Rainbow Trout Serum Research Articles

Influence of lactic-acid bacteria feed supplementation on free amino acid levels in serum and feces of rainbow trout (Oncorhynchus mykiss, Walbaum 1792)

IntroductionThe influence of intestinal microbiota, particularly lactic-acid bacteria, on digestion and nutrient metabolic activities has been extensively studied. In this research, we investigated the effects of supplementing the feed of rainbow trout (O. mykiss) with P. acidilactici or L. lactis probiotics. A Control group without probiotic supplementation was also included. The study aimed to evaluate growth, feed conversion indices, free amino acid levels in serum and feces, and the relative gene expression of amino acid solute carrier transporters (SLC).MethodsRainbow trout weighing 73.9±10 g were fed with a basal commercial diet supplemented with mono-strains of P. acidilactici or L. lactis at a dosage of 5 x 108 CFU per gram of feed for 60 days. The trout were reared in 100-liter tanks with independent closed recirculation systems, with a water replacement rate of 80% of the total volume daily and a controlled temperature of 15 ± 0.3 °C. The proximate composition of the basal diet consisted of crude protein (49.8%), fat (32.1%), fiber (1.31%), ash (13%), and moisture (8.3%) on a dry matter basis. The diet was provided at a rate of 2% of bodyweight per day.ResultsNo significant differences (p > 0.05) were observed in growth and feed conversion indices between the experimental groups. However, significant differences in free amino acid levels in feces and serum samples (p ≤ 0.05) were observed. The P. acidilactici group exhibited significantly higher levels of glutamate, lysine, proline, and tyrosine in feces samples, and higher levels of arginine, iso/leucine, phenylalanine, proline, serine, threonine, and valine in serum samples compared to the Control group. Additionally, the supplemented probiotic groups showed significant downregulation (p ≤ 0.05) of theslc6a19 and slc7a9 genes, which encode transporters for neutral and cationic amino acids.DiscussionThe autochthonous rainbow trout strain, P. acidilactici, demonstrated more pronounced effects on amino acid levels in feces and serum compared to the non-autochthonous L. lactis strain. However, the increased amino acid levels, particularly in the serum, resulting from P. acidilactici supplementation, did not lead to improved trout growth or feed conversion. Nonetheless, lactic-acid bacteria supplementation induced measurable metabolic effects, as evidenced by elevated levels of specific amino acids in the serum and feces.Future research should focus on evaluating these probiotic effects using specialized diets and considering the observed metabolic effects in this study.

Impact of dietary glutamate and glycine on growth and nutrient utilization in rainbow trout (Oncorhynchus mykiss)

A feeding trial was conducted to test the effect of dietary glutamate and glycine supplementation on growth potential, nutritional utilization and digestion, and energy metabolism of rainbow trout (Oncorhynchus mykiss). Four isonitrogenous and isolipidic diets (2 × 2 factorial design) were formulated with glutamate (Glu) and glycine (Gly) as the main factors. The protein source consisted mainly of black soldier fly larvae meal (40% inclusion in all diets) for its low-level content of glutamate and glycine. Triplicate groups of rainbow trout (30 fish/tank with mean initial body weight of 87.5 g ± 0.9) were restrictively fed by hand twice a day for 6 weeks. Dietary Glu and Gly supplementation did not affect feed intake, specific growth rate, or the feed conversion ratio. Whole body protein, lipid, amino acid and fatty acid composition were also unaffected by the supplementation of Glu and Gly in the diets. Dietary Gly supplementation increased the apparent digestibility of amino acids. Furthermore, supplementation with Gly, increased the concentration of serine, glycine, tryptophane, tyrosine and citrulline, while supplementation with Glu in the diets increased the concentration of hydroxy-proline and β-alanine in the serum of rainbow trout. Both dietary Glu and Gly supplementation improved the digestibility of the fatty acids. The expression of genes involved in the hepatic bile acid synthesis (e.g., apical sodium dependent bile acid transporter and organic solute transporter) were affected by supplementation of Gly in the diets. In general, this study showed that dietary supplementation with Gly and Glu improved the nutrient digestibility in rainbow trout.

