Comparative genome analysis reveals important genetic differences among serotype O1 and serotype O2 strains of Y. ruckeri and provides insights into host adaptation and virulence.

Despite the existence of a commercial vaccine routinely used to protect salmonids against Yersinia ruckeri, outbreaks still occur, mainly caused by nonmotile and lipase-negative strains (serotype O1 biotype 2). Moreover, epizootics caused by other uncommon serotypes have also been reported. At the moment, one of the main concerns for the aquaculture industry is the expanding range of hosts of this pathogen and the emergence of new biotypes and serotypes causing mortality in fish farms and against which the vaccine cannot protect. The comparative analysis of the genome sequences of five Y. ruckeri strains (150, CSF007-82, ATCC29473, Big Creek 74, and SC09) isolated from different hosts and classified into different serotypes revealed important genetic differences between the genomes analyzed. Thus, a clear genetic differentiation was found between serotype O1 and O2 strains. The presence of 99 unique genes in Big Creek 74 and 261 in SC09 could explain the adaptation of these strains to salmon and catfish, respectively. Finally, the absence of 21 genes in ATCC29473 which are present in the other four virulent strains could underpin the attenuation described for this strain. The study reveals important genetic differences among the genomes analyzed. Further investigation of the genes highlighted in this study could provide insights into the understanding of the virulence and niche adaptive mechanisms of Y. ruckeri.

Whole genome analysis of Yersinia ruckeri isolated over 27 years in Australia and New Zealand reveals geographical endemism over multiple lineages and recent evolution under host selection.

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.

Analysis of Yersinia ruckeri strains isolated from trout farms in northwest Germany.

Enteric redmouth disease (ERM), caused by Yersinia ruckeri, is among the most important infectious diseases in rainbow trout Oncorhynchus mykiss aquaculture in Europe. Our aim was to analyse the persistence of Y. ruckeri strains in trout farms in northwest Germany and their dissemination between farms based on a detailed molecular and phenotypical characterisation scheme. The data on identification and characterisation of Y. ruckeri strains and examining the distribution of these strains in the field could serve as a basis for preventive disease monitoring plans. During the observation period from June 2011 until June 2012, we collected 48 Y. ruckeri isolates from 12 different rainbow trout hatcheries. In total, 44 (91.7%) of the isolates were non-motile; in particular, all isolates recovered during the sampling period in winter and early spring were non-motile. In several trout farms, characteristic farm-specific Y. ruckeri isolates from particular typing groups were isolated throughout the year, while in other farms, which had a trading relationship between each other, ERM outbreaks were caused by Y. ruckeri from the same typing group. Our data indicate that in some farms, the causative Y. ruckeri strains persisted in the respective trout farm. The presence of Y. ruckeri from the same typing group in farms with a trading relationship indicates a dissemination of the infection between the farms.

Yersinia ruckeri Biotypes 1 and 2 in France: presence and antibiotic susceptibility.

Yersinia ruckeri is the causative agent of yersiniosis, a disease reported in a number of fish species, especially rainbow trout. This study was undertaken to describe the phenotypes of Y. ruckeri on French rainbow trout farms. More than 100 isolates, collected during recent outbreaks on trout farms, were characterized by phenotypic tests, namely using biochemical tests of the API 20E system, serotyping, biotyping (tests for motility and lipase activity) and by describing the pattern of susceptibility to several antibiotics. The isolates showed a low phenotypic diversity with a prevalent serotype (O1) and API 20E profile 5 1(3)07 100. As in other European countries, Biotype 2 (BT2), which lacks both motility and secreted lipase activity, was found to be present in France. The emergence of 'French' BT2 was different than that observed for other European countries (Finland, Spain, Denmark and the UK). The antibiotic pattern was uniform for all isolates, regardless of the geographical area studied. The results indicate that no resistance has yet emerged, and the efficacy of the antibiotic generally used against yersiniosis in France, trimethoprim/sulfamethoxasol, is not compromised (minimum inhibitory concentrations [MIC] of between 0.016 and 0.128 µg ml-1). Enrofloxacin and doxycycline, not used as a first-line treatment in fish diseases, have reasonably good efficacies (with MICs ≤0.128 and 0.256, respectively).

