An experimental vaccine against Aeromonas hydrophila can induce protection in rainbow trout,Oncorhynchus mykiss (Walbaum)

A candidate vaccine against Aeromonas hydrophila in rainbow trout, Oncorhynchus mykiss, was developed using a bacterial lysate. To test the strength of protection, A. hydrophila challenge models were compared using injection into both the intraperitoneal (IP) cavity and the dorsal sinus (DS) with selected doses of live bacteria washed in saline or left untreated. Unlike the IP route, injection into the DS with either saline washed or unwashed cells resulted in consistent cumulative mortality and a dose response that could be used to establish a standard challenge having an LD₅₀ of approximately 3 × 10⁷ colony forming units per fish. Survivors of the challenge suffered significantly lower mortality upon re‐challenge than naïve fish, suggesting a high level of acquired resistance was elicited by infection. Passive immunization using serum from hyper‐immunized fish also resulted in significantly reduced mortality indicating protection can be transferred and that some portion of resistance may be antibody mediated. Vaccination of groups of rainbow trout with A. hydrophila lysate resulted in significant protection against a high challenge dose but only when injected along with Freund’s complete adjuvant. At a low challenge dose, mortality in all groups was low, but the bacterial lysate alone appeared to offer some protection.     

Use of garlic, Allium sativum, to control Aeromonas hydrophila infection in rainbow trout, Oncorhynchus mykiss (Walbaum)

Garlic, Allium sativum , which was fed at 0, 0.05, 0.1, 0.5 and 1.0 g per 100 g of feed for 14 days to rainbow trout, Oncorhynchus mykiss (Walbaum), led to control of experimental infection with Aeromonas hydrophila. At doses of 0.5 and 1.0 g garlic per 100 g of feed, there was a reduction in mortalities to 4% compared with the controls (88%). Moreover, there was a significant increase in growth, feed conversion and protein efficiency. There was stimulation of the number of erythrocytes and leucocytes, a significantly higher haematocrit, enhancement of phagocytic activity, respiratory burst, lysozyme, anti-protease and bactericidal activities following feeding with garlic.     

Use of dietary ginger, Zingiber officinale Roscoe, as an immunostimulant to control Aeromonas hydrophila infections in rainbow trout, Oncorhynchus mykiss (Walbaum)

Ginger, Zingiber officinale, which was fed at 0, 0.05, 0.1, 0.5 and 1.0 g per 100 g of feed for 14 days to rainbow trout, Oncorhynchus mykiss (Walbaum), led to control of experimental infection with Aeromonas hydrophila . At 0.5 g ginger per 100 g of feed, there was a reduction in mortalities to 0% compared with the controls (64%). Moreover, there was a significant increase in growth, feed conversion and protein efficiency. There was proliferation in the number of neutrophils, macrophages and lymphocytes, and enhanced phagocytic, respiratory burst, lysozyme, bactericidal and anti-protease activities compared with the controls.     

The garlic component,allicin, prevents disease caused by Aeromonas hydrophila in rainbow trout,Oncorhynchus mykiss (Walbaum)

Allicin was fed at 0 (= control), 0.5 and 1.0 mL of Allimed^® liquid 100 g^?1 of feed for 14 days to rainbow trout, Oncorhynchus mykiss (Walbaum), fingerlings before infection with Aeromonas hydrophila with a resultant reduction in mortalities from 80% in the controls to 8% [relative percentage survival (RPS) = 90%] and 0% (RPS = 100%) among the treated fish. Allicin was strongly antibacterial compared to the control, with a minimum inhibitory concentration (MIC) of >400 μL mL^?1 of Allimed^® liquid. Use of allicin led to a lower number of white blood cells (132.0 ± 0.4 × 10³) compared to 175.0 ± 0.1 × 10³ in the controls, but elicited increased phagocytic activity, i.e. a phagocytic value of 39.2% compared to 13.6% in the controls, and serum lysozyme activity, which showed significant (P > 0.05) differences compared to the control at 15 and 30 min after the first reading at 0 min of incubation.     

Virulence of Flavobacterium psychrophilum isolates in rainbow trout Oncorhynchus mykiss (Walbaum)

Flavobacterium psychrophilum, the causative agent of bacterial cold‐water disease (BCWD) in freshwater‐reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 10⁸ cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 10⁶, 10⁷ or 10⁸ cfu/fish of F. psychrophilum FPG 101, the 10⁸ cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R² = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety‐seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log₁₀ gene/copies and >3.0 log₁₀ gene copies/reaction, respectively, following infection with 10⁸ cfu/fish.     