Investigation of Some Biochemical Parameters and Mineral Values in the Blood of Rainbow trout Growing in Dam and Sea water

In this study, it was aimed to investigate the effect of the difference in the breeding environment (pH, temperature, salinity, oxygen content and saturation) of rainbow trout grown in dam and sea water on the level of some minerals (calcium, magnesium, zinc, iron) and biochemical parameters in the serum. The study material consisted of 20 rainbow trout (Oncorhynchus mykiss) weighing about 800-1000 gr in the dam water of Samsun (Derbent Dam) and the Black Sea water (Yakakent). Blood samples were taken from both groups on the same day in December and the pH, temperature, oxygen content and saturation level of the water were determined by YSK oxygen meter, and salinity was determined by refractometry. Total protein (TP), albumin (Alb), cholesterol (TK), glucose (Glu), urea, creatinine (Cre), uric acid (UA), Aspartate transaminase (AST), Alanine Aminotransferase (ALT), triglyceride (TG) Calcium, magnesium, iron and zinc and ALT, AST enzyme activities were determined by spectrophotometric method in an autoanalyzer. It was observed that the salinity, oxygen and saturation levels of the sea water were higher than those of the dam water, and the pH and temperature were close to each other. It was determined that TP, Alb, Alb/Glo, TK, TG, UA and Ca levels in rainbow trout raised in the dam were significantly higher than those raised in the sea (P<0.05). Glu, Cre, Mg and Fe levels were found to be higher in the blood serum of rainbow trout (P<0.05). Zn, urea, AST and ALT levels of rainbow trout raised in sea and dam were similar. In our study where the same fish species were provided with the same feeding conditions, it was observed that the differences in salinity and dissolved oxygen levels of fish farming waters caused changes in some mineral and blood parameter levels in fish. It was concluded that determining the supplementation of the missing minerals in fish according to the salinity and dissolved oxygen level in the environment may be important for the development of the fish.

Association of Gut Microbiota With Metabolism in Rainbow Trout Under Acute Heat Stress.

Global warming is one of the most common environmental challenges faced by cold-water fish farming. Heat stress seriously affects the feeding, growth, immunity, and disease resistance of fish. These changes are closely related to the destruction of intestinal barrier function, the change of intestinal microbiota, and metabolic dysfunction. However, the causal relationship between the phenotypic effects of heat stress as well as intestinal and metabolic functions of fish is unknown. In the current study, the optimal growth temperature (16°C) of rainbow trout was used as the control group, while the fish treated at 22.5°C, 23.5°C, and 24.5°C for 24 h, respectively, were the treatment groups. The 16S rRNA gene sequencing analysis showed that with the increase in temperature, the relative abundance and diversity of intestinal microbiota decreased significantly, while the number of Mycoplasma, Firmicutes, and Tenericutes increased significantly. Non-targeted metabolomics analysis by liquid chromatography-mass spectrometry analysis and correlation analysis showed that the changes of metabolites related to amino acids, vitamins, and short-chain fatty acids in serum of rainbow trout under acute heat stress were strongly correlated with the decrease of relative abundance of various intestinal microbiota, especially Morganella, Enterobacter, Lactobacillus, Lawsonia, and Cloacibacterium. In addition, we also found that acute heat stress seriously affected the intestinal structure and barrier function, and also caused the pathological damage of epithelial cells. These results indicate that the gut microbiome of acute heat-stressed rainbow trout could mediate metabolite transfer through the gut barrier by affecting its integrity. Significant changes in gut morphology, permeability, antioxidant capacity, and pro-inflammatory cytokine levels were observed. Therefore, it is necessary to explore the changes of intestinal microbiota under heat stress to help understand the regulatory mechanism of heat stress and protect the intestinal health of rainbow trout from the negative effects of rising water temperature.

