In vivo and in vitro cell-mediated immune responses of rainbow trout, Oncorhynchus mykiss (Walbaum), against Cryptobia salmositica Katz, 1951 (Sarcomastigophora: Kinetoplastida)

Abstract. Delayed-type hypersensitivity (DTH) reaction (an in vivo manifestation of cell-mediated immunity) was detected in Oncorhynchus mykiss maintained on a pantothenic-acid-supplemented diet 2 weeks after infection with Cryptobia salmositica. The reaction was similar to that in mammals with mononuclear cell infiltration into the dermis and muscle layers and the presence of oedema. DTH reaction was also displayed by fish on a pantothcnic-acid-supplemented diet that had recovered from the infection and were protected against further infection. The reaction was less marked in infected or protected fish on a pantothenie-acid-deficient diet. Inhibition of macrophage migration (an in vitro expression of cell-mediated immunity) was observed when head kidney cell suspensions from protected fish maintained on either pantothenic acid supplemented or deficient diets were incubated with Cryptobia antigen. No inhibition of migration was evident when head kidney cell suspensions from the above fish were incubated without antigen, nor was it evident when cells from uninfected fish were used. The occurrence of a typical DTH reaction in rainbow trout and the feasibility of assessing it by measuring the thickness of the induration provides a simple and practical method for assessing cell-mediated immunity in large scale vaccination programmes against pathogens.     

Impact of a pathogenic haemoflagellate, Cryptobia salmositica Katz, on the metabolism and swimming performance of rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Oxygen consumption of juvenile rainbow trout (5 g at 13°C) at moderate swimming speeds did not change significantly when infected with Cryptobia salmositica. However, significant reductions of as much as 44% of the maximum aerobic scope for activity and 24% of the critical swimming speed were observed when the parasitaemia reached a maximum of 57.6 × 10⁶ ml⁻¹ fish blood at 3 weeks post- infection. Blood haematocrit was significantly reduced from the initial 34.1 to 19.7% at 4 weeks post- infection, probably as a result of haemolysis by the parasite. The destruction of red blood cells clearly led to lower oxygen carrying capacity, and reduced respiratory and swimming performance.     

The immune response of rainbow trout, Salmo gairdneri Richardson, to the haemoflagellate, Cryptobia salmositica Katz, 1951

Abstract. Rainbow trout that recovered from experimental Cryptobia salmositica infection 6 and 10 weeks earlier were protected against multiple intraperitoneal challenges of 50 000 and 10 000 parasites isolated from infected fish. The immunity was non-sterile; low parasitaemias were detected following a larger challenge (112 000 parasites). The indirect haemagglutination test was used to detect C. salmositica -specific agglutimns. Antibody titers increased during the first 18 weeks of infection. The infectivity of cultured C. salmositica was neutralized by incubation in heat-inactivated immune plasma. Infectivity of C, salmositica from infected fish was not neutralized by similar treatment. Complement fixing antibody was detected using the in vitro immune lysis test. Immune lysis occurred when cultured C. salmositica were used. Adoptive transfer of both leucocytes and plasma from immune fish conferred partial protection against the parasite in naive recipients. Complement fixing antibody may be important during early acute infection while phagocytosis may be important during the later chronic phase.     

Immunosuppression in rainbow trout, Salmo gairdneri Richardson, caused by the haemoflagellate Cryptobia salmositica Katz, 1951

Abstract. An experimental Cryptobia salmositica infection in rainbow trout, Salmo gairdneri Richardson, produced suppression of the humoral response against sheep red blood cells as measured by direct haemagglutination. Two-month and 5-month infections produced equal suppression. The parasite also produced suppression of the humoral response against a bacterial pathogen, Yersinia ruckeri . Anti- Y. ritckeri titres were significantly lower in most fish infected with C. salmositica than in non-infected fish. Immunosuppression became evident when C. salmositica first appeared in the blood (first 2 weeks of infection), Immunosuppression was confirmed by challenge with Y. ruckeri . Mortality at challenge occurred in 64·3% to 83·3% of the fish already infected with C. salmositica at the time of initial Y. ruckeri exposure. There was no mortality at challenge if fish were not infected with C. salmositica at initial bacterial exposure, nor in those concurrently infected with both pathogens. Antigenic competition may have caused the immunosuppression.     

