Protect Rainbow Trout Research Articles

Oral immunization with attenuated Salmonella Typhimurium as a carrier of DNA vaccine against infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) is a serious pathogen in the salmonid aquaculture industry and leads to economic losses in the world. This study aimed to develop a new oral DNA vaccine designed to protect rainbow trout against infection by IHNV. Fish were administered via the oral route by the attenuated Salmonella enterica serovar Typhimurium as a carrier of pcDNA3.1-IHNG (glycoprotein (G)) plasmid for 7 days and finally, fish were challenged by 105pfuml-1 IHNV. The results revealed that the antigen gene was identified in different tissues of rainbow trout at 15-, 30-, and 45 days post-vaccination (dpv). Also, the recombinant vaccine elicited both an innate and specific immune response, resulting in a significant upregulation of the expression levels of ifn-1, mx-1, vig-1, igm, and igt. In addition, serum levels of neutralizing antibodies were observed to be elevated in the vaccinated fish, in contrast to the unvaccinated fish, following 30 dpv. Compared to trout that received empty S. Typhimurium, notable differences in cumulative percentage mortality were evident among the vaccinated fish. The relative percent survival (RPS) was recorded at 58.2% for the group that received oral vaccine, while the group that received empty S. Typhimurium exhibited an RPS of 18.2%. Therefore, our results showed that this bacterial vector can be a candidate carrier for pcDNA3.1-IHNG plasmid that may be employed to confer protection to rainbow trout against IHNV.

Behavioral, haematological, and physiological effects of oxygen-rich spray after simulated catch and release in rainbow trout (Oncorhynchus mykiss).

This study investigated the impact of an oxygen-rich spray on rainbow trout subjected to short-term air exposure episodes. These episodes can be due to sampling procedures or catch-and-release (C&R) practices, focusing on behavioral, haematological, and physiological responses. For this, 12 rainbow trout were divided into two groups: control and oxygen-rich sprayed fish. The fish were chased for 3 min and exposed to air for 6 s, and blood and behavior parameters were assessed. Simulated C&R resulted in partial physiological reflex impairment across groups. However, sprayed fish exhibited significant differences during recovery in head complex reflex and faster tendencies to regain orientation compared to control. Haematological analyses revealed that the treated group displayed reduced erythrocyte swelling and maintenance of red blood cell (RBC) counts, indicating reduced need for compensatory responses to hypoxia. Meanwhile, stress-related indicators, including cortisol, glucose, and lactate, remained unaffected by the treatment, suggesting no interference of spraying with the fish ability to launch a healthy stress response. No adverse effects were observed on skin surfaces or gills after an exploratory analysis. Overall, the oxygen-rich spray exhibited favorable effects, indicating potential benefits in protecting rainbow trout during C&R practices or sampling episodes. Moreover, there is a possible wider application of this methodology to benefit other species of fish subjected to stressors such as aquaculture, research, and fisheries management.

Bilayer coatings for extension of the shelf life of fish fillets: Incorporating seaweed sulfated polysaccharides in chitosan-alginate LbL structures.

The aim of this study was to develop a new active coating of layer-by-layer (LbL) structure composed of alginate (as polyanions) and chitosan (as a polycation) containing sulfated polysaccharide (fucoidan) from Sargassum angustifolium, to protect rainbow trout fillets during refrigerated storage. Chitosan and alginate do not combine with each other as a homogeneous solution, so they are suitable for multilayer coatings. The results demonstrated that coating samples with chitosan and fucoidan significantly improved the quality of fish fillets and extended their shelf life from 6 to 16 days. The chemical values (TBARS and TVB-N) and bacterial growth (total viable count (TVC), total psychrophilic count (PTC), and lactic acid bacteria (LAB)) indicated lower levels in the LbL coating samples containing fucoidan compared to the alginate and control samples. Among the different coating samples, the LbL coating with fucoidan (AChF1) exhibited lower weight loss, improved chromaticity (L*, a*, and b*), and minimal changes in mechanical and sensory evaluations. Based on the findings, AChF1 was the most effective treatment for increasing the shelf life of rainbow trout fillets during refrigerated storage. Therefore, it has potential applications in the food packaging industry.

Limited Probiotic Effect of Enterococcus gallinarum L1, Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3 to Protect Rainbow Trout against Saprolegniosis.