Analysis of indicators of lysozyme activity of blood serum of rainbow trout (Oncorhynchus mykiss) when using the drug "Smartbiotic"

In this study, to activate the humoral link of nonspecific resistance of the fish organism, as well as to stimulate growth and development, prevent infectious and invasive diseases, the drug "SmartBiotic" was used, which contains humic acids. The research was carried out on the basis of LLC "Lososevo - whitefish nursery" Kingisepsky district, Leningrad region, the village of Velkota, which is the largest nursery in the city of Kingisepp for breeding rainbow trout. For the study, three groups of underyearlings of the rainbow trout (Oncorhynchus mykiss) were formed. One group served as control, and the other two were experimental. The first experimental group of young of the year received the drug at a concentration of 0.15%, and the second experimental group - at a dose of 0.25%. In the control group of rainbow trout, SmartBiotic was not used. All three groups were formed using the analog pairing method and raised in three different pools. Blood serum samples were taken twice: before giving postbiotic and one and a half months after using the drug. In the blood serum of fish in all three groups, the lysozyme activity of blood serum was determined according to the generally accepted method.

Dietary supplementation of lemon verbena (Aloysia citrodora) improved immunity, immune-related genes expression and antioxidant enzymes in rainbow trout (Oncorrhyncus mykiss)

The use of veterinary drugs for the treatments of fish diseases has often health and environmental side effects and thus alternative more sustainable strategies are needed. In this study we have explored the effect of dietary administration of lemon verbena (Aloysia citrodora) leaves powder on growth, immune parameters and antioxidant enzyme activity of rainbow trout (Oncorrhyncus myskiss). The results show that adding lemon verbena (LV) leaves powder (0.5, 1 and 2%), as feed supplement does not affect significantly rainbow trout growth, but increases the levels of some immune parameters. All enriched-diets induced an increase in the levels of lysozyme activity and total immunoglobulin in rainbow trout serum (P < 0.05), whilst 1 and 2% enriched-diets increased lysozyme and immunoglobulin in skin mucus. Expression of interleukin-1β (IL-1β) was enhanced in all fish supplemented with LV leaves powder (0.5, 1 and 2%) whilst only the diet with the highest dose (2%) induced a significantly higher expression of IL-8 and tumour necrosis factor (TNF-α). Fish fed with LV leaves powder at 1 and 2% had significantly lower expression of transforming growth factor (TGF-β, P < 0.05). Furthermore, all enriched-diets significantly increased the activity of super oxide dismutase (SOD, P < 0.05) and diets containing 1 and 2% of LV leaves powder also increased activity of glutathione-S-transferase (GST) and glutathione peroxidase (GPx). In conclusion, LV leaves powder administered as feed supplement at 2% enhances the expression of some immune-related genes (IL-1β, IL-8 and TNF- α), rises the levels of lysozyme and total immunoglobulin in serum and skin mucus and increases the activity of antioxidant enzymes (SOD, GST and GPx) in rainbow trout.

Characterization of a novel Yersinia ruckeri serotype O1-specific bacteriophage with virulence-neutralizing activity.

A lytic bacteriophage (φNC10) specific to serotype O1 Yersinia ruckeri has been identified and evaluated as a model to assess the potential use of bacteriophages and their products for disease control in aquaculture. Electron microscopy of purified φNC10 revealed a virion particle with a small (70nm) polyhedral head and short tail. φNC10 infected only serotype O1 strains of Y.ruckeri and failed to bind a defined Y.ruckeri mutant strain lacking O1 lipopolysaccharides (O1-LPS), suggesting that φNC10 uses O1-LPS as its receptor. In addition, spontaneous φNC10-resistant mutants of Y.ruckeri exhibited defects in O1-LPS production and were sensitive to rainbow trout serum. Purified φNC10 displayed a polysaccharide depolymerase activity capable of degrading Y.ruckeri O1-LPS and thereby sensitizing Y.ruckeri to the bactericidal effects of rainbow trout serum. The φNC10-associated polysaccharide depolymerase activity also reduced the ability of Y.ruckeri cells to cause mortality following intraperitoneal injection into rainbow trout. These data demonstrate a potential utility of φNC10 and its associated polysaccharide depolymerase activity for Y.ruckeri disease prevention.

