Microbiology is the scientific study of microorganisms such as bacteria, viruses and fungi. Bovine tuberculosis (bTB) is caused by infection with the bacterium Mycobacterium bovis. Knowledge of the microbiology of M. bovis is useful to provide context for understanding how bTB affects a population (epidemiology) and how it is controlled. The purpose of this article is to highlight and explain these aspects and their relevance.

 

History

The organisms causing tuberculosis and leprosy in man were first recognised by the famous German physician and microbiologist Robert Koch in 1882. Although not named by him, others proposed the name Mycobacterium for the genus in 1886. Whilst the organism causing tuberculosis in cattle was recognised by Koch as being different to that causing TB in man, surprisingly it was not formally named Mycobacterium bovis until 1970.

Robert Koch

Robert Koch

Genus

The genus Mycobacterium is very large, containing nearly 200 species, most of which do not cause disease or only do so in persons with weakened immunity.  A group of genetically related species causing tuberculosis in man and other animals is known as the M. tuberculosis complex (MTC) and it is thought that they evolved from soil-living organisms then spreading and diverging with human migrations and with the domestication of cattle [1] [2]. This group contains both M. tuberculosis and M. bovis.  Another group, the M. avium complex (MAC), contains M. avium subsp. avium primarily found in birds and M. avium subsp. paratuberculosis, the cause of Johne’s disease in cattle, sheep and goats. Rarely, other species of Mycobacteria may be involved in cases of TB in animals and humans.

 

Relationships

The close microbiological relationship between these organisms has important implications for public and animal health. TB in humans is primarily caused by M. tuberculosis, but M. bovis can also be involved in a minority of cases.  So, it is important that the infecting species is identified in order to determine the source of TB in people. 

Secondly, the immune response arising from infection with Mycobacteria can cross react with similar species and strains of the genus. Crucially, cattle can be infected by M. avium subsp. avium or M. avium subsp. paratuberculosis (and indeed more rarely, other Mycobacteria) which can cause a cross-reacting immune response.  This can compromise the effectiveness of the diagnostic tests for M. bovis leading to both false positives and false negatives in different circumstances [3].  In practice, cattle infected with M. bovis and also affected by or vaccinated against Johne’s disease are less likely to be detected by the tuberculin skin test.

Bacillus Calmette–Guérin (BCG) is a strain of M. bovis artificially developed at the beginning of the 20th century with reduced virulence.  It has been widely used as a vaccine in people globally, as it provides partial protection against tuberculosis.  BCG has been proposed for use in cattle to increase their resistance to bovine tuberculosis, but its use is currently banned in the EU because it interferes with the diagnostic tests for TB in cattle and there is currently no validated test to differentiate infected among vaccinated animals [4].

 

Culture and typing

M. bovis can be grown in vitro in the laboratory from clinical samples such as milk or urine, but more usually from tissue samples collected post mortem in the abattoir. However it is very slow growing and requires special expertise and safety facilities. This means that culture can only be carried out in specialist laboratories and the results of tests necessary to confirm disease may take several weeks to be reported. It is only after growth in the laboratory that the species of Mycobacterium isolated can be identified.

Testing for M bovis

 

Genotyping and molecular epidemiology

If M. bovis is isolated and identified in the laboratory, it can be further typed using genetic methods looking at different target sequences of the bacterium’s DNA so that individual strains can be identified.  One of the most commonly used techniques is spoligotyping [5] which when combined with VNTR typing provides the genotype currently routinely reported for strains isolated in the UK [6].  This information can be used to trace the origin of outbreaks, particularly when spread occurs through trade of infected animals [7].  Genotypes of M. bovis in cattle and wildlife tend to be geographically localised in stable clusters in the West of England and parts of Wales, reflecting a persistent environmental/wildlife reservoir of the bacterium and local spread between cattle in those areas (Fig. 1). Genotype data can be used to trace the probable geographical origin of infection in herds that have purchased cattle, particularly from non-local farms. Genotyping can also be used to suggest the source of human infections or to check for cross contamination in the laboratory. In the future, other more discriminatory methods such as whole genome sequencing of isolates may be used which will be much more powerful in inferring epidemiological links during outbreaks [8].

Spoligotype map

A map showing the geographical distribution of major spoligotypes in GB

 

Nature of Mycobacteria

Mycobacteria are unusual amongst bacteria in their robustness, resilience and slow growth characteristics and the chronic and insidious nature of the diseases that they cause. M. bovis is a facultative intracellular ‘parasite’, meaning that it can survive and indeed thrive inside the host’s macrophages (cells of the immune system that are meant to engulf and destroy the invading bacteria). It has many adaptations to intracellular life and may become quiescent (dormant) or divide very slowly, which enhances its survival and it has a tendency to become walled off in granulomas (small nodules of chronic inflammation) in the tissues.