Non-adjuvanted flagellin elicits a non-specific protective immune response in rainbow trout (Oncorhynchus mykiss, Walbaum) towards bacterial infections.

Enteric redmouth disease, caused by Yersinia ruckeri, may result in high mortalities in farmed salmonids. Prophylaxis has been achieved with an immersion vaccine comprised of inactivated serovar 1 biotype 1 (motile) Y. ruckeri cultures. However, there has been a growing number of enteric redmouth outbreaks in vaccinated livestock associated with serovar 1 biotype 2 (non-motile) Y. ruckeri strains which do not produce flagellin. It was the aim of this study to evaluate the protective role of flagellin in enteric redmouth vaccines. Results showed that flagellin in the inactivated whole-cell vaccine were not the main immunoprotective molecule in eliciting a protective immune response towards infection. However, use of non-adjuvanted flagellin as a sub-unit vaccine, both in the native and recombinant form, resulted in a potent non-specific protective function towards challenge with biotype 1 (flagellin-producing) and biotype 2 (flagellin-devoid) Y. ruckeri. This vaccine can also protect rainbow trout against other microbial fish pathogens, for example Aeromonas salmonicida. Thus non-adjuvanted flagellin may have potential as a non-specific vaccine for fish towards bacterial pathogens.

Comparative evaluation of administration methods for a vaccine protecting rainbow trout against Yersinia ruckeri O1 biotype 2 infections.

Numerous outbreaks of enteric red mouth disease (ERM) caused by Yersinia ruckeri O1 biotype 2 in rainbow trout farms are currently being recorded despite established vaccination procedures against this disease. This could indicate that the currently used application of single immersion vaccination (using a commercial vaccine AquaVac(®) RELERA™) does not provide full protection. We elucidated by a controlled duplicated experiment if different vaccine administration methods can improve level and extent of protection. Rainbow trout, Oncorhynchus mykiss were vaccinated by: (1) a single immersion in bacterin diluted 1:10 for 30s (only primary vaccination); (2) two times 30s immersion (primary immersion vaccination followed by booster immersion vaccination 1 month later); (3) a single i.p. injection (only primary vaccination); (4) immersion vaccination followed by injection booster 1 month later; (5) a single 1h bath in bacterin diluted 1:2000; and (6) immersion (30s, 1:10) plus booster (1h in diluted 1:2000 vaccine) 5 months later). Injection challenge experiments were performed 3, 5 and 7 months post primary vaccination with 8.5×10(6) CFU/fish, 10.6×10(6) CFU/fish and 1×10(8) CFU/fish, respectively. In the first challenge trial, control fish exhibited a mortality of 76%, one time immersion vaccination had a mortality of 37%, two times immersion vaccinated fish had a 4% mortality, the one-time injection vaccinated group showed a mortality of 2% and the immersion plus injection boostered fish showed no mortality at all. When rainbow trout were challenged 5 months post primary vaccination, 26% mortality occurred in control fish, 21% in one time immersion group, 12% in two times immersion group, 5% in the one-time injection vaccinated group whereas immersion plus injection boostered fish again showed no mortality at all. When challenged 7 months post vaccination, one-time immersion vaccinated were not protected at all compared to the control group whereas injection vaccinated fish showed lower mortality (17%) compared to booster immersed fish (32% mortality) which was still better than un-vaccinated controls (44% mortality). It was noteworthy that a diluted bacterin (1:2000 for 1h after 5 months post primary vaccination) booster showed the same effect as a booster with 1:10 bacterin dilution for 30s applied 1 month after primary vaccination. Antibody levels showing significant elevations 28 days post challenge in vaccinated fish point to this immune parameter as a protective element. The superior and extended protection offered by booster vaccination or simply injection is noteworthy and may be applied in future vaccination strategies at farm level.

A polyphasic approach to study the intraspecific diversity of Yersinia ruckeri strains isolated from recent outbreaks in salmonid culture.

