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.     

Isolation of Yersinia ruckeri Bacteriophages

Eight bacteriophages effective against Yersinia ruckeri, the enteric redmouth disease bacterium, were isolated. Phage YerA41, a tailed icosahedral virus isolated from sewage enrichments, lysed 34 of 35 strains of Y. ruckeri serovar I, but was inactive against 15 strains belonging to three other serological groups. Six other phages lysed strains of serovars II, V, and I′, a subgroup of serovar I. YerL62, a phage obtained by mitomycin C induction, was specific for one of three serovar V strains. These bacteriophages, particularly YerA41, have potential value for fish disease diagnostic work.     

Evidence that Yersinia ruckeri possesses a high affinity iron uptake system

Abstract: This work represents the first evidence of the presence of an iron uptake system siderophore mediated in the bacterial fish pathogen Yersinia ruckeri . A group of 20 strains representative of this species, with different serotype and origin were examined. All of them were able to grow at high concentrations (from 0.7 to 1.1 mM) of the iron chelator EDDA. Although the Y. ruckeri isolates failed to cross-feed the indicator strains for enterobactin and aerobactin production, the chemical tests revealed the presence in the culture supernatants of phenolate siderophores. At least three outer membrane proteins were induced in iron limiting conditions. All the strains showed a similar pattern of induced membrane proteins regardless their serotype or origin, which suggests a similarity in the iron uptake system. This system could have an important role in the pathogenicity of Y. ruckeri for fish.     

Evidence that Yersinia ruckeri possesses a high affinity iron uptake system.

This work represents the first evidence of the presence of an iron uptake system siderophore mediated in the bacterial fish pathogen Yersinia ruckeri. A group of 20 strains representative of this species, with different serotype and origin were examined. All of them were able to grow at high concentrations (from 0.7 to 1.1 mM) of the iron chelator EDDA. Although the Y. ruckeri isolates failed to cross-feed the indicator strains for enterobactin and aerobactin production, the chemical tests revealed the presence in the culture supernatants of phenolate siderophores. At least three outer membrane proteins were induced in iron limiting conditions. All the strains showed a similar pattern of induced membrane proteins regardless their serotype or origin, which suggests a similarity in the iron uptake system. This system could have an important role in the pathogeneicity of Y. ruckeri for fish.     

The physiological effects of salmon lice infection on post-smolt of Atlantic salmon

The physiological effects of salmon lice infections on post-smolt of Atlantic salmon were examined by experimentally infecting hatchery reared post-smolts with infective copepodids. Even at high infection intensities, ranging from 30–250 lice per fish, early chalimus stages did not have severe, physiological effects on the fish. There was a sudden increase in fish mortality after the appearance of preadult I stages. Infected fish were then suffering due to lesions and osmoregulatory failure. Plasma chloride level increased significantly and total protein, albumin and haematocrit decreased significantly in infected compared to uninfected fish. All infected fish became moribund before adult lice appeared. Infection intensities above 30 salmon lice larvae per fish thus appear to cause death of Atlantic salmon post-smolt soon after the lice reach their pre-adult stage.     

An epizootic in chinook salmon (Oncorhynchus tshawytscha) caused by a sorbitol-positive serovar 2 strain of Yersinia ruckeri

Enteric redmouth disease is described in chinook salmon (Oncorhynchus tshawytscha) at a state hatchery in Sand Ridge, Illinois. Biochemical, isoenzyme, and serological data indicated that the epizootic was caused by a sorbitol-fermenting Serovar 2 strain of Yersinia ruckeri. In laboratory experiments the isolate was pathogenic for both brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar).     

The Fish Pathogen Yersinia ruckeri Produces Holomycin and Uses an RNA Methyltransferase for Self-resistance

Holomycin and its derivatives belong to a class of broad-spectrum antibacterial natural products containing a rare dithiolopyrrolone heterobicyclic scaffold. The antibacterial mechanism of dithiolopyrrolone compounds has been attributed to the inhibition of bacterial RNA polymerase activities, although the exact mode of action has not been established in vitro. Some dithiopyrrolone derivatives display potent anticancer activities. Recently the biosynthetic gene cluster of holomycin has been identified and characterized in Streptomyces clavuligerus. Here we report that the fish pathogen Yersinia ruckeri is a holomycin producer, as evidenced through genome mining, chemical isolation, and structural elucidation as well as genetic manipulation. We also identified a unique regulatory gene hom15 at one end of the gene cluster encoding a cold-shock-like protein that likely regulates the production of holomycin in low cultivation temperatures. Inactivation of hom15 resulted in a significant loss of holomycin production. Finally, gene disruption of an RNA methyltransferase gene hom12 resulted in the sensitivity of the mutant toward holomycin. A complementation experiment of hom12 restored the resistance against holomycin. Although the wild-type Escherichia coli BL21(DE3) Gold is susceptible to holomycin, the mutant harboring hom12 showed tolerance toward holomycin. High resolution liquid chromatography (LC)-ESI/MS analysis of digested RNA fragments demonstrated that the wild-type Y. ruckeri and E. coli harboring hom12 contain a methylated RNA fragment, whereas the mutated Y. ruckeri and the wild-type E. coli only contain normal non-methylated RNA fragments. Taken together, our results strongly suggest that this putative RNA methyltransferase Hom12 is the self-resistance protein that methylates the RNA of Y. ruckeri to reduce the cytotoxic effect of holomycin during holomycin production.     