In vitro and in vivo effects of rabbit anti-thymocyte serum on circulating leucocytes and production of complement fixing antibodies in thymectomized Oncorhynchus mykiss (Walbaum) infected with Cryptobia salmositica Katz 1951

Rabbit anti-thymocyte serum (RATS) against thymocytes of rainbow trout was toxic to leucocytes from intact and thymectomized rainbow trout at 10 °C under in vitro conditions. The total number of leucocytes decreased significantly in 24 h after RATS was injected intraperitoneally into intact rainbow trout, but the number returned to pre-injection level within 1 week. RATS destroyed a lower percentage of leucocytes in thymectomized fish than in intact fish under both in vitro and in vivo conditions and the recovery in the number of leucocytes was slower in thymectomized fish. The parasitaemia, packed cell volume and production of complement fixing antibody in thymectomized and intact fish (injected with RATS before Cryptobia salmositica infection) were not significantly different from control fish (not injected with RATS), and they both acquired protective immunity against cryptobiosis on recovery. This indicates that RATS is not cytotoxic to B-like cells in the lymphoid tissue which produce complement fixing antibody against C. salmositica and that the protective antigen in C. salmositica seems to be thymus-independent.     

Identification of a localized mucosal immune response in rainbow trout, Oncorhynchus mykiss (Walbaum), following immunization with a protein-hapten antigen

The ability of rainbow trout, Oncorhynchus mykiss (RBT), to produce a localized mucosal immune response was investigated following intraperitoneal (i.p.) or peranal (p.a.) immunization with a protein-hapten carrier, fluorescein isothiocyanate conjugated to keyhole limpet haemocyanin (FITC/KLH). Antibody levels in serum, mucus, tissue culture supernatant from blood and spleen leucocytes, and excised skin, intestine and gill tissues were determined by ELISA. Significantly, elevated antigen-specific antibodies were elicited in both serum and mucus of fish immunized i.p. Mucosal antibody responses, in general, paralleled serum responses over time. Leucocytes isolated from spleen and blood of i.p. immunized fish at week 10 produced significantly elevated antibody levels against FITC when cultured in vitro. Excised skin, intestine and gill tissues from these fish also exhibited significantly elevated antibody responses indicating localized production in the mucosa from tissue-specific B cells. A localized mucosal immune response was elicited only after i.p. and not p.a. immunization, suggesting that systemically stimulated B cells migrate to mucosal tissues where they produce antibodies locally.     

Identification and pathogenicity to rainbow trout, Oncorhynchus mykiss (Walbaum), of some aeromonads

Twelve strains of fish pathogenic aeromonads were identified by 16S rRNA sequencing as Aeromonas bestiarum , A. hydrophila , A. hydrophila subsp. dhakensis , A. salmonicida subsp. salmonicida , A. sobria biovar sobria and A. veronii biovar sobria. Following intramuscular injection, A. hydrophila subsp. dhakensis caused dark liquefying, raised furuncle-like lesions in rainbow trout within 48 h. Extracellular products of all cultures contained gelatinase and lecithinase, and most revealed lipase. Congo red absorption and siderophore production was recorded, but not so the suicide phenomenon or slime production. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profile of the outer membrane proteins (OMP) revealed 10–25 bands, of which major bands were seen in the region of 32.5–47.5 and 62–83 kDa. Marked heterogenicity of the OMP and whole cell protein (WCP) profiles within and among the species was observed. Polypeptides of 83–173 kDa were detected in the WCP profile of the cultures, but they were not expressed in OMP fractions.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the pathogenic haemoflagellate, Cryptobia salmositica Katz, and protection against cryptobiosis in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), inoculated with a live vaccine

Abstract. An enzyme-linked immunosorbent assay using dried blood on filter paper, was developed for the detection of antibodies against the haemoflagellate Cryptobia salmositica in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum). Each fish (average weight about 5g) in three experimental groups was either inoculated with 20000 attenuated live C. salmositica vaccine, or inoculated with 2000 or 20000 pathogenic parasites per fish. The vaccine was effective in protecting juvenile trout 4 weeks after vaccination and antibody titers were higher in vaccinated and challenged fish than in unvaccinated and infected ones. Specific antibodies were detected one week post-infection (w.p.i.) with the pathogen and declined to low levels at 6 w.p.i. The high-dose group (20000 per fish) had antibody titres comparable to those of the vaccinated and challenged fish.     