A polyphasic analysis was carried out on Yersinia ruckeri strains isolated from recently outbreaks in vaccinated fish using a combination of different phenotypic and molecular typing methods in order to study their variability and epidemiological relationships. Eighty strains were subjected to biotyping with conventional tests and API 20E system, serotyping, outer membrane protein (OMP) and lipopolysaccharide (LPS) profiling, and genetic fingerprinting by ERIC-PCR and REP-PCR techniques. The strains showed a high diversity, as evidenced by the formation of different phenotypic groups mainly related to the serotypes, LPS and OMP profiles. The diversity among all isolates, calculated as Simpson's diversity index (Di), varied between 0.35 (REP-PCR) and 0.70 (OMP). The most discriminative values (Di value ≥0.86) were obtained from any combination of three methods including biotype, serotype, API 20E profile, LPS or OMP. With the combination of all typing methods used a Di value of 0.90 was obtained. Association between different groups to the host species was evidenced. Furthermore, it seems that strains with similar characteristics are associated with recent outbreaks occurred in vaccinated fish in certain geographical areas. Our results emphasize the usefulness of using a combination of several different typing methods for epidemiological and bacterial diversity studies.

Multilocus sequence typing reveals high genetic diversity and epidemic population structure for the fish pathogen Yersinia ruckeri.

Yersinia ruckeri is the causative agent of enteric redmouth in fish and one of the major bacterial pathogens causing losses in salmonid aquaculture. Previously typing methods, including restriction enzyme analysis, pulsed-field gel electrophoresis and multilocus enzyme electrophoresis (MLEE) have indicated a clonal population structure. In this work, we describe a multilocus sequence typing (MLST) scheme for Y.ruckeri based on the internal fragment sequence of six housekeeping genes. This MLST scheme was applied to 103 Y.ruckeri strains from diverse geographic areas and hosts as well as environmental sources. Sequences obtained from this work were deposited and are available in a public database (http://publmst.org/yruckeri/). Thirty different sequence types (ST) were identified, 21 of which were represented by a single isolate, evidencing high genetic diversity. ST2 comprised more than one-third of the isolates and was most frequently observed among isolates from trout. Two major clonal complexes (CC) were identified by eBURST analysis showing a common evolutionary origin for 94 isolates forming 21 STs into CC1 and for 6 isolates of 6 STs in the CC2. It was also possible to associate some unique ST with isolates from recent outbreaks in vaccinated salmonid fish.

Identification of Yersinia ruckeri from diseased salmonid fish by Fourier transform infrared spectroscopy.

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), which mainly affects salmonid fish. Isolates of Y. ruckeri from diseased salmonid fish were obtained over a 6-year period from eight fish farms in the State of Baden-Württemberg, Southwest Germany. The strains were characterized by biochemical methods and Fourier transform infrared spectroscopy (FT-IR) combined with artificial neural network analysis. These methods were complemented by 16S rDNA sequencing for several isolates. The set of strains from these fish farms included sorbitol-positive, gelatinase-positive and non-motile Y. ruckeri. These variants were differentiated with an advanced FT-IR module, which is part of a higher-ranking method including more than 200 well-defined Yersinia strains against a background of more than 1000 other Gram-negative isolates. Validation of the newly constructed method yielded 97.4% of Y. ruckeri identified correctly on the species level. Thus, the FT-IR analysis enables distinction of all Y. ruckeri from other Yersinia species (e.g. fish-borne Y. enterocolitica) and other Enterobacteriaceae typically misidentified because of similar biochemical reaction profiles, especially Hafnia alvei. The differentiation of sorbitol-positive variants of Y. ruckeri using FT-IR was demonstrated.

Phenotypic and molecular characterization of Yersinia ruckeri isolates from rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) in Turkey.

The aim of the study was the phenotypic and molecular characterization of Yersinia (Y) ruckeri strains, the causative agent of Enteric Redmouth Disease (ERM), by antibiotyping, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of whole cell proteins. For this aim, a total of 97 Y ruckeri isolates were analyzed. The isolates were distinguished into ten antibiotypes and six phenotypes according to their resistance properties and whole cell protein profiles, respectively. Also, a glycoprotein band of approximately 25.5 kDa was observed in all Y ruckeri strains tested. In all strains, six different RAPD types were observed. In conclusion, Y ruckeri strains isolated from rainbow trout of fish farms in Turkey showed variation according to their phenotypic and genotypic characteristics, and the use of these three typing techniques in double and triple combinations could be more useful for discriminating the strains.

