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.     

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.     

Construction of a virulent, green fluorescent protein-tagged Yersinia ruckeri and detection in trout tissues after intraperitoneal and immersion challenge

A green fluorescent protein (GFP) expressing strain of Yersinia ruckeri was created by the transposition of a Tn10-GFP-kan cassette into the genome of Y. ruckeri Strain YRNC10. The derivative, YRNC10-gfp, was highly GFP fluorescent, retained the gfp-km marker in the absence of kanamycin selection, and exhibited in vitro growth kinetics similar to those of the wild type strain. YRNC10-gfp colonized and caused mortality in immersion and intraperitoneally challenged rainbow trout Oncorhynchus mykiss, although it was modestly attenuated compared to the wild type strain. The distribution and location of YRNC10-gfp in infected fish was visualized by epifluorescence microscopy. Abundant extracellular bacteria and a small number of intracellular bacteria were observed in the kidney, spleen and peripheral blood. To determine the percentage of trout cells containing intracellular bacteria, GFP fluorescence was measured by flow cytometry. A small population of GFP positive leukocytes was detected in peripheral blood (1.6%), spleen (1.1%) and anterior kidney (0.4%) tissues. In summary, this is the first report of the construction of a virulent, GFP-tagged Y. ruckeri, which may be a useful model for detecting and imaging the interactions between an aquatic pathogen and the natural salmonid host.     

Lack of uptake of Renibacterium salmoninarum by gill epithelia of rainbow trout

The role of rainbow trout ( Oncorhynchus mykiss , Walbaum) gill cells in uptake of the salmonid pathogen Renibacterium salmoninarum was examined using in vitro , whole animal and isolated perfused head models. Uptake of the pathogen was observed only when dissected gill tissue was exposed to the live pathogen. In contrast, live cells of Yersinia ruckeri interacted with the gill epithelia in the isolated perfused head model, confirming the findings of previous studies with whole animal and in vitro systems. These results are discussed in relation to the role of gill tissue in bacterial kidney disease (BKD) pathogenesis and in antigen trapping.     

Temperature-dependent expression of immune-relevant genes in rainbow trout following Yersinia ruckeri vaccination

The gene expression of immune-relevant genes in rainbow trout Oncorhynchus mykiss following vaccination with a bacterin of Yersinia ruckeri, a bacterial pathogen causing enteric red mouth disease (ERM), was investigated at 5, 15, and 25 degrees C. Rainbow trout were immunized by i.p. injection of a water-based Y. ruckeri (serotype O1) bacterin, and gene expression profiles were compared to control groups injected with phosphate buffered saline (PBS). Blood and tissue samples (spleen and head kidney) were taken for subsequent analysis using solid phase enzyme-linked immunosorbent assay (ELISA) and real-time PCR, respectively. The up-regulation of cytokine genes was generally faster and higher at high water temperature, with major expression at 25 degrees C. The proinflammatory cytokine interleukin (IL)-1beta and interferon (IFN)-gamma were significantly up-regulated in all immunized groups, whereas the cytokine IL-10 was only up-regulated in fish kept at 15 and 25 degrees C. The gene encoding the C5a (anaphylatoxin) receptor was expressed at a significantly increased level in both head kidney and spleen of immunized fish. The secreted immunoglobulin M (IgM)-encoding gene was significantly up-regulated in the head kidney of immunized trout reared at 25 degrees C, and a positive correlation (r = 0.663) was found between gene expression of secreted IgM in the head kidney and Y. ruckeri-specific antibodies in plasma measured by ELISA. However, no regulation of the teleost specific immunoglobulin T (IgT), which was generally expressed at a much lower level than IgM, could be detected. The study indicated that expression of both innate and specific adaptive immune-response genes are highly temperature-dependent in rainbow trout.     

Dietary administration of beta-mercapto-ethanol treated Saccharomyces cerevisiae enhanced the growth, innate immune response and disease resistance of the rainbow trout, Oncorhynchus mykiss

The effects of dietary whole cell yeast (Saccharomyces cerevisiae), n-3 HUFA-enriched yeast and treated yeast cells with beta-mercapto-ethanol (2ME) on immunity, growth performance and disease resistance to Yersinia ruckeri were investigated in Oncorhynchus mykiss. During 30 days, juvenile rainbow trout were fed diets supplemented with different forms of yeast at 5 × 10(7) CFU g(-1) or a control diet. After the feeding trial, remaining fish of each treatment were challenged by pathogenic Yersinia ruckeri and kept under observation for 14 days to record clinical signs and daily mortality rate. Yeast supplementation in all treatment groups significantly promoted the growth performance compared to control group. A significantly increase was also observed in immune responses in juvenile fish fed 2ME-treated yeast diet. More ever, the lowest fish mortality was obtained in this treatment group. The present results show that a diet supplemented with 2ME-treated yeast stimulates the immune system and growth of juvenile rainbow trout thus enhancing their resistance against Y. ruckeri.     