Previous studies have demonstrated that the strains Enterococcus gallinarum L1, Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3 are probiotics against vibriosis or lactococosis in sea bass or rainbow trout. In this study, the utility of these bacterial strains in the control of saprolegniosis was evaluated. For this purpose, both in vitro inhibition studies and competition for binding sites against Saprolegnia parasitica and in vivo tests with experimentally infected rainbow trout were carried out. In the in vitro tests, the three isolates showed inhibitory activity upon mycelium growth and cyst germination and reduced the adhesion of cysts to cutaneous mucus; however, this effect depended on the number of bacteria used and the incubation time. In the in vivo test, the bacteria were administered orally at 108 CFU g-1 in the feed or at 106 CFU ml-1 in the tank water for 14 days. None of the three bacteria showed protection against S. parasitica infection either through water or feed, and the cumulative mortality reached 100% within 14 days post infection. The obtained results show that the use of an effective probiotic against a certain disease in a host may not be effective against another pathogen or in another host and that the results obtained in vitro may not always predict the effects when used in vivo.

Protection of rainbow trout (Oncorhynchus mykiss) against VHSV genotype Ia and IHNV by immunization with VHSV genotype IVa backbone-based single-cycle viruses

To evaluate the protective effect of viral hemorrhagic septicemia virus genotype IVa (VHSV IVa) genome-based single-cycle viruses against VHSV genotype Ia (VHSV Ia) and infectious hematopoietic necrosis virus (IHNV) in rainbow trout, three kinds of single-cycle VHSVs were rescued using reverse genetic technology: i) rVHSV-IaGΔTM-IVaG containing the transmembrane and cytoplasmic region-deleted G protein (GΔTM) of VHSV Ia instead of VHSV IVa full G gene ORF and having VHSV IVa G proteins on the envelope; ii) rVHSV-IaGΔTM-IaG containing VHSV Ia GΔTM instead of VHSV IVa full G gene ORF and having VHSV Ia G proteins on the envelope; iii) rVHSV-IaGΔTM-ihnvGΔTM-IVaG containing not only VHSV Ia GΔTM instead of full G gene but also IHNV GΔTM instead of NV gene and having VHSV IVa G proteins on the envelope. Rainbow trout immunized with rVHSV-IaGΔTM-IaG and rVHSV-IaGΔTM-IVaG showed significantly higher serum antibody titers against both VHSV Ia and VHSV IVa, and showed no mortality against VHSV Ia infection, while fish in the control groups showed 100% mortalities. Fish immunized with rVHSV-IaGΔTM-ihnvGΔTM-IVaG showed significantly higher serum antibody titers against VHSV IVa, VHSV Ia, and IHNV compared to fish in the control group. Immunization with rVHSV-IaGΔTM-ihnvGΔTM-IVaG induced significantly higher protection against not only VHSV Ia but also IHNV. These results suggest that the present single-cycle rVHSV-based system can be used as a platform to produce combined vaccines that can protect fish from multiple pathogenic species. However, the mechanism of the high protection against IHNV despite comparatively low antibody titer remains to be investigated.

Efficacy of DNA Vaccines in Protecting Rainbow Trout against VHS and IHN under Intensive Farming Conditions.

Despite the negative impact of viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) on European rainbow trout farming, no vaccines are commercially available in Europe. DNA vaccines are protective under experimental conditions, but testing under intensive farming conditions remains uninvestigated. Two DNA vaccines encoding the glycoproteins (G) of recent Italian VHSV and IHNV isolates were developed and tested for potency and safety under experimental conditions. Subsequently, a field vaccination trial was initiated at a disease-free hatchery. The fish were injected intramuscularly with either the VHS DNA vaccine or with a mix of VHS and IHN DNA vaccines at a dose of 1 µg/vaccine/fish, or with PBS. At 60 days post-vaccination, fish were moved to a VHSV and IHNV infected facility. Mortality started 7 days later, initially due to VHS. After 3 months, IHN became the dominant cause of disease. Accordingly, both DNA vaccinated groups displayed lower losses compared to the PBS group during the first three months, while the VHS/IHN vaccinated group subsequently had the lowest mortality. A later outbreak of ERM caused equal disease in all groups. The trial confirmed the DNA vaccines to be safe and efficient in reducing the impact of VHS and IHN in farmed rainbow trout.

