ABSTRACT
Brachyspira hampsonii causes dysentery-like disease in infected pigs. Serial passage of a virulent swine isolate (P13) one-hundred times in laboratory culture medium was conducted to produce an attenuated strain, and to identify genomic determinants of virulence through comparison of genome sequences of the original and passaged strains. The resulting strain, P113, did not differ from P13 in terms of diagnostic biochemical characteristics but had an enhanced growth rate in culture, indicating laboratory adaptation. Whole genome sequencing of P113 revealed several single-nucleotide changes including a T to C transition that results in an R to G amino acid change in a putative mannose-1-phosphate guanylytransferase that is implicated in production of lipo-oligosaccharide. P113 was partially attenuated in a mouse model of infection, indicated by significantly fewer observations of abnormal feces in mice infected with P113 relative to P13. No differences were detected in bacterial shedding in feces, demonstrating that the ability of the organism to colonize mice was not affected. Passage through a mouse did not further alter the virulence of P113. Results of this study provide insight into genomic determinants of virulence in B. hampsonii and a live attenuated vaccine candidate.
Subject(s)
Brachyspira/pathogenicity , Gram-Negative Bacterial Infections/microbiology , Gram-Negative Bacterial Infections/pathology , Animals , Brachyspira/genetics , Brachyspira/growth & development , DNA Mutational Analysis , Disease Models, Animal , Mice , Microbiological Techniques , Nucleotidyltransferases/genetics , Polymorphism, Single Nucleotide , Serial Passage , Virulence , Whole Genome SequencingABSTRACT
BACKGROUND: The development of a mouse model as an in vivo pathogenicity screening tool for Brachyspira spp. has advanced the study of these economically important pathogens in recent years. However, none of the murine models published to date have been used to characterize the clinical signs of disease in mice, instead focusing on pathology following oral inoculation with various Brachyspira spp. The experiments described herein explore modifications of published models to characterize faecal consistency, faecal shedding and pathology in mice challenged with "Brachyspira hampsonii" clade II (Bhamp). METHODS AND RESULTS: In Experiment 1, 24 CF-1 mice were randomly allocated to one of three inoculation groups: sham (Ctrl), Bhamp, or B. hyodysenteriae (Bhyo; positive control). Half of each group was fed normal mouse chow (RMH) while the other received a low-zinc diet (TD85420). In Experiment 2, eight CF-1 mice and nine C3H/HeN mice were divided into Ctrl or Bhamp inoculation groups, and all fed TD85420. In Experiment 1, mice fed TD85420 demonstrated more severe mucoid faeces (P = 0.001; Kruskal Wallis) and faecal shedding for a significantly greater number of days (P = 0.005; Kruskal Wallis). Mean faecal scores of Bhamp inoculated mice trended higher than Ctrl (P = 0.06; Wilcoxon rank-sum) as did those of Bhyo mice (P = 0.0; Wilcoxon rank-sum). In Experiment 2, mean faecal scores of inoculated CF-1 mice were significantly greater than in C3H mice (P = 0.049; Kruskal Wallis) but no group differences in faecal shedding were observed. In both experiments, mice clustered based on the severity of colonic and caecal histopathology but high lesion scores were not always concurrent with high fecal scores. CONCLUSION: In our laboratory, CF-1 mice and the lower-zinc TD85420 diet provide a superior murine challenge model of "Brachyspira hampsonii" clade II.