The effect of sublethal endrin exposure on rainbow trout, Salmo gairdneri Richardson. I. Evaluation of serum cortisol concentrations and immune responsiveness

Two-hundred-and-forty rainbow trout, Salmo gairdneri , were exposed for 30 days to sublethal concentrations of the pesticide endrin. On day 30, ten fish from each treatment group were sacrificed and examined for the ability of peritoneal macrophages to phagocytize latex beads. The remaining fish were immunized with 10 μg of Yersinia ruckeri O-antigen, and exposure to endrin continued. The migration inhibition factor assay (MIF), plaque forming cell assay (PFC), and serum agglutination titres (SAG) were performed 2, 14, and 30 days post-antigen inoculation. Serum was collected from all fish for serum cortisol concentrations. Endrin exposure had no effect on the phagocytic ability of peritoneal macrophages. However, the MIF, PFC and SAG responses were significantly reduced from control values. Serum cortisol concentrations were found to be significantly elevated in endrin-exposed fish. Serum cortisol concentrations were found to be significantly higher on days 44 and 60 (192 and 194 ng ml⁻¹, respectively) when compared to days 30 and 32 (159 and 141 ng ml⁻¹, respectively). Cortisol values for days 30 and 32 did not differ significantly, nor did those of days 44 and 60. The relationship between elevated serum cortisol concentrations and endrin exposure on the immune response is discussed.     

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.     

Induction of antibody-producing cells in rainbow trout, Salmo gairdneri Richardson; by flush exposure

Splenic antibody-producing cells were produced by rainbow trout that had been exposed to O-antigens extracted from Yersinia ruckeri and Aeromonas salmonicida by adding the concentrated antigen preparation directly into the water of the tank holding the fish for a flush exposure. This method was compared with the proven techniques of exposure: intraperitoneal injection or a 2 minute immersion of the fish in the antigen preparation. Dosage experiments showed that the production of antibody-producing cells was induced by the immersion of trout for 2 minutes in water with 5.0 μg/ml-1 (or more) with the Y. ruckeri O-antigen, or 500 μg ml-1 (or more) of the A. salmonicida O-antigen. Similar differences were evident when the respective antigens were added directly to the water.     

Induction of vitellogenin production in male tilapia (Oreochromis mossambicus) by commercial fish diets

Mozambique tilapia, (Oreochromis mossambicus), are a euryhaline teleost and an important biological model species. Captive male tilapia frequently have high levels of the estrogen-induced yolk precursor protein vitellogenin (Vg), a common indicator of exposure to estrogenic compounds. Sex steroids are found in commercial fish diets, but relatively few studies have examined the relationship between commercial diets and Vg production. In a fasting experiment to ascertain a dietary role in male Vg production, plasma Vg was reduced to negligible levels after 2 weeks of fasting, while no change in estrogen receptor (ER) expression was seen. When male tilapia were fed a squid-based diet that replaced the commercial trout diet, plasma Vg was reduced to undetectable levels over 40 days, concomitant with significant reductions in hepatic expression of Vgs A, B, and C, and ERβ, compared with control fish fed commercial trout diet. Female tilapia fed the squid-based for 20 days had no change in these parameters. When male tilapia were fed a defined, soy-based diet, plasma Vg reduced to 20% of levels in fish given either commercial trout diet or a defined, fishmeal-based diet. Overall, results from these studies suggest that estrogens in a commercial trout diet induce vitellogenin production by increasing expression of Vg, but not ER genes in male tilapia.     

Estrogenic effect of propylparaben (propylhydroxybenzoate) in rainbow trout Oncorhynchus mykiss after exposure via food and water

