- 1 Context and importance
- 2 Epidemiology
- 3 Pathogenesis
- 4 Pathology
- 4.1 No visible lesions
- 4.2 Pathology in African buffaloes
- 4.3 Pathology in rhinoceroses
- 4.4 Pathology in greater kudus
- 4.5 Pathology in lions
- 4.6 Pathology in leopards
- 4.7 Pathology in chacma baboons
- 4.8 Pathology in warthogs
- 4.9 Pathology in rhinoceroses
- 4.10 Pathology in impala
- 4.11 Pathology in lechwe
- 4.12 Pathology in civets
- 4.13 Pathology in bushbuck
- 4.14 Pathology in olive baboons
- 5 Diagnosis
- 6 Management and control
- 7 References
- 8 External links
Context and importance
Mycobacterium bovis is known to infect a very large range of species, including humans, in which it causes bovine tuberculosis (Bovine TB; bTB; BTB). Bovine TB is becoming increasingly important in wildlife populations across the world, and in ensuring the persistence of the disease in livestock. Bovine tuberculosis has been eradicated from cattle populations in some of the developed countries. But in some of them eradication is impossible because of the presence of bovine tuberculosis in wildlife species that play an increasing role in sustaining the disease as the prevalence of the disease in cattle declines
At times, and particularly during the 20th century, M. bovis was an important zoonosis, but because of public health measures, its zoonotic threat has declined substantially in developed countries. It is, however, recognised as an existing threat to pastoral populations living in close association with their animals and in people suffering from immuno-incompetence such as induced by HIV-AIDS and related conditions in humans, particularly those in the developing world. From a practical point of view, the half-life of mycobacteria in unpasteurised fresh milk and souring dairy products is 12 hours, giving ample time for infection to occur should the milk or milk products be comsumed within a day following milking 
There are varying, often subjective and diametrically opposed opinions about the impact of bovine tuberculosis on wildlife populations. By and large, insufficient data are available to support either opinion as most of them are based on data sets extending over short periods of time, and these are used to anticipate population trends and effects of a disease with an exceedingly long epidemiological cycle. As an example, the disease in the Kruger National Park seems to have entered the Park during the course of the 1950s or early '60s, and it took more than 30 years for it to reach a dimension where its presence was detected. It will still take many years to fully establish itself in the Park, and into the extended trans-frontier Park and conservation areas of which it now forms a part, before its final impact will be measurable. Most of us who are actively involved in studying the disease, and arguing about its impact, will not live long enough to assess whether our very subjective assumptions and modelling were correct. Similarly, its role as an emerging and neglected zoonosis remains mainly speculative. Whether, with some regional exceptions, it will ever pose a major threat to human health in Africa, remains to be seen, even within the context of the immunosuppressive effects of the human HIV infection that is prevalent on the continent
It is assumed that Mycobacterium bovis was absent from the African continent (at least sub-Saharan Africa) until it was introduced during colonial times in the 1800s with imported cattle from the United Kingdom, the Netherlands and Australia. Since then the infection in cattle spread throughout the continent.
The first record of the infection and disease in wildlife in Africa was in the late 1920s when it was diagnosed in common (Sylvicapra grimmia) and in (Tragelaphus strepsiceros) from South Africa (Paine & Martinaglia, 1928)(Martinaglia, 1930), and (Thorburn & Thomas 1940). Later, in the 1960s, the disease was diagnosed in free-ranging African buffaloes (Syncerus caffer) in Uganda   . It has now been recorded in a variety of wildlife species in different countries throughout Africa and has become a critical issue when dealing with the interaction between wildlife, humans and their domesticated animals at their interfaces and diseases that may be transmitted between the various species.
The epidemiology of bTB varies according to species affected, regional occurrence, and the number of species affected in an ecosystem. The complexity of the various patterns of manifestation has not been determined for African wildlife and extensive research is required to identify the various determinants and drivers of the disease. Group dynamics and dispersal patterns will no doubt have a substantial influence on the way and rate of spread of infection in an area or ecosystem. Dealing with complex matters such as these is always difficult, particularly when situations arise where if opinions are repeated often enough, they tend to become entrenched and accepted as fact.
Tuberculosis is sustained in populations because of the presence of maintenance, or reservoir, hosts. From these maintenance hosts (source populations), infection can spill over into other species (the target populations), a process known as pathogen spillover or spillover effects, and then results in the development of spillover hosts; spillover hosts may under certain circumstances, also become maintenance hosts. Maintenance hosts only develop when sufficient intra-species transmission of the infection occurs. This sustained transmission is dependent on the basic reproduction rate (R0), and a critical community size (CCS); when these conditions do not persist, the prevalence will decline and the disease will eventually disappear. Even though these conditions may not prevail in populations, it must be kept in mind that within specific populations, groups or clusters of animals within the larger group may still satisfy these requirements and sustain the infection.
