Bovine tuberculosis (bTB) is one of the globe’s most common, multihost zoonoses and results in substantial socioeconomic costs for governments, farming industries, and tax payers. Despite decades of surveillance and research, surprisingly, little is known about the exact mechanisms of transmission. In particular, as a facultative intracellular pathogen, to what extent does survival of the causative agent,
A comprehensive understanding of how transmission occurs is required in order to control the disease and to design and deploy effective control measures. Here, we focus on the bovine tuberculosis (bTB) episystems in Western Europe, but the concepts discussed are readily applicable to other jurisdictions. bTB eradication in Britain and Ireland is proving to be extremely difficult [
In the recent past, as with human TB, it was largely assumed that the transmission of bTB between hosts was mostly facilitated by direct contact and inhalation of bacilli in relatively large (>5
Indirect transmission from a contaminated environment has been hypothesised to involve generation of bacilli droplet nuclei from fomites such as soil, pasture, slurry, excreta, and the built environment either through host animal inhalation or ingestion [
In the following, we review the evidence for environmental persistence of tuberculous bacilli which may constitute an infection risk and detail the methods and approaches that have been used to detect them and inform on the likely epidemiological risk.
Mycobacteria are common environmental microorganisms, but some species are significant human and animal pathogens.
The slow-growing species tend to be more associated with an intracellular lifestyle and pathogenicity: rapid-growing species tend to be mostly environmental saprophytes and include only a limited number of opportunistic pathogens [
The globally important
The MTBC has recently been stratified, using molecular methods, into a number of very closely related lineages; lineages 1–7 comprise human-adapted lineages, whereas various animal-adapted pathogens (ecotypes) group together in a separate lineage [
Survival on various matrices is, however, only one part of the puzzle; how those surviving bacilli can then infect new hosts from those matrices is another. For years, the received wisdom was that these organisms infected hosts which were in direct, close respiratory contact. However, indirect mechanisms of transmission should not be excluded.
In host-pathogen systems where environmental transmission pathways may occur, complex interacting factors will influence pathogen transmission, including host susceptibility, environmental pathogen persistence, infectiousness and the mechanisms of host exposure, variables still largely unknown for many hosts, and MTBC bacteria. The
A number of pathogens, including some more associated with being transmitted directly, such as foot-and-mouth disease virus (FMDV), norovirus, and influenza virus, also use indirect transmission via a contaminated environment. Even for influenza, which has been intensely studied for decades, the relative importance of droplet and bioaerosol transmission and transmission from touching contaminated objects or surfaces is not clear. This seriously complicates and confounds disease epidemiology and control options, allowing spread from several nonhost sources [
Due to the multifactorial nature of bTB transmission dynamics, it is challenging to understand bTB epidemiology in host communities and the environment [
Mounting evidence [
In the following, we review evidence from multiple MTBC host-pathogen systems, including bTB in the UK and Ireland. These data add weight to the hypothesis that a contaminated environment may be playing an important and underappreciated epidemiological role in MTBC maintenance.
There is now relatively high-quality scientific evidence, including studies using the guinea pig and mouse infection models, that supports environmental presence, persistence, and infectivity and a role for environmental contamination [
Although recently recreated landmark guinea pig experiments from the 1950s demonstrated that airborne-expelled droplet nuclei from infectious TB patients were the main route of transmission, they did not exclude a role for other transmission routes, including via the environment [
Researchers who demonstrated how
In Michigan, evidence suggests that contamination of cattle feed via inadvertent supplemented feeding of wild bTB-infected deer, maintained for hunting, contributes to bTB incidence in cattle [
In the UK and Ireland, the hypothesis of the preeminence of direct respiratory spread in close proximity hosts in shared airspace, resulting in the predominance of tuberculous lesions in the upper respiratory tracts of both hosts, has been challenged by proximity collar data [
Other significant veterinary pathogens are transmissible from such inhalable environmental sources.
Cattle may act to contaminate pasture and housing through shedding in excreta, respiratory droplets, and nasal mucus. Shedding in nasal mucus seems to be an extremely rare and intermittent occurrence; the stress of movement, testing, calving, etc., may play a role, but data are sparse [
While the generation of respiratory droplets may be the primary route by which cattle
Solid manure (faeces) was not considered a major risk factor in spread, providing it was properly composted (for at least 30 days) before land spreading and did not produce droplet nuclei or other bioaerosols [
While not conclusive, the fact that some studies suggest a potential role for slurry-based contamination of the environment may be important; it is perhaps wise then to consider mitigation strategies. Chemical disinfection of slurry could be considered [
The relative importance of spread of
The potential role of infectious wildlife in any environmental contamination needs to be considered. Badgers are an accepted wildlife reservoir for
Alongside potentially infectious urine deposits, another known part of badger social behaviour and ecology is the digging of faecal latrines at territorial boundaries. Culture and molecular detection [
A further relevant aspect of badger ecology, and of mustelids more generally, is the scent marking employed to mark territory via anal and caudal glands [
Although many animals are susceptible, relatively little is known about the status and potential of other wildlife in the UK to contribute significantly to
It has been suggested that cats might be exposed by contact with infectious cattle in the farmed environment, for example, where there is bTB mastitis, cats might become infected via ingestion of contaminated raw milk. That said, bTB mastitis in cattle is rare these days [
There are very few reports of confirmed
There is little evidence of these companion animals shedding bacilli to the environment, but the possibility cannot be excluded.