The estrogenic effect of propylparaben was investigated in a rainbow trout Oncorhynchus mykiss test system. Propylparaben was administered orally to sexually immature rainbow trout every second day for up to 10 days in doses between 7 and 1830 mg kg(-1) 2 d(-1) and in the water at 50 and 225 microg l(-1) for 12 days. Plasma vitellogenin was measured before and during the exposures and the concentrations of propylparaben in liver and muscle were determined at the end of experiments. Increases in average plasma vitellogenin levels were seen at oral exposure to 33 mg propylparabenkg(-1) 2 d(-1); the most sensitive fish responded to 7 mg kg(-1). The ED(50) values for increase in vitellogenin synthesis were 35, 31 and 22 mg kg(-1) 2 d(-1) at day 3, 6 and 11, respectively. Exposure to 225 microg propylparabenl(-1) increased vitellogenin synthesis, but exposure to 50 microg l(-1) did not. Propylparaben showed little tendency to bioaccumulation in rainbow trout; less than 1 per thousand of the total amount of propylparaben administered orally at 1830 mg kg(-1) 2 d(-1) over the 10-d experimental period was retained in muscle and liver 24 h after the end of the experiment. Exposure to 225 microg propylparabenl(-1) for 12 d led to concentrations of 6700 and 870 microg propylparabenkg(-1) liver and muscle, respectively. Half lives for propylparaben were 8.6 h in liver and 1.5 h in muscle.     

Invasion and initial replication of ultraviolet irradiated waterborne infective stages of Myxobolus cerebralis results in immunity to whirling disease in rainbow trout

Myxobolus cerebralis is a microscopic metazoan parasite (Phylum Myxozoa: Myxosporea) associated with salmonid whirling disease. There are currently no vaccines to minimise the serious negative economical and ecological impacts of whirling disease among populations of salmonid fish worldwide. UV irradiation has been shown to effectively inactivate the waterborne infective stages or triactinomyxons of M. cerbralis in experimental and hatchery settings but the mechanisms by which the parasite is compromised are unknown. Treatments of triactinomyxons with UV irradiation at doses from 10 to 80 mJ/cm(2) either prevented (20-80 mJ/cm(2)) or significantly inhibited (10 mJ/cm(2)) completion of the parasite life cycle in experimentally exposed juvenile rainbow trout (Oncorhynchus mykiss). However, even the highest doses of UV irradiation examined (80 mJ/cm(2)) did not prevent key steps in the initiation of parasite infection, including attachment and penetration of the epidermis of juvenile rainbow trout as demonstrated by scanning electron and light microscopy. Furthermore, replication of UV-treated parasites within the first 24h following invasion of the caudal fin was suggested by the detection of concentrations of parasite DNA by quantitative PCR comparable to that among fish exposed to an equal concentration of untreated triactinomyxons. Subsequent development of parasites treated with an 80 mJ/cm(2) dose of UV irradiation however, was impaired as demonstrated by the decline and then lack of detection of parasite DNA; a trend beginning at 10 days and continuing thereafter until the end of the study at 46 days post parasite exposure. Treatments of triactinomyxons with a lower dose of UV irradiation (20 mJ/cm(2)) resulted in a more prolonged survival with parasite DNA detected, although at very low concentrations, in fish up to 49 days post parasite exposure. The successful invasion but only short-term survival of parasites treated with UV in rainbow trout resulted in a protective response to challenges with fully infective triactinomyxons. Prior treatments of juvenile rainbow trout with UV-treated triactinomyxons (10 and 20 mJ/cm(2)) resulted in a reduced prevalence of infection and significantly lower concentrations of cranial myxospores (two direct measures of the severity of whirling disease) compared with trout receiving no prior treatments when assessed 5 months post parasite exposure to fully infective triactinomyxons.     

Bacterial-binding activity and plasma concentration of ladderlectin in rainbow trout (Oncorhynchus mykiss)

Soluble, defense lectins bind conserved microbial patterns leading to pathogen opsonization, enhanced phagocytosis and activation of complement. These immune functions, however, vary widely among individuals due to genetic and acquired differences affecting binding capacity or plasma concentration. Most evidence for the defensive function of soluble lectins is based on mammals, but several functionally homologous, but less well-characterized, lectins have been identified in fish. In this study, we compared binding of rainbow trout plasma ladderlectin to relevant, intact bacterial targets. A polyclonal antiserum raised against a synthetic peptide identical to the 20 N-terminal amino acids of the reduced 16 kDa rainbow trout ladderlectin subunit was used to detect plasma ladderlectin in immunoblots and indirect enzyme-linked immunosorbent assay (ELISA). Ladderlectin binding to Aeromonas salmonicida subsp. salmonicida, Aeromonas hydrophila, Yersinia ruckeri and Pseudomonas sp. was detected by PAGE and immunoblots of saccharide elutions from intact bacteria incubated in the presence of normal trout plasma. Although plasma concentrations of immunoreactive ladderlectin were low in the majority of trout, significant (P < 0.0001) variation between individual fish was observed in two separate populations. In addition, one population demonstrated a subset of individuals whose ladderlectin levels were approximately seven-fold higher than the population median. These findings indicate that rainbow trout have variable amounts of plasma ladderlectin capable of binding to the surfaces of several relevant bacterial targets.     