The nature of the disease and the way in which the pathogens are excreted and transmitted will also determine the way in which it spreads and whether it is sustainable. Individual factors that play a role here include: excretion potential, mortality rate, dose, and route of infection
A number of maintenance hosts have been identified in Africa. The African buffalo has been identified as a maintenance host in South Africa and Uganda, the Kafue lechwe in Zambia, and greater kudu in South Africa, and warthogs, perhaps, in Uganda. Host behaviour has a pronounced effect on the way in which the infection is transmitted within and between species and these are obvious when considering the different maintenance species that may play a role in the dissemination of the disease in Africa. This behaviour may also be changed by human intervention that may increase the complexity of the dynamics of the disease. The epidemiology will also become increasingly complex in multi-host systems in which a number of maintenance hosts may be present in a single ecosystem, such as the case in Africa.
In South Africa some work has been done and a pattern is starting to emerge. The initial publication by De Vos et al, 2001, records the early observations in the Kruger National Park in which the disease has become endemic and is still spreading through the Park and into the adjacent parts of the recently established Mozambican and Zimbabwean parts of the Greater Limpopo Transfrontier Park and Conservation Areas. There is little doubt that in the Kruger National Park, buffaloes are the main maintenance hosts and that by the nature of the extent of their lesions and the massive numbers of mycobacteria that can occur in lesions in advanced cases of the disease, that they play a significant role in disseminating mycobacteria in the environment. They have the ability to spread the infection by aerosol, environmental contamination, and as a source for predators and scavengers by ingestion of diseased tissues, particularly when they feed on animals with generalised disease.
Spread of the disease in buffaloes is facilitated by their habits. They are typically gregarious, remain in closely knit family groups and have a tendency to wallow that would facilitate droplet dispersal and spread of the infection. Infected buffaloes often die close to or in pools where they contaminate the environment with large numbers of mycobacteria
Recently genetic polymorphism has been detected that appears to be associated with the TB status of African buffaloes. These genes may have the potential for marker-assisted breeding programmes with the aim of breeding buffaloes that have an increased resistance to tuberculosis 
Lions appear to initially contract the disease by ingestion of infected tissues although, based on the nature and spread of lesions, they can also contract the disease per-cutaneously because of intra-specific conflict, and by inhalation of infected tissues while eating. Spread of the disease within prides of lions is most likely to be by aerosol dissemination of bronchial exudate in which large numbers of bacteria are usually present. Small and isolated lion populations are vulnerable and the effect of the infection on the group is exacerbated by in-breeding and its consequences (Trinkel et al., 2011) . Contrary to expectations, co-infection by M. bovis and feline immunodeficiency virus, did not manifest any synergy and appears to be of little importance in determining the outcome of the mycobacterial infection in this species. Whether lions constitute a maintenance host, remains undecided and there are varying opinions about this matter that will only be resolved once sufficient information is collected over time.
Greater kudus in South Africa have been known to be infected with tuberculosis since the early 1900s but little attention has been given to their potential role in maintaining and disseminating the infection in livestock and in wildlife. They are likely to sustain the infection because of the close association of the family groups in which they live. The way in which they contract and disseminate the infection is markedly different from the other species. It has been speculated for a long time that they contract the disease per cutaneously through scarification of the skin of the head and ears by the thorns of Acacia spp trees and shrubs, exposure to contaminated vegetation, and the development of initial lesions in lymph nodes of the parotid area from which it disseminated haematogenously or lymphogenously throughout the body. Fistulous tracts develop in the parotid region from which exudate containing M. bovis is shed onto the vegetation from where contamination of skin wounds occur, or it is ingested by browsers, such as kudu. Epidemiologically, M bovis strains are found that are common to kudus and buffaloes, depending on the part of the country in which they occur. However, strains have also been isolated from kudus that do not occur in buffaloes and it is possible that there may be a different epidemiological cycle in kudus thus qualifying them as a maintenance host.
The epidemiology of the disease in warthogs is vague. The distribution of lesions ais indicative of "per os" infection (Woodford 1982).
As is the case in olive baboons, it is assumed that chacma baboons contract the initial M. bovis infection through scavenging and ingesting diseased tissues, or other contaminated material. Once infected, and within the context of the extent and nature of the pulmonary lesions, they appear to spread the infection within troops by droplet infection. It does seem that chacma baboons are not maintenance hosts, and that, provided they are not exposed to a constant source of infection, that the disease disappears from an infected group within months.
Terrestrial small mammals
A comprehensive survey of small mammals on cattle farms in Tanzania, did not detect the presence of M. bovis, and their potential role in the epidemiology of the this mycobacterium in domesticated animals and wildlife remains unclear
In cattle the pathogenesis of the disease remains unclear, and even the main routes of infection, the infective dose, and incubation period remain hypothetical. Contrary to the situation in experimental disease, the natural disease is assumed to develop following multiple exposures to the organisms, and that the interaction of more than one infectious strain may play a role.
Very few data are available for African wildlife species on which to base any assumption to explain the development of lesions and progression of the disease to full-blown disease, recovery, or the existence of innate resistance.
