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

Abstract

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 Yersinia ruckeri 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 Y. ruckeri strain may rapidly be placed in a global epizootiological context.

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

“…The consistent repeat sizes of the ten VNTR loci, as well as their in vitro and in vivo stability, have previously been verified in the original study based upon this protocol 14 . Briefly, this was done using Sanger sequencing (repeat size), and by MLVA typing of multiple isolates following serial passages (in vitro) and from within individual disease outbreaks (in vivo).…”

“…If occurring to an extent where final (rounded) VNTR repeat counts become affected, this means the locus-specific variables s and i ( Table 1), used to determine VNTR repeat counts, must be re-calibrated. This involves linear regression on plots comparing accurate sequence sizes versus CE size calls, as described by Gulla et al 2018 14 .…”

“…Through a comprehensive MLVA based population study involving 484 Y. ruckeri isolates recovered from a range of spatiotemporal origins and habitats (host fish, environment, etc. ), our understanding regarding the epizootiology and population structure of this important fish pathogen was substantially increased 14 . MLVA typing enabled the tracing of clones disseminated anthropogenically over decades, presumably through transport of fish, as well as identification of locally confined strains.…”

“…The ten-loci MLVA protocol presented here, which recently formed the basis for an extensive Y. ruckeri population study 14 , involves extraction of DNA from agar-cultivated colonies, multiplex PCR and capillary electrophoresis (CE), followed by downstream in silico applications. For each examined isolate, two multiplex PCRs, both containing five fluorescently labelled primer pairs (6FAM, NED or VIC) each targeting individual VNTR regions, are run in parallel under identical conditions.…”

A pan genome reverse vaccinology approach to prevent Streptococcus agalactiae infection in farmed tilapia

“…These services are often not widely available in developing economies and lack of diagnostics has been attributed as one contributing factor in misdiagnosis and misuse of antibiotics in tilapia aquaculture in Vietnam and aquaculture in general (Boerlage et al, 2017;Henriksson et al, 2017). Whilst tools such as next generation sequencing technologies and bioinformatics can provide rapid and low cost genomic and metagenomic data that allow more accurate surveillance and detection of causative agents of disease (Barnes et al, 2016;Brynildsrud et al, 2014;Gardy and Loman, 2018;Gulla et al, 2018;Kawasaki et al, 2018), the establishment of readily available basic diagnostic and strain collection capability locally in aquaculture growth areas is a first priority.…”