Mycobacterium bovis

The pathogenesis of M. bovis infection in cattle involves the interplay of both host and bacterial factors. Genetic susceptibility of the host is thought to be important in determining the outcome of infection (Allen et al. 2010) as is magnitude of the infectious dose and route of infection. Infection is usually via the respiratory and intestinal tracts. However, the number of organisms required to produce infection in cattle is many times greater via the oral compared to the respiratory route. The organism has not been shown to produce toxic factors, rather virulence appears to reside largely in the lipids and waxes of the cell wall. Mycosides, phospholipids and sulpholipids are thought to protect the tubercle bacilli against phagocytosis. Glycolipids cause a granulomatous response and enhance the survival of phagocytosed mycobacteria. Waxes and various tuberculoproteins induce the delayed-type hypersensitivity detected in the tuberculin test. In previously unexposed animals, multiplication of the mycobacteria occurs within macrophages as phago-lysosome fusion is inhibited. Resistance to phagocytic killing allows continued intracellular and extracellular replication. Infected host cells and mycobacteria can reach local lymph nodes and from there may pass to the thoracic duct followed by general dissemination.

After the first week, cell-mediated immune reactions begin to modify the host response and activated macrophages are able to kill some mycobacteria. The cytokines TNF-α and IFN-γ are essential in the activation of macrophages and are produced by both the innate and adaptive immune responses. The aggregation of macrophages contributes to the formation of a tubercle or granuloma (Fig. 11.1) and a fibrous layer may encompass the lesion. Caseous necrosis occurs at the centre of the lesion and this may proceed to calcification (cattle) or liquefaction. Once cell-mediated immunity is established, lymphatic spread is retarded but T-lymphocyte-mediated reactions cause tissue damage and mycobacterial spread occurs by contiguous extension or via the erosion of bronchi, blood vessels or viscera to new areas. Haematogenous dissemination may produce miliary tuberculosis in animals such as deer, characterized by multifocal granuloma formation in major organs or on serosal surfaces.

Mycobacterium avium

Transmission is usually by the faecal–oral route and in birds the lesions are characteristically found in the liver and can also be present in the intestines, spleen and bone marrow. Infection with M. avium may sensitize cattle to the tuberculin test.

Mycobacterium tuberculosis

Mycobacterium tuberculosis is transmitted by aerosols or fomites and lesions are principally found in the lungs and lymph nodes. Non-human primates, dogs, canaries and psittacine birds are susceptible to human tuberculosis.

Mycobacterium lepraemurium

Mycobacterium lepraemurium is thought to cause both feline and murine leprosy. The disease in cats is characterized by the formation of single and multiple nodules or granulomas in the skin, often with the development of non-healing ulcers. Less commonly, skin lesions in cats can be caused by some of the atypical mycobacteria.

Mycobacterium avium subspecies paratuberculosis

Cell-mediated immune phenomena are involved in the pathogenesis of disease caused by M. avium subsp. paratuberculosis. The organisms are found within macrophages, which are unable to kill them, in the submucosa of the ileocaecal area and adjacent lymph nodes. The ileum and colon are usually involved with extension to the rectum. The mucous membrane becomes thickened and permanently corrugated as a result of cellular infiltration. Lesions may be of either of two types, multibacillary (lepromatous) or paucibacillary (tuberculoid) and appear to be correlated with host immune response. Extensive pathology is correlated with high levels of IL-10 production whereas subclinical disease is associated with increased IFN-γ production (Sweeney et al. 1998, Khalifeh and Stabel 2004). Clinical signs include profuse diarrhoea and the disease is progressive, leading to emaciation and death. Diarrhoea may not necessarily be seen in sheep and goats. Mortality is ultimately caused by malabsorption of nutrients and loss of protein into the intestine.

Large numbers of mycobacteria are present in epithelioid and giant cells in the mucosa of clinically affected cattle and are shed in the faeces. Animals are thought to be infected in the neonatal period, primarily through faecally contaminated milk. Organisms are also shed in colostrum and in utero infection may occur. Not all infected animals become clinical cases but remain subclinical excretors and these animals shed small numbers of mycobacteria intermittently. The incubation period is long, usually 18–24 months.