 

Cellular structure

Mycobacteria have a unique and complex cell wall structure comprising layers of protein, polysaccharide (carbohydrate) and particularly lipid (fat) components that confer structural strength and great resistance both to attack in the intracellular environment and enhance survival outside the host.

 

Pathogenesis and intracellular life

The pathogenesis of M. bovis is complex and largely attributed to its ability to evade or manipulate the host’s immune response. These strategies facilitate the establishment of the primary infection, its persistence, quiescence and reactivation under favourable conditions. The organism deploys multiple remarkable biochemical, immunological, and genetic strategies to gain access to host macrophages and then resist the usual bacterial killing mechanisms, reprogramming them and indeed thriving in this hostile environment.

Of particular note and importance is the tolerance of Mycobacteria to acidic conditions. This is needed because the host acidifies the phagosomes, small envelopes within the macrophages containing the engulfed bacteria intended to kill them. Mycobacteria have evolved to resist this, which has consequences for the ability of M. bovis to survive in other acidic environments such as silage.  The host’s response of granuloma formation (so called ‘walling off of infection’) in its attempt to limit bacterial dissemination also serves to provide a secure niche protected from the immune response.  This reduced exposure to the host’s immune cells also means that a diagnostically detectable antibody response, which is common for other bacterial and in viral diseases, is often delayed for a considerable period. Therefore, tests that measure cell-mediated immune responses, such as the tuberculin skin and interferon-gamma tests are more suitable for the diagnosis of TB in animals and man.

As an obligate aerobe requiring oxygen for survival, M. bovis survives the low-oxygen environment within the granuloma by entering a state of non-replicating quiescence in which it can survive for months or years until conditions become more favourable, perhaps when host immunity wanes through stress or intercurrent disease. The quiescent state is brought about following the increased storage of nutrients, reduction in metabolic pathways and changes in the structure of the cell wall, particularly in its lipid component.  These attributes also confer on M. bovis the ability to survive harsh conditions in the environment for considerable periods, maintaining its ability to infect susceptible hosts.

Conclusion

M. bovis, is exquisitely adapted for survival in the host for long periods and establishing chronic infections with a long incubation period that are difficult to detect in the live animal. These same characteristics also allow the organism to survive in the environment for long periods posing a risk to cattle and wildlife from this environmental exposure.

A basic knowledge of the microbiology of Mycobacteria is crucial for an understanding of the on-farm risks and the development of strategies for their minimisation.

 

References

[1] Gutierrez MC, Brisse S, Brosch R, Fabre M, Omaïs B, et al. (2005) Ancient Origin and Gene Mosaicism of the Progenitor of Mycobacterium tuberculosis . PLOS Pathogens 1(1): e5. https://doi.org/10.1371/journal.ppat.0010005

[2] Wirth T, Hildebrand F, Allix-Béguec C, Wölbeling F, Kubica T, et al. (2008) Origin, Spread and Demography of the Mycobacterium tuberculosis Complex. PLOS Pathogens 4(9): e1000160. https://doi.org/10.1371/journal.ppat.1000160

[3] Coad, M., Clifford, DJ., Vordermeier, HM., Whelan, AO. (2013) The consequences of vaccination with the Johne’s disease vaccine, Gudair, on diagnosis of bovine tuberculosis Veterinary Record 172, 266

[4] Chambers, MA., Carter, SP., Wilson, GJ., Jones, G., Brown, E., Hewinson, RG., Vordermeier, M.

(2014) Vaccination against tuberculosis in badgers and cattle: an overview of the challenges, developments and current research priorities in Great Britain Veterinary Record 175, 90-96.

[5] J Kamerbeek, L Schouls, A Kolk, M van Agterveld, D van Soolingen, S Kuijper, A Bunschoten, H Molhuizen, R Shaw, M Goyal, J van Embden

Journal of Clinical Microbiology Apr 1997, 35 (4) 907-914

[6] Smith N. H., Dale J., Inwald J., Palmer S., Gordon S. V., Hewinson R. G. & Smith J. M. (2003) The population structure of Mycobacterium bovis in Great Britain: clonal expansion. Proceedings of the National Academy of Sciences of the United States of America 100, 15271–15275

[7] Skuce, RA., Mallon, TR., McCormick, CM., McBride, SH., Clarke, G., Thompson, A., Couzens, C., Gordon, AW., McDowell, SWJ.

(2010) Mycobacterium bovis genotypes in Northern Ireland: herd-level surveillance (2003 to 2008)

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[8] Biek et al., journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1003008