As is the case in humans and other species, there is a marked intra- and interspecies variation in the morphology, both macroscopically and histologically, of the lesions cause by M. bovis infections (Ridley 1983). The lesions (depending on the route of infection), have a characteristic distribution and may occur as solitary granulomas, multiple lesions in a single organ, multiple lesions in multiple organs, or generalised in which case the lesions have a milliary distribution in many organs, and occur on the peritoneum and pleura (Guilbride et al. 1963; Thurlbeck, Butas, Mankiewicz, & Laws, 1965). It has become customary to compare the lesions caused by M. bovis infections in the various species to the lesions caused in cattle suffering from this disease.
Mycobacterium bovis infection in cattle causes a typical granulomatous inflammatory reaction. These granulomas are characteristically a focal accumulation of predominantly macrophages, epithelioid cells, multinucleated giant cells, lymphocytes, and a peripheral connective tissue capsule.
The pathogenesis of the lesions in cattle is poorly defined. Wangoo et al. 2005 , classified lesions in experimentally infected calves on the basis of their extent and cellular composition when killed 29 weeks after being infected. They categorised the lesions histologically into four stages:
- The first or initial stage comprised an unencapsulated cluster of epithelioid macrophages, small lymphocytes and a few neutrophils sometimes a few Langhans' giant cells but without necrosis. Few or no mycobacteria (per section) can be detected in these lesions, and when they occurred, they were seen in the cytoplasm of macrophages or giant cells
- The second or solid stage reflected the development of a thin connective tissue capsule and sometimes, necrosis;
- Stage three, or the stage with minimal necrosis reflected full encapsulation of the lesion, with centrally located necrosis in which the necrotic debris may undergo mineralisation. In these lesions the granulomatous inflammatory reaction was limited to the periphery of the lesion and extended to the capsule. At this stage, the cells were an admixture of epithelioid cells, Langhans giant cells and scattered neutrophils.
- The last stage, stage four, or the stage with necrosis and mineralisation reflected the presence of large, multicentric areas of caseous necrosis containing islands of mineralisation, and an admixture of peripherally located epithelioid cells and Langhans' giant cells. At this stage numerous mycobacteria may be present in the lesions and they may then be seen in the cellular debris in the caseous necrotic material.
The earliest experimental lesions detected were between 14-42 days following infection. Here the lesions were pale yellowish plaques of 1-3 mm diameter on mucous membranes, while the lesions in the lungs were plum-coloured consolidation of varying extent. Within these areas of consolidation, multiple 1-4 mm diameter nodules containing caseous necrotic centres were present  It is unlikely that the sequence of lesion development, and the morphology of the lesions in buffaloes, will consistently fit into these patterns given the marked variation in the macro- and microscopical appearance of the lesions in different animals.
Lesions at different stages of their development may occur in the same lymph node and within the tissues of a single animal at the same time. This variation appears to reflect a continuing process of infection, granuloma formation, necrosis, and re-seeding. Each lesion appears to be an autonomous micro-environment that may be subject to different modulatory responses. The development of these granulomas over time is unclear since all of the experimental animals were killed at the same time, and certain assumptions and inferences are thus built into the interpretation of the experimental results. The granulomas are considered to be dynamic and their cell populations, and cytokine reactions will change over time to reflect the developmental stage of the lesion. It is assumed too that this reaction will be different in different species since the known variation in the appearance and progression in the nature of the lesions in the various species are too varied to reflect a simple uniform pathogenetic mechanism common to the various species. It is assumed too that the variation in the morphological appearance of lesions may similarly be influenced by innate resistance, the nature of immunological reaction in the various species, immune competence of individuals and the strain of the organism that they are exposed to. The classification for lesions in cattle may thus be used as a broad guideline to classify lesions in the various species, but an attempt to classify all lesions in these species according to this scheme will be impossible given the variation that has been seen. Much more work is needed, before a unifying classification can be developed, if such a classification is indeed necessary. The tendency to extrapolate between species is dangerous and should be avoided.
No visible lesions
It is not always possible to detect granulomatous lesions in all animals infected with M. bovis. These so-called no visible lesion cases vary in number according to the techniques used for examining carcases and tissues and on the experience and diligence of the person doing the examination. Gavier-Widen et al., 2009 reviewed this issue in wildlife, and ignorance of this situation, may be critical when dealing with tuberculous infections in wildlife in general, and particularly when managing the risk of introducing the disease into disease-free groups of animals.
This matter is dealt with below under the heading of Diagnosis in dead animals
Pathology in African buffaloes
In African buffaloes the lesions often resemble those seen in cattle with tuberculosis (characterised by a fibrous capsule, marked caseation and some calcification) , and the various developmental stages of the granulomas can be classified similarly to those in cattle, but some are atypical and have a neoplastic, fibro-lardaceous appearance containing areas of caseous necrosis lacking calcification . Pus may be present in some of the lesions in buffaloes and it is invariably of a lighter colour than that seen in tuberculous lesions in cattle. The caseous necrotic centres my become liquefied in lesions both in the lungs and in the lymph nodes. In advanced cases of the diseases, the carcases are cachectic .