Long-Term Protection Elicited by an Inactivated Vaccine Supplemented with a Water-Based Adjuvant against Infectious Hematopoietic Necrosis Virus in Rainbow Trout (Oncorhynchus mykiss).

Previous inactivated vaccines against infectious hematopoietic necrosis (IHN) usually had a strong early immune protective effect but failed to provide long-term protection in rainbow trout (Oncorhynchus mykiss). To find a method for stabilizing the desired protective effect of IHN vaccines, we assessed the immune enhancement effect of four adjuvants on formaldehyde inactivated vaccine for IHN at 60 days postvaccination (dpv). The efficacy of a two-dose vaccination with the candidate adjuvant-formaldehyde inactivated vaccine for IHN was evaluated in terms of early protection and long-term protection (30 to 285 dpv). Neutralizing antibody titers were also measured at each time point. The Montanide GEL 02 PR (Gel 02) adjuvant significantly enhanced the immune protection provided by the IHN inactivated vaccine, whereas the immune-boosting effect of the other tested adjuvants lacked statistical significance. Both tested Gel 02-adjuvanted IHN inactivated vaccine dosages had a strong immune protection effect within 2 months postvaccination, with a relative percent of survival (RPS) of 89.01% to 100%, and the higher dosage provided complete protection at 204 dpv and a RPS of 60.79% on 285 dpv by reducing viral titers in rainbow trout. The neutralizing antibodies were observed only in vaccinated fish on 30 and 60 dpv. Through compatibility with an appropriate adjuvant, the highly immune protective effect of an IHN inactivated vaccine was prolonged from 60 dpv to at least 284 dpv; this novel adjuvant-IHN inactivated vaccine has promise as a commercial vaccine that provides the best available and longest duration of protection against IHN to rainbow trout. IMPORTANCE Infectious hematopoietic necrosis virus (IHNV) is one of the most serious pathogens threatening the global salmon and trout industry. However, there is currently only one commercialized infectious hematopoietic necrosis (IHN) vaccine, and it is inadequate for solving the global IHN problem. In this study, a promising adjuvanted inactivated vaccine with long-term protection was developed and comprehensively studied. We confirmed the presence of a late antiviral response stage in vaccinated rainbow trout that lacked detectable neutralizing antibodies, which are commonly recognized to be responsible for long-term specific protection in mammals. These findings further our understanding of unique features of fish immune systems and could lead to improved prevention and control of fish diseases.

Immunological Effects of Recombinant Lactobacillus casei Expressing IHNV G Protein and Rainbow Trout (Oncorhynchus mykiss) Chemokine CK6 as an Oral Vaccine.

IHNV is a virus that infects salmonids and causes serious economic damage to the salmonid farming industry. There is no specific treatment for the disease caused by this pathogen and the main preventive measure is vaccination, but this is only possible for small groups of individuals. Therefore, it is important to investigate new oral vaccines to prevent IHNV. In this study, the CK6 chemokine protein of rainbow trout and the truncated G protein of IHNV were used to construct a secretory expression recombinant L.casei vaccine for rainbow trout. The results showed that the levels of IgM and IgT antibodies in rainbow trout reached the highest level on the 15th day after the secondary immunization, and the antibodies exhibited high inhibitory activity against viral infection. Furthermore, the expression of relevant cytokines in different tissues was detected and found to be significantly higher in the oral vaccine group than in the control group. It was also found that pPG-612-CK6-G/L.casei 393 could stimulate splenic lymphocyte proliferation and improve mucosal immunity with significant differences between the immunized and control groups. When infected with IHNV, the protection rate of pPG-612-CK6-G/L.casei 393 was 66.67% higher than that of the control group. We found that pPG-612-CK6-G/L.casei 393 expressed and secreted the rainbow trout chemokine CK6 protein and IHNV truncated G protein, retaining the original immunogenicity of rainbow trout while enhancing their survival rate. This indicates that recombinant L.casei provides a theoretical basis and rationale for the development of an oral vaccine against IHNV and has important practical implications for the protection of rainbow trout from IHNV infection.