Rapid genotyping assays for the identification and differentiation of Yersinia ruckeri biotype 2 strains.

AIMS: To establish PCR-based assays for the rapid identification and differentiation of each of four known biotype 2 (BT2) phenotype-causing alleles in Yersinia ruckeri strains currently circulating in Europe and the United States. METHODS AND RESULTS: Novel assays were developed relying on detection of mutant allele-specific changes in restriction enzyme cleavage sites within targeted PCR products. The developed assays were validated against isolates previously genotyped by DNA sequencing. CONCLUSIONS: The described methods were specific, rapid and simple to perform and interpret. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed genotyping assays provide a valuable tool for identification and differentiation of specific BT2 strains of Y. ruckeri. These assays will be critical for the design and validation of new vaccines or other measures meant to control BT2 strains.

Serological and molecular heterogeneity among Yersinia ruckeri strains isolated from farmed Atlantic salmon Salmo salar in Chile.

We investigated 11 strains of Yersinia ruckeri, the causative agent of enteric redmouth disease (ERM), that had been isolated from Atlantic salmon Salmo salar L. farmed in Chile and previously vaccinated against ERM. Phylogenetic analysis of the 16S rRNA gene sequences confirmed the identification of the salmon isolates as Y. ruckeri. A comparative analysis of the biochemical characteristics was made by means of traditional and commercial miniaturised methods. All studied isolates were motile and Tween 80 positive, and were identified as biotype 1. In addition, drug susceptibility tests determined high sensitivity to sulphamethoxazole/trimethroprim, oxytetracycline, ampicillin and enrofloxacin in all isolates. Serological assays showed the presence of O1a, O1b and O2b serotypes, with a predominance of the O1b serotype in 9 strains. Analysis of the lipopolysaccharide profiles and the correspondent immunoblot confirmed these results. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the outer membrane proteins revealed that all Chilean strains had profiles with a molecular weight range between 34 and 55 kDa, with 3 distinct groups based on differences in the major bands. Genotyping analyses by enterobacterial repetitive intergenic consensus (ERIC-) and repetitive extragenic palindromic (REP-)PCR techniques clearly indicated intraspecific genetic diversity among Chilean Y. ruckeri strains.

Antigenic and cross-protection studies of biotype 1 and biotype 2 isolates of Yersinia ruckeri in rainbow trout, Oncorhynchus mykiss (Walbaum).

AIMS: The study investigated antigen characteristics of biotype (bt) 1 and bt 2 isolates of Yersinia ruckeri. METHODS AND RESULTS: The cell surface characteristics of Y. ruckeri were compared for their antigenic characteristics using polyclonal antibodies that revealed that both biotypes had a homogenous whole-cell protein antigenic profile. Notable differences in the antigenic properties were observed in the lipopolysaccharide profile of both biotypes. Two iron-regulated outer membrane proteins (IROMP) of c.90 and 100 kDa were shown to be major specific antigens. The results demonstrate for the first time differences in antigens between bt 1 and bt 2 isolates of serotype O1 isolates of Y. ruckeri. The protection induced in rainbow trout by a commercial monovalent, and bivalent inactivated vaccine was tested with the outcome that the ability of isolates to cause mortality in vaccinated fish varied with geographical location. In this context, vaccination studies suggested that the O antigen was the dominant immunogenic molecule involved in protection against the disease. CONCLUSIONS: The O antigen of Y. ruckeri was the dominant immunogenic molecule involved in the protection of rainbow trout against enteric redmouth disease. SIGNIFICANCE AND IMPACT OF THE STUDY: There are distinct phenotypic and antigenic differences in Y. ruckeri bt 1 and bt 2 with O antigen recognized as the dominant immunogenic molecule. The data have significance in explaining the lack of success of the earlier monovalent vaccine and demonstrate the effectiveness of the newer bivalent vaccine.

Novel non-motile phenotypes of Yersinia ruckeri suggest expansion of the current clonal complex theory.