The prevalence of infection appears to increase incrementally in animals up to 14-years-of-age, and then decline ). The distribution of the lesions is quite consistent in that in most buffaloes with tuberculosis, lesions occur in the lungs (50% of cases had only pulmonary lesions) in most buffaloes, while in the largest proportion of infected buffaloes lesions occurred in the bronchial and/or mediastinal lymph nodes. Rarely, lesions occurred exclusively in the retropharyngeal and cervical lymph nodes ), and in the palatine tonsils. Animals with generalised tuberculosis also manifested a granulomatous peritonitis while a granulomatous pleuritis and pericarditis occurred in only a quarter of the buffaloes examined. Individual buffaloes contained lesions in the liver, spleen, and uterus, and in the gastro-hepatic nodes. Peripheral nodes were rarely involved but they do develop lesions in advanced cases of the disease.
Pulmonary lesions may occur throughout the lungs but they most often occur in the dorsal-caudal aspects of the diaphragmatic lobes; often, only single lesions are detected, and they may be quite small and are the most easily detected by thorough palpation. The lesions vary in appearance from small almost undetectable, encapsulated, discrete, fibro-caseous granulomas to numerous coalescing foci, up to 30 cm in diameter ). The granulomas commonly undergo limited to extensive caseous necrosis and the necrotic exudate may become partially liquefied . Other organs and tissues are rarely affected, but when they do, the lesions have the characteristic appearance of the lesions seen in lymph nodes, palatine tonsils, and the lungs .
The lesions in the lymph nodes vary substantially in appearance. The range of lesions is depicted in the series of images below.
Lesions in the tonsils may be difficult to detect and they are most easily located by palpating the organ. The granulomas are often not encapsulated and the reaction extends into the surrounding normal tissue, and sometimes into the adjacent skeletal muscle.
Intestinal lesions are rarely seen in buffaloes, except in advanced cases due to expectoration of large numbers of bacteria from advanced lesions in the lungs. The intestinal lesions are then more commonly seen in the distal portion of the small intestine and at the ileo-caecal junction. Here the lesions had a linear configuration and were mostly within the mucosa where their presence caused the development of small irregular ulcers. Where these lesions occurred, the draining mesenteric nodes was always affected.
The histologically appearance of the lesions in African buffaloes differs little from those seen in cattle: the inflammatory reaction being characterised by a granulomatous reaction, the presence of Langhans’ giant cells, central caseous necrosis with some calcification , epithelioid cells, and a pronounced peripheral fibrous capsule(). Not all lesions are encapsulated . In some of the lesions masses of neutrophils are present in the centre of the necrotic portions and these undergo liquefaction as a consequence of this inflammatory reaction. The numbers of acid-fast bacteria seen in sections differ substantially; in some instances, numerous bacteria are scattered throughout the inflammatory reaction, while in others they are very sparse, and, in a small percentage of the cases, acid-fast organisms are not detectable ).
Pathology in rhinoceroses
The lesions described here are those of a single black rhinoceros in which the cause was confirmed by culture . It was euthanised because of old age and being in a poor condition, and necropsied to determine the cause of death. Numerous age-related changes were detected. In addition, it had a multifocal, granulomatous pneumonia characterized by the presence of two well-defined, non-encapsulated firm granulomatous masses in the caudo-dorsal portion of the left lung.
Histologically the necrogranulomatous inflammatory reaction was characterised by the presence of epithelioid cells, Langhans' giant cells, lymphocytes and plasma cells. Similar histological lesions were present in adjacent lung tissue. No acid-fast bacteria could be detected microscopically in smears from the exudate or in histopathological sections of the lesions.
Pathology in greater kudus
Infected animals may be in good condition, in spite of suffering from advanced tuberculosis. The first description of the tuberculous lesions in kudu was by Paine and Martinaglia, 1928. They reported the lesions in a number of specimens, one being that of a head, and the other in an animal that was shot for a different purpose. The lesions in the head are typically seen in the parotid and pharyngeal area where the lymph nodes were markedly enlarged and the exudate was purulent. In another animal, no lesions occurred in the parotid region and were only seen in the vertebral column as a spinal tuberculoma. Thorburn and Thomas, 1940 provided a more comprehensive review of the lesions in kudus. The nature of the inflammatory reaction is dissimilar to that seen in buffaloes and cattle in that it is yellow, soft and creamy. Isolated lesions may be difficult to distinguish from abscesses caused by pyogenic bacteria. 
Pathology in lions
The lesions caused by Mycobacterium bovis in lions are substantially different in their macroscopical and histological appearance when compared to the lesions seen in other wildlife species in which this infection occurs. Histologically the lesions are characterised by homogenous infiltrates of macrophages lacking necrosis, calcification and Langhans giant cells. Normally mycobacteria are absent from these macrophages. In the lungs the inflammatory reaction is limited initially to the interstitial tissue while a mixed macrophage and neutrophil exudate may be present in the alveoli. The areas of granulomatous inflammation are interspersed with foci of an acute inflammatory reaction containing fibrin, a neutrophil exudate, and a marked inflammatory oedema. The alveolar walls are thickened and infiltrated by a mixed inflammatory exudate consisting of macrophages, fibroblasts, and proliferating type 2 pneumocytes. Marked bronchiectasis is seen in which there is a marked mucopurulent inflammatory exudate in which large numbers of mycobacteria can be seen following staining with the Ziehl-Neelsen staining technique.
