Bovine tuberculosis is a contagious disease of cattle and potentially other animals, some of which may act as additional reservoirs of infection, like the badger in parts of GB. Transmission between different hosts need not require direct contact as viable organisms excreted by infected animals in their sputum, ruptured abscesses, faeces and urine can survive for variable amounts of time in the environment on pasture, foodstuffs and surfaces of buildings and equipment. The purpose of this document is not to give definitive survival times for Mycobacterium bovis in different situations as this is not possible.  It is however intended to highlight the underlying factors that enable M. bovis to survive in the environment in order to support the assessment of risk of different circumstances encountered on farm.

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Nature of Mycobacteria

M. bovis is a slow growing, very resilient organism notable in its adaptations for survival in the host. It causes an insidious, chronic disease with a variable but often long incubation period with very few symptoms in the early stages of the disease. Depending on the host and other factors, M. bovis is shed by infected animals in sputum, urine, faeces and wound discharges. It is readily transmitted between cattle and susceptible wildlife if they come into close direct contact.  However, spread may also occur indirectly if these infected fluids contaminate the environment and then come in to contact with susceptible animals in the right conditions.  There is evidence from some depopulated farms kept free of cattle for long periods which then suffer recurrence of TB, that M. bovis may persist for prolonged periods in the environment under certain environmental conditions [1].

Adaptation for survival

It must be remembered that pathogenic Mycobacteria (those that cause disease) are likely to have arisen from harmless soil dwelling organisms and will have retained many of the adaptations needed for life outside of an animal [2]. M. bovis has further become exquisitely adapted to surviving and indeed flourishing in the hostile environment within macrophages (immune cells). Such adaptations include a waxy cell wall, rich in unique lipid (fat), polysaccharide (carbohydrate) and protein components, and the ability to adopt a quiescent state. This quiescent state, often incorrectly referred to as dormancy, is a state in which bacterial metabolism, replication and its interaction with the exterior is reduced to a minimum. The structure of the cell wall is fluid and changes in order to adopt these different states.  Such adaptations are complex, multi-layered and comprehensive but result in M. bovis managing to survive within host cells for prolonged periods. These adaptations also confer the ability to survive in the harsh conditions experienced in the environment.  Nevertheless M. bovis does not multiply outside the animal host and it only grows in vitro in selective culture media under special laboratory conditions and at a very slow rate.

Environmental contamination and survival

Numbers of M. bovis organisms excreted from various sources in a variety of species is variable and intermittent. However, as a guide, studies of infected badgers have found 105–106 (106 = one million) organisms/ml in lung exudates and 102–105 organisms/ml in urine and faeces.  These numbers are reasonably high, but not as high as may be found with the excretion of other species of bacteria in other diseases.  Experimental studies have shown that the infective dose required to establish disease in cattle via the respiratory route may be as low as a single organism [3]. Moreover, there was no significant difference in the likelihood of becoming infected, the time taken to become skin test positive or the degree of pathology developed between animals receiving a small or larger number of organisms [3].  However, higher doses are believed to be necessary to cause infection via the oral (digestive) route, e.g. when cattle ingest grass, fodder, concentrates, contaminated with the bacterium, or when calves are fed raw milk from infected cows with TB mastitis. Therefore, it would appear that cattle are quite susceptible and at risk of becoming infected after contact with viable M. bovis cells in the environment, but that the portal of entry and amount of viable bacteria present in the contaminated substrate are important factors.

Mycobacteria are very resilient organisms and can survive and remain infective in conditions that would readily kill other bacteria. For example, they are relatively resistant to acid and alkaline conditions and desiccation (drying) [4]. However, exposure to UV light and high temperatures from sunlight tend to kill the organisms, whereas cool, moist, dark conditions are protective.  Due to the variety of routes of excretion by cattle and wildlife across the farm, contamination of a wide variety of substrates can pose a risk.  In general, this will include pasture, fodder and forage, buildings, handling equipment and transport.  M. bovis is more likely to be exposed to the desiccating effects of sunlight on exposed solid surfaces but survive longer when contamination of softer material such as grass, hay or silage occurs.

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Evidence and interpretation of research

There have been a large number of published studies reporting the survival of M. bovis in various substrates and locations [5].  However, it is important to interpret these reports with great care.  The experiments are often highly artificial involving, for example, the inoculation of unnaturally large numbers of organisms grown in culture in the laboratory [6].  Such organisms are growing in quite a different biochemical state suited to growth in culture and cannot rapidly adapt to the state required for survival in the environment.  In natural circumstances, pathogenic Mycobacteria surviving outside a host are likely to be in a quiescent state, protected by an enhanced cell wall that has changed in structure in readiness for this ecological niche [7].  

