title: Leptospirosis in UK vet visiting dogs, wild rodents and the pathogenomics of Leptospira species
Creators: name: Ball, Christopher
Publisher: name: University of Liverpool Repository
Year: 2014

description: Canine infection from pathogenic Leptospira serovars remains an issue within the UK, despite the availability of a canine vaccine. Canine leptospirosis cases are non-reportable and data regarding current levels for both suspected and confirmed cases is limited. A questionnaire based survey was undertaken to determine the number of canine leptospirosis cases within UK practices over a 12 month period. Average canine vaccination coverage across responding practices was determined as 60%, with 1669 vaccines administered per practice on average within the responding practices. No significant difference was witnessed between doses administered in either mixed or dedicated small animal practices (1692.40 and 1653.38 respectively), demonstrating that vaccination habits vary between individual clinicians rather than practice type. Diagnosing leptospirosis remains an issue, particularly relating to vague clinical signs during early infection. Survey results emphasised the priority that clinicians base on initially vague signs, with leptospirosis being typically considered once icteric signs present, where mortality rates are greater and treatment is less effective. Despite well documented associations linking leptospirosis and rodents, a current uncertainty remains regarding serovars maintained within the UK. In an attempt to clarify the situation, 283 wild rodents were sampled from rural (n=7) and urban sites (n=8). Infection was identified within 23 (8.13%) samples belonging to wood mice (n=16/152), bank voles (n=5/47) and field voles (n=2/10). Initial Leptospira identification using direct sequencing of PCR amplicons showed a single infecting pathogenic species (Leptospira interrogans). Serology data was obtained for 71 rodents using the microscopic agglutination test (MAT). Positive samples from pooled antigen testing (n=7/71; 9.86%) were further tested using four individual antigens. Data further confirmed a single infecting species (L. interrogans) and serogroup (Australis). Interestingly, we did not detect Leptospira within the portion of 67 rat kidney samples investigated. Stained kidney sections (n=11) showed limited association between inflammation and leptospire presence, indicating that rodents may shed the bacteria asymptomatically. Multi-locus sequence typing (MLST) schemes have been successfully applied to identify the sequence types (STs) of pathogenic Leptospira strains. The PCR positive rodent samples were tested using MLST (n=23). All samples with a full profile (n=11) were shown to belong within ST-24, with five partial profiles also likely to belong to the same ST. To date, three serovars are within ST-24 (Jalna, Bratislava and Muenchen), that belong to the Australis serogroup. This was the first study to utilise DNA extracted directly from kidney tissue to perform Leptospira MLST analysis. MLST data further emphasises a single infecting species and presents evidence for a single infecting serogroup. Further work involved full genome sequencing of ten strains not previously investigated, covering pathogenic, intermediate and saprophytic species. Sequence data for each strain was obtained using the MiSeq platform. The ‘core’ genome was identified across 17 strains (n=1,095; 28.76%), with pathogenic strains being more conserved with a greater shared core genome (n=2,859; 69.30%). Single nucleotide polymorphism (SNP) data was generated for strains (n=6), with an average of 35,346 SNPs per strain (range=686 to 55,303). L. interrogans serovar Icterohaemorrhagiae had the lowest SNP count (686) and was the only strain with a greater number of non-synonymous SNPs compared to synonymous (1.8:1); indicating close relatedness between serovars Icterohaemorrhagiae and Copenhageni. Coding sequences were identified within genome regions that may relate to antigenic differences (high SNP variation) or relate to key cellular processes (low SNP variation). Due to poor gene characterisation, hypothetical proteins make up a high proportion of coding sequences within such regions. Further work to characterise identified coding sequences may identify future therapeutic or diagnostic targets. This project aimed to investigate the current situation concerning canine and rodent Leptospira research within the UK. Results presented within this thesis demonstrate several wild rodent species within England are capable of maintaining and potentially shedding pathogenic strains known to infect both humans and dogs. Serogroup Australis (found infecting rodents) is now protected within a tetravalent canine vaccine; however annual booster vaccinations are required for optimal immunity. Extended urban sampling would be of great benefit considering the absence of positive urban samples. Suspected and confirmed canine cases are still witnessed within UK practices despite the reported majority having current vaccinations. Continued monitoring of serogroups would benefit vaccination strategies, and an emphasis on early detection within infected dogs would allow for the greatest chance of survival.