Lions affected by this disease, manifest marked emaciation (Fig.1). The initial lesions in the lungs are nondescript nodules of a few centimetres in diameter that bulge slightly above the pleural surface (Fig. 2). In more advanced cases these lesions become confluent and affect large tracts of the lung (Fig. 3). The nodules, when cut into, are ill-defined and contain a semi-fluid mucopurulent exudate (Fig. 4) that originates from the dilated portions of the bronchi that are subject to the consequences of bronchiectasia (Fig. 5). The tissue surrounding the dilated bronchi contains a granulomatous inflammatory reaction seen as a collar of pale, tan-coloured tissue that blends with the surrounding normal parenchyma (Fig. 5).
The lymph nodes of these animals may be enlarged, but it is not possible to detect lesions macroscopically because of the nature of the inflammatory reaction that lacks the normal features of a mycobacterial infection (Fig. 6). In many instances the lymph nodes may also contain large cystic spaces that extend throughout the node (Fig. 8.), but these lesions too have no diagnostic significance when dealing with tuberculosis.
Lions suffering from BTB often manifest large cutaneous granulomas (Fig. 13 and 14) and arthritis caused by the infection. The joints in live animals may be substantially enlarged (Fig. 9 and 10). When cut into the joints reflect the changes of a chronic arthritis (Fig. 11) and may contain numerous fibrin globules as part of the exudate (Fig. 10). Hypertrophic osteopathy (Fig. 12) may be seen in the long bones of some of the infected animals in association with joint lesions. These lesions may, or may not be the direct consequence of BTB. Occasionally panophthalmitis (Fig. 15) as a consequence of the infection may be seen.
In many of the terminal cases of BTB, renal amyloidosis may be detected. This lesion may, or may not be directly related to the mycobacterial infection, though it is also seen in free-ranging lions that do not suffer from the disease
Pathology in leopards
The lesions seen in leopards are different to those seen in bovids, and are more outspoken than those seen in lions.
The macroscopical lesions in leopard are more outspoken than those seen in lions, but they do have the same characteristics in that there is limited necrosis, if any, no encapsulation or calcification
Pathology in chacma baboons
The macroscopic lesions are typical tuberculous granulomas that may occur throughout the body; the physical appearance of the lesions suggest that there is early embolic spread following the initial infection. Lesions appear consistently in the mesenteric lymph node (Fig. 1.), spleen (Fig. 2.) and lungs (Fig. 3). The lymph nodes of the head and neck are inconsistently affected as are others such as the mammary, inguinal, and axillary, and in individual cases lesions may also be present in the liver (Fig. 8.) and kidneys (Fig. 9.). Tuberculous granulomas may occur in some of the vertebra.
The lesions are commonly well-circumscribed with a multifocal to confluent distribution. Their consistency varies from solid in those with fibrosis, to soft when the content becomes liquefied. The initial lesions in the lungs have a typical milliary distribution (Fig. 3.) reflecting an embolic origin. In time the become larger, confluent (Fig. 6.), and the exudate then may become liquefied (Fig. 7.) and cavities in the lung tissue may develop.
Histologically the lesions are characterised by areas of central caseation, containing aggregates of necrotic neutrophils within multifocal to coalescing granulomatous pneumonia. Infiltrates of macrophages, epithelioid cells and multi-nucleated Langhans giant cells, lymphocytes and plasma cells surrounded the necrotic centres of the granulomas. The lesions were poorly encapsulated miliary spread can be seen. Liquefied exudate within granulomas may drain into adjacent bronchioles and bronchi that contain a multifocal caseous granulomatous inflammation that ulcerated in areas. Granulomatous lesions may also be present in the liver, spleen, lymph nodes. Many acid-fast bacteria occur within the lesions.
Pathology in warthogs
Woodford described the lesions in warthogs infected with M. bovis. They were seen as calcified abscesses in the submaxillary lymph nodes and lungs. The lesions in the lungs may be extensive and consist of a caseo-calcific consolidation of the entire lung due to coalescing masses of small granulomas with an embolic distribution. In generalised cases lesions may also be seen on serosal surfaces and there may be dissemination to many lymph nodes. Both M. bovis and atypical mycobacteria were isolated from these cases.
Pathology in rhinoceroses
Pathology in impala
de Vos, 1977. 
Pathology in lechwe
Pathology in cheetahs
The appearance of lesions in a single cheetah was recorded. Macroscopically the only lesions occurred in the lungs as extensive areas of necrosis of a multifocal to confluent granulomatous pneumonia. Histologically the lesions were characterises by the presence of multiple aggregates of neutrophils, occasional foci of coagulative necrosis, limited fibrin exudation, and limited calcification of a poorly encapsulated expanding granulomatous inflammatory reaction. The alveoli surrounding the inflammatory response contained macrophages and epithelioid cells and associated terminal bronchioles were plugged with a necrotic exudate containing many neutrophils. Scattered acid-fast bacteria occur in the exudate and intra-cytoplasmically in macrophages at the periphery of the lesions
Pathology in civets
Pathology in bushbuck
Pathology in olive baboons
The known lesions in this species have been recorded in free-ranging olive baboons from the same troop. .
