Detecting and understanding mycobacterial infections in domestic cats and their role in the risk of zoontoci, intra- and inter-species spread

Mycobacterial infections are a global concern in humans and animals. In the UK, tuberculosis (TB) in animals is not confined to cattle and badgers; it is a serious concern in deer, camelids and cats. We have shown that cats are significantly at risk of TB as ~1% of feline biopsies have changes consistent with mycobacteriosis; 15% due to M. bovis, 19% M. microti (the vole/rodent bacillus). Some M. bovis infections progress extremely rapidly - a major concern given the newly recognised cat to human zoonotic risk, and notifiable status (M. bovis spreads between many different mammalian species). We hypothesise that the outcome of a cats exposure to Mycobacteria is determined by the nature of the pathogen and the host's immune response. However, a major bottleneck is failure to culture in ~50% of cases (despite having ZN+ bacteria). Rapid diagnosis is key to determining zoonotic risk and management options.

Currently, identifying the Mycobacteria species depends on a) detecting ZN+ bacteria b) IFN-gamma ELISA c) culture. These tests can detect Mycobacteria and the ELISA differentiates (with some specifity) M. bovis, M. microti and M. avium. Culture is a gold standard method to confirm live bacteria but is lengthy and frequently negative (despite tissue being ZN+). To rapidly identify infecting bacteria will impact significantly on knowing the zoonotic, inter- and intra-species risk, management options, affect survival (likelihood of clearance or latency) and inform ongoing monitoring. It could also monitor in-contact animals. We need to invesitgate the host respone to these infections if we are to understand Mycobacterial diseases more fully, and so elucidate the role of the host's response in determining which cases are likely to spread disease or, where appropriate, respond to treatment.

The project has 5 objectives:
1) Collect samples from Mycobacteria-infected cats and controls. Confirm Mycobacterial presence using current tests (culture, ZN, IFN-gamma ELISA) (RDSVS, Roslin, Biobest).
2) Isolate Mycobacterial DNA using established protocols from fresh frozen tissue or FFPE blocks. Determine the Mycobacterial sequences using the latest second and third generation sequencing methods (Illumina HiSeq 2500/MiSeq; Oxford Nanopore MinION) and compare them to published genome sequences (Roslin/Edinburgh Genomics).
3) Perform comparative analysis of the genome sequences of the isolated strains to identify sequence similarities and differences. Identify unique sequences for the development of strain-specific PCRs and develop multiplex PCR assays for the rapid diagnosis of feline TB and other Mycobacterial infections (Biobest).
4) Monitor cases long-term to correlate specific sequences with disease outcomes, and any transmission (RDSVS).
5) The nature of host-pathogen interaction between macrophages and Mycobacteria defines infection outcome in a number of species, but has not been investigated in cats. Understanding the basis for immunity or disease progression will inform veterinary medicine, and help to suggest appropriate management regimens for infected cats. We will identify cats with M. bovis TB and compare macrophage distribution, function and phenotype between infected and control tissues (Roslin).

This is a new collaboration bringing together expertise in feline medicine and tuberculosis, with macrophage biology, and pathogen genome sequencing incorporating both Roslin/RDSVS and Biobest Laboratories.