Detection of Yersinia ruckeri in rainbow trout blood by use of the polymerase chain reaction

We evaluated a polymerase chain reaction (PCR) method for detecting Yersinia ruckeri, the bacterial pathogen causing enteric redmouth disease (ERM), in blood of rainbow trout Oncorhynchus mykiss. Identification of the PCR product was confirmed by Southern blot hybridization with a 32P-labeled oligonucleotide probe matching a sequence within the small subunit ribosomal RNA gene of Y. ruckeri. Following a 1 h immersion of rainbow trout in water with 4.5 x 10(6) colony-forming units of Y. ruckeri l(-1), the PCR was positive for all blood samples from 1 h (first sample) to 5 d and was negative from 9 to 30 d (last sample). Fish in this experiment did not show signs of disease, probably because they had been vaccinated against Y. ruckeri. To test this method with naturally infected fish, 42 rainbow trout from hatcheries were examined. Four of these fish had clinical signs of ERM and were infected with Y. ruckeri based on bacteriological culture. The PCR method detected Y. ruckeri in blood, intestine, liver, and trunk kidney from the 4 fish with ERM and from 5 additional rainbow trout that were bacteriologically negative for Y. ruckeri. Three of 5 rainbow trout from streams receiving effluent from hatcheries were positive for Y. ruckeri when tested with PCR, although there was no growth of Y. ruckeri on culture plates inoculated with the same samples. Samples were successfully stored for 1 wk in lysis buffer at 25 degrees C. This study demonstrated that a non-lethal blood sample can be used with PCR to detect Y. ruckeri.     

Occurrence and phenotypic characterization of Yersinia ruckeri strains with biofilm-forming capacity in a rainbow trout farm

The presence of Yersinia ruckeri in a French fish farm was investigated. Y. ruckeri was isolated mainly from algae and sediment samples rather than from water. Twenty-two Y. ruckeri isolates were obtained, and three strains were distinguished by enterobacterial repetitive intergenic consensus PCR amplification. These strains were able to adhere to solid supports. This characteristic was correlated with flagellum-mediated motility. Killing experiments showed that sessile cells were more resistant to oxolinic acid than their planktonic counterparts. Our results demonstrate that surface colonization of fish farm tanks by Y. ruckeri biofilms is a potential source of recurrent infection for extended periods of time.     

Association between plasma antibody response and protection in rainbow trout Oncorhynchus mykiss immersion vaccinated against Yersinia ruckeri

A key hallmark of the vertebrate adaptive immune system is the generation of antigen-specific antibodies from B cells. Fish are the most primitive gnathostomes (jawed vertebrates) possessing an adaptive immune system. Vaccination of rainbow trout against enteric redmouth disease (ERM) by immersion in Yersinia ruckeri bacterin confers a high degree of protection to the fish. The immune mechanisms responsible for protection may comprise both cellular and humoral elements but the role of specific immunoglobulins in this system has been questioned and not previously described. The present study demonstrates significant increase in plasma antibody titers following immersion vaccination and significantly reduced mortality during Y. ruckeri challenge.Rainbow trout were immersion-vaccinated, using either a commercial ERM vaccine (AquaVac™ ERM vet) or an experimental Y. ruckeri bacterin. Half of the trout vaccinated with AquaVac™ ERM vet received an oral booster (AquaVac™ ERM Oral vet). Sub-groups of the fish from each group were subsequently exposed to 1x10⁹ CFU Y. ruckeri/ml either eight or twenty-six weeks post vaccination (wpv). All vaccinated groups showed 0% mortality when challenged, which was highly significant compared to the non-vaccinated controls (40 and 28% mortality eight and twenty-six weeks post vaccination (wpv), respectively) (P<0.0001). Plasma samples from all groups of vaccinated fish were taken 0, 4, 8, 12, 16 and 26 wpv. and Y. ruckeri specific IgM antibody levels were measured with ELISA. A significant increase in titers was recorded in vaccinated fish, which also showed a reduced bacteremia during challenge. In vitro plasma studies showed a significantly increased bactericidal effect of fresh plasma from vaccinated fish indicating that plasma proteins may play a role in protection of vaccinated rainbow trout.     

Temperature-dependent protection against Ichthyophthirius multifiliis following immunisation of rainbow trout using live theronts

Rainbow trout Oncorhynchus mykiss Walbaum, 1792 fingerlings were vaccinated by intraperitoneal (i.p.) injection using live theronts of the skin parasitic ciliate Ichthyophthirius multifiliis Fouquet, 1876 at 2 temperatures (12 and 20 degrees C), and protection against challenge infections was subsequently evaluated by bath exposure to live theronts. Vaccination conferred a relative protection (evaluated as the decrease in the number of established theronts) at 12 degrees C and almost complete immunity at 20 degrees C. Significantly increased immobilisation titers (using plasma immobilisation of live theronts) were found in immunised fish at Week 2 and 4 post-vaccination. Lysozyme activity of plasma from vaccinated fish increased from Week 1 to 4. Both immobilisation titers and lysozyme activity were significantly higher at 20 degrees C. This study demonstrated that live theronts are good candidates for an antigen source for development of effective vaccines against white spot disease in this fish host, and further indicated that the protection of rainbow trout against I. multifiliis infection is highly temperature dependent and may be associated with both adaptive and innate response mechanisms.     

