multi-locus-variable-number-tandem-repeat-analysis-fish-pathogenic

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium <em>Yersinia ruckeri</em> by Multiplex PCR and Capillary Electrophoresis

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, ...

Norwegian Veterinary Institute

Inverse autotransporters comprise the recently identified type Ve secretion system and are exemplified by intimin from enterohaemorrhagic Escherichia coli and invasin from enteropathogenic Yersiniae. These proteins share a common domain architecture and promote bacterial adhesion to host cells. Here, we identified and characterized two putative inverse autotransporter genes in the fish pathogen Yersinia ruckeri NVH_3758, namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin-like molecule). When trying to clone the highly repetitive genes for structural and functional studies, we experienced problems in obtaining PCR products. PCR failures and the highly repetitive nature of inverse autotransporters prompted us to sequence the genome of Y. ruckeri NVH_3758 using PacBio sequencing, which produces some of the longest average read lengths available in the industry at this moment. According to our sequencing data, YrIlm is composed of 2603 amino acids (7812bp) and has a molecular mass of 256.4kDa. Based on the new genome information, we performed PCR analysis on four non-sequenced Y. ruckeri strains as well as the sequenced. Y. ruckeri type strain. We found that the genes are variably present in the strains, and that the length of yrIlm, when present, also varies. In addition, the length of the gene product for all strains, including the type strain, was much longer than expected based on deposited sequences. The internal repeats of the yrInv gene product are highly diverged, but represent the same bacterial immunoglobulin-like domains as in yrIlm. Using qRT-PCR, we found that yrIlm and yrInv are differentially expressed under conditions relevant for pathogenesis. In addition, we compared the genomic context of both genes in the newly sequenced Y. ruckeri strain to all available PacBio-sequenced Y. ruckeri genomes, and found indications of recent events of horizontal gene transfer. Taken together, this study demonstrates and highlights the power of Single Molecule Real-Time technology for sequencing highly repetitive proteins, and sheds light on the genetic events that gave rise to these highly repetitive genes in a commercially important fish pathogen.

The repeat structure of two paralogous genes, Yersinia ruckeri invasin (yrInv) and a "Y. ruckeri invasin-like molecule", (yrIlm) sheds light on the evolution of adhesive capacities of a fish pathogen.

A multilocus variable-number tandem-repeat analysis (MLVA) assay was developed for epizootiological study of the internationally significant fish pathogen , which causes yersiniosis in salmonids. The assay involves amplification of 10 variable-number tandem-repeat (VNTR) loci in two five-plex PCRs, followed by capillary electrophoresis. A collection of 484  isolates, originating from various biological sources and collected from four continents over 7 decades, was analyzed. Minimum-spanning-tree cluster analysis of MLVA profiles separated the studied population into nine major clonal complexes and a number of minor clusters and singletons. The major clonal complexes could be associated with host species, geographic origin, and serotype. A single large clonal complex of serotype O1 isolates dominating the yersiniosis situation in international rainbow trout farming suggests anthropogenic spread of this clone, possibly related to transport of fish. Moreover, subclustering within this clonal complex indicates putative transmission routes and multiple biotype shift events. In contrast to the situation in rainbow trout,  strains associated with disease in Atlantic salmon appear as more or less geographically isolated clonal complexes. A single complex of serotype O1 exclusive to Norway was found to be responsible for almost all major yersiniosis outbreaks in modern Norwegian salmon farming, and site-specific subclustering further indicates persistent colonization of freshwater farms in Norway. Identification of genetically diverse  isolates from clinically healthy fish and environmental sources also suggests the widespread existence of less-virulent or avirulent strains. This comprehensive population study substantially improves our understanding of the epizootiological history and nature of an internationally important fish-pathogenic bacterium. The MLVA assay developed and presented represents a high-resolution typing tool particularly well suited for  infection tracing, selection of strains for vaccine inclusion, and risk assessment. The ability of the assay to separate isolates into geographically linked and/or possibly host-specific clusters reflects its potential utility for maintenance of national biosecurity. The MLVA is internationally applicable and robust, and it provides clear, unambiguous, and easily interpreted results. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed from a harvested colony within a single working day. As the resulting MLVA profiles are readily portable, any  strain may rapidly be placed in a global epizootiological context.

Multilocus Variable-Number Tandem-Repeat Analysis of Yersinia ruckeri Confirms the Existence of Host Specificity, Geographic Endemism, and Anthropogenic Dissemination of Virulent Clones.

A recently described typing system based on sequence variation in the virulence array protein (vapA) gene, encoding the A-layer surface protein array, allows unambiguous subtyping of Aeromonas salmonicida. In the present study, we compile A-layer typing results from a total of 675 A. salmonicida isolates, recovered over a 59-year period from 50 different fish species in 26 countries. Nine novel A-layer types (15-23) are identified, several of which display a strong predilection towards certain fish hosts, including e.g. Cyprinidae and Pleuronectidae species. Moreover, we find indications that anthropogenic transport of live fish may have aided the near global dissemination of two cyprinid-associated A-layer types. Comparison of whole genome phylogeny and A-layer typing for a subset of strains further resulted in compatible tree topologies, indicating the utility of vapA as a phylogenetic as well as an epizootiological marker in A. salmonicida. A Microreact project (microreact.org/project/r1pcOAx9m) has been created, allowing public access to the vapA analyses and relevant metadata. In sum, the results generated provide valuable insights into the global population structure of A. salmonicida, particularly in relation to its piscine host spectrum and the geographic distribution of these hosts.

Snorre Gulla

Fish Health Research Group

Norwegian Veterinary Institute

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Snorre Gulla

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Norwegian Veterinary Institute

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Fish Health Research Group