA staff.
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A tuberculosis antibody enzyme immunoassay (EIA) (CSL Limited, Melbourne, Victoria, Australia) was carried out on serum samples collected from: 1) ungulate species sampled for rinderpest surveillance activities and as part of the year 2000 Ikorongo–Grumeti meat cropping program (Table 1), and 2) 184 Serengeti lions (Panthera leo) and 19 lions from the nearby Ngorongoro Crater collected between 1984 and 2000 as part of long term ecological and epidemiological studies. Samples collected between 1984 and 1993 were part of a genetic survey and thus constitute a random sample with respect to tuberculosis. Beginning in 1994, additional samples were collected from individuals showing clinical signs of disease as part of a veterinary surveillance program conducted by Tanzania National Parks.
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Individual lions within the Serengeti study area (Fig. 1
) have been monitored from birth as part of a long term demographic research program (Packer et al., 1999). For lions sampled outside the study area, ages were estimated from body size, mane development (for males), and nose coloration (Whitman et al., 2004). Age-prevalence curves were calculated to investigate age-related patterns of infection.
Mycobacterium bovis was cultured from 2/18 (11%) carcasses of apparently healthy wildebeest, and from 1/9 (11%) topi culled as part of a meat cropping program in the Ikorongo-Grumeti game reserves adjacent to the Serengeti National Park. None of these carcasses showed visible lesions on post-mortem examination carried out by qualified veterinarians. Of the carcasses with gross lesions sampled in the sport hunting camps, M. bovis was isolated from one out of three wildebeest and the one kudu (Tragelaphus imberbis) from Mdori, adjacent to the Tarangire National Park. No Mycobacteria species were cultured from any of the lesion-positive samples collected in the Winget (Longido) or Intercon hunting areas (Monduli District) (Table 2, Fig. 1).
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The five M. bovis isolates identified by biochemical tests also tested positive for M. bovis by PCR. These isolates all produced a 245bp DNA product from the IS986 insertion sequence but failed to generate a 396bp amplification product from the mtp40 genomic fragment, identifying the strains as M. bovis (Sinclair et al., 1995).
Overall, eight of 184 (4%) Serengeti lions were seropositive for tuberculosis (Fig. 2). Although seroprevalence was highest in recent years, the difference was not significant (
2 = 1.69, d.f. = 1, p = 0.19), with four of 45 (9%) animals seropositive since 1997 and four of 139 (3%) seropositive from 1984–1996. Furthermore, the latter period included six animals that were sampled because they showed obvious clinical signs of disease. Including only those lions sampled "randomly" as part of genetic studies, the seroprevalence was similar in both periods, with one of 39 (3%) seropositive animals detected after 1997 (2 = 0.58, d.f. = 1, p = 0.45). The Ngorongoro lions were all sampled between 1984 and 1991, and none were seropositive. However, with an overall low seroprevalence of 4% in the Serengeti, the sample size from the Crater population might have been too small to detect positive individuals.
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). Four of the seropositive lions were obviously ill when sampled, and a fifth became ill within 2 months; none of these individuals survived for more than 6 months after sampling. Clinical signs in these individuals included dyspnea, bilateral sub-mandibular swelling, ataxia, hypermetria, and debilitation. The other three all appeared to be in good health when sampled, and their subsequent survival was 6 months, 18 months, and 37 months. Although seropositive animals had somewhat shorter survival than seronegative animals of the same age, the sample size is too small to detect statistical significance. Only two of 212 (0.9%) ungulate samples were seropositive for tuberculosis, including one of 41 (3%) wildebeest in the Serengeti and one of 17 (6%) buffalo in the Tarangire ecosystem.
This study provides the first confirmation of Mycobacterium bovis in Tanzanian wildlife, with infection detected in wildebeest, topi, and kudu. Serological results indicate that buffalo might also be infected with M. bovis. Although sample sizes were small, the isolation of M. bovis from 11% of culled wildebeest suggests that infection might be widespread because the Serengeti wildebeest population exceeds one million individuals (Tanzania Wildlife Conservation Monitoring, 2000). These infected wildebeest are part of migratory herds that range throughout the Serengeti ecosystem, including the Masai Mara National Reserve, Kenya.
None of the M. bovis-infected wildebeest showed detectable lesions at postmortem examination and were therefore unlikely to be excreting high levels of Mycobacteria in aerosol, urine, or feces, as has been described for wildlife maintenance hosts of M. bovis in other areas (de Lisle et al., 2002). Nonetheless, it is possible that the disease could be transmitted to carnivores through the consumption of infected tissue. Migratory wildebeest spend considerable periods outside the protected areas of the Serengeti National Park, and transmission of M. bovis between livestock and wildlife might also be possible. Intradermal skin testing of Masai cattle has demonstrated a prevalence of bovine tuberculosis of 2% in villages on the southern and eastern Serengeti plains, where wildebeest graze in the wet season (Shirima, unpubl. data).
Serological tests have often been considered too insensitive to detect tuberculosis in cattle as well as in wildlife (Goodger et al., 1994; Costello et al., 1997). It has been shown in previous investigations in the Kruger National Park that the EIA used in this study has a very high specificity and can reliably detect M. bovis infection at a population level. Evaluation of this EIA in comparison with bacterial culture and histopathology in buffalo in the Kruger National Park demonstrated a sensitivity of 10.4% and a false positive reactor rate of 1%. In lions, the sensitivity of the EIA was 25% with no false positive reactions in culture-negative specimens (Michel, unpubl. data). Similarly, the EIA detected 27% of deer with gross lesions and culture-confirmed M. bovis (Jones, unpubl. data).