Protective Effects of Non-Encapsulated and Microencapsulated Lactobacillus Delbrueckii Subsp. Bulgaricus in Rainbow Trout (Oncorhynchus Mykiss) Exposed to Lead (Pb) Via Diet

Abstract The present study was designed to investigate the effects of dietary non-encapsulated and microencapsulated Lactobacillus delbrueckii subsp. bulgaricus on growth performance, intestinal enzymatic activities, antioxidant capacity and hepato-biochemical parameters of rainbow trout before or after exposure to lead via diet. Fingerling fish (16 ± 4 g) were divided into four groups: negative control (NC), positive control (PC), probiotic (PR) and encapsulated probiotic (EN-PR). During the pre-exposure period (days 0–45), fish in the NC and PC groups received the basal diet, whereas fish in the PR and EN-PR groups were fed with basal diet containing 108 CFU g−1 feed of non-encapsulated and microencapsulated probiotic, respectively. During the exposure period (days 46–66), the fish in the probiotic and PC groups were co-treated with 500 μg g−1 feed of lead nitrate. Blood, liver and gut samples were taken at days 0, 45, 52, 59 and 66. The results revealed that growth performance and intestinal enzymatic activities were significantly (P<0.05) improved in the probiotic groups compared to the NC group (day 45). Dietary exposure to lead resulted in the highest levels of liver aspartate aminotransferase (AST), liver alkaline phosphatase (ALP) and serum malondialdehyde (MDA), and the lowest activities of serum superoxide dismutase (SOD) and catalase (CAT) in the PC group (day 66). The levels of liver ALP were significantly (P<0.05) lower in the probiotic groups compared to the NC and PC groups prior to and after exposure to dietary lead. Serum levels of total protein, albumin, SOD, CAT and glutathione (GSH) were significantly increased in fish fed with both non-encapsulated and microencapsulated probiotics (P<0.05). However, microencapsulated probiotic showed the greatest potential for alleviation of the disturbed activities of intestinal and hepatic enzymes, and improvement of serum biochemical and antioxidant parameters. Our findings suggest that L. delbrueckii subsp. bulgaricus, particularly in the microencapsulated form, can be used as a potential probiotic to protect rainbow trout from dietborne lead toxicity.

Evaluation of the effect of fish yersiniosis on the course of antioxidant protection processes in trout

Indicators that characterize the state of lipid peroxidation and antioxidant protection in rainbow trout grown in the North-West of the country in normal conditions have been determined. It was found that the amount of malondialdehyde, antioxidant enzymes and total phospholipids had no significant changes in the samples taken in the farms of the Leningrad region and the Republic of Karelia, while the concentration level depended on the age of the fish. The maximum content of the final products of lipid peroxidation malondialdehyde was recorded in fish weighing 600-720 g, which is associated with a high fat content in the diet and the inability of the body to metabolize them completely. The developing pathological process in the body of fish when infected with Yersinia accelerated the intensity of lipid peroxidation, as a result, toxic lipid peroxides accumulated in organs and tissues, and the concentration of malondialdehyde increased from 65.4 to 95.2 %. There were also decreases in the activity of antioxidant protection: the value of catalase decreased from 6.7 to 23.7 %, peroxidase 7.1-19.5 %, the maximum drop in the activity of ceruloplasmin 9.5 %.

Effect of a novel postbiotic containing lactic acid bacteria on the intestinal microbiota and disease resistance of rainbow trout (Oncorhynchus mykiss).

This study was aimed to assess the effect of a novel postbiotic on bacterial community composition and structure within the intestinal ecosystem of rainbow trout (Oncorhynchus mykiss), as well as evaluate its capacity to protect rainbow trout from Lactococcus garvieae infection. After 30 days of dietary postbiotic supplementation, high-throughput 16S rRNA gene sequencing revealed that bacterial community composition, diversity and richness were significantly higher in treated fish than in control fish. The proportion of sequences affiliated to the phylum Tenericutes, and to a lesser extent, the phyla Spirochaetes and Bacteroidetes was increased in fish fed a postbiotic-enriched diet compared to control fish, whereas the abundance of Fusobacteria was higher in control fish. Moreover, the treated fish showed significantly (p < 0.05) improved protection against L. garvieae compared to control fish. These findings suggest that dietary postbiotic supplementation may represent an environmentally friendly strategy for preventing and controlling diseases in aquaculture.

