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Vibrio vulnificus is a zoonotic pathogen that can cause death by septicaemia in farmed fish (mainly eels) and humans. The zoonotic strains that have been isolated from diseased eels and humans after eel handling belong to clade E (or serovar E (SerE)), a clonal complex within the pathovar (pv.) piscis. The aim of this study was to evaluate the accuracy of MALDI-TOF mass spectrometry (MS) in the identification of SerE, using the other two main pv. piscis-serovars (SerA and SerI) from eels as controls. MALDI-TOF data were compared with known serologic and genetic data of five pv. piscis isolates or strains, and with the non pv. piscis reference strain. Based on multiple spectra analysis, we found serovar-specific peaks that were of ~3098 Da and ~ 4045 Da for SerE, of ~3085 Da and ~ 4037 Da for SerA, and of ~3085 Da and ~ 4044 Da for SerI. Therefore, our results demonstrate that MALDI-TOF can be used to identify SerE and could also help in the identification of the other serovars of the species. This means that zoonosis due to V. vulnificus could be prevented by using MALDI-TOF, as action can be taken immediately after the isolation of a possible zoonotic V. vulnificus strain., (© 2023 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Lumpfish (Cyclopterus lumpus) is a transatlantic marine fish displaying large population sizes and a high potential for dispersal and gene-flow. These features are expected to result in weak population structure. Here, we investigated population genetic structure of lumpfish throughout its natural distribution in the North Atlantic using two approaches: I) 4,393 genome wide SNPs and 95 individuals from 10 locations, and II) 139 discriminatory SNPs and 1,669 individuals from 40 locations. Both approaches identified extensive population genetic structuring with a major split between the East and West Atlantic and a distinct Baltic Sea population, as well as further differentiation of lumpfish from the English Channel, Iceland, and Greenland. The discriminatory loci displayed ~2-5 times higher divergence than the genome wide approach, revealing further evidence of local population substructures. Lumpfish from Isfjorden in Svalbard were highly distinct but resembled most fish from Greenland. The Kattegat area in the Baltic transition zone, formed a previously undescribed distinct genetic group. Also, further subdivision was detected within North America, Iceland, West Greenland, Barents Sea, and Norway. Although lumpfish have considerable potential for dispersal and gene-flow, the observed high levels of population structuring throughout the Atlantic suggests that this species may have a natal homing behavior and local populations with adaptive differences. This fine-scale population structure calls for consideration when defining management units for exploitation of lumpfish stocks and in decisions related to sourcing and moving lumpfish for cleaner fish use in salmonid aquaculture., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Jansson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), can be transmitted both horizontally and vertically and there is no available cure or prophylaxis. The control of BKD requires continuous surveillance, which is challenging in aquaculture as well as in programs for conservation and restoration of salmonid fish strains. BKD is a notifiable disease in Sweden and is monitored through the mandatory health control program using a polyclonal ELISA for detection of the Rs p57 protein in kidney. Fish must be killed for sampling, an obvious disadvantage especially regarding valuable broodfish. The present study shows that gill-/cloacal swabs collected in vivo for real-time PCR (qPCR gc ), allow a sensitive and specific detection of Rs. The sensitivity of qPCR gc was estimated to 97.8% (credible interval (ci) 93.8%-100%) compared to 98.3% (ci 92.7%-100%) and 48.8% (ci 38.8%-58.8%) of kidney samples for qPCR (qPCR k ) and ELISA (ELISA k ) respectively, by use of the Bayesian Latent Class Analysis (BLCA). Since the goal of the program is eradication of BKD the most sensitive test is preferrable. Using qPCR gc instead of ELISA k will result in a lower false negative rate and can be useful for surveillance in aquaculture and in breeding programs with valuable fish. However, a higher false positive rate warrants confirmatory lethal testing before a previously Rs negative farm is subject to restrictions., (© 2022 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Bacterial kidney disease (BKD) can be a devastating bacterial infection in salmonids, and it is present in aquaculture throughout the world. BKD is caused by the Gram-positive facultative intracellular bacterium Renibacterium salmoninarum (R. salmoninarum) that is spread both horizontally and vertically. Disease signs include external ulcerations and blisters and internal signs such as organ swelling, granulomas, petechiae and ascites. In Sweden, BKD accounts for a significant income loss in aquacultures due to expensive decontamination of the facility and increased disease susceptibility for the immunocompromised fish leading to higher mortality rates. In addition, uncontrolled spread in aquaculture may threaten the survival of wild fish populations. The aim of our study was to investigate the prevalence of R. salmoninarum in wild salmonids caught in Swedish waters where net pen farms with a recent history of BKD are present. Four rivers with at least one BKD-positive or recently BKD-positive farm were selected. In addition, we evaluated the use of environmental DNA (eDNA) for surveillance and monitoring of ongoing infections at these locations. In total, 1058 fish were sampled from four different river systems, and of them 52 (4.9%) were positive for R. salmoninarum by antigen ELISA. Surprisingly, these fish were not evenly distributed between the four river systems, but 50 were caught in the same river (Ljungan). This accounts for an alarmingly high rate of 17% R. salmoninarum-positive samples in wild salmonids in this area. This number is far above what was expected and clearly shows the risk with an open farming system as well as the importance of effective health monitoring programmes to avoid an uncontrolled spread of the disease. The use of eDNA for monitoring BKD is somewhat difficult to evaluate. Few of the water samples analysed were PCR positive for R. salmoninarum (2 of 38) and those were collected where no ELISA positive fish were identified. In addition to water, sediment samples were collected under a net pen farm that had recently slaughtered all fish due to ongoing R. salmoninarum infections. Sediment samples are more promising than water as 4 of 5 samples at one farming facility where positive for R. salmoninarum. Thus, sediment samples may be valuable for monitoring potential ongoing BKD in farms, without the need to sacrifice valuable fish., (© 2022 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