The extent of the lesions and their spread indicated generalised disease with lesions present in the lungs, spleen, liver, and in various lymph nodes including those of the head and neck, thorax, and in the mesenteric and inguinal lymph nodes.
In animals in which the lungs were affected, multifocal small granulomas of not more than 1 cm diameter were scattered throughout all the lobes of the lungs, or were limited to the cranial lobes. In some of the cases, there were localised to extensive fibrous adhesions between the lungs and the thoracic pleura. The consistency of the granulomas varied and their content may be either liquified or inspissated, and variably calcified. The lesions in the lymph nodes cause enlargement of the nodes (up to 3 cm in diameter) and on cut surface they may contain a greyish-white fluid exudate.
Histologically the lesions vary according to their stage of development. The early miliary lesions were characterised by the presence of centrally located epithelioid cells containing clusters of Langhans' giant cells surrounded by small mononuclear cells. In older lesions necrosis of the centres of the granulomas occurred, with or without calcification. Small numbers of acid-fast, rod-shaped bacteria can be seen on smears made from the exudate, or in histological sections stained with the Ziehl-Neelsen stain.
Confirming a diagnosis of M. bovis tuberculosis in the various wildlife species that have or may become infected, may be fairly difficult: in general, the easy ones are easy, the difficult ones are exceedingly challenging.
The ease with which the diagnosis is made, and confirmed, varies according to whether the animal is alive or dead. In live animals the challenge lies in the diversity of tests that are available but have not been validated for use in wildlife, and in the general lack of sensitivity and specificity of these tests when used in live animals.
The recent publication by Maas et al., (2013) provides a good overview of the challenges and dilemmas confronting practitioners  when they have to select tests for diagnostic purposes in live animals. The value of these tests vary according to the purpose for which they are used (surveillance, monitoring, single-animal diagnostics, certification of bTB status, or research). Tests based on the cell-mediated immune reactions (INF-γ) (the single or comparative intra-dermal tuberculin test, and the INF-γ test) are still the most suitable to detect early reactors but they suffer, as most other tests do, from the lack of sensitivity and specificity, and the lack of validation for use in most of the wildlife species in which their application is required. Serological tests, are with few exceptions, even less specific, but they can be used more efficiently to detect late and advanced cases of tuberculosis at times when the CMI-immune response has faded and animals with advanced disease become anergic and fail to respond to these tests. Using different tests in combination appears to be beneficial in that they in combination, detect more positives than any one test on its own; they must, however, be selected carefully taking into consideration species variations and idiosyncrasies. Given their lack of sensitivity and specificity, the current tests are valid as herd tests but not as definitive test to certify the disease status of individual animals, particularly when the TB status of the herd or group of animals from which they originate, is not known. Recently, IP-10 was shown to be a sensitive marker of antigen recognition and that measurement of this cytokine in antigen-stimulated whole blood might increase the sensitivity of conventional IGRAs in African buffaloes.
The intradermal skin test is unsuitable for use in rhinoceroses, and the reaction to bovine and avian PPD is excessive and non-specific. A gamma interferon assay is being developed, and can be used experimentally. It needs to be validated for use before it can be reliably used for diagnostic purposes in these species
The accuracy of testing using a number of testing techniques in meerkats varied (Drewe et al., 2009) . The use of tracheal washes for diagnostic purposes has been advocated; in meerkats the procedure was found to be highly specific but lacked sensitivity to be of value. The combination of the results of MAPIA and RT produce a sensitivity and specificity of adequate levels to allow its use for screening purposes in wild meerkats.
Validation of tests for use in wildlife species is particularly important since interpretation of the intra-dermal and of the INF-γ tests is dependent on the species in which it is used, the epidemiological setting, and whether it is a known-positive group or cohort. In addition, the interpretation that can be very subjective, can be modified to increase the sensitivity (but decrease the specificity) of the test result. Other test and host-related factors also influence the sensitivity and specificity . Combining the test results of the intra-dermal and INF-γ tests, increases both the sensitivity and specificity of the diagnostic exercise as there is a lack of correlation between the outcomes of the two tests . It must be kept in mind that the lower the prevalence of the infection in a group of animals, the more difficult it becomes to determine presence or absence of infection, and the final judgement as to whether groups of animals are free from infection, remains challenging. 