It is becoming apparent that invertebrate hosts in the shared farmed environment might act as vectors for
Similarly, environmental single-celled organisms, such as free-living amoebae (FLA), have long been hypothesised to be a “nursery” for intracellular bacteria within the wider environment [
Arthropod (ecto)parasites are rarely reported on UK and Ireland badgers. Consequently, they would appear to be unlikely vectors of transmission between badgers and cattle [
Intriguingly, the obligate intracellular pathogen
These findings indicate that our sole focus on livestock and wildlife hosts such as deer, badgers, wild boar, and possums may be underappreciating an important source of epidemiological risk and that a wider “ecosystem health” approach to bTB may be more appropriate. The complex cycling of intracellular pathogens between these nonbovine vertebrate and invertebrate reservoir hosts and the environment may also warrant further research.
In human TB epidemiology and pathogenesis, at least, there is probably a need to make a clearer distinction between true bioaerosol transmission and that of the so-called “droplet nuclei,” containing expelled TB bacteria; there is considerable uncertainty and ambiguity in the literature. The term “aerosol” tends to be used interchangeably with “droplet nuclei” when they are not quite the same thing, and there may be important implications for transmission and control. The importance of clarifying such ambiguity is illustrated clearly in the current COVID-19 pandemic.
In general, aerosols refer to the very small droplet sizes generated (<3
Improved understanding of aerosol science should allow rational explanation and intervention selection for infectious diseases. Mycobacteria can be shielded from environmental stresses in multibacillary aggregates generated from some hosts and environments; this improves their resilience further and suggests short distance transmission between close-contact predominates [
A prerequisite for the efficacy of indirect transmission from the environment is the longevity of pathogen survival in environmental matrices and fomites. Recently, the relevance of MTBC members’ persistence in a variety of environmental matrices has been revisited. While the potential role of the environment in bTB epidemiology is plausible and a reemerging area of research with more modern methods, classical epidemiology studies, which investigated bTB risk pre- and post-FMD restocking, concluded that cattle-cattle transmission was still potentially the most important source. However, there was an associated “stationary” breakdown risk for bTB persistence on the farm, outside of cattle, which decayed with “time since last breakdown.” Further cohort studies of continuously stocked and restocked herds following FMD indicated that an observed lower risk of herd breakdown in the first year after restocking might be due to a temporary reduction in the force (load) of infection on the farm. However, this reduction did not persist following the (re)introduction of cattle, suggesting that cattle themselves were contributing significantly to the observed persistence [
Studying
No direct studies have been carried out to investigate
Contaminated silage is another potential environmental source, although the ensiling process should reduce available oxygen, reduce pH, and raise the temperature (20°–30°C). However, pH 4-5 and temperatures obtained are well tolerated by
A field experiment in Michigan revealed that
Cattle faeces, containing
In an Australian mid-1980s study,
Prevailing weather is likely to dictate the viability of
Although historically not considered relatively high risk, cattle-cattle transmission has been demonstrated experimentally outdoors at pasture. However, the mechanism of transmission (whether direct or indirect or some combination) was not clear. Stocking density influenced the probability of transmission at pasture; reducing it should proportionately reduce the probability that cattle contact contaminated grass before any bacteria are destroyed by germicidal sunlight. Soil can be ingested by cattle grazing pasture, comprising ∼5–10% of the fresh weight intake and 10–15% of the dry weight intake. The movement of soil-contaminated fodder between farms may also be risky [
Maddock [
A study of African buffaloes, the maintenance host for
An important and necessary next step towards hypothesis-driven research on environmental persistence of Culture- and molecular-based (culture-dependent and culture-independent methods, respectively) methods for detecting Establishment of
In the following, we review the literature pertaining to these methods and identify potential difficulties/limitations from both a methodological and inferential standpoint. Investigations into environmental persistence and transmission are hampered by the lack of standardised and validated methods, whose performance characteristics are understood for reliably detecting bacilli in different matrices and fomites.
