Mycobacterium bovis

Mycobacterium bovis is a slow-growing (16- to 20-hour generation time) aerobic bacterium and the causative agent of tuberculosis in cattle (known as bovine TB). It is related to Mycobacterium tuberculosis, the bacterium which causes tuberculosis in humans. M. bovis can jump the species barrier and cause tuberculosis-like infection in humans and other mammals.

Bacterium morphology and staining The bacteria are curved or straight rods. They sometimes form filaments, which fragment into bacilli or cocci once disturbed. In tissues, they form slender rods, straight or curved, or club-shaped. Short, relatively plump bacilli (rods) are seen in tissue smears, and large slender beaded rods in culture. They have no flagella or fimbria, and no capsule. Mycobacterium tuberculosis group bacteria are 1.0–4.0 μm long by 0.2–0.3 μm wide in tissues. In culture, they may appear as cocci, or as bacilli up to 6–8 μm long. The bacteria stain Gram-positive, acid-fast. The cell wall contains as high as 60% lipid, giving the mycobacteria their hydrophobic characteristics, slow growth, and resistance to desiccation, disinfectants, acids, and antibodies. (Mycobacterium family). They are not easy to stain with aniline dyes; although they are Gram-positive, confirming this may be difficult. Ziehl–Neelsen staining results in stain pink with hot carbol fuscin and then resist decolourisation with 3% hydrochloric acid in 95% alcohol (i.e. they are acid-alcohol fast); following washing, the slide is counterstained with e.g. methylene blue. They are nonspore-forming.

Culture and biochemical features

Growth requirements M. bovis is a facultative intracellular parasite. For in vitro growth, special culture media are required; for example, Dorset's egg medium incorporates egg yolk, phosphate buffer, magnesium salts, and sodium pyruvate; amino acids may be added, but glycerol is not included, as it is inhibitory. Culture generally requires several weeks at 37 °C to reach colonies visible to the unaided human eye. It is strictly aerobic and grows at 37, but not at 25 °C. Optimal growth occurs at 37-38 °C. The species does not reduce nitrate or niacin, and is resistant to pyrazinamide. It is sensitive to thiophene-2-carboxylic acid hydrazide.

Appearance of colonies Initially (after 3–4 weeks), its minute, dull flakes, begin to thicken to form dry, irregular masses standing high above the culture medium surface. Eventually, confluent growth is seen over the whole culture surface, forming a rough, waxy blanket, becoming thick and wrinkled and reaching up the sides of the container. Colonies are yellow when first visible, darkening to deep yellow and eventually brick red, if exposed to light. In fluid media, growth is on the surface only, unless a wetting agent (e.g. Tween 80) is added to the medium.

Cell structure and metabolism M. bovis is similar in structure and metabolism to M. tuberculosis. M. bovis is a Gram-positive, acid-fast, rod-shaped, aerobic bacterium. Unlike M. tuberculosis, M. bovis lacks pyruvate kinase activity, due to pykA containing a point mutation that affects binding of Mg2+ cofactor. Pyruvate kinase catalyses the final step of glycolysis, the dephosphorylation of phosphorenolpyruvate to pyruvate. Therefore, in M. bovis, glycolytic intermediates cannot enter oxidative metabolism. Although no specific studies have been performed, M. bovis seemingly must rely on amino acids or fatty acids as an alternative carbon source for energy metabolism.

Pathogenesis

During the first half of the 20th century, M. bovis is estimated to have been responsible for more losses among farm animals than all other infectious diseases combined. Infection occurs if the bacterium is ingested or inhaled. M. bovis is usually transmitted to humans by consuming raw milk from infected cows, although it can also spread via aerosol droplets. Actual infections in humans are nowadays rare in developed countries, mainly because pasteurisation kills M. bovis bacteria in infected milk. In the UK, cattle are tested for the disease as part of an eradication program and culled if they test positive. Such cattle can still enter the human food chain, but only after a meat inspector or a government veterinary surgeon has inspected the carcass and certified that it is fit for human consumption. However, in areas of the developing world where pasteurisation is not routine, M. bovis is a relatively common cause of human tuberculosis. Bovine tuberculosis is a chronic infectious disease that affects a broad range of mammalian hosts, including humans, cattle, deer, llamas, pigs, domestic cats, wild carnivores (foxes, coyotes) and omnivores (common brushtail possum, mustelids and rodents); it rarely affects equids or sheep. The disease can be transmitted in several ways; for example, it can be spread in exhaled air, sputum, urine, faeces, and pus, so the disease can be transmitted by direct contact, contact with the excreta of an infected animal, or inhalation of aerosols, depending on the species involved.

Application to biotechnology M. bovis is the ancestor of the most widely used vaccine against tuberculosis, M. bovis bacillus Calmette-Guérin (BCG). The attenuated BCG strain was produced by serial passage starting from an initial virulent strain, subculturing on glycerine potato medium 230 times over a span of 13 years.

In non-human animals

Control

Testing Tuberculin skin testing is possible in cattle. Casal et al. 2012 tried both recombinant protein and overlapping peptide provocation, finding the peptide test to be less sensitive. The tuberculin test causes false positives in BCG-vaccinated cattle. The Detect Infected among Vaccinated Animals (DIVA) skin test is designed to detect only infected animals among vaccinated ones. Phase 1 trials were completed in the UK in 2022, and phase 2 by 2025. (Technically speaking, DIVA is a descriptive term for all tests able to perform this kind of differentiation. The specific form of test being tested in the UK is DST-F, a fusion protein of three selected antigens not found in BCG. In this context, it makes BCG a marker vaccine.)

Vaccination The BCG vaccine can be used in cattle. The CattleBCG vaccine, based on the BCG Danish strain, is the leading candidate in the UK. Its future implementation depends on the success of the DST-F trials.

Epidemiology and responses

China M. bovis is prevalent among the Chinese deer population at 16.1%. It even more common among yaks with a seroprevalence of 48.7%.

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