Atypical mycobacteria

Cases of disease involving these mycobacteria are comparatively rare. Predisposing factors may include a large infective dose and/or immunosuppression in the host. Some atypical mycobacteria can sensitize cattle to the tuberculin test.

Specimens

Specimens from live animals might include aspirates from cavities, lymph nodes, biopsies, tracheobronchial lavages and the centrifuged deposit from about 50 mL milk in the case of suspected tuberculous mastitis. In the case of dead animals, fresh and fixed (in 10% formalin for histopathology) samples of lesions or a selection of lymph nodes from a tuberculin-reactor with no visible lesions are taken.

Direct microscopy

The Ziehl–Neelsen (ZN) stain is used to stain smears from lesions and other specimens. The mycobacteria appear as slender, often beaded, red-staining rods against a blue background (if methylene blue is the counter-stain). These can only be visualized if at least 5 × 10⁴ mycobacteria/mL of material are present. The numbers of M. bovis are often low in bovine specimens but lesions of avian tuberculosis in poultry (Fig. 11.2) and M. bovis lesions in animals such as deer (Fig. 11.3) and badgers usually yield large numbers of mycobacteria.

Smears stained by fluorescent dyes, such as auramine, acridine orange or fluorochrome, can be examined using a fluorescence microscope. These stains allow the mycobacteria to be seen more easily if relatively small numbers are present.

Isolation

Details of isolation procedures for M. bovis are given in Cousins (2009). Several preliminary procedures are necessary in order to recover the comparatively slow-growing mycobacteria:

Mycobacteria are comparatively resistant to acids, alkalis and quaternary ammonium compounds. Decontaminating agents, such as 5% oxalic acid, 2–4% NaOH and 1% quaternary ammonium compound can be used on specimens. The mildest decontaminatng procedure that gives sufficient control over contaminants is desirable but even under optimal conditions 80–90% of the mycobacteria in the specimen may be killed by the decontaminating agent. Each laboratory will need to find the best compromise between destruction of contaminants and survival of sufficient mycobacteria for isolation. Examples of the procedures for recovering mycobacteria from specimens are given in Figure 11.4.

Media for the mycobacteria

The egg-based Lowenstein–Jensen and Stonebrinks media are most commonly used in veterinary bacteriology. Lowenstein–Jensen medium can be obtained commercially and the formula and method of preparation of Stonebrinks medium is given in Appendix 2. The media are prepared as solid slants in screw-capped bottles. Malachite green dye (0.025 g/100 mL) is commonly used as the selective agent. Mycobacterium tuberculosis, M. avium and many of the atypical mycobacteria require glycerol for growth. However, glycerol is inhibitory to M. bovis while sodium pyruvate (0.4%) enhances its growth. Thus media with glycerol and without glycerol (but with Na pyruvate) should be inoculated. The media can be made more selective by the addition of cycloheximide (400 µg/mL), lincomycin (2 µg/mL) and nalidixic acid (35 µg/mL). Each new batch of culture medium should be inoculated with stock strains of mycobacteria to ensure that the medium supports satisfactory growth. The inoculated media may have to be incubated at 37°C for up to eight weeks for the mycobacteria in the tuberculosis group. Mycobacterium tuberculosis and M. avium prefer the caps on the culture media to be loose while M. bovis grows best in airtight containers. Liquid culture media may also be used for the isolation of mycobacteria and commercially available systems suitable for use in specialized laboratories processing large numbers of samples have been developed. Detection of growth of mycobacteria in these systems is based on radiometric or fluorometric methods. Examples include the BACTEC™ and MGIT™(Becton Dickinson) systems.

Most of the atypical mycobacteria will grow on media suitable for the tuberculosis-causing mycobacteria. Mycobacterium leprae and the mycobacterium causing bovine skin tuberculosis have not yet been cultured in vitro.