Tissue-specific differences in detection of Yersinia ruckeri carrier status in rainbow trout (Oncorhynchus mykiss).

Effective monitoring for subclinical infections is a cornerstone of proactive disease management in aquaculture. Salmonid fish that survive enteric redmouth disease (ERM) can carry Yersinia ruckeri as a latent infection for several months, potentially facilitating cryptic spread between facilities that exchange fish. In this study, fingerling rainbow trout (Oncorhynchus mykiss) were infected by immersion and sampled for up to 14 weeks post-infection. Yersinia ruckeri was cultured from the posterior kidney of more than 89% of fish up to 4 weeks post-infection, but from 2% or fewer of fish sampled at later time points. In contrast, qPCR-based detection of the Y. ruckeri 16s rRNA gene in intestine and spleen extracts revealed a much higher rate of infection: at 14 weeks post-infection Y. ruckeri was detected in nearly 50% of spleens and 15% of intestines. The difference between spleen and intestine is likely due at least in part to technical limitations of qPCR on intestinal DNA extracts; accordingly, we propose that qPCR of spleen DNA ought to be considered the preferred standard for detection of carriers of Y. ruckeri.

Absolute quantification of priority bacteria in aquaculture using digital PCR.

Modern aquaculture systems are designed for intensive rearing of fish or other species. Both land-based and offshore systems typically contain high loads of biomass and the water quality in these systems is of paramount importance for fish health and production. Microorganisms play a crucial role in removal of organic matter and nitrogen-recycling, production of toxic hydrogen sulfide (H2S), and can affect fish health directly if pathogenic for fish or exerting probiotic properties. Methods currently used in aquaculture for monitoring certain bacteria species numbers still have typically low precision, specificity, sensitivity and are time-consuming. Here, we demonstrate the use of Digital PCR as a powerful tool for absolute quantification of sulfate-reducing bacteria (SRB) and major pathogens in salmon aquaculture, Moritella viscosa, Yersinia ruckeri and Flavobacterium psychrophilum. In addition, an assay for quantification of Listeria monocytogenes, which is a human pathogen bacterium and relevant target associated with salmonid cultivation in recirculating systems and salmon processing, has been assessed. Sudden mass mortality incidents caused by H2S produced by SRB have become of major concern in closed aquaculture systems. An ultra-sensitive assay for quantification of SRB has been established using Desulfovibrio desulfuricans as reference strain. The use of TaqMan® probe technology allowed for the development of multi-plex assays capable of simultaneous quantification of these aquaculture priority bacteria. In single-plex assays, limit of detection was found to be at around 20 fg DNA for M. viscosa, Y. ruckeri and F. psychrophilum, and as low as 2 fg DNA for L. monocytogenes and D. desulfuricans.

Multiplex droplet digital PCR assay for detection of Flavobacterium psychrophilum and Yersinia ruckeri in Norwegian aquaculture.

We report the development of ddPCR assays for single and simultaneous detection of the bacterial pathogens Flavobacterium psychrophilum and Yersinia ruckeri in water from land-based recirculation aquaculture systems (RAS), producing Atlantic salmon (Salmo salar) smolt. The method was tested and verified for use in water analyses from RAS production sites, and proved to be specific and with sensitivity 0.0011 ng DNA for F. psychrophilum and 1.24 ng for Y. ruckeri. These bacteria are important fish pathogens that have caused reoccurring salmonid infection disease in RAS. Monitoring pathogen levels in water samples could be a useful alternative surveillance strategy to evaluate operational risk assessment connected to stress factors. Water quality is essential for fish health and growth in RAS production in general, and high or increasing levels of these pathogens in the RAS water may generate an early indication of unfavourable conditions in the RAS environment, and give directions to operational actions. This approach may reduce fish mortality, reduce production loss, and offer more effective and targeted preventive measures within RAS production.

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

Yersina ruckeri is an enterobacteria responsible for Enteric redmouth disease (ERM), which causes significant economic losses in the aquaculture industry worldwide. Two biotypes have been described within Y. ruckeri: biotype 1 (BT1) and biotype 2 (BT2). Unlike BT1, BT2 is negative for motility and lipase secretion. The emergence of BT2 Y. ruckeri has been associated with disease outbreaks in vaccinated fish in several countries, notably France in the early 2000s. In this study, 15 BT2 strains (14 BT2 strains isolated in France and the BT2 reference strain EX5) were studied to compare the phenotypic characters of the BT1 and BT2 strains and to determine the genetic origin of the emergence of BT2 in France. BT1 bacteria are significantly longer in size than BT2 bacteria (a difference of 0.222 µm). The loss of motility of some French BT2 strains could be due to the loss of their ability to produce flagella caused by three mutations within the fliG, flhC and flgA genes. In the light of these results, the emergence of BT2 Yersinia ruckeri in France is discussed.