Recovery of a new biogroup of Yersinia ruckeri from diseased rainbow trout (Oncorhynchus mykiss,Walbaum)

Cultures of a new biogroup of Yersinia ruckeri, the causal agent of enteric redmouth (ERM), were recovered in England from diseased rainbow trout (Oncorhynchus mykiss, Walbaum), which had been previously vaccinated with a commercial ERM vaccine. The bacterial isolates were confirmed as Y. ruckeri by the results of sequencing the 16S rRNA, but differed from the characteristics of the taxon by positivity for the Voges Proskauer reaction and a general lack of motility, and could not be equated with any of the existing serovars. Cultures were pathogenic in laboratory-based infectivity experiments with 100% mortalities occurring in juvenile rainbow trout (average weight = 10 g) within 4-days of intraperitoneal or intramuscular injection with 10(5) cells/fish. Protection against disease was achieved using a formalin-inactivated whole vaccine prepared against a representative isolate.     

Temperature-dependent expression of sarcolemmal K(+) currents in rainbow trout atrial and ventricular myocytes

Temperature has a strong influence on the excitability and the contractility of the ectothermic heart that can be alleviated in some species by temperature acclimation. The molecular mechanisms involved in the temperature-induced improvement of cardiac contractility and excitability are, however, still poorly known. The present study examines the role of sarcolemmal K(+) currents from rainbow trout (Oncorhynchus mykiss) cardiac myocytes after thermal acclimation. The two major K(+) conductances of the rainbow trout cardiac myocytes were identified as the Ba(2+)-sensitive background inward rectifier current (I(K1)) and the E-4031-sensitive delayed rectifier current (I(Kr)). In atrial cells, the density of I(K1) is very low and the density of I(Kr) is remarkably high. The opposite is true for ventricular cells. Acclimation to cold (4 degrees C) modified the two K(+) currents in opposite ways. Acclimation to cold increases the density of I(Kr) and depresses the density of I(K1). These changes in repolarizing K(+) currents alter the shape of the action potential, which is much shorter in cold-acclimated than warm-acclimated (17 degrees C) trout. These results provide the first concrete evidence that K(+) channels of trout cardiac myocytes are adaptable units that provide means to regulate cardiac excitability and contractility as a function of temperature.     

Temperature-dependent genotypic selection and embryonic survival of rainbow trout

The survival of rainbow trout (Salmo gairdneri) with different developmental rates and genotypes at eight polymorphic loci was compared in two experiments. The embryos were reared at temperatures colder (5 and 8°C) and warmer (12°C) than normally experienced by the strain (9.5°C). Embryo survival in five different hatching groups (representing the sequential order of hatching) was compared at 8 and 12°C. Embryos in the center of the hatching distribution (groups 2, 3, and 4) showed a greater survival to yolk sac resorption than those in the extremes (groups 1 and 5). These differences are significant in the embryos reared at 12°C in both experiments. Embryo survival from hatching to yolk sac resorption was poorer at 12 compared to 8°C. In the one experiment where it was examined, significantly fewer embryos hatched at 5 and 12 than at 8°C. Significant deviations from genotypic expectations (based upon parental allele frequencies) were detected in the progeny (23 of 34 cases). In contrast, few deviations from Hardy-Weinberg expectations were detected (5 of 34 cases) when progeny allele frequencies are used. Few significant differences in genotype frequencies were detected among the progeny reared at the different temperatures. This suggests that nonequal contributions of parental gametes rather than differential selection of genotypes account for the deviations from expectations in the progeny.This research was supported by National Science Foundation Grant BSR-8300039 awarded to Dr. Fred W. Allendorf. Moira M. Ferguson was supported by a postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada.     

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.     

Time-dependent changes in protein expression in rainbow trout muscle following hypoxia

Adaptation to hypoxia is a complex process, and individual proteins will be up- or down-regulated in order to address the main challenges at any given time. To investigate the dynamics of the adaptation, rainbow trout (Oncorhynchus mykiss) was exposed to 30% of normal oxygen tension for 1, 2, 5 and 24 h respectively, after which muscle samples were taken. The successful investigation of numerous proteins in a single study was achieved by selectively separating the sarcoplasmic proteins using 2-DE. In total 46 protein spots were identified as changing in abundance in response to hypoxia using one-way ANOVA and multivariate data analysis. Proteins of interest were subsequently identified by MS/MS following tryptic digestion. The observed regulation following hypoxia in skeletal muscle was determined to be time specific, as only a limited number of proteins were regulated in response to more than one time point. The cellular response to hypoxia included regulation of proteins involved in maintaining iron homeostasis, energy levels and muscle structure. In conclusion, this proteome-based study presents a comprehensive investigation of the expression profiles of numerous proteins at four different time points. This increases our understanding of timed changes in protein expression in rainbow trout muscle following hypoxia.     

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.     

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.