Effects of salmon lice infection and salmon lice protection on fjord migrating Atlantic salmon and brown trout post-smolts

Effects of artificial salmon lice infection and pharmaceutical salmon lice prophylaxis on survival and rate of progression of Atlantic salmon (n = 72) and brown trout post-smolts (n = 72) during their fjord migration, were studied by telemetry. The infected groups were artificially exposed to infective salmon lice larvae in the laboratory immediately before release in the inner part of the fjord to simulate a naturally high infection pressure. Groups of infected Atlantic salmon (n = 20) and brown trout (n = 12) were also retained in the hatchery to control the infection intensity and lice development during the study period. Neither salmon lice infection nor pharmaceutical prophylaxis had any effects on survival and rate of progression of fjord migrating Atlantic salmon post-smolts compared to control fish. Atlantic salmon spent on average only 151.2 h (maximum 207.3 h) in passing the 80 km fjord system and had, thus, entered the ocean when the more pathogenic pre-adult and adult lice stages developed. The brown trout, in comparison to Atlantic salmon, remained to a larger extent than Atlantic salmon in the inner part of the fjord system. No effect of salmon lice infection, or protection, was found in brown trout during the first weeks of their fjord migration. Brown trout will, to a larger extent than Atlantic salmon, stay in the fjord areas when salmon lice infections reach the more pathogenic pre-adult and adult stages. In contrast to Atlantic salmon, they will thereby possess the practical capability of returning to freshwater when encountering severe salmon lice attacks.     

Dietary vitamin E and the response of rainbow trout, Oncorhynchus mykiss (Walbaum), to infection with Yersinia ruckeri

Rainbow trout fed diets containing 7, 86 or 806 mg vitamin E kg⁻¹ for 22 weeks were exposed to virulent Yersinia ruckeri by bath or injection. Mortalities were always least among those fed the highest concentration of vitamin E but serum antibody production was not affected by vitamin E levels.     

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 is the etiologic agent of enteric redmouth disease, a disease of salmonid fish species that is found worldwide in areas where salmonid fish species are farmed (3, 6, 18, 20). Vaccines for enteric redmouth disease have been used successfully for nearly 3 decades and consist of immersion-applied, killed whole-cell preparations of motile serovar 1 Y. ruckeri strains (22). Recently though, outbreaks have been reported in vaccinated fish at trout farms in the United Kingdom (2), Spain (9), and the United States (1). The Y. ruckeri strains isolated from these outbreaks are uniformly atypical serovar 1 isolates lacking both flagellar motility and secreted lipase activity. These variants have been classified as Y. ruckeri biotype 2 (BT2) and are believed to have a reduced sensitivity to immersion vaccination (2). The objective of this study was to obtain a better understanding of the emergence of BT2 Y. ruckeri by identifying genetic elements necessary for expression of the Y. ruckeri flagellum and determining the role that the flagellum plays in virulence by using a rainbow trout infection model.     

Genetic changes in an Atlantic salmon population resulting from escaped juvenile farm salmon

The study was undertaken on three adjacent rivers in NW Ireland, on one of which an Atlantic salmon Salmo salar freshwater juvenile rearing unit is situated. Two markers which distinguished farm and wild populations were used. An Ava II-B RFLP in the ND1 region of mtDNA was at a frequency of 0.58 in the farm strain but absent in the wild populations. Allele E at minisatellite locus Ssa- A45/2/l was at a frequency of 0.91 in farm samples, but at a maximum of 0.41 in the populations in the two rivers adjacent to the one with the juvenile rearing unit. The farm strain showed a significant reduction in mean heterozygosity (0.281 ± 0.057), over three minisatellite loci examined, compared to wild samples (0.532 ± 0.063). The occurrence of farm genotypes and the independent occurrence of mtDNA and minisatellite markers in several parr samples from the river indicated that escaped juvenile salmon completed their life cycle, bred and interbred with native fish, upon their return to the river. Escaped fish homed accurately, as adults, to the site of escape, i.e. the area adjacent to the hatchery outflow in the upstream part of the river. Breeding of males in the lower part of the river was also indicated but this could have been due to mature male parr which had moved downstream. The return of adults of farm origin to the river to breed was indicated by the presence of the Ava II-B haplotype in adults netted in the estuary.     

High Gyrodactylus salaris infection rate in triploid Atlantic salmon Salmo salar

We describe an unusually high infection rate of Gyrodactylus salaris Malmberg in juvenile Atlantic salmon Salmo salar L. of Baltic Sea origin, which are generally believed to be more resistant to G. salaris than East Atlantic salmon populations. Based on analyses of mitochondrial (complete cytochrome oxidase 1 [CO1] gene, 1548 bp) and nuclear (ADNAM1, 435 bp; internal transcribed spacer [ITS] rDNA region, 1232 bp) DNA fragments, the closest relatives of the characterized Estonian G. salaris strain were parasites found off the Swedish west coast and in Raasakka hatchery, Iijoki (Baltic Sea, Finland). Analyses of 14 microsatellite loci of the host S. salarrevealed that approximately 40% of studied fish were triploids. We subsequently identified triploid Atlantic salmon of Baltic origin as more susceptible to G. salaris infection than their diploid counterparts, possibly due to compromised complement-dependent immune pathways in triploid salmon. This is in accordance with earlier studies that have shown elevated susceptibility of triploids to various viral or bacterial pathogens, and represents one of the first reports of increased susceptibility of triploid salmonid fish to an ectoparasite. However, further experimental work is needed to determine whether triploid Atlantic salmon is generally more susceptible to G. salaris compared to their diploid counterparts, irrespective of the particular triploidization method and population of origin.