Recovery of Bacillus mycoides,B. pseudomycoides and Aeromonas hydrophila from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) with gill disease

During a 3‐month period from June to the end of August 2016, ~5% mortalities were observed in a farm with rainbow trout (Oncorhynchus mykiss Walbaum) and one farm of common carp (Cyprinus carpio L.) in Bulgaria. The disease was manifested by gill ulcers/rot, asphyxiation and bloody ascites. Aeromonas hydrophila was isolated from the internal organs of all the diseased fish. Bacillus mycoides or B. pseudomycoides were recovered from the gill lesions on diseased carp and rainbow trout, respectively, with identification achieved by conventional phenotyping and by sequencing of the 16S rRNA gene. In vivo experiments confirmed that all three organisms were pathogenic to rainbow trout.     

Efficacy of a polyvalent injectable vaccine against Flavobacterium psychrophilum administered to rainbow trout (Oncorhynchus mykiss L.)

Flavobacterium psychrophilum is one of the most important pathogens affecting cultured rainbow trout (Oncorhynchus mykiss). Recent information from UK salmonid farms showed country‐wide distribution of genetically and serologically divergent clones, which has hampered the development of a vaccine for rainbow trout fry syndrome. The current study assessed the efficacy of an injectable polyvalent vaccine containing formalin‐inactivated F. psychrophilum in rainbow trout. The vaccine was formulated with an oil adjuvant (Montanide ISA 760VG) or formalin‐killed cells alone. Duplicate groups of trout (60 ± 13 g) were given phosphate‐buffered saline or vaccine formulated with Montanide by intra‐peritoneal (i.p.) injection and challenged by intra‐muscular (i.m.) injection with a homologous and a heterologous isolate of F. psychrophilum at 525 degree days post‐vaccination (dd pv). Significant protection was achieved in vaccinated fish (p = 0.0001, RPS 76% homologous, 88% heterologous). Efficacy of the adjuvanted vaccine was also demonstrated by heterologous challenge at 1155 dd pv resulting in 100% protection, whereas survival in the un‐adjuvanted group was not significantly different from control fish. Levels of specific antibody at 1155 dd pv, as measured by ELISA, were significantly higher in the fish vaccinated with adjuvant when compared with unvaccinated fish.     

Immunohistochemical evaluation of experimental Vagococcus salmoninarum infection in rainbow trout (Oncorhynchus mykiss,Walbaum 1792)

The aim of this study was to investigate the pathogenesis and histopathological and immunohistochemical findings in rainbow trout (Oncorhynchus mykiss) following experimental vagococcosis. For this purpose, 60 rainbow trout were used. The experimental study used the pathogen Vagococcus salmoninarum. The fish were intraperitoneally (IP) administered with an inoculate containing 0.1 mL of the bacteria, resulting in a dose of 1.2 × 10⁹ cfu mL^?1 per fish. For histopathological observations, tissue samples were taken from fish that died during the experiment and fish that survived until the end of the trial (60th day). All the tissue samples were immunohistochemically stained by the avidin–biotin–peroxidase complex and immunofluorescence methods using polyclonal antibody to detect V. salmoninarum antigens. In immunoperoxidase staining, positive reactions to bacterial antigens were most commonly seen in the kidney, heart and liver. In the immunofluorescence analysis, the distribution of antigens in the tissue and organs was similar to that observed with the immunoperoxidase staining. The results reveal an important correlation between histochemical and immunohistochemical staining in demonstrating the distribution of V. salmoninarum antigens in the affected tissues.     