Potential Role of Rainbow Trout Erythrocytes as Mediators in the Immune Response Induced by a DNA Vaccine in Fish.

In recent years, fish nucleated red blood cells (RBCs) have been implicated in the response against viral infections. We have demonstrated that rainbow trout RBCs can express the antigen encoded by a DNA vaccine against viral hemorrhagic septicemia virus (VHSV) and mount an immune response to the antigen in vitro. In this manuscript, we show, for the first time, the role of RBCs in the immune response triggered by DNA immunization of rainbow trout with glycoprotein G of VHSV (GVHSV). Transcriptomic and proteomic profiles of RBCs revealed genes and proteins involved in antigen processing and presentation of exogenous peptide antigen via MHC class I, the Fc receptor signaling pathway, the autophagy pathway, and the activation of the innate immune response, among others. On the other hand, GVHSV-transfected RBCs induce specific antibodies against VHSV in the serum of rainbow trout which shows that RBCs expressing a DNA vaccine are able to elicit a humoral response. These results open a new direction in the research of vaccination strategies for fish since rainbow trout RBCs actively participate in the innate and adaptive immune response in DNA vaccination. Based on our findings, we suggest the use of RBCs as target cells or carriers for the future design of novel vaccine strategies.

Bacteriophage‐based cocktail modulates selected immunological parameters and post‐challenge survival of rainbow trout (Oncorhynchus mykiss)

Recently, a rapid increase in the resistance of pathogenic bacteria to antibiotics and chemotherapeutics admitted for use in aquaculture has been observed. This happens especially often in intensive breeding. The use of drugs in closed circuits is problematic because it can damage biological filters. Therefore, in recent years, there has been a growing interest in natural methods of combating pathogens. These include bacteriophages. The aim of the study was to determine the safety of the new BAFADOR® bacteriophage-based preparation, its effect on selected immunological parameters and the effectiveness of prophylactic and therapeutic use after experimental infections with pathogenic bacteria Aeromonas hydrophila and Pseudomonas fluorescens. The use of BAFADOR® increased the activity of lysozyme, total protein level and immunoglobulin level. The level of ceruloplasmin in the rainbow trout serum remained unchanged regardless of the route of administration of the preparation. Potential killing activity and metabolic activity of spleen phagocytes and proliferation of pronephros lymphocytes were higher compared to the control group. Both therapeutic and prophylactic application of the preparation after mixed experimental infection of A.hydrophila and P.fluorescens limited the mortality of rainbow trout.

In vitro investigations on extracellular proteins secreted by Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome

BackgroundProteases produced by many microorganisms, including oomycetes, are crucial for their growth and development. They may also play a critical role in disease manifestation. Epizootic ulcerative syndrome is one of the most destructive fish diseases known. It is caused by the oomycete Aphanomyces invadans and leads to mass mortalities of cultured and wild fish in many countries. The areas of concern are Australia, China, Japan, South and Southeast Asian countries and the USA. Extracellular proteases produced by this oomycete are believed to trigger EUS pathogenesis in fish. To address this activity, we collected the extracellular products (ECP) of A. invadans and identified the secreted proteins using SDS-PAGE and mass spectrometery. A. invadans was cultivated in liquid Glucose-Peptone-Yeats media. The culture media was ultra-filtered through 10 kDa filters and analysed using SDS-PAGE. Three prominent protein bands from the SDS gel were excised and identified by mass spectrometery. Furthermore, we assessed their proteolytic effect on casein and immunoglobulin M (IgM) of rainbow trout (Oncorhynchus mykiss) and giant gourami (Osphronemus goramy). Antiprotease activity of the fish serum was also investigated.ResultsBLASTp analysis revealed that the prominent secreted proteins were proteases, mainly of the serine and cysteine types. Proteins containing fascin-like domain and bromodomain were also identified. We could demonstrate that the secreted proteases showed proteolytic activity against the casein and the IgM of both fish species. The anti-protease activity experiment showed that the percent inhibition of the common carp serum was 94.2% while that of rainbow trout and giant gourami serum was 7.7 and 12.9%, respectively.ConclusionsThe identified proteases, especially serine proteases, could be the potential virulence factors in A. invadans and, hence, are candidates for further functional and host–pathogen interaction studies. The role of identified structural proteins in A. invadans also needs to be investigated further.