The general principle of applying molecular- and culture-based methods to real-world samples to detect pathogens or other environmental bacteria has an acceptable pedigree. A considerable amount of work on developing direct detection methodologies for the human pathogen,
The direct detection “issues” mentioned above hold true for detection of other members of the MTBC and related mycobacteria, including
An additional, significant caveat in all molecular-based
Next, we review the methods and variations used by several international groups in their application of the basic molecular detection methodology, in pursuit of developing
Researchers from Warwick University and Ireland used direct detection molecular methods to propose that
In theory, such PCR tests could be used to attempt to detect infected badger groups from badger faeces sampled at latrines. However, since not all group members would be infected and not all infected would be shedding, this requires extensive and resource-intensive repeat sampling. DEFRA recently have allowed private use of additional nonvalidated tests, such as PCR, but at the herd keepers’ own risk and expense. Potentially, such PCR tests could be used to attempt detection in various matrices and environments on and around farms. The test performance characteristics need to be considered, and sampling frames need to be designed accordingly. However, despite detecting pathogen DNA, whether
Subsequently, a validation ring trial of the qPCR test was reported [
Despite encouraging results using the same sample panels, the issue remained that detecting
In GB, DEFRA has recently allowed the application of additional, nonvalidated tests at the herd keepers’ own risk and expense. One such test uses a generalist bacteriophage and specific PCR or isothermal amplification to detect viable
In Spain, Gortazar and colleagues used molecular detection methods, together with other metadata, to investigate multihost systems [
The same Spanish group proposed that indirect transmission might be facilitated if MTBC bacilli persisted in the environment sufficiently long to provide an exposure risk to sympatric domestic and wild animals [
The methods this group employed were a simple DNA extraction using the phenol/chloroform solvent extraction protocol and the IS
More recently, the Gortazar group advocated the use of environmental DNA sampling and interpretation for bTB risk assessment in multihost episystems [
Contemporaneously to these Spanish groups, French laboratories have also been exploring
An alternative to the more straightforward quantitative or endpoint molecular detection methods described above is to apply whole-genome sequencing-based methods to environmental matrices. There is an increasing appreciation of the importance of complex microbiomes in the health of humans, animals, and the environment. Microbiomes can now be surveyed in a relatively unbiased fashion using high-throughput genome sequencing protocols and associated bioinformatics pipelines. Rapid developments in whole-genome sequencing chemistry, throughput, and cost now make it feasible to nonselectively index the microorganisms present in clinical or, potentially, environmental samples in unbiased sequencing surveillance [
Zhou et al. [
A recent biomedical example [
In a first pilot study of building-dust microbiomes, protocols were developed for microbiome indexing, based on 16S rRNA sequencing, using both short-read technology (Illumina) and long-read technology (MinION). Despite the well-documented, relatively low read accuracy of the MinION, the outputs, in terms of genera and species detected, were remarkably similar, with MinION reporting greater taxonomic resolution [
A complimentary way to investigate persistence is through the establishment of
Aside from providing a controlled environment in which to optimise detection methods,
Barbier et al. [
Additionally, incorporating vertebrate, invertebrate, and/or microbial hosts of
In larger-scale mesocosms, one could envisage addition of other hosts such as earthworms, arthropods, and even potentially small mammals (mice, guinea pigs, or ferrets). However, it is still “early days” with these methods. Starting small with a simple, base model that is a good proof of concept is important. From there, complexity could be increased to embrace differing soil types and simulation of differing climatological factors, such as UVA intensity, precipitation, and temperature.
bTB is currently the most costly, complex, and indeed controversial multihost, endemic zoonosis in the UK and Ireland. While other countries do not have the same degree of a problem with the disease, it is a potent animal health concern as well as a recognized zoonosis and potential barrier to trade. Evidence across multiple international territories supports the existence of a shared, colocalised, and relatively stable epidemic involving infectious livestock and wildlife hosts. The predominance of local risk factors in most classical and molecular epidemiology studies suggests either that intraspecies transmission occurs in all hosts and that some level of interhost transmission occurs or that sympatric hosts are all equally exposed to an infectious environment. Local epidemiology may differ by region and over time, so it would be unwise to generalise or extrapolate without comprehensive surveillance data.
Surprisingly, despite living with this troubling and costly disease for decades and significant investment in surveillance and research, the actual sources of infection and routes of transmission are not well understood. This limits the options for rational intervention to break chains of transmission. Due to well-documented limitations in diagnostic test performance characteristics, much of the epidemic remains unobserved. Most bTB reactors are now asymptomatic. While direct inhalation is the main route for
Studies suggest how
Culture-dependent methods and culture-independent molecular methods do exist to attempt to demonstrate viable
Leading research groups have been able to develop experimental simulations of the environment (micro- or mesocosms), which can be seeded with
It seems plausible that
Further development of this field of study may well help to uncover transmission mechanisms that until now have remained opaque and also reveal the relative importance of an environmental reservoir of infection for disease persistence. Furthermore, refinement of direct detection methodologies and downstream analyses has use beyond the bTB episystem. Other pathogens such as antimicrobial resistant (AMR) bacteria, which are believed to accumulate in the environment, may be indexed using similar approaches [
No data were used to support this study.
The authors declare that they have no conflicts of interest.
The authors gratefully acknowledge the useful discussions and input from Dr. Maria Laura Boschiroli, Dr. Alain Hartmann, Dr. Franck Biet, Dr. Lorainne Michelet, and Ciriac Charles of the Institut National de la Recherche Agronomique (INRA) and the Agence Nationale de Sécurité sanitaire de l’alimentation, de l’Environnement et du travail (ANSES), and Dr. Andrew Robertson, University of Exeter. The authors acknowledge the assistance of librarian Noirin Dobson. This work was commissioned and funded by the Department of Agriculture, Environment and Rural Affairs for Northern Ireland (DAERA-NI) under Evidence and Innovation Project 18/03/01.