Co‐infection of rainbow trout (Oncorhynchus mykiss) with infectious hematopoietic necrosis virus and Flavobacterium psychrophilum

Co‐infection of rainbow trout with infections haematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum is known to occur, and it has been speculated that a combined infection can result in dramatic losses. Both pathogens can persist in fish in an asymptomatic carrier state, but the impact of co‐infection has not been well characterized or documented. In this study, it was hypothesized that fish co‐infected with F. psychrophilum and IHNV would exhibit greater mortality than fish infected with either pathogen alone. To test this, juvenile rainbow trout were co‐infected with low doses of either IHNV or F. psychrophilum, and at 2 days post‐initial challenge, they were given a low dose of the reciprocal pathogen. This combined infection caused high mortality (76.2%–100%), while mortality from a single pathogen infection with the same respective dose was low (5%–20%). The onset of mortality was earlier in the co‐infected group (3–4 days) when compared with fish infected with F. psychrophilum alone (6 days) or IHNV (5 days), confirming the synergistic interaction between both pathogens. Co‐infection led to a significant increase in the number of F. psychrophilum colony‐forming units and IHNV plaque‐forming units within tissues. This finding confirms that when present together in co‐infected fish, both pathogens are more efficiently recovered from tissues. Furthermore, pathogen genes were significantly increased in co‐infected groups, which parallel the findings of increased systemic pathogen load. Extensive tissue necrosis and abundant pathogen present intracellularly and extracellularly in haematopoietic tissue. This was pronounced in co‐infected fish and likely contributed to the exacerbated clinical signs and higher mortality. This study provides novel insight into host–pathogen interactions related to co‐infection by aquatic bacterial and viral pathogens and supports our hypothesis. Such findings confirm that mortality in fish exposed to both pathogens is greatly elevated compared to a single pathogen infection.     

Experimental Lactococcus garvieae infection in rainbow trout,Oncorhynchus mykiss,Walbaum 1792: a comparative histopathological and immunohistochemical study

The aim of this study was to induce Lactococcus garvieae infection in young and adult fish through different routes [intraperitoneal (IP) and immersion (IM)] and to investigate the pathogenesis and histopathological and immunohistochemical findings comparatively. For this purpose, a total of 180 rainbow trout (90 young, 20 ± 5 g and 90 adult, 80 ± 10 g) obtained from a commercial fish farm were used. The fish were divided into eight groups, four experimental groups (Young‐Adult IP groups and Young‐Adult IM groups, each contain 30 fish) and four control groups (Young‐Adult IP Control groups and Young‐Adult IM control groups, each contain 15 fishes). The experimental study was conducted using L. garvieae, and confirmatory identification was performed by PCR. The sequence result of the PCR amplicon of 16S rDNA from isolate L. garvieae LAC1 was determined and deposited in the GenBank database under accession number KC883976 . Fish in the IP groups were intraperitoneally administered an inoculate containing 10⁶ cfu mL^?1 bacteria 0.1 mL. In the IM groups, fish were kept in inoculated water containing 10⁸ cfu mL^?1 bacteria for 20 min. Mortality as well as clinical and pathological findings was recorded daily, and significant differences in macroscopic and microscopic results were observed between the IP and IM administration groups. All tissue samples were immunohistochemically stained by the avidin‐biotin‐peroxidase complex and immunofluorescence (IF) methods using polyclonal antibody to detect L. garvieae antigens. In immunoperoxidase staining in the IP groups, positive reactions to bacterial antigens were most commonly seen in the spleen, kidney, heart, liver, peritoneum and swim bladder. In the IM groups, bacterial antigens were most commonly found in the eye, gill, spleen and kidney. In the IF method, the distribution of antigens in tissue and organs was similar to the reactions with immunoperoxidase staining. Finally, in this experimental study, an important correlation was seen between the distribution of L. garvieae antigens and lesions developing in many organ and tissues.     

Immunization of rainbow trout Oncorhynchus mykiss (Walbaum) with a crude lipopolysaccharide extract from Flavobacterium psychrophilum

Control methods for Flavobacterium psychrophilum are limited and oftentimes ineffective; hence, research efforts have focused on vaccine development. This study tested the hypothesis that a crude lipopolysaccharide (LPS) extract from F. psychrophilum will elicit a protective immune response in rainbow trout Oncorhynchus mykiss (Walbaum) against F. psychrophilum challenge. Rainbow trout (mean weight, 3 g) were immunized intraperitoneally with the following treatment and control preparations: 10 μg of crude LPS with or without Freund's complete adjuvant (FCA), 25 μg of crude LPS with or without FCA and saline with or without FCA. Immunization of fish with 10 or 25 μg of crude LPS/FCA resulted in significant antibody responses against F. psychrophilum using ELISA with a whole‐cell lysate as the coating antigen, but only minimal levels of protection were conferred following F. psychrophilum challenge at 14 weeks post immunization. Western blot analyses demonstrated that fish exhibited antibodies specific for low‐molecular mass proteins present in the crude LPS extract, but did not exhibit antibodies specific for F. psychrophilum LPS. The results indicate that higher immunization doses and/or the use of an alternative extraction method that yields larger LPS molecules (23–70 kDa) may be necessary to elicit specific antibody responses against F. psychrophilum LPS.     