Hemato - Immunological and biochemical parameters, skin antibacterial activity, and survival in rainbow trout (Oncorhynchus mykiss) following the diet supplemented with Mentha piperita against Yersinia ruckeri.

This study was aimed to assess the potential effects of Mentha piperita on the hemato - immunological and biochemical parameters, skin antibacterial activity and protection against Yersinia ruckeri infection in rainbow trout Oncorhynchus mykiss. Fish were divided into 4 groups before being fed diets supplemented with 0, 1, 2 and 3% of Mentha piperita (MP) plant extract for 8 weeks. Dose-dependent increases immune (both in skin mucus and blood serum) and hematological parameters (number of red and white cells, hematocrit and hemoglobin contents), as well as in respiratory burst activity, total protein, albumin, and neutrophil levels in fish fed supplemented diets compared to the control fish. Furthermore, dietary MP plant extract supplements have no significant effect on blood biochemical parameters and enzymatic activities of liver determined in serum of rainbow trout. After 8 weeks the cessation of feeding with MP plant extract, survival rates of 54.4%, 63.6% and 75.2% were recorded in groups which received 1, 2 and 3% of MP plant extract of feed, respectively, compared to 34.6% survivals in the control. This study underlying several positive effects of dietary administration of MP plant extract to farmed fish.

Comparative analysis of innate immune responses to Streptococcus phocae strains in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss).

Streptococcus phocae subsp. salmonis is a Gram-positive bacterium that causes mortality only in Atlantic salmon (Salmo salar) farmed in Chile, even when this species is co-cultured with rainbow trout (Oncorhynchus mykiss). This susceptibility could be determined by innate immune response components and their responses to bacterial infection. This fish pathogen shares subspecies status with Streptococcus phocae subsp. phocae isolated from seals. The present study compared innate immune system mechanisms in Atlantic salmon and rainbow trout when challenged with different S.phocae, including two isolates from Atlantic salmon (LM-08-Sp and LM-13-Sp) and two from seal (ATCC 51973(T) and P23). Streptococcus phocae growth was evaluated in the mucus and serum of both species, with rainbow trout samples evidencing inhibitory effects. Lysozyme activity supported this observation, with significantly higher (p<0.01) expression in rainbow trout serum and mucus as compared to Atlantic salmon. No differences were found in phagocytic capacity between fish species when stimulated with ATCC 51973(T) and P23. Against all S.phocae strains, rainbow trout and Atlantic salmon showed up to two-fold increased bactericidal activity, and rainbow trout demonstrated up to three-fold greater reactive oxygen species production in macrophages. In conclusion, the non-specific humoral and cellular barriers of Atlantic salmon were immunologically insufficient against S.phocae subsp. salmonis, thereby facilitating streptococcosis. Moreover, the more robust response of rainbow trout to S.phocae could not be attributed to any specific component of the innate immune system, but was rather the consequence of a combined response by the evaluated components.

Comparison of plasma and tissue disposition of enrofloxacin in rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio) after a single oral administration

The aim of the study was to investigate the serum and tissue disposition of enrofloxacin and its active metabolite ciprofloxacin in rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio) after a single oral administration at a dose of 10 mg kg–1. Concentrations of enrofloxacin in the serum of rainbow trout showed high variability with two peaks at the third and 24th hour after administration. The highest concentrations were found in the liver. The curves of liver levels showed similar changes to the respective serum samples. In the muscles, enrofloxacin concentrations were also higher compared with the respective serum samples. Ciprofloxacin concentrations were lower and showed smaller variations in all investigated tissues. The serum and tissue concentrations of enrofloxacin and ciprofloxacin in common carp showed two peaks, with the first Cmax at the third hour after drug administration as in rainbow trout. Concentrations of both investigated substances were higher in the liver than in the serum. The differences in common carp were less pronounced in comparison with rainbow trout. Relatively high levels of both substances were found in the muscles. Seven days after treatment enrofloxacin concentrations in the serum and tissues were within the therapeutic levels for most of the sensitive microorganisms in trout. Lower concentrations of its metabolite ciprofloxacin were found in the investigated tissues at the last sampling point. Lower levels of both substances were found in carp.