On the other hand, failure to culture M. bovis in the laboratory is no guarantee that no infectious organisms are present. For example, some reports using culture suggest that pathogenic Mycobacteria may only remain viable for less than three months in soil, even in winter [4].  However, experiments using genomic detection methods have detected M. bovis in soil 21 months after inoculation [8] or are capable of infecting mice after at least one year [9].

Different substrates

The most common substrates that M. bovis may been found in as a result of contamination by infected cattle or wildlife are pasture, concentrates, hay, silage, soil, faeces and water.  Conditions in most of these materials are quite favourable for the survival of M. bovis with a near neutral pH and protection from desiccation, excessive heat and UV radiation. Contamination of silage however will expose the organism to pH below 4 and very hypoxic (low oxygen) conditions.  M. bovis is well adapted to surviving such conditions as it can rapidly respond, in part by adopting its quiescent state.   In this state, the organisms can survive for long periods whilst remaining viable and able to revert to an active infective state within hours [10]. 

An interesting and potentially significant factor in the successful persistence of Mycobacteria in the environment is their potential to survive and replicate within amoeba and other single celled organisms that live in the soil, in water and in the surface water film on grass.  These organisms often form resistant cysts as protection against desiccation, extreme pH and other stressors in the environment and so serve as a further protective layer for Mycobacteria within.  This potential risk has only recently been recognised and evidence that it is a significant factor in the epidemiology of bovine tuberculosis is yet to emerge.  However, there have been several reports that amoebae are implicated in a number of Mycobacterial outbreaks in a human health care setting [11].

Contamination of hard surfaces such as yards, buildings, handling equipment and transport occurs, but the survival of M.bovis in such situations exposed to sunlight, desiccation and cleaning/disinfection is likely to be short.

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Conclusion

Pathogenic Mycobacteria are thought to have evolved from soil-dwelling organisms and they retain the ability to resist the harsh conditions that they may encounter. They are unusually resistant to low pH and desiccation, although the high temperatures and UV of sunlight will reduce survival.  Survival may involve the adoption of a quiescent state from which they can readily emerge and become infective. Infection of protozoa might also play a part in survival in the environment, but this needs further investigation. Relatively large numbers of M. bovis excreted by cattle and wildlife can contaminate a range of substrates and the ability of M. bovis to persist for long periods in an infective state under the right conditions rendering the indirect transmission of bovine tuberculosis a substantial risk.

References

[1] Rodrıguez-Hernandez et al., Reviews in Medical Microbiology 2016, 27:20–24

[2] Smith et al., Nat Rev Microbiol. 2009 Jul;7(7):537-44. doi: 10.1038/nrmicro2165. Epub 2009 Jun 1

[3] Dean et al., Infect Immun. 2005 Oct; 73(10): 6467–6471

[4] Bourne , wildpro.twycrosszoo.org/S/0zM_Firmicutes/Mycobacterium/Mycobacterium_bovis/Mycobacterium_bovis/10MbovisPhysSuscpt.htm

[5] http://www.tbhub.co.uk/biosecurity/tb-in-the-environment/

[6] Fine, Amanda E. et al. “A Study of the Persistence of Mycobacterium Bovis in the Environment under Natural Weather Conditions in Michigan, USA.” Veterinary Medicine International 2011 (2011): 765430. PMC. Web. 17 Aug. 2018.

[7] Alderwick, et al., Cold Spring Harb Perspect Med 2015;5:a021113

[8] Young JS, Gormley E, Wellington EMH. Molecular Detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in Soil. Applied and Environmental Microbiology. 2005;71(4):1946-1952. doi:10.1128/AEM.71.4.1946-1952.2005.

[9] Ghodbane R, Medie F, Lepidi H, Nappez C, Drancourt M. Microbiology 160(3):496-501 doi:10.1099/mic.0.073379-0

[10] Gengenbacher and Kaufmann, Mycobacterium tuberculosis: success through dormancy, FEMS Microbiology Reviews, Volume 36, Issue 3, 1 May 2012, Pages 514–532, https://doi.org/10.1111/j.1574-6976.2012.00331.x

[11] Drancourt, M., Microbial Pathogenesis 77 (2014) 119e124