The biochemical and cell surface characteristics of 63 non-motile isolates of Yersinia ruckeri from various sources were compared using the API 20E rapid identification system and conventional phenotypic methods. Eight individual phenotypic groups from a variety of fish species were observed from the data set. Non-motile isolates were not exclusively observed from serogroup O1; membership of biotype 2 was recorded for representatives from serogroups O2-O7. Variations in phenotypes highlights that new clonal groups are arising and that the current typing scheme requires expansion. Previously, it was hypothesized that disease was caused by a few virulent clones; data in this paper suggests that this assumption is not the case. The lipopolysaccharide (O antigen) type in the non-motile biotype was different from other isolates of Y. ruckeri.

Identification of flagellar motility genes in Yersinia ruckeri by transposon mutagenesis.

Here we demonstrate that flagellar secretion is required for production of secreted lipase activity in the fish pathogen Yersinia ruckeri and that neither of these activities is necessary for virulence in rainbow trout. Our results suggest a possible mechanism for the emergence of nonmotile biotype 2 Y. ruckeri through the mutational loss of flagellar secretion.

Yersinia ruckeri biotype 2 isolates from mainland Europe and the UK likely represent different clonal groups.

There have been increased reports of outbreaks of enteric redmouth disease (ERM) caused by Yersinia ruckeri in previously vaccinated salmonids in Europe, with some of these outbreaks being attributed to emergent non-motile, Tween 80-negative, biotype 2 isolates. To gain information about their likely origins and relationships, a geographically and temporally diverse collection of isolates were characterised by serotyping, biotyping, pulsed-field gel electrophoresis (PFGE) and outer membrane protein (OMP) profiling. A total of 44 pulsotypes were identified from 160 isolates by PFGE, using the restriction enzyme NotI. Serotype O1 isolates responsible for ERM in rainbow trout in both the US and Europe, and including biotype 2 isolates, represented a distinct subgroup of similar pulsotypes. Biotype 2 isolates, responsible for outbreaks of the disease in rainbow trout in the UK, Denmark and Spain, had different pulsotypes, suggesting that they represented different clones that may have emerged separately. Danish biotype 2 isolates recovered since 1995 were indistinguishable by PFGE from the dominant biotype 1 clone responsible for the majority of outbreaks in Denmark and the rest of mainland Europe. In contrast, US biotype 2 isolate YRNC10 had an identical pulsotype and OMP profile to UK biotype 2 isolates, suggesting that there had been exchange of these isolates between the UK and the US in the past. UK Atlantic salmon isolates were genetically and serologically diverse, with 12 distinct pulsotypes identified among 32 isolates.

Yersinia ruckeri infections in salmonid fish.

Yersinia ruckeri is the causative agent of yersiniosis or enteric redmouth disease leading to significant economic losses in salmonid aquaculture worldwide. Infection may result in a septicaemic condition with haemorrhages on the body surface and in the internal organs. Despite the significance of the disease, very little information is available on the pathogenesis, hampering the development of preventive measures to efficiently combat this bacterial agent. This review discusses the agent and the disease it causes. The possibility of the presence of similar virulence markers and/or pathogenic mechanisms between the Yersinia species which elicit disease in humans and Y. ruckeri is also examined.

First report of Yersinia ruckeri biotype 2 in the USA.

A polyphasic characterization of atypical isolates of Yersinia ruckeri (causative agent of enteric redmouth disease in trout) obtained from hatchery-reared brown trout Salmo trutta in South Carolina was performed. The Y. ruckeri isolates were biochemically and genetically distinct from reference cultures, including the type strain, but were unequivocally ascribed to the species Y. ruckeri, based on API 20E, VITEK, fatty acid methyl ester profiles, and 16S rRNA gene sequencing analysis. These isolates were nonmotile and unable to hydrolyze Tween 20/80 and were therefore classified as Y. ruckeri biotype 2. Genetic fingerprint typing of the isolates via enterobacterial repetitive intergenic consensus (amplified by polymerase chain reaction) and fragment length polymorphism showed biotype 2 as a homogeneous group distinguishable from other Y. ruckeri isolates. This is the first report of Y. ruckeri biotype 2 in the USA.