VACCINATION of Atlantic salmon leads to long-lasting higher levels of serum immunoglobulin and possible skewed ratios of two distinct IgM isotypes

Primary and secondary antibody responses in blood of teleost fish are mainly IgM mediated, as they lack an IgG homolog and a class switch mechanism. Thus, the major serum immunoglobulin is tetrameric IgM. A unique antibody class in teleosts; named IgT, appears to be specialized for mucosal immunity and is present in low concentrations in serum. A third class; IgD was recently detected in serum of rainbow trout, but is less abundant than IgT. In the present study, relative quantification of total IgM showed that Atlantic salmon (Salmosalar) which had received an oil-adjuvanted commercial vaccine maintained about 2-fold higher levels of IgM; 14 and 17 months after intraperitoneal injection, in comparison to unvaccinated fish kept in the same cage. Similar results were obtained by ELISA (serum IgM) and reverse transcription real time PCR (spleen mRNA). Analyses conducted in our lab have shown that several salmonid species possess two slightly different IgM isotypes as a result of ancestral tetraploidy. In Atlantic salmon, two distinct IgM subpopulations (A and B) can be separated by anion exchange chromatography. The IgM-B tetramer exhibits a higher degree of disulfide cross-linking than IgM-A, presumably due to an extra cysteine near the C-terminus of the heavy chain. The typical A/B ratio is approximately 40/60. Anion exchange elution profiles of serum IgM from vaccinated fish indicate that prolonged triggering of the immune system can lead to a skewed ratio of IgM-A/IgM-B. In the context of recent results from rainbow trout (Onchorhynchusmykiss), showing that high affinity antibodies are more highly polymerized and have a longer half life time, Atlantic salmon is an interesting model to elucidate these aspects further since tools are available to distinguish IgM-A and IgM- B in this species.

Species-dependent effects of the phenolic herbicide ioxynil with potential thyroid hormone disrupting activity: modulation of its cellular uptake and activity by interaction with serum thyroid hormone-binding proteins

Ioxynil, a phenolic herbicide, is known to exert thyroid hormone (TH) disrupting activity by interfering with TH-binding to plasma proteins and a step of the cellular TH-signaling pathway in restricted animal species. However, comparative studies are still lacking on the TH disruption. We investigated the interaction of [125I]ioxynil with serum proteins from rainbow trout, bullfrog, chicken, pig, rat, and mouse, using native polyacrylamide gel electrophoresis. Candidate ioxynil-binding proteins, which included lipoproteins, albumin and transthyretin (TTR), differed among the vertebrates tested. Rainbow trout and bullfrog tadpole serum had the lowest binding activity for ioxynil, whereas the eutherian serum had the highest binding activity. The cellular uptake of, and response to, ioxynil were suppressed by rat serum greater than by tadpole serum. The cellular uptake of [125I]ioxynil competed strongly with phenols with a single ring, but not with THs. Our results suggested that ioxynil interferes with TH homeostasis in plasma and with a step of cellular TH-signaling pathway other than TH-uptake system, in a species-specific manner.

