Abstract
Introduction: Bovine tuberculosis (bTB), caused by Mycobacterium bovis infection, is the most significant infectious disease of UK cattle. Badgers (Meles meles) can also be infected by M. bovis and transmission can circulate between them and farmed cattle. Management of infection risks could be supported by the availability of rapid (animal-side) tests or noninvasive (faecal) tests for bTB in badgers.Objectives: This thesis aims to investigate the blood metabolome and faecal microbiome of wild M. bovis infected badgers. These next generation techniques (untargeted metabolomics and 16S amplicon sequencing) have not before been employed in this species, and could identify novel biomarkers that could lead to improved diagnostic performance for bTB in this species.
Methods: Flow infusion electrospray - high-resolution mass spectrometry (FIE-HRMS) was used to assess two types of sample. Firstly, to determine potential metabolite differences between infected and non-infected badgers based on thoracic blood samples obtained from badgers found dead in Wales (n=12), as well as to assess biochemical variation (the metabotype) of the bTB-free badger population (n=285). Secondly, using the peripheral blood of live trapped wild badgers for metabolite changes linked to bTB infection status (n=148), based on culture, Dual Path Platform (DPP) and interferon-gamma (IFN-g) tests.
Then, investigating the faecal microbiome of the badgers found dead in Wales by sequencing the V3-V4 region of the 16S rRNA gene, and undertaking a sex-matched age comparison of faecal microbiome variations between the adult and cub microbiomes (n=12). Comparisons were also made against other wild, omnivorous, mammals’ faecal microbiomes using publicly available data.
Results: The metabotype of uninfected badgers was most significantly affected by the sex of the animal and to a much lesser extent, it’s age, as indicated by tooth wear. Within the female population, variation was also seen between lactating and non-lactating individuals. No significant variation was linked to the presence of bite wounds, lymph node lesions, or geographical region of origin. The major differences in blood metabolomes from this population with M. bovis infection were changes to sphingolipid metabolism and glycerophospholipid metabolism, highlighting biological pathways that could hold novel biomarkers.
In the live, trapped badgers metabolomics was shown to be able to differentiate between badger blood samples based on their corresponding disease status (DPP, IFNᵧ or culture positive). Once again sex of the animal had a major influence, as the significant differences that existed differed greatly between the metabolomes of infected male and female badgers.
The faecal microbiome from this population, not infected with M. bovis, showed Fusobacteria as more abundant in the microbiomes of the cubs than the adults although no significant difference was seen in the diversity of bacteria within or between (alpha or beta diversity) the adult and cub badger microbiomes. When compared to the faecal microbiome of other wild, omnivorous mammals the badger microbiome was significantly different in bacterial populations (beta diversity).
Conclusion: Assessing the blood metabolome of badgers in Wales without bTB highlighted that sex is a major factor contributing towards variation in the metabolites present in the blood of each sex. This sex bias was still a major source of variation when comparing blood metabolomes of M. bovis infected badgers. Metabolomics was shown to be able to differentiate between badgers based on their infection status and sex, and this was the case even after autoclaving the blood so that it could be handled in a CL2 lab. The diagnostic accuracy, based on receiver operating characteristic curve (ROC) analyses, of individual metabolites were shown to fall below the threshold required to provide a useful novel
diagnostic approach. However specific pathways that were consistently altered with infection were identified. Future metabolomic work when making comparisons between bTB infected and non-infected badger samples should be sex-matched, given the different shifts seen with infection in the two sexes. While future investigations into the faecal microbiome of bTB infected and non-infected badgers using whole genome sequencing will build on what has been identified in non-infected badgers using 16S sequencing.
| Date of Award | 2024 |
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| Original language | English |
| Awarding Institution |
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| Sponsors | Llywodraeth Cymru KESS-2 |
| Supervisor | Luis Mur (Supervisor) |