Development of a live vector vaccine against infectious pancreatic necrosis virus in rainbow trout

Infectious pancreatic necrosis virus (IPNV) causes severe viral disease in salmonids, with major economic losses in the global rainbow trout aquaculture industry following from a high mortality rate. In the present study, we aimed to develop a safe and efficient vaccine to protect rainbow trout against IPNV infection. Administered via the immersion route, a live vector vaccine containing the coding region of the IPNV VP2 induced protective immune responses in rainbow trout. For best protection, juvenile rainbow trout should be immunized with a 1:100 dilution of the titer with 1 × 1010.0 ml−1 TCID50 of the live vector vaccine, via 10-min immersion. Expression of the IPNV VP2 gene was confirmed in spleens of vaccinated rainbow trout, and reached the highest level at 3 days post-vaccination (dpv) and thereafter gradually decreased between 3 and 15 dpv. The expression of TLR-3, TLR-7 and TLR-8 was upregulated after vaccination, and the highest expression levels of IFN-1, Mx-1, Mx-3, Vig-1 and Vig-2 were detected at 3 dpv. Four markers of the adaptive immune response (CD4, CD8, IgM and IgT) were continuously increased in the spleens of vaccinated fish as compared with controls, between 3 and 15 dpv. The cumulative percentage mortality differed significantly between the vaccinated fish and the controls (empty-plasmid-vaccinated). The results of the IPNV challenge showed that the live vector vaccine had a high protection rate against IPNV (relative percent survival value of 88.24), and the vaccinated rainbow trout displayed high levels of serum antibodies against IPNV infection. Taken together, our results demonstrate that this is a promising live vector vaccine that could be used to protect rainbow trout against IPNV infection.

Comparative transcriptome analysis reveals the mechanism of β-glucan in protecting rainbow trout (Oncorhynchus mykiss) from Aeromonas salmonicida infection

To study the mechanism of β-glucan in immune protection, rainbow trout were fed diets with or without 0.2% β-glucan for 42 days and then infected with Aeromonas salmonicida. After that, spleen tissues were sampled on 4- and 6-days post infection (dpi). Transcriptome analysis was compared between control group (CG, without β-glucan addition) and 0.2% β-glucan group (BG). In CG vs BG, 378 and 406 DEGs were identified on 4 dpi and 6 dpi respectively; furthermore, 46 DEGs were shared on 4 dpi and 6 dpi, enriching in GO terms, such as complement activation, inflammatory response, and metabolic process. KEGG pathway analysis revealed that some DEGs in CG vs BG were involved in immune or metabolic signaling pathways such as complement and coagulation cascades, toll-like receptor signaling pathway, NF-κB signaling pathway, antigen processing and presentation, and platelet activation on 4 or 6 dpi. DEGs, such as fgg, fgb, f5, c9, c3, c5, tlr5, and myd88, were analyzed in CG vs BG on 4 dpi and 6 dpi, implying their potential roles in β-glucan-modulated immunity. These results are beneficial to understand the mechanism of β-glucan in resisting bacteria in fish.

A chimeric recombinant infectious hematopoietic necrosis virus induces protective immune responses against infectious hematopoietic necrosis and infectious pancreatic necrosis in rainbow trout

Infectious pancreatic necrosis virus (IPNV) and infectious hematopoietic necrosis virus (IHNV) are two common viral pathogens that cause severe economic losses in all salmonid species in culture, but especially in rainbow trout. Although vaccines against both diseases have been commercialized in some countries, no such vaccines are available for them in China. In this study, a recombinant virus was constructed using the IHNV U genogroup Blk94 virus as a backbone vector to express the antigenic gene, VP2, from IPNV via the reverse genetics system. The resulting recombinant virus (rBlk94-VP2) showed stable biological characteristics as confirmed by virus growth kinetic analyses, pathogenicity analyses, indirect immunofluorescence assays and western blotting. Rainbow trout were immunized with rBlk94-VP2 and then challenged with the IPNV ChRtm213 strain and the IHNV Sn1203 strain on day 45 post-vaccination. A significantly higher survival rate against IHNV was obtained in the rBlk94-VP2 group on day 45 post-vaccination (86%) compared with the PBS mock immunized group (2%). Additionally, IPNV loads decreased significantly in the rBlk94-VP2 immunized group in the liver (28.6-fold to 36.5-fold), anterior kidney (21.7-fold to 44.2-fold), and spleen (14.9-fold to 22.7-fold), as compared with the PBS mock control group. The mRNA transcripts for several innate and adaptive immune-related proteins (IFN-γ, IFN-1, Mx-1, CD4, CD8, IgM, and IgT) were also significantly upregulated after rBlk94-VP2 vaccination, and neutralizing antibodies against both IHNV and IPNV were induced on day 45 post-vaccination. Collectively, our results suggest that this recombinant virus could be developed as a vaccine vector to protect rainbow trout against two or more diseases, and our approach lays the foundations for developing live vaccines for rainbow trout.