Chitosan nanoparticle to carry vitamin C through the gastrointestinal tract and induce the non-specific immunity system of rainbow trout (Oncorhynchus mykiss)

Chitosan is a polysaccharide that has gained interest in recent to encapsulate active compounds due to its non-toxicity, biodegradability, biocompatibility and immunity system induction. The chitosan nanoparticle was prepared by adding vitamin C to tripolyphosphate (TPP) solution and then blending it stepwise to the chitosan solution under stirring. Nanoparticle characterizations were analyzed by scanning electron microscopy (SEM) and Zetasizer. The release profile of vitamin C into the gastrointestinal and serum of rainbow trout (Oncorhynchus mykiss) was portrayed and its effect on the immunity system of the fish was determined as well. The results showed that the chitosan nanoparticle had spherical shape; positive charge with particle size of 185nm and in vivo controlled release until 48h. Lysozyme and complement contents in the fish serum increased as long as they were provoked by the nanoparticle. This nanoparticle is a promising technique to boost immunity system of fish.

The yctCBA Operon of Yersinia ruckeri , Involved in In Vivo Citrate Uptake, Is Not Required for Virulence

A three-gene operon, named yctCBA (Yersinia citrate transporter), induced by citrate and repressed by glucose was identified from a previously selected in vivo-induced (ivi) clone in the fish pathogen Yersinia ruckeri. Interestingly, despite being an ivi clone, the drastic growth reduction of the yctC mutant in the presence of citrate, and the relatively high content of this compound in rainbow trout serum, the operon was not required for virulence.

Comparative susceptibility of Atlantic salmon and rainbow trout to Yersinia ruckeri: Relationship to O antigen serotype and resistance to serum killing

A study was undertaken to compare the virulence and serum killing resistance properties of Atlantic salmon and rainbow trout Yersinia ruckeri isolates. Five isolates, covering heat-stable O-antigen O1, O2 and O5 serotypes, were tested for virulence towards fry and juveniles of both species by experimental bath challenge. The sensitivity of 15 diverse isolates to non-immune salmon and rainbow trout serum was also examined. All five isolates caused significant mortality in salmon fry. Serotype O1 isolate 06059 caused the highest mortality in salmon (74% and 70% in fry and juveniles, respectively). Isolate 06041, a typical ERM-causing serotype O1 UK rainbow trout strain, caused mortalities in both rainbow trout and salmon. None of the salmon isolates caused any mortalities in 150–250g rainbow trout, and only serotype O2 isolate 06060 caused any significant mortality (10%) in rainbow trout fry. Disease progression and severity was affected by water temperature. Mortality in salmon caused by the isolates 06059 and 05094 was much higher at 16°C (74% and 33%, respectively) than at 12°C (30 and 4% respectively). Virulent rainbow trout isolates were generally resistant to sera from both species, whereas salmon isolates varied in their serum sensitivity. Convalescent serum from salmon and rainbow trout that had been infected by serotype O1 isolates mediated effective classical pathway complement killing of serotype O1 and O5 isolates that were resistant to normal sera. Overall, strains recovered from infected salmon possess a wider range of phenotypic properties (relative virulence, O serotype and possession of serum-resistance factors), compared to ERM-causing rainbow trout isolates.

In vitro markers for virulence in Yersinia ruckeri

In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non-virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE-214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non-virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin-D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non-virulent strains were generally serum sensitive.

Leptin mRNA and Protein Immunoreactivity in Adipose Tissue and Liver of Rainbow Trout (Oncorhynchus mykiss) and Immunohistochemical Localization in Liver

Leptin is an important modulator of energy balance and metabolism in mammals, but for evolutionary older vertebrates like fish, the first reports on leptin expression were only recently characterized and the functional role scarcely. In this study, we demonstrated leptin immunoreactivity in liver tissue of rainbow trout (Oncorhynchus mykiss) by immunohistochemistry using three different polyclonal antibodies against mammalian leptin. Immunoreactivity was observed in hepatocytes and also in parts of the biliary system. Using Western blot, we detected an immunoreactive band of about 16 kDa in serum and visceral adipose tissue (AT) of rainbow trout. The presence of leptin in fish AT has been doubted in other studies. Besides the immunoreactivity, leptin mRNA was detected in trout AT albeit not in all animals sampled. Our observations add further evidence to the concept of AT being a source of leptin in trouts. Moreover, the cellular localization of leptin immunoreactivity in liver opens up new vistas for understanding the functional role of leptin in teleosts.