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., (© FEMS 2019.)

The pathogen Flavobacterium psychrophilum is a major problem for the expanding salmonid fish farming industry in Sweden as well as worldwide. A better understanding of the phylogeography and infection routes of F. psychrophilum outbreaks could help to improve aquaculture profitability and the welfare of farmed fish while reducing the need for antibiotics. In the present study, high-throughput genome sequencing was applied to a collection of F. psychrophilum isolates (n=38) from outbreaks on fish farms in different regions of Sweden between 1988 and 2016. Antibiotic susceptibility tests were applied to a subset of the isolates and the results correlated to the presence of genetic resistance markers. We show that F. psychrophilum clones are not regionally biased and that new clones with a higher degree of antibiotic resistance have emerged nationwide during the study period. This supports previous theories of the importance of live fish and egg trade as a route of infection. Continuous monitoring of recovered isolates by high-throughput sequencing techniques in the future could facilitate tracing of clones within and between countries, as well as the detection of emergent virulent or antibiotic-resistant clones. This article contains data hosted by Microreact.

Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD), which affects a variety of freshwater-reared salmonid species. A large-scale study was performed to investigate the genetic diversity of F. psychrophilum in the four Nordic countries: Denmark, Finland, Norway, and Sweden. Multilocus sequence typing of 560 geographically and temporally disparate F. psychrophilum isolates collected from various sources between 1983 and 2012 revealed 81 different sequence types (STs) belonging to 12 clonal complexes (CCs) and 30 singleton STs. The largest CC, CC-ST10, which represented almost exclusively isolates from rainbow trout and included the most predominant genotype, ST2, comprised 65% of all isolates examined. In Norway, with a shorter history (<10 years) of BCWD in rainbow trout, ST2 was the only isolated CC-ST10 genotype, suggesting a recent introduction of an epidemic clone. The study identified five additional CCs shared between countries and five country-specific CCs, some with apparent host specificity. Almost 80% of the singleton STs were isolated from non-rainbow trout species or the environment. The present study reveals a simultaneous presence of genetically distinct CCs in the Nordic countries and points out specific F. psychrophilum STs posing a threat to the salmonid production. The study provides a significant contribution toward mapping the genetic diversity of F. psychrophilum globally and support for the existence of an epidemic population structure where recombination is a significant driver in F. psychrophilum evolution. Evidence indicating dissemination of a putatively virulent clonal complex (CC-ST10) with commercial movement of fish or fish products is strengthened.

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), is a serious threat to salmon in aquaculture as well as to wild populations. We have developed a real-time polymerase chain reaction (PCR) for detection of Rs in kidney samples. The PCR is based on detection of unique parts of the 16S rRNA gene of Rs and DNA equivalent to 1-10 Rs genomes was detected per reaction. No cross-reactivity with other fish pathogenic or related bacteria could be demonstrated. Analysis of individual kidney samples collected from BKD classified populations identified 39.9% of the fish as positive by real-time PCR compared with 28.0% by polyclonal enzyme-linked immunosorbent assay (ELISA). The real-time PCR assay was found to be well suited for complementary use with ELISA for diagnosis of BKD, with the ability to detect clinical as well as covert Rs infections. The infection level determined by the polyclonal ELISA and by real-time PCR was significantly correlated.