Whirling disease: host specificity and interaction between the actinosporean stage of Myxobolus cerebralis and rainbow trout Oncorhynchus mykiss

Scanning electron microscopic studies were conducted on rainbow trout Oncorhynchus mykiss in the first 60 min after their exposure to the triactinomyxon spores of Myxobolus cerebralis. The results demonstrated that as early as 1 min post exposure the whole process, from the attachment of the triactinomyxon spores to the complete penetration of their sporoplasm germs, had occurred. The triactinomyxon spores sought out the secretory openings of mucous cells of the epidermis, the respiratory epithelium and the buccal cavity of trout and used them as portals of entry. Exposure experiments of the triactinomyxon spores of M. cerebralis to non-salmonid fish, such as goldfish Carassius auratus, carp Cyprinus carpio, nose Chondrostoma nasus, medaka Oryzias latipes, guppy Poecilia reticulata and also the amphibian tadpole Rana pipiens as well as to rainbow trout fry indicated a specificity for salmonids. Attempts to activate the triactinomyxon spores by exposure to mucus prepared from cyprinid and salmonid fish showed no significant differences from those conducted in tap water. The results suggest that the simultaneous presence of both mechano- and chemotactic stimuli was required for finding the salmonid fish host.     

Susceptibility of two strains of rainbow trout (one with suspected resistance to whirling disease) to Myxobolus cerebralis infection

The susceptibility of 2 strains of rainbow trout Oncorhynchus mykiss, 1 from North America (TL) and 1 from Germany (GR), to Myxobolus cerebralis (the cause of salmonid whirling disease) was assessed following exposure to the infectious stages (triactinomyxons). Two laboratory experiments were conducted with age-matched rainbow trout of each strain. At the beginning of the study, the 2 trout strains were aged ca. 570 degree-days in Expt 1, and ca. 999 degree-days in Expt 2. In both experiments, replicate groups of each trout strain were exposed to 10, 100, 1000 or 10000 triactinomyxons (TAMs) fish(-1) for 2 h. The fish were then held in aquaria receiving 15 degrees C well-water. Severity of infection was evaluated 5 mo after exposure by presence of clinical signs (whirling and/or black tail), prevalence of infection, severity of microscopic lesions, and spore counts. Clinical signs of whirling disease were evident only in the younger fish exposed in Expt 1: These occurred first among TL rainbow trout at the highest dose at 6 to 7 wk post exposure and then 2 wk later in fish at the 1000 TAMs dose. Black tail was also observed among GR rainbow trout at the 10000 TAMs dose only, but in fewer fish. The prevalence of infection, spore numbers, and severity of microscopic lesions due to M. cerebralis among GR rainbow trout were less at all doses compared to TL rainbow trout. Risk of infection analyses showed that TL rainbow trout were more prone to infection at the lower doses than GR trout. Mean spore counts were consistently (10- to 100-fold) less in GR than TL trout at doses of 1000 TAMs or lower. Microscopic lesions increased with increasing dose in both strains of rainbow trout. The mechanisms underlying the greater resistance of the GR strain to M. cerebralis infections are unknown, but are under investigation as part of a long-term project to determine the basis for resistance and susceptibility of salmonid fishes to whirling disease.     

The effect of sublethal endrin exposure on rainbow trout, Salmo gairdneri Richardson. II. The effect of altering serum cortisol concentrations on the immune response

The effect of altering serum cortisol concentrations on the immune response was elucidated in endrin- and non-endrin-exposed rainbow trout, Salmo gairdneri. Fish were immunized with 10 μg of Yersinia ruckeri O-antigen following 30 days of treatment. The migration inhibition factor assay (MIF), plaque-forming cell assay (PFC) and serum agglutination titres (SAG) were performed 2, 14 and 30 days post-antigen inoculation. Endrin exposure was continued subsequent to antigen inoculation. Control fish were fed 20 and 35 mg kg⁻¹ body weight day⁻¹ of cortisol and metyrapone, respectively. Endrin-exposed fish received 35 mg kg⁻¹ body weight day⁻¹ of metyrapone in their diet. Control fish receiving cortisol had significantly reduced MIF, PFC and SAG responses. The MIF response was completely restored in endrin-exposed fish receiving dietary metyrapone. The PFC response and SAG titres were partially restored, 61 and 69% respectively, in endrin-exposed fish receiving metyrapone. The results indicate that elevated serum cortisol concentration obtained in endrin-exposed fish has a central role in repression of the immune response.