Whole-Genome sequencing in routine Mycobacterium bovis epidemiology - scoping the potential.

Mycobacterium bovis the main agent of bovine tuberculosis (bTB), presents as a series of spatially-localised micro-epidemics across landscapes. Classical molecular typing methods applied to these micro-epidemics, based on genotyping a few variable loci, have significantly improved our understanding of potential epidemiological links between outbreaks. However, they have limited utility owing to low resolution. Conversely, whole-genome sequencing (WGS) provides the highest resolution data available for molecular epidemiology, producing richer outbreak tracing, insights into phylogeography and epidemic evolutionary history. We illustrate these advantages by focusing on a common single lineage of M. bovis (1.140) from Northern Ireland. Specifically, we investigate the spatial sub-structure of 20 years of herd-level multi locus VNTR analysis (MLVA) surveillance data and WGS data from a down sampled subset of isolates of this MLVA type over the same time frame. We mapped 2108 isolate locations of MLVA type 1.140 over the years 2000-2022. We also mapped the locations of 148 contemporary WGS isolates from this lineage, over a similar geographic range, stratifying by single nucleotide polymorphism (SNP) relatedness cut-offs of 15 SNPs. We determined a putative core range for the 1.140 MLVA type and SNP-defined sequence clusters using a 50 % kernel density estimate, using cattle movement data to inform on likely sources of WGS isolates found outside of core ranges. Finally, we applied Bayesian phylogenetic methods to investigate past population history and reproductive number of the 1.140 M. bovis lineage. We demonstrate that WGS SNP-defined clusters exhibit smaller core ranges than the established MLVA type - facilitating superior disease tracing. We also demonstrate the superior functionality of WGS data in determining how this lineage was disseminated across the landscape, likely via cattle movement and to infer how its effective population size and reproductive number has been in flux since its emergence. These initial findings highlight the potential of WGS data for routine monitoring of bTB outbreaks.

Estimation of the Prevalence of Antimicrobial Resistance in Badgers (Meles meles) and Foxes (Vulpes vulpes) in Northern Ireland.

Antimicrobial resistant (AMR) bacteria can be shared between humans and animals, through food, water, and the environment. Wild animals are not only potential reservoirs of AMR, but are also sentinels mirroring the presence of AMR zoonotic bacteria in the environment. In Northern Ireland, little is known about levels of AMR in bacteria in wildlife, thus the current study aimed to estimate the prevalence of AMR bacteria in wildlife using wildlife species from two ongoing surveys as a proxy. Nasopharyngeal swabs and faecal samples from European badgers (Meles meles) (146 faecal samples; 118 nasal samples) and red foxes (Vulpes vulpes) (321 faecal samples; 279 nasal samples) were collected throughout Northern Ireland and were used to survey for the presence of extended spectrum beta lactamase resistant and AmpC-type beta lactamases Escherichia coli (ESBL/AmpC), Salmonella spp. (only in badgers) and methicillin resistant Staphylococcus aureus (MRSA). ESBLs were detected in 13 out of 146 badger faecal samples (8.90%) and 37 out of 321 of fox faecal samples (11.53%), all of them presenting multi-drug resistance (MDR). Fourteen out of 146 (9.59%) badger faecal samples carried Salmonella spp. [S. Agama (n = 9), S. Newport (n = 4) and S. enterica subsp. arizonae (n = 1)]. Overall, AMR was found only in the S. enterica subsp. arizonae isolate (1/14, 7.14%). No MRSA were detected in nasopharyngeal swabs from badgers (n = 118) and foxes (n = 279). This is the first attempt to explore the prevalence of AMR in the two common wildlife species in Northern Ireland. These findings are important as they can be used as a base line for further research exploring the origin of the found resistance. These results should encourage similar surveys where environmental samples are included to bring better understanding of AMR dynamics, and the impact on wildlife, domestic livestock and humans.

The First Whole Genome Sequence and Characterisation of Avian Nephritis Virus Genotype 3.

Avian nephritis virus (ANV) is classified in the Avastroviridae family with disease associations with nephritis, uneven flock growth and runting stunting syndrome (RSS) in chicken and turkey flocks, and other avian species. The whole genome of ANV genotype 3 (ANV-3) of 6959 nucleotides including the untranslated 5' and 3' regions and polyadenylated tail was detected in a metagenomic virome investigation of RSS-affected chicken broiler flocks. This report characterises the ANV-3 genome, identifying partially overlapping open reading frames (ORFs), ORF1a and ORF1b, and an opposing secondary pseudoknot prior to a ribosomal frameshift stemloop structure, with a separate ORF2, whilst observing conserved astrovirus motifs. Phylogenetic analysis of the Avastroviridae whole genome and ORF2 capsid polyprotein classified the first complete whole genome of ANV-3 within Avastroviridae genogroup 2.

A metagenomic comparison of endemic viruses from broiler chickens with runting-stunting syndrome and from normal birds.

Runting-stunting syndrome (RSS) in broiler chickens is an enteric disease that causes significant economic losses to poultry producers worldwide due to elevated feed conversion ratios, decreased body weight during growth, and excessive culling. Of specific interest are the viral agents associated with RSS which have been difficult to fully characterize to date. Past research into the aetiology of RSS has implicated a wide variety of RNA and DNA viruses however, to date, no individual virus has been identified as the main agent of RSS and the current opinion is that it may be caused by a community of viruses, collectively known as the virome. This paper attempts to characterize the viral pathogens associated with 2-3-week-old RSS-affected and unaffected broiler chickens using next-generation sequencing and comparative metagenomics. Analysis of the viromes identified a total of 20 DNA and RNA viral families, along with 2 unidentified categories, comprised of 31 distinct viral genera and 7 unclassified genera. The most abundant viral families identified in this study were the Astroviridae, Caliciviridae, Picornaviridae, Parvoviridae, Coronaviridae, Siphoviridae, and Myoviridae. This study has identified historically significant viruses associated with the disease such as chicken astrovirus, avian nephritis virus, chicken parvovirus, and chicken calicivirus along with relatively novel viruses such as chicken megrivirus and sicinivirus 1 and will help expand the knowledge related to enteric disease in broiler chickens, provide insights into the viral constituents of a healthy avian gut, and identify a variety of enteric viruses and viral communities appropriate for further study.