Protective effects of a synbiotic against experimental Saprolegnia parasitica infection in rainbow trout (Oncorhynchus mykiss)

A combination of probiotics and prebiotics as synbiotics allows assessing their synergistic effects. This study evaluated the effects of a synbiotic supplement on growth performance, haematological parameters and resistance to Saprolegnia parasitica in rainbow trout, Oncorhynchus mykiss (Walbaum) fingerlings. Fish fed a dietary synbiotic in three levels of 0.5, 1.0 and 1.5 g kg^?1 thrice a day. The fingerlings were challenged with Saprolegnia parasitica after 60 days post feeding and their mortalities recorded up to 15 days. The fingerlings at all three experimental treatments showed significant (P < 0.05) increases in final mean weights and specific growth rates (SGR). The best feed conversion ratio (FCR), feed conversion efficiency (FCE) and maximum survival rate were also obtained by the fish fed 1.0 g synbiotic kg^?1 diet. Furthermore, supplementation with synbiotic significantly increased blood factors at all treatments. After challenges with Saprolegnia parasitica, the synbiotic‐fed groups showed significantly higher survival rates compared with the control group. These results reveal that a dietary synbiotic of 1.0 g kg^?1 fed for 60 days leads to increased growth performance and survival rate as well as improved feeding efficiency in rainbow trout fingerling, rendering them more resistant against infection by Saprolegnia parasitica.     

Prevention of Saprolegniasis in rainbow trout (Oncorhynchus mykiss) eggs using oregano (Origanum onites) and laurel (Laurus nobilis) essential oils

The present study investigated the antifungal effects of essential oils of oregano (Origanum onites) and laurel (Laurus nobilis) on Saprolegniasis, a disease that occurs in rainbow trout eggs during the incubation period. Oregano and laurel were ground after drying, and essential oils were obtained by water distillation method using a Clevenger apparatus. The essential oils were added to potato dextrose agar (PDA) at the rates of 1–1000 ppm, and the minimum inhibitory concentration (MIC) was determined as 250 ppm whereas the minimum lethal concentration (MLC) was determined to be 500 ppm for both plants. In the in vivo trials, fertilized eggs were treated with predetermined doses either by bathing during water hardening and incubation period or only during incubation period, and death rates were monitored during embryological development. The best larvae hatching rate was determined in 500 ppm oregano and 500 ppm laurel groups treated during water hardening plus daily as 82.11% and 79.87%, respectively. According to the results, it was determined that oregano and laurel essential oils exhibited better results in all doses compared with the negative control group, and 500 ppm dose had a better effect than the positive control group treated with formalin.     

Erythromycin and florfenicol treatment of rainbow trout Oncorhynchus mykiss (Walbaum) experimentally infected with Flavobacterium psychrophilum

Flavobacterium psychrophilum is responsible for significant economic losses in rainbow trout aquaculture. Antimicrobial treatment remains the primary means of control; however, there are limited choices available for use. The objectives of the study were therefore to determine the minimum inhibitory concentrations for erythromycin and florfenicol in selected F. psychrophilum isolates and to evaluate their clinical treatment efficacy in experimentally infected rainbow trout. All isolates tested had moderate susceptibility to florfenicol and erythromycin except one isolate, which had low susceptibility to erythromycin. Two isolates (one with moderate and one with low susceptibility to erythromycin) were used in an experimental infection trial. Rainbow trout juveniles were injected intraperitoneally with 10⁸ cfu/fish and after mortality had begun, fish were given erythromycin‐ and florfenicol‐medicated feed at a rate of 75 mg kg^?1 day^?1 and 10 mg kg^?1 day^?1 fish body weight, respectively, for 10 consecutive days. The splenic F. psychrophilum load was determined using an rpoC quantitative PCR throughout the 30‐day trial. Relative to antibiotic‐free controls, erythromycin treatment significantly (p < 0.05) reduced mortality of rainbow trout juveniles infected with FPG101, even when treatment was initiated after clinical signs developed.     

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.