Development of a live vector vaccine against infectious hematopoietic necrosis virus in rainbow trout

Infectious hematopoietic necrosis virus (IHNV) leads to serious disease and economic losses in the salmonid aquaculture industry. The present study aimed to develop an effective and efficient vaccine to protect rainbow trout (Oncorhynchus mykiss) against IHNV infection. Administered via the immersion route, a live vector vaccine containing the regions of the IHNV glycoprotein (G) induced immune responses in rainbow trout. Use of the immersion route induced more-efficient mucosal immunity than intramuscular injection vaccination. IHNV G gene expression was detected in the spleens of rainbow trout at 3, 7 and 15 days post-vaccination (dpv). The G gene expression continuously decreased between 3 and 15 dpv. In addition, the expression of TLR-3, TLR-7 and TLR-8 was upregulated after vaccination, and the highest expression levels of IFN-1, Mx-1, Mx-3, Vig-1 and Vig-2 were observed at 3 dpv. Four markers of the adaptive immune response (CD4, CD8, IgM and IgT) gradually increased. When experimental fish were challenged with IHNV by immersion, significant differences in cumulative percentage mortality were observed in the vaccinated fish and the unvaccinated (empty-plasmid-vaccinated) fish. The relative survival rate was 92% and 6% in the vaccinated group and empty-plasmid group, respectively. Serum antibody levels gradually increased in the vaccinated fish, unlike in the unvaccinated fish, after 7 dpv. Our results suggest there was a significant increase in fish immune responses and resistance to infection with IHNV following administration of the live vector vaccine. Therefore, this live vector vaccine is a promising vaccine that may be utilized to protect rainbow trout against IHNV.

VP2 (PTA motif) encoding DNA vaccine confers protection against lethal challenge with infectious pancreatic necrosis virus (IPNV) in trout

IPNV in Atlantic salmon is represented by various strains with different virulence and immunogenicity linked to various motifs of the VP2 capsid. IPNV variant with P217, T221, A247 (PTA) motif is found to be avirulent in Atlantic salmon, but virulent in rainbow trout, and other salmonid species. This study describes a DNA vaccine delivered intramuscularly encoding the VP2 protein of infectious pancreatic necrosis virus (IPNV) with PTA motif that confers high protection in rainbow trout (Oncorhynchus mykiss). Intramuscular injection of 2, 5 and 10 μg of DNA (pcDNA3.1-VP2) in rainbow trout fry (4–5 g), confers relative protection of 75–83% in the different vaccine groups at 30 days post vaccination (450° days). The VP2 gene is expressed in spleen, kidney, muscle and liver at day 30 post-vaccination (RT-PCR), and IFN-1 and Mx-1 mRNA are upregulated at early time post vaccination, and so also for IgM, IgT, CD4 and CD8 in the head kidney of vaccinated fish compared to controls, 15 and 30 days post vaccination. Significant increase of serum anti-IPNV antibodies was found 30–90 days post-vaccination that was correlated with protection levels. Mortality corresponded with viral VP4 gene expression were significantly decreased in vaccinated and challenged fish. This shows for the first time that a VP2-encoding DNA vaccine delivered intramuscularly elicits a high level of protection alongside with high levels of circulating antibodies in rainbow trout and a lowered viral replication.