Comparative bioinformatic and proteomic approaches to evaluate the outer membrane proteome of the fish pathogen Yersinia ruckeri.

Yersinia ruckeri is the aetiological agent of enteric redmouth (ERM) disease and is responsible for significant economic losses in farmed salmonids. Enteric redmouth disease is associated primarily with rainbow trout (Oncorhynchus mykiss, Walbaum) but its incidence in Atlantic salmon (Salmo salar) is increasing. Outer membrane proteins (OMPs) of Gram-negative bacteria are located at the host-pathogen interface and play important roles in virulence. The outer membrane of Y. ruckeri is poorly characterised and little is known about its composition and the roles of individual OMPs in virulence. Here, we employed a bioinformatic pipeline to first predict the OMP composition of Y. ruckeri. Comparative proteomic approaches were subsequently used to identify those proteins expressed in vitro in eight representative isolates recovered from Atlantic salmon and rainbow trout. One hundred and forty-one OMPs were predicted from four Y. ruckeri genomes and 77 of these were identified in three or more genomes and were considered as "core" proteins. Gel-free and gel-based proteomic approaches together identified 65 OMPs in a single reference isolate and subsequent gel-free analysis identified 64 OMPs in the eight Atlantic salmon and rainbow trout isolates. Together, our gel-free and gel-based proteomic analyses identified 84 unique OMPs in Y. ruckeri. SIGNIFICANCE: Yersinia ruckeri is an important pathogen of Atlantic salmon and rainbow trout and is of major economic significance to the aquaculture industry worldwide. Disease outbreaks are becoming more problematic in Atlantic salmon and there is an urgent need to investigate in further detail the cell-surface (outer membrane) composition of strains infecting each of these host species. Currently, the outer membrane of Y. ruckeri is poorly characterised and very little is known about the OMP composition of strains infecting each of these salmonid species. This study represents the most comprehensive comparative outer membrane proteomic analysis of Y. ruckeri to date, encompassing isolates of different biotypes, serotypes, OMP-types and hosts of origin and provides insights into the potential roles of these diverse proteins in host-pathogen interactions. The study has identified key OMPs likely to be involved in disease pathogenesis and makes a significant contribution to furthering our understanding of the cell-surface composition of this important fish pathogen that will be relevant to the development of improved vaccines and therapeutics.

Yersinia ruckeri Isolated from Common Mudpuppy Necturus maculosus.

During a routine health inspection of apparently healthy wild-caught common mudpuppies Necturus maculosus, the bacteria Yersinia ruckeri was isolated and the identity confirmed using biochemical and molecular methods. This represents the first isolation of Y. ruckeri from an amphibian. This finding increases the known host range capable of harboring this important fish pathogen and could have serious management implications for aquaculture. Furthermore, addressing wild amphibians in fish hatchery biosecurity plans is discussed.

pYR4 From a Norwegian Isolate of Yersinia ruckeri Is a Putative Virulence Plasmid Encoding Both a Type IV Pilus and a Type IV Secretion System.

Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae, pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.

A highly sensitive, non-invasive qPCR-based strategy for direct quantification of Yersinia ruckeri in fish faeces.

Finfish with asymptomatic Yersinia ruckeri infections pose a major risk as they can transmit the pathogen and cause clinical outbreaks in stock populations. Current tools have insufficient quantitative ability for accurately detecting the trace levels of Y. ruckeri typically associated with asymptomatic infection, necessitate invasive or lethal sampling, or require long processing times. This study presents a highly sensitive qPCR-based method, targeting part of the Y. ruckeri 16S rRNA sequence, that is capable of detecting extremely low levels of Y. ruckeri in noninvasively collected faecal samples. Quantitative precision and accuracy of faecal sample analysis was consistent, despite the complexity of the faecal matrix. The assay demonstrated linearity over a six log-wide dynamic range. Its limit of detection (LOD) and limit of quantification (LOQ) were 4 and 10 copies of the target sequence, respectively. Sensitivity of the assay was comparable to other qPCR-based methods without requiring invasive or lethal sampling. Applicability as a screening strategy was tested using passively collected faecal samples. Asymptomatic Y. ruckeri infection was detected in all samples, although none of the fish exhibited overt infection. This method will be beneficial for finfish disease management if developed further as a noninvasive, screening tool against asymptomatic Y. ruckeri infection.

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.

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.

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.

Global 3D imaging of Yersinia ruckeri bacterin uptake in rainbow trout fry.