The 16S rRNA genes from eight isolates of Renibacterium salmoninarum with different origins and dates of isolation were sequenced to evaluate the possibility to construct a diagnostic PCR system with target sites within this gene. The sequences were found to be identical but for one single position in one of the isolates, and two regions with an adequate number of nucleotide differences as compared to closely related species were identified. Species-specific fluorescent PCR primers complementary to these regions were constructed as well as oligonucleotides for DNA preparation by sequence capture. A mimic molecule was constructed to be used as an internal control. The PCR was specific and allowed the detection of DNA equivalent to 1-10 R. salmoninarum genomes per reaction. The DNA preparation with sequence capture and analysis by PCR with a mimic was found to be a reliable method for analysis of kidneys from fish with BKD. The amount of PCR inhibiting substances present in the tissue was reduced, and the relevant DNA was concentrated in the capture step. Furthermore, the use of the mimic molecule in the system assured that false negative results could be identified.

A cell mediated immune (CMI) response was measured in vitro to heat-killed and to paraformaldehyde fixed Renibacterium salmoninarum (Rs) in rainbow trout (Oncorhynchus mykiss) experimentally challenged with live Rs. The mitogenic response to the T lymphocyte mitogen Concanavalin A (Con A) was reduced during samplings 4 to 6 weeks after immersion, but no effect of the response to the B lymphocyte mitogen lipopolysaccharide (LPS) was detected. The subpopulation of lymphocytes, detected by the monoclonal antibody 1C2, was decreased from the 4th week to the 5th week of infection, and remained at the decreased level up to 10 weeks post immersion. The proportion of Immunoglobulin (Ig) bearing lymphocytes was not affected during the Rs infection period. The humoral antibody level to heat-stable Rs-antigens was increased up to 10 weeks after immersion but after 27 weeks was reduced to a level similar to that of the non-challenged fish. An anamnestic response was demonstrated in challenged fish, as intraperitoneal injection of heat-treated Rs bacteria into Rs challenged fish elicited a stronger humoral antibody response compared with injection into non-challenged fish.

Humoral antibodies to heat-stable antigens of Renibacterium salmoninarum (Rs) were detected by enzyme-linked immunosorbent assay (ELISA) in rainbow trout Oncorhynchus mykiss and in Atlantic salmon Salmo salar challenged by immersion. A slow antibody response was found: 3% (1/30) was positive 4 wk after immersion and 72% (26/36) was positive after 8 wk. All 30 fish sampled after 4 wk were found to be infected, as determined by bacterial culture and/or the presence of soluble antigens in the kidney. At 6, 8 and 12 wk after immersion the proportion of positives indicated by ELISA was 58%. The Rs infection was detected by cultivation in 36% of sampled fish collected on the same occasion. Elevated antibody titres to Rs were detected in samples from both Atlantic salmon (59% in 1 farm) and from rainbow trout (20% in 1 of 5 sampled farms) in naturally exposed populations all of which classified positive for bacterial kidney disease (BKD). Elevated antibody titres were detected among sampled fish from populations of rainbow trout and salmon with clinical BKD. Samples collected from farm populations of rainbow trout, salmon and brown trout Salmo trutta, exposed to Rs but without clinical BKD, were negative in the ELISA, although Rs bacteria or soluble antigens were detected at the same sampling. The antibody ELISA method cannot be recommended for general fish health monitoring purposes, but may be a valuable tool for monitoring the disease progression during controlled experiments.

Bacterial kidney disease in salmonid fish is caused by the slow-growing Gram-positive rod, Renibacterium salmoninarum. The partial sequence of 16S rRNA from R. salmoninarum was determined and compared with published bacterial 16S rRNA sequences. From this sequence information, a 30-bases-long oligonucleotide was designed and used as a specific probe for identification of R. salmoninarum in filter hybridization experiments. Strong specific hybridization signals were observed for all strains of R. salmoninarum tested. Furthermore, no cross-hybridization could be seen against 22 other bacterial species, among them other salmonid fish pathogens. The detection limit for the probe in direct filter hybridization by the dot-blot technique was 2.5 x 10(4) bacteria. It was also possible to detect R. salmoninarum in clinical samples by direct filter hybridization.

This article focuses on the detection of Renibacterium salmoninarum and Yersinia ruckeri by the peroxidase-antiperoxidase immunohistochemical technique in melanin-containing cells of fish tissues. Immunohistochemical enzyme techniques are important tools for detection of microbial antigens in tissues. Such methods may substantially aid diagnostic work, especially for organisms which are slow-growing or difficult to culture. Since detection of infectious agents in fixed postmortem material is often readily accomplished by those techniques, they may be excellent for retrospective studies. Thus, enzyme immunohistochemistry is increasingly used in work on infectious fish diseases; for example, viral diseases and bacterial infections.