Pulsed-field gel electrophoresis and multi locus sequence typing for characterizing genotype variability of Yersinia ruckeri isolated from farmed fish in France

Abstract

Yersinia ruckeri is a pathogen that has an impact on aquaculture worldwide. The disease caused by this bacterial species, yersiniosis or redmouth disease, generates substantial economic losses due to the associated mortality and veterinary costs. For predicting outbreaks and improving control strategies, it is important to characterize the population structure of the bacteria. The phenotypic and genetic homogeneities described previously indicate a clonal population structure as observed in other fish bacteria. In this study, the pulsed-field gel electrophoresis (PFGE) and multi locus sequence typing (MLST) methods were used to describe a population of isolates from outbreaks on French fish farms. For the PFGE analysis, two enzymes (NotI and AscI) were used separately and together. Results from combining the enzymes showed the great homogeneity of the outbreak population with a similarity > 80.0% but a high variability within the cluster (cut-off value = 80.0%) with a total of 43 pulsotypes described and an index of diversity = 0.93. The dominant pulsotypes described with NotI (PtN4 and PtN7) have already been described in other European countries (Finland, Germany, Denmark, Spain and Italy). The MLST approach showed two dominant sequence types (ST31 and ST36), an epidemic structure of the French Y. ruckeri population and a preferentially clonal evolution for rainbow trout isolates. Our results point to multiple types of selection pressure on the Y. ruckeri population attributable to geographical origin, ecological niche specialization and movements of farmed fish.

Yersinia ruckeri Isolates Recovered from Diseased Atlantic Salmon (Salmo salar) in Scotland Are More Diverse than Those from Rainbow Trout (Oncorhynchus mykiss) and Represent Distinct Subpopulations

“…Historically, the majority of disease in rainbow trout in Europe and North America has been caused by serotype O1 strains of biotype 1 (the Hagerman strain) or, more recently, by biotype 2 variants (42). The association of a limited number of clones with a large proportion of disease in rainbow trout fits with the epidemic population structure model proposed for Y. ruckeri (34,57) and is consistent with the belief that Y. ruckeri is a recently emerged pathogen that was originally geographically isolated but quickly became widely disseminated (3,42). These strains presumably represent hypervirulent clones (58) that have adapted to a new niche (intensively cultured rainbow trout) (42) and consequently "exploded" in number and become widely distributed by extensive fish movements (34,57).…”

“…Conversely, non-O1 serotypes are usually associated with nonsalmonid species or with the environment, leading to the suggestion that different Y. ruckeri isolates exhibit various degrees of host specificity and virulence or adaptation for survival in the environment (16,31,63). Different pulsotypes and multilocus sequence types of Y. ruckeri are also associated with specific host species, strengthening the proposal for adaptive niche specialization (34,57). Indeed, experimental challenge studies have also highlighted the differences in virulence between different serotypes and clonal groups of Y. ruckeri (33,58); in particular, serotype O1 strains recovered from species other than rainbow trout were all avirulent in this species (33,58).…”

“…The association of a limited number of clones with a large proportion of disease in rainbow trout fits with the epidemic population structure model proposed for Y. ruckeri (34,57) and is consistent with the belief that Y. ruckeri is a recently emerged pathogen that was originally geographically isolated but quickly became widely disseminated (3,42). These strains presumably represent hypervirulent clones (58) that have adapted to a new niche (intensively cultured rainbow trout) (42) and consequently "exploded" in number and become widely distributed by extensive fish movements (34,57). In contrast, the evidence presented here and by others (24) clearly shows that Y. ruckeri strains that are associated with diseased Atlantic salmon are more diverse than those recovered from rainbow trout.…”

Mutations involved in the emergence of Yersinia ruckeri biotype 2 in France

“…For this study, 14 of these French strains, all belonging to BT2, were selected to examine as much diversity as possible. The selection criteria consisted of the strains' geographical origin and year of isolation, their phenotypic differences (serotypes and API20E profiles), and their genetic differences (pulsotypes and sequence types) (Table ) (Calvez et al, , ). Two reference strains were also included: ATCC 29473 and TUTI EX 5, respectively belonging to BT1 and BT2.…”

“…No mutation within fliR, flhA and flhB was revealed (Calvez et al, ). French BT1 and BT2 strains were also very similar when the serotypes, API20E profiles, antibiotic susceptibilities and genetic profiles were compared (Calvez, Fournel, Douet, & Daniel, ; Calvez et al, ). The aim of this study is to further characterize the phenotypic differences between the BT1 and BT2 strains and the genetic origin of the emergence of BT2 isolated in France.…”