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) in rainbow trout, and the first commercially available fish vaccine was an immersion vaccine against ERM consisting of Y. ruckeri bacterin. The ERM immersion vaccine has been successfully used in aquaculture farming of salmonids for more than 35 years. The gills and the gastrointestinal (GI) tract are believed to be the portals of antigen uptake during waterborne vaccination against ERM; however, the actual sites of bacterin uptake are only partly understood. In order to obtain insight into bacterin uptake during waterborne vaccination, optical projection tomography (OPT) together with immunohistochemistry (IHC) was applied to visualize bacterin uptake and processing in whole rainbow trout fry. Visualization by OPT revealed that the bacterin was initially taken up via gill lamellae from within 30 seconds post vaccination. Later, bacterin uptake was detected on other mucosal surfaces such as skin and olfactory bulb from 5 to 30 minutes post vaccination. The GI tract was found to be filled with a complex of bacterin and mucus at 3 hours post vaccination and the bacterin remained in the GI tract for at least 24 hours. Large amounts of bacterin were present in the blood, and an accumulation of bacterin was found in filtering lymphoid organs such as spleen and trunk kidney where the bacterin accumulates 24 hours post vaccination as demonstrated by OPT and IHC. These results suggest that bacterin is taken up via the gill epithelium in the earliest phases of the bath exposure and from the GI tract in the later phase. The bacterin then enters the blood circulatory system, after which it is filtered by spleen and trunk kidney, before finally accumulating in lymphoid organs where adaptive immunity against ERM is likely to develop.

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).

Yersiniosis in Atlantic cod, Gadus morhua (L.), characterization of the infective strain and host reactions.

A disease outbreak in farmed Atlantic cod caused by Yersinia ruckeri is reported. Mortality started following vaccination of cod reared in two tanks (A and B). The accumulated mortality reached 1.9% in A and 4.8% in B in the following 30 days when treatment with oxytetracycline was applied. Biochemical and molecular analysis of Y. ruckeri isolates from the cod and other fish species from fresh and marine waters in Iceland revealed a high salinity-tolerant subgroup of Y. ruckeri serotype O1. Infected fish showed clinical signs comparable with those of Y. ruckeri -infected salmonids, with the exception of granuloma formations in infected cod tissues, which is a known response of cod to bacterial infections. Immunohistological examination showed Y. ruckeri antigens in the core of granulomas and the involvement of immune parameters that indicates a strong association between complement and lysozyme killing of bacteria. Experimental infection of cod with a cod isolate induced disease, and the calculated LD50 was 1.7 × 10(4) CFU per fish. The results suggest that yersiniosis can be spread between populations of freshwater and marine fish. Treatment of infected cod with antibiotic did not eliminate the infection, which can be explained by the immune response of cod producing prolonged granulomatous infection.

In vivo monitoring of Yersinia ruckeri in fish tissues: progression and virulence gene expression.

In this study, the utilization of bioluminescence imaging (BLI) allowed us to define the progression of Yersinia ruckeri during the infection of rainbow trout. A luminescent Y. ruckeri 150 strain was engineered using the pCS26-Pac plasmid containing the lux operon from Photorhabdus luminescens. Two different models of infection of rainbow trout were defined depending on the route in which bacteria were administered, being the gut the major organ affected following bath immersion. This indicates that this organ is important for bacterial dissemination inside the fish and the establishment of the infection. Moreover, the expression of three previously selected operons by in vivo expression technology (IVET) was analysed, the yhlBA involved in the production of a haemolysin, the cdsAB related to the uptake of cysteine and the yctCBA implicated in citrate uptake. Apart from these factors, the expression of yrp1 encoding a serralysin metalloprotease involved in pathogenesis was also analysed. The results indicated that all of the assayed promoters were expressed during infection of rainbow trout. In addition to these findings, the methodology described in this work constitutes a useful model for studying the infection process in other fish pathogenic bacteria.

Tissue specific uptake of inactivated and live Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss): visualization by immunohistochemistry and in situ hybridization.

Understanding of uptake and invasion routes of Yersinia ruckeri, causing Enteric Red Mouth Disease (ERM) in rainbow trout (Oncorhynchus mykiss), is essential for improved understanding of the pathogenicity and immune response mechanisms associated this disease. The present work shed light on areas of invasion in rainbow trout by the use of immunohistochemistry and in situ hybridization techniques. Fish were exposed to live or formalin inactivated bacteria and samples were subsequently taken for histology from various outer and inner surfaces. We applied a specific monoclonal antibody and specific oligonucleotide probes binding to Y. ruckeri (serotype O1, biotype 2) in tissue sections and were able to demonstrate a tissue specific uptake of this bacterium (both formalin inactivated and live form). Uptake and subsequent translocation dynamics at various surfaces demonstrated different site specific propensities between the formalin inactivated and live bacterial organisms. Lateral lines, dorsal fin, epidermis and gastro-intestinal tract mucosal tissue were the primary areas where bacterial uptake was demonstrated readily after exposure. The fate of internalized bacterial organisms within the host suggested that central immune organs are involved in the final antigen processing.

Isolation of Yersinia ruckeri strain H01 from farm-raised Amur Sturgeon Acipenser schrencki in China.