Grobler et al commented on the application of some of these tests for diagnostic purposes
Diagnosis in dead animals
In dead animals, the diagnosis is dependent on detecting the presence of lesions, recognition of the lesions that may vary substantially in appearance within and between species, collecting appropriate specimens to confirm the diagnosis, and the experience of the person doing the post-mortal examination. The success rate in specimens submitted for diagnostic purposes is dependent on selecting appropriate specimens; those that do not contain lesions are unlikely to render a positive culture. Further challenges to obtain an accurate diagnosis, lie within the laboratory infrastructure where identification of cultured mycobacteria is influenced by the availability of sophisticated techniques by which to classify the organisms down to species, clade, and type levels, and these are not available in routine diagnostic laboratories, particularly in Africa. For an accurate diagnosis it is recommended that specimens be submitted to research laboratories that are suitably equipped to execute the required diagnostic procedures to identify the specific mycobacteria.
Lesions detected macroscopically and histopathologically may be typical for mycobacterial infections, but their presence only justifies a presumptive diagnosis; the diagnosis needs to be confirmed by a positive culture result. The macroscopic appearance of tuberculous lesions is not diagnostic and they may be confused with similar lesions caused by a host of other unrelated causes . All lesions detected macroscopically and assumed to be tuberculous granulomas, should be submitted for histopathological confirmation of the nature of the reaction and relationship to cause and to confirm the lesion is indeed one caused by a mycobacterium . The presence of acid-fast bacteria in infected tissues examined histologically may be used as further support of the diagnosis of tuberculosis. They are, however, not always detectable, and the ability to detect them in tissue sections and smears from the exudate obtained from infected organs, will depend on the concentration of organisms in the specific specimen. The inability to detect organisms histologically thus does not justify elimination of a mycobacterial infection as the cause of the lesion.
Routine abattoir examinations and necropsies
Routine abattoir examination of carcases to detect tuberculous lesions is not an accurate way of detecting the disease. Depending on the person examining the carcases, up to 50% of positive animals may be missed and if the prevalence is low, this may result in erroneously classifying specific herds as not infected. Similarly, cursory post-mortal examination of carcases in the veld, is risky, since lesions may be small and localised, or atypical, and are then easily missed. Suitable techniques that should be used if it is critical to make a correct diagnosis, have been described by Corner (1994). It is equally important to collect a full set of specimens for culture and histopathological examination to increase the accuracy of the procedures. It is also important to handle specimens for culture in the best possible way. If they can be transported to a laboratory within 24 hours, they should be kept cool at temperatures of between 4 and 6 degrees °C; if this cannot be done, they should be frozen as soon as possible and kept frozen until the specimens are required for culture . A similar protocol has been developed for badgers, that can also be used in smaller mammals to accurately survey populations for the presence of infection. Including histopathological examination of a collection of tissues, substantially increases the sensitivity of the detection method.
No visible lesions (NVL)
In many of the various wildlife species that become infected, and contract the disease, certain animals may contain no visible lesions.
This matter has been reviewed by Gavier-Widén et al. (2009) and it is advisable to peruse this article for the detail pertaining to individual species. No visible lesion infections are those in which the lesions cannot be detected macroscopically but where lesions are detectable on histopathological examination, and where it is possible to culture M. bovis from the tissues. In cattle 10% of positive skin-reactors may be in this category, and up to 30% of in-contact cattle with a negative skin test may be similarly apparently free from infection. This reality may have a substantial impact on the sensitivity of the diagnostic techniques used to detect cases of tuberculosis particularly in cohorts of animals in which the BTB status is unknown.
Numerous wildlife species have been recorded not to manifest macroscopically detectable lesions while infected with M. bovis. These include badgers, feral ferrets, brushtail possums, wild mink, coyotes, raccoons, black bears, red foxes, various deer, wild boar, and a number of rodents including brown rats, wood mouse, yellow-necked mouse, and field voles. Up to 80% or more of some of the species when infected with M. bovis, may not develop macroscopic lesions.
The inability to detect lesions is not only dependent on the pattern of infection in specific species, but also on the methods used to examine the animals at necropsy, and the analyses used to confirm the diagnosis.
Improving the techniques of macroscopically examining tissues such as lymph nodes, skin lesions, oropharyngeal tonsils, and the lungs increases the sensitivity of the examination. Similarly, given the variation in histological appearance of lesions caused by M. bovis infection in the various species, the experience of the investigator will have a substantial impact on the success of the investigation.
The results of culture for the presence of mycobacteria for gauging the prevalence of the disease is considered to be the gold standard for diagnostic purposes. Here too, the sensitivity of the method is influenced by the protocol used for specimen collection and culture.
