title: Bovine tuberculosis in a multi-host system in Northern Ireland: spatial distribution, molecular epidemiology and rapid diagnosis
Creators: name: Akhmetova, Assel
Publisher: name: University of Glasgow
Year: 2023

description: Bovine tuberculosis (bTB) has a complex epidemiology and a wide range of host species. United Kingdom and Ireland are one of the examples where control of bovine tuberculosis (bTB) is complex and very desirable for the farming and economy sectors. Despite the increasing implementation of control measures, such as rigorous farms surveillance, control of animal movements, testing of individual animals, and effective contact tracing are implemented for reducing the bTB spread on a national scale as the prevalence of the disease in cattle herds remains high (9.95% in 2021) (DAERA, 2021). The presence of a wildlife reservoir, the Eurasian badger Meles meles and its possible role in bTB persistence and spread in cattle species make bTB epidemiology very complex. Test-and-vaccinate or remove (TVR) wildlife intervention study was implemented in years 2014-2018 to control bTB levels in badgers and prevent disease transmission to livestock in Northern Ireland. Chapter 1 included literature review about bovine tuberculosis, its pathogenesis and diagnosis in animals, summary of previous research in the field of detection and molecular epidemiology as well as recent control system of bTB in Great Britain and Northern Ireland. This Chapter also covered recent issues and research gaps in relation to bTB laboratory diagnosis tests and epidemiology and thesis outline. Chapter 2 focused on Mycobacterium bovis spatial and molecular analysis of data from 1248 cattle and badger individuals within the TVR region (100 km2 and 2 km buffer zone) south eastern area in Northern Ireland with high bTB prevalence and high badger density (DAERA, 2018a). The study aimed to estimate the association between the spatial distribution of M. bovis multiple locus variable number of tandem repeats analysis (MLVA) types found in cattle and in badgers in the TVR area. Kernel density estimates of the shared MLVA types between cattle and badgers were evaluated to understand the spatial structure of the data. Contours in 95% density levels were estimated to demonstrate the spatial overlap of the main MLVA types, for each of the major strains. Two spatial models were built to assess the spatial distribution associations of M. bovis MLVA types in relationship to badger-cattle and cattle-badger transmissions. Thirty-seven MLVA types were found in cattle (n=31) and badger (n=6) species, with four of them (004, 006, 122 and 297) shared between two host species. Genotype 006 was identified as the most frequent and represented &gt;51% M. bovis isolates; it was indicated as a founder MLVA type for other genotypes using goeBURST algorithm. Strong association between spatial distribution of MLVA types in cattle and badgers was identified using kernel discriminant analysis (KDA). Combining whole genome sequence (WGS) data analysis with the associated epidemiological metadata provided an opportunity for a thorough genomic epidemiological analysis of M. bovis transmission in the TVR area. These results were shown in Chapter 3 of current PhD thesis and aimed to explain the relative importance of within and between species transmissions for bTB persistence. In total 619 M. bovis isolates collected between years 1986 and 2018 from cattle and badgers were sequenced. From this dataset, previously studied endemic clade (MLVA 006 genotype) comprising of 302 isolates was used to study bTB transmission dynamics using Bayesian coalescent-based methods, Discrete Ancestral Trait Mapping (DATM) approach to reconstruct ancestral states of M. bovis collected from cattle and badgers, and outbreaker2 software to reconstruct M. bovis transmission tree and outbreak reconstruction (Campbell et al., 2018). Estimated M. bovis substitution rate (mean 0.36-0.37 substitutions per genome per year) and most recent common ancestor (1970-1980s) was similar with other studies for bTB genomic epidemiology (Crispell et al., 2019, Salvador et al., 2019). Results obtained from the transmission trees reconstruction demonstrated high levels of between cattle transmissions and transmissions from cattle to badgers, and within badgers. The evidence of inter-species was also demonstrated in reconstructed phylogenetic trees. Overall, the results of this chapter showed that genetic and genomic M. bovis data obtained from historical isolates and the TVR intervention study can provide exceptional resolution for the genomic epidemiology of bTB, shedding light into the role of livestock and wildlife in the transmission of M. bovis in the region. In Chapter 4, I developed a molecular bacterial load assay for rapid quantification of M. bovis directly from infected animals’ tissues collected in Northern Ireland. Molecular bacterial load assay is currently used as a research method to monitor anti-TB therapy for human tuberculosis and quantifies M. tuberculosis bacterial load decline in response to treatment. I optimised this laboratory protocol on bTBfree bovine tissues spiked with M. bovis BCG and evaluated the performance of MBLA for bTB detection. This demonstrated that MBLA assay is efficient for bTB diagnostics in animal tissues. MBLA identifying Mycobacterium tuberculosis complex (MTBC) specific ribosomal RNA can quantify viable M. bovis in animal tissues with the range of concentrations between 1.59E+09 CFU/ml to 1.68E+02 CFU/ml. MBLA was then applied for the analysis of 214 culture-positive bTB infected bovine tissue samples. Using MBLA, M. bovis was detected in 90% of cases and bacterial loads were reliably quantified in 73% of positive samples. Work with hazard group 3 highly contagious bacilli as M. bovis, requires the use of facilities with higher biosafety level (containment level 3, CL3) that are not always available in diagnostic and research laboratories. Therefore, I conducted heat inactivation experiments to quantify M. bovis BCG using both 16S rRNA and DNA to determine the possibility of working with infected tissues outside a CL3 laboratory, i.e. in a CL2 laboratory after inactivation. This showed that infected samples can be used for RNA-based techniques such as MBLA after heat killing of bacilli without any impact on mycobacterial load in clinical specimens. To enable the MBLA to be used in the field, I also tested the stability of M. bovis BCG RNA at room temperature for up to one month. I demonstrated that if tested RNA samples were stored and transported at room temperature within at least one month, it would not affect the quantification of mycobacteria. The results of the MBLA assay in Chapter 4 used for the infected bovine tissue samples collected within the TVR study area suggested that this molecular technique can be used as diagnostic method for rapid (results obtained within the same day as sample collection as opposed to weeks of culture testing) and sensitive detection of bovine tuberculosis directly in animal tissue samples. Overall, these findings aided to our understanding of bTB transmission within cattle and badgers in an endemic area and offered tools for rapid molecular identification of M. bovis in this setting. The discussion of these results was presented in Chapter 5.