Antimicrobial effect of the Biotronic® Top3 supplement and efficacy in protecting rainbow trout (Oncorhynchus mykiss) from infection by Aeromonas salmonicida subsp. salmonicida

Demand for more environmentally friendly practices have led to the adoption of several feed supplements by the fish farming industry. In the present study, we investigated a commercially available formula that includes a mixture of three compounds: organic acids, a phytochemical and Biomin® Permeabilizing Complex. This mixture demonstrated antimicrobial properties in vitro and was able to inhibit growth of multiple species of aquatic bacterial pathogens, including Aeromonas salmonicida.Bacterial challenge was performed using A. salmonicida and three exposure routes: intra-peritoneal injection, immersion, and cohabitation. Mortality rates following infection by injection were significantly decreased in the fish that had received the supplemented feed. Fish infected through the other routes did not show a significant difference in mortality.In term of farming performance, while the fish that had received the feed supplement showed an improvement in weight gain and final weight, these changes were not found to be statistically significant. Similarly, no significant difference was observed in the feed conversion ratio. The results of this study suggest that this feed supplement may be effective at protecting rainbow trout from fish furunculosis.

Protection of rainbow trout against yersiniosis by lpxD mutant Yersinia ruckeri.

Yersinia ruckeri is a Gram negative bacteria causing yersiniosis in freshwater and marine fish. Lipid A, important for pathogenesis of Gram negative bacteria, biosynthesis pathway requires nine enzyme catalyzed steps. Although there are nine genes encoding lipid A biosynthesis in bacteria, biosynthesis of lipopolysaccharides relies on lpxD gene that encodes the third pathway enzyme. The roles of LpxD in Y.ruckeri virulence have not been studied. In the present study, in-frameshift deletion of lpxD gene and their role in Y.ruckeri virulence in rainbow trout were determined. For this purpose, 92% of the Y.ruckeri lpxD genes were deleted by homologous recombination. After running in SDS-PAGE and staining with silver stain, no LPS was detectable in the Y.ruckeri ΔlpxD mutant. Virulence and immunogenicity of the Y.ruckeri ΔlpxD mutant (YrΔlpxD) were determined in rainbow trout. Rainbow trout immunized with YrΔlpxD with immersion, or intraperitoneal injection method displayed superior protection (relative percentage survival≥84%) after exposure to wild type Y.ruckeri. In conclusion, our results indicated that deletion of the lpxD gene causes significant attenuation of Y.ruckeri in rainbow trout, and LPS deficient YrΔlpxD could be used as a live attenuated vaccine against Y.ruckeri in rainbow trout. This vaccine can protect fish and it can be applied to fish with different methods such as immersion or injection.

Protection of rainbow trout (Onchorynchus mykiss L.) against Flavobacterium psychrophilum by immersion and injection vaccination using a novel polyvalent vaccine

Rainbow Trout Fry Syndrome (RTFS) is a disease caused by the Gram-negative bacterium Flavobacterium psychrophilum, responsible for economic losses in the trout industry worldwide. The diversity of F. psychrophilum strains, inherent difficulties in vaccinating juvenile fish and lack of a reproducible immersion challenge model has hampered the development of a vaccine for this disease. Disease episodes tend to occur between 10-14 oC, with necrotic lesions seen on the skin surrounding the dorsal fin and tail; in small fry often no clinical signs are apparent and death occurs due to septicaemia. At present no commercial vaccines are available, leaving antibiotics as the only course of action to contain disease outbreaks. The development of a vaccine is required, due to the potential risk of resistance developing in the antibiotics presently licensed for use in aquaculture.

Temporary protection of rainbow trout gill epithelial cells from infection with viral haemorrhagic septicaemia virus IVb.

The branchial epithelium is not only a primary route of entry for viral pathogens, but is also a site of viral replication and subsequent shedding may also occur from the gill epithelium. This study investigated the potential of agents known to stimulate innate immunity to protect rainbow trout epithelial cells (RTgill-W1) from infection with VHSV IVb. RTgill-W1 cells were pretreated with poly I:C, FuGENE(®) HD+poly I:C, lipopolysaccharide (LPS), LPS+poly I:C or heat-killed VHSV IVb and then infected with VHSV IVb 4 days later. Cytopathic effect (CPE) was determined at 2, 3, 4, 7 and 11days post-infection. Virus in cells and supernatant was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All of the treatments delayed the onset of CPE (per cent of monolayer destruction), compared with untreated controls; however, killed VHSV or poly I:C combined with LPS was the most effective. Similarly, the detection of viral RNA in the supernatant was delayed, and the quantity was significantly (P<0.05) reduced by all treatments with the exception of LPS alone (4 days). Unlike many of the other treatments, pretreatment of RTgill-W1 with heat-killed VHSV did not upregulate interferon 1, 2 or MX 1 gene expression.