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Here, we report the first complete genomes of three cultivable treponeme species from bovine digital dermatitis (DD) skin lesions, two comparative human treponemes, considered indistinguishable from bovine DD species, and a bovine gastrointestinal (GI) treponeme isolate. Key genomic differences between bovine and human treponemes implicate microbial mechanisms that enhance knowledge of how DD, a severe disease of ruminants, has emerged into a prolific, worldwide disease. Bovine DD treponemes have additional oxidative stress genes compared to nearest human-isolated relatives, suggesting better oxidative stress tolerance, and potentially explaining how bovine strains can colonize skin surfaces. Comparison of both bovine DD and GI treponemes as well as bovine pathogenic and human non-pathogenic saprophyte Treponema phagedenis strains indicates genes encoding a five-enzyme biosynthetic pathway for production of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, a rare di-N-acetylated mannuronic acid sugar, as important for pathogenesis. Bovine T. phagedenis strains further differed from human strains by having unique genetic clusters including components of a type IV secretion system and a phosphate utilisation system including phoU, a gene associated with osmotic stress survival. Proteomic analyses confirmed bovine derived T. phagedenis exhibits expression of PhoU but not the putative secretion system, whilst the novel mannuronic acid pathway was expressed in near entirety across the DD treponemes. Analysis of osmotic stress response in water identified a difference between bovine and human T. phagedenis with bovine strains exhibiting enhanced survival. This novel mechanism could enable a selective advantage, allowing environmental persistence and transmission of bovine T. phagedenis. Finally, we investigated putative outer membrane protein (OMP) ortholog families across the DD treponemes and identified several families as multi-specific adhesins capable of binding extra cellular matrix (ECM) components. One bovine pathogen specific adhesin ortholog family showed considerable serodiagnostic potential with the Treponema medium representative demonstrating considerable disease specificity (91.6%). This work has shed light on treponeme host adaptation and has identified candidate molecules for future diagnostics, vaccination and therapeutic intervention., Author summary Bovine digital dermatitis (DD) is a severe infectious disease causing cattle lameness, which is now endemic in many countries across the world. This lameness results from inflamed lesions between the heel bulbs and is very painful resulting in poor animal welfare and substantially reduced production. There remains no single cure for DD and whilst topical antibiotic treatment enables some healing, lesions frequently reappear. Current evidence implicates bacteria known as Treponema in the pathogenesis of DD. Here we characterise the genomes of several bovine DD treponeme species as well as related bacteria from humans and the bovine gastrointestinal tract. Comparative analyses demonstrate that production of a novel mannuronic acid sugar is a key feature of bovine pathogens and several survival mechanisms were identified which likely enable the bovine pathogens to inhabit the skin surface and be transmitted within the farm environment. Studies investigating putative outer membrane proteins which are potential vaccine candidates identified that the majority have a role in host attachment, with one family of proteins exhibiting particular promise as serodiagnostic antigens. This increased understanding of the considered causal pathogens of bovine DD, together with the genomic and proteomic resources produced by this study should underpin future diagnostic, vaccination and therapeutics studies to combat this severe disease of ruminants.

Whilst there has been considerable research focusing on the kinship of wolves, data on free-ranging dogs was sparse and there has been a long standing controversial debate over their ability to form packs. One of the aims of this project was to reconstruct kinship relationships in a population of free-ranging dogs, assessing the genetic variability and inbreeding level. For this purpose, I studied a population inhabiting a nature reserve at the outskirts of Rome in Italy. Analysis of twelve microsatellite loci revealed low number of alleles per locus, low levels of heterozygosity and difficulties in assigning parentage, possibly resulting from high levels of inbreeding in the population. Results from parentage analysis suggested multiple breeding individuals to be present in the social groups. One explanation for this is a result of the domestication process as free-ranging dogs no longer follow seasonal reproductive behaviour and have a plentiful supply of human waste to scavenge reducing competition. Although parentage analysis suggested multiple paternity for two litters, results had low statistical support and could be due to low genetic variability in the population. Recent research has found MARCH7 as a common candidate gene under diversifying selection between free-breeding dogs and either East Asia or European dog breeds, with a SNP labelled in the intronic region of the gene. MARCH7 belongs to the membrane-associated RING-CH (MARCH) family, a RING finger protein family of E3 ubiquitin ligases, consisting of 11 members in mammals. The second aim of this study was to test for the possible signals of diversifying selection between free-ranging dogs, pure-breed dogs and wolves in the MARCH7 gene. This was achieved through three main routes: Sanger sequencing of a targeted region previously identified as being under selection, evolutionary comparison through investigation of nonsynonymous and synonymous patterns and phylogenetic analysis of mammalian species and ab initio prediction of protein structure . Sequence analysis demonstrated the possibility of copy number variation and alternative splicing in MARCH7 but failed to show polymorphism at the previously identified intronic SNP. Comparative analysis demonstrated MARCH7 to have highly conserved regions, most notably the RING-CH domain, but also polymorphic regions, where a multitude of both synonymous and nonsynonymous mutations are present across mammalian species studied. Comparison of nonsynonymous and synonymous mutations demonstrated MARCH7 to be under purifying selection across mammalian species. Ab initio prediction of protein structure indicated a highly disordered structure across the majority of the gene, with the exception of the RING-CH domain.

Trichomonosis, caused by the protozoan Trichomonas gallinae, is an emerging infectious disease in finches, and is more commonly found in columbids and raptors. Infections can be sub-clinical or cause morbidity and mortality, but the parasite is currently only detectable by incubation of an oral swab. Here, we test whether T. gallinae parasites can be detected by PCR from faecal or non-cultured samples from the oral cavity and gastrointestinal tract of infected Turtle Doves (Streptopelia turtur). PCR did not detect T. gallinae parasites in any faecal samples screened, and in only 1 of 11 oral/gastrointestinal samples (from the mouth of a nestling suspected to have died from trichomonosis). We conclude that both oral swabs and parasite culture are still necessary to detect the sub-clinical presence of T. gallinae infection in birds.