Yersinia ruckeri is the causative agent of enteric redmouth disease or yersiniosis, which affects salmonids and several other species of fish. However, there are no reports on the characteristics and pathogenicity of Y. ruckeri isolated from farm-raised Amur Sturgeon Acipenser schrencki. Here, we isolated and characterized Y. ruckeri strain H01 from the diseased Amur Sturgeon in China. The phenotypic and genotypic characteristics of Y. ruckeri were observed, and its virulence was tested by examining experimentally infected sturgeons. Examination of the flagellar morphology of Y. ruckeri by transmission electron microscopy showed five to eight peritrichous flagella located on the cell body. Actively dividing cells with an obvious cell membrane were approximately 0.64 µm in diameter and between 1.7 and 2.5 µm in length. The dose that was lethal to 50% of the test fish after intraperitoneal injection was determined to be 7.2×10(6) CFU, and Y. ruckeri could be reisolated from the liver and kidneys of infected sturgeon. Antimicrobial susceptibility tests showed that H01 was susceptible to 10 antimicrobial agents. Part of the 16S rRNA sequences (563 base pair) was amplified and sequenced to study the genotypic characterization in Y. ruckeri (GenBank accession number JQ657818). The phylogenetic tree revealed H01 was clustered together with Y. ruckeri strains. Together, this study describes the isolation, characterization, and phenotypic-genotypic analysis of a Y. ruckeri strain isolated from farm-raised Amur Sturgeon. The results discovered may provide some theoretical basis for the prevention and control of yersiniosis in Amur Sturgeon.

Highly sensitive detection and quantification of the pathogen Yersinia ruckeri in fish tissues by using real-time PCR.

Yersinia ruckeri is the causative agent of enteric redmouth diseases (ERM) and one of the major bacterial pathogens causing losses in salmonid aquaculture. Since recent ERM vaccine breakdowns have been described mostly attributed to emergence of Y. ruckeri biotype 2 strains, rapid, reproducible, and sensitive methods for detection are needed. In this study, a real-time polymerase chain reaction (PCR) primer/probe set based on recombination protein A (recA) gene was designed and optimized to improve the detection of Y. ruckeri. The primer/probe set proved to have a 100 % analytical specificity and a sensitivity of 1.8 ag µl(-1), equivalent to 1.7 colony-forming units (CFU) ml(-1), for purified DNA, 3.4 CFU g(-1) for seeded liver, kidney, and spleen tissues, and 0.34 CFU/100 µl(-1) for seeded blood, respectively. The assay was highly reproducible with low variation coefficient values for intra- and inter-run experiments (2.9 % and 9.5 %, respectively). Following optimization, the assay was used to detect changes in the bacterial load during experimental infection. Rainbow trout (Onchorhynchus mykiss) were exposed to two strains of Y. ruckeri (biotype 1 and biotype 2) by intraperitoneal inoculation. Internal organs (liver, kidney, spleen) and blood were biopsied from dead fish daily for 15 days to quantify copies of pathogen DNA per gram of tissue. The findings showed the efficacy of this real-time PCR assay to quantify Y. ruckeri cells in the fish tissues and also confirmed this assay as a non-lethal method for the detection of this pathogen in blood samples.

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.

Denaturing gradient gel electrophoresis for nonlethal detection of Aeromonas salmonicida in salmonid mucus and its potential for other bacterial fish pathogens.

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA was used to nonlethally detect Aeromonas salmonicida and other bacteria in salmonid skin mucus. Mucus samples from wild spawning coho salmon (Oncorhynchus kisutch) with endemic A. salmonicida and from cultured lake trout (Salvelinus namaycush) were tested by PCR-DGGE and were compared with mucus culture on Coomassie brilliant blue agar and internal organ culture. PCR-DGGE gave a highly reproducible 4-band pattern for 9 strains of typical A. salmonicida, which was different from other Aeromonas spp. Aeromonas salmonicida presence in mucus was evident as a band that comigrated with the bottom band of the A. salmonicida 4-band pattern and was verified by sequencing. PCR-DGGE found 36 of 52 coho salmon positive for A. salmonicida, compared with 31 positive by mucus culture and 16 by organ culture. Numerous other bacteria were detected in salmonid mucus, including Pseudomonas spp., Shewanella putrefaciens, Aeromonas hydrophila and other aeromonads. However, Yersinia ruckeri was not detected in mucus from 27 lake trout, but 1 fish had a sorbitol-positive Y. ruckeri isolated from organ culture. Yersinia ruckeri seeded into a mucus sample suggested that PCR-DGGE detection of this bacterium from mucus was possible. PCR-DGGE allows nonlethal detection of A. salmonicida in mucus and differentiation of some Aeromonas spp. and has the potential to allow simultaneous detection of other pathogens present in fish mucus.