No abstract

Yersinia ruckeri is a salmonid pathogen with widespread distribution in cool-temperate waters including Australia and New Zealand, two isolated environments with recently developed salmonid farming industries. Phylogenetic comparison of 58 isolates from Australia, New Zealand, USA, Chile, Finland and China based on non-recombinant core genome SNPs revealed multiple deep-branching lineages, with a most recent common ancestor estimated at 18 500 years BP (12 355–24 757 95% HPD) and evidence of Australasian endemism. Evolution within the Tasmanian Atlantic salmon serotype O1b lineage has been slow, with 63 SNPs describing the variance over 27 years. Isolates from the prevailing lineage are poorly/non-motile compared to a lineage pre-vaccination, introduced in 1997, which is highly motile but has not been isolated since from epizootics. A non-motile phenotype has arisen independently in Tasmania compared to Europe and USA through a frameshift in fliI, encoding the ATPase of the flagella cluster. We report for the first time lipopolysaccharide O-antigen serotype O2 isolates in Tasmania. This phenotype results from deletion of the O-antigen cluster and consequent loss of high-molecular-weight O-antigen. This phenomenon has occurred independently on three occasions on three continents (Australasia, North America and Asia) as O2 isolates from the USA, China and Tasmania share the O-antigen deletion but occupy distant lineages. Despite the European and North American origins of the Australasian salmonid stocks, the lineages of Y. ruckeri in Australia and New Zealand are distinct from those of the northern hemisphere, suggesting they are pre-existing ancient strains that have emerged and evolved with the introduction of susceptible hosts following European colonization.

A real-time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10(4) ± 1 × 10(4) CFU g(-1) (without enrichment) and 40 ± 10 CFU g(-1) (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real-time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real-time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.

In an effort to develop a rapid diagnostic test for the fish pathogen Aeromonas salmonicida, the performance of 2 polymerase chain reaction (PCR) primer sets (AP and PAAS) targeting the fish pathogen A. salmonicida and 1 PCR primer set (MIY) targeting A. salmonicida subsp. salmonicida were evaluated. Initially, the PCR assays were used to screen purified DNA extracted from 308 A. salmonicida isolates. The AP and PAAS PCR tests were demonstrated to be 100% specific for the species A. salmonicida and did not cross-react with any of the non-target organisms (bacterial species other than A. salmonicida) used in this study. The combined sensitivity of the AP and PAAS tests was 99.4% and offered the best coverage in terms of identifying the target organism. The MIY PCR appeared to be 100% sensitive and specific for A. salmonicida subsp. salmonicida. Studies with tissues, spiked with known quantities of bacteria, were conducted to determine the lower detection limit of the PCR tests, and then the ability of these PCR tests to detect A. salmonicida in experimentally infected salmonids was assessed.