Management and control
The management and control of bovine TB in wildlife presents major challenges not only in respect of the control and management of the disease in wildlife itself, but because of its role as maintenance hosts in maintaining the infection in livestock that impedes the global attempt to eradicate the disease and to eliminate its zoonotic threat at the interface . Strategies to manage and control the disease should be based on adequate data relevant to the disease in the specific area, they should be scientifically sound, and there should be good reason to believe that the outcome will be successful. Theoretical models, based in thumb-suck information, are likely to fail dismally. Recently Gortazar et al. identified and discussed six specific research needs to address the issues of control: 1) complete the world map of wildlife M. tuberculosis complex (MTC) reservoirs and describe the structure of each local MTC host community; 2) identify the origin and behaviour of generalized diseased individuals within populations, and study the role of factors such as co-infections, re-infections and individual condition on TB pathogenesis; 3) quantify indirect MTC transmission within and between species; 4) define and harmonize wildlife disease monitoring protocols, and apply them in a way that allows proper population and prevalence trend comparisons in both space and time; 5) carry out controlled and replicated wildlife TB control experiments using single intervention tools; 6) analyse cost-efficiency and consider knowledge transfer aspects in promising intervention strategies.</ref>
Mycobacterium bovis has the ability to infect multiple hosts in the same ecosystem, and the spread of the infection to other species create major difficulties to eliminate the disease from such an ecosystem, particularly because it is unknown which and how many of the species in the system can become infected, and act as maintenance hosts. The complexity of these multi-species systems dictates that the system must be considered in its entirety when devising management and control strategies; one cannot only focus on a single species in such a system with the hope of managing the disease. Within this context, the monetary value of wildlife species, and opinion of society pertaining to the management of populations, or groups of animals within populations, have a major impact on whether control measures can be implemented.
In captivity, the dynamics are different: the density of animals, aggregation in certain areas such as housing, feeders, and water troughs, and being subject to stress. Control strategies under these circumstances are different, and perhaps easier to apply that to free-ranging animals.
A conceptual framework for control and management
O'Brien et al.  provided a conceptual framework to manage bovine TB in free-ranging wildlife. They suggest that control strategies should follow a logical sequence of discovery/detection, epidemiological characterization, initial control, simulation and forecast (modeling), focused control, and verification of the control process or eradication. These processes often overlap, or may even run consecutively. It is critical to keep in mind when dealing with these situations, is that the disease, and the animals affected, are part of a complex, integrated, multi-host ecosystem in which all the components should be accounted for when devising an intervention.
As part of the epidemiological characterization, at least the following matters should be taken into consideration: the extent of the problem; the reservoir population; the size of the reservoir population; the maintenance host(s); spill-over species and the likelihood that some of them may become maintenance hosts, the routes of transmission; and potential zoonotic impact. The dynamics at the interface with livestock and humans should equally be assessed since transmission at this interface my take place in a bi-directional way. The source of infection may be outside of the conservation area and this should equally be taken into consideration when designing management and control strategies.
The initial control addresses the basics: density reduction and attempts to reduce/limit further spread of the disease. What needs to be done here is dependent on the characteristics of the specific case dealt with.
The next stage, that of modeling, is dependent on the availability of reliable surveillance data allowing the process to commence. This will allow predictions of how the disease will manifest itself in the ecosystem, its eventual effects on the species within that system, the success or failure of the application of management and control mechanisms, and cost-benefit analyses.
Focused management and control of the disease in a specific environment can only be implemented once modeling is complete and the intervention agreed on. These interventions include targeted culling, fencing, removal of the young, etc., depending on the objectives of the intervention.
Verification that the objectives of the intervention have been met. Ongoing surveillance throughout the process and following apparent resolution of the problem, remains critical and should be pursued on an ongoing basis as part of managing wildlife systems.
To cull or not to cull...
Australia is the only country that successfully eradicated bovine tuberculosis, and they did this by strict and intensive culling of the wildlife host, the water buffalo. The New Zealand authorities are implementing the same management procedure to eradicate bovine tuberculosis. Both these countries could follow this route since the wildlife species were both introduced and they had no intrinsic value. This is not the case in South Africa, for instance, where African buffaloes, and other susceptible species have a high intrinsic value and culling is not necessarily an acceptable option; public opinion would also oppose this approach
Culling to reduce the buffalo population to low densities has been considered as a possible way to curtail the spread of the disease to other species in extensive ecosystems. Culling on a regular basis to reduce the prevalence of the diseases in conservation areas, has been applied in the Hluhluwe-Mfolozi game reserve (a relatively small reserve) has been successful, but it is not anticipated that this practice will eventually lead to the elimination of the disease in that Park. Modelling the effect of random culling in large ecosystems, revealed a lack of effectiveness of the process (Rodwell, 2000).
The use of an effective vaccine for tuberculosis in cattle and wildlife susceptible to M. bovis infection, is likely to be the only rational control mechanism to control and manage bTB. This applies particularly to those countries in which multiple species within an ecosystem are infected and in which culling would not be effective because of residual numbers of infected species. With few exceptions (possums, badgers, white-tailed deer, and ferrets) the use of currently available vaccines remain ineffective. Intensive research for new and more effective vaccines is ongoing, particularly in humans, and there is an increasing likelihood that an effective vaccine will become available in due course (Buddle et. al. 2011). Based on the lack of efficacy of the vaccine (De Klerk et al.), and modelling the use of the current BCG vaccine in African buffaloes have shown that it lacks the ability to control the disease (Cross & Getz, 2006)
Selective breeding for increased resistance to BTB in buffaloes may be a viable method of BTB management in the future, particularly if genetic information can be incorporated into these schemes. Different strategies can be employed in selective breeding programmes, and these may be consider in the implementation of genetic improvement schemes
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