Due to the limited and variable sensitivity of the EIA, the true prevalence of infection in different species is difficult to determine, and seroprevalence estimates reported here are likely to provide only an indication of minimum prevalence. Furthermore, the detection of antibodies to the MPB70 antigen indicates exposure to Mycobacteria species in the M. tuberculosis complex, but does not discriminate between different species in the group, which is comprised of M. tuberculosis, M. bovis, M. microti, M. africanum, and M. canettii (Brosch et al., 2002). However, with the isolation of M. bovis in common prey species of lions, we consider it likely that seropositivity in lions reflects exposure to M. bovis through consumption of infected prey.
Despite the limitations of the EIA, this assay proved a useful tool in this study, indicating, first, the presence of Mycobacteria infection in the Serengeti lion population, second, that infection has been present in the lion population since at least 1984, and third, the presence of infection in buffalo (for which culture-positive samples have not yet been obtained).
As with wild ungulates, we do not yet know the impact of tuberculosis on the Serengeti lions. Preliminary data from a study currently carried out in the Kruger National Park indicate that tuberculosis spreads rapidly in lion populations; 90% of lions monitored in an area with a high herd prevalence of M. bovis in buffalo have become infected (South Africa Veterinary Foundation, http://www.savf.org.za/rhino.htm). Over 97% of the animals sampled in this study were known to be positive for feline immunodeficiency virus, FIV (Packer et al., 1999). However, FIV is absent in the lions of Hluhluwe–Umfolozi Park (Van Vuuren et al., 2003), yet bTB has caused severe pathology in the HUP lions (Dave Cooper, pers. comm.), suggesting that FIV might not play an important role in the pathogenicity of infection.
Other Mycobacterium species isolated from wildlife in this study included M. avium, M. phlei, and M. terrae, which are in a group described as nontuberculous mycobacteria. Mycobacterium avium can cause clinical disease in some ungulate species (Thorel et al., 2001) and, in this study, was associated with pathology in the infected buffalo in the Serengeti. Mycobacterium phlei and M. terrae generally are considered non-pathogenic in most species, but in recent studies in Tanzania these Mycobacteria species have been associated with granulomatous lesions in both cattle and humans (Kazwala et al., 2002). Transmission of non-tuberculous Mycobacteria is generally considered to occur through environmental contamination. However, the isolation of M. phlei and M. terrae from lesions in the lung and thoracic lymph nodes in cattle raises the possibility of direct animal-to-animal transmission.
It has been suggested that bTB has the potential to have a major impact on the wildlife of Kruger National Park because it is an alien pathogen that has only recently been introduced into naïve wildlife populations (de Vos et al., 2001). Results from this study indicate that bTB has been present in the Serengeti for several decades, reinforcing earlier findings from Uganda and Kenya, which have demonstrated bTB as a disease problem in buffalo in Uganda (Woodford, 1982a, b) and in baboons (Papio cynocephalus) in the Masai Mara, Kenya (Tarara et al., 1985). In southern Africa, livestock and wildlife are usually separated by fences, and the intensive control measures in South African livestock might have limited the opportunities for transmission of bTB to wildlife. In contrast, the continued practice of traditional (fenceless) livestock husbandry in East Africa is likely to have facilitated the spread and establishment of infection in wildlife over a much longer period, perhaps permitting a more stable endemic pattern of infection.
Consumption of infected buffalo is considered the predominant route by which lions in the Kruger National Park become infected with M. bovis (Keet et al., 2000b). With M. bovis confirmed in the wildebeest population, Serengeti lions also are clearly at risk of infection through eating infected prey. We suggest that bovine tuberculosis has the potential to be a mortality factor in Serengeti lions because seropositivity was associated with clinical signs of disease and shortened survival times in several individuals. However, clinical signs associated with seropositivity were inconsistent with those recorded in tuberculosis-infected lions in the Kruger National Park (Keet et al., 2000b) and this hypothesis can only be examined when post-mortem and culture findings become available for Serengeti lions and the data linked with survival data. Without longitudinal data on known individuals, it is also impossible to assess the severity of infection in ungulate populations.
In summary, M. bovis has been confirmed in a range of wild ungulate species in the Serengeti and Tarangire ecosystems in northern Tanzania. Serological surveys also demonstrate exposure of Serengeti lions to M. bovis since at least 1984. The limitations of this data set do not allow an accurate estimate of the prevalence or impact of infection in different species. Further investigation is clearly needed to determine the impact on tuberculosis on wildlife populations in the Serengeti and the role of different species in maintenance and transmission of infection.
We thank the Department for International Development Animal Health Programme (UK) for financial support of this study (Grants R7229 and R7357). E.E. was supported by a grant from the TAN091–SUA–NORAD frame agreement. We would like to thank staff in the Mdori, Intercon, and Winget hunting camps, and field staff of the Serengeti Regional Conservation Programme (SRCP) for assistance with collection of wildlife samples. Mr. C. Luziga provided invaluable assistance with laboratory diagnosis and PCR at Sokoine University of Agriculture.
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Received for publication 29 April 2004.
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