Dietary modulation of digestive enzymes by the administration of feed additives to rainbow trout,Oncorhynchus mykiss Walbaum

The aim of this study was to assess the metabolic and digestive enzyme activities in rainbow trout, Oncorhynchus mykiss Walbaum fed dietary supplements. The biometric indices and the following enzymes activities were measured: acid protease and pepsin from the stomach homogenates, alkaline phosphatase (AP) from the small intestine and the brush border membrane, total proteolytic enzymes and trypsin from the small intestine and liver/pancreas. Dietary garlic induced a higher pepsin activity in the stomach than lipopolysaccharide (LPS) and ginger. Conversely, dietary ginger and LPS showed a significant difference (P > 0.05) in acid protease activity compared to the controls. There was not any significant difference observed between treated groups and the controls for hepato‐pancreas proteolytic enzyme activity, except for trypsin. AP activity increased significantly with dietary garlic, ginger and LPS compared to the controls.     

The effect of glycyrrhizin on rainbow trout, Oncorhynchus mykiss (Walbaum), leucocyte responses

Abstract. Treatment of rainbow trout macrophages with glycyrrhizin (GL), an aqueous extract of licorice, Glycyrrhiza glabra , enhanced their respiratory burst activity. Maximal effects were seen using concentrations of 10–100 μml⁻¹. GL also modulated trout lymphocytes, increasing proliferation responses to the mitogen phytohaemagglutinin some two-fold over a range of GL concentrations. In addition, GL elicited the release of a macrophage activating factor (MAF) from head kidney leucocytes, as assessed by the ability of generated supernatants to increase respiratory burst activity of target macrophages. MAF activity was most apparent using 100 μg ml⁻¹ GL to induce MAF release and a 48-h incubation period with the target macrophages. Finally, GL was shown to enhance the release of MAF in response to the mitogen concanavalin A. The possible use of GL as a stimulant of fish innate defences is discussed.     

Characterization of a monoclonal antibody against the 47- kDa antigen of Cryptobia salmositica Katz and its use in an antigen-capture enzyme-linked immunosorbent assay for detection of parasite antigen in infected rainbow trout, Oncorhynchus mykiss (Walbaum)

A monoclonal antibody (designated MAb-007) was produced against the pathogenic haemoflagellate Cryptobia salmositica Katz. This IgG3 antibody recognized the 47-kDa antigenic polypeptide of C. salmositica (SDS-PAGE and Western immuno-blotting). The antibody did not agglutinate live parasites, and there was no change in the staining intensity of the 47-kDa band on Western immunoblots after immunoabsorption of MAb-007 with live intact parasites. The 47-kDa antigen recognized by MAb-007 was localized in the cytoplasm of the parasite (immunogold labelling and electron microscopy). The monoclonal antibody cross- reacted with the 47-kDa polypeptides of C. bullocki Srrout and C. catostomi Bower & Woo. It was used in an antigen-capture ELISA for the detection of parasite antigen in the plasma of rainbow trout inoculated with the parasite, or with an attenuated vaccine strain of C. salmositica. All pre-infection plasma were negative while all infected fish with detectable parasitaemias were positive for antigen at 1–9 weeks after infection. Parasite antigen was even detected in vaccinated fish that were negative for parasites using the wet mount microscopic technique. The antigen-capture ELISA detected C, salmositica antigen in whole cell lysate preparations at concentrations as low as 0.5 μg ml^-1. Fifty microlitres of fish plasma was required in the antigen-capture ELISA, and the use of a plate reader and 96-well plates facilitated rapid analysis of a large number of plasma samples. The sensitivity of the assay makes it a potentially useful tool for detection of Cryptobia infections.     

Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.     

Bacterial pathogens in rainbow trout, Oncorhynchus mykiss (Walbaum), reared at Danish freshwater farms

During a 2-year period, bacterial fish pathogens were monitored on five rainbow trout, Oncorhynchus mykiss (Walbaum), freshwater farms in Denmark. A total of 1206 fish were examined and 361 bacterial isolates were identified phenotypically. Enteric redmouth disease, furunculosis and rainbow trout fry syndrome/coldwater disease were recorded. Infections caused by Flavobacterium psychrophilum occurred most frequently, but only one outbreak of enteric redmouth disease caused by Yersinia ruckeri serotype O1 and one of furunculosis caused by Aeromonas salmonicida were recorded during the monitoring period. Flavobacterium psychrophilum was isolated on all farms, both during disease outbreaks and from fish without any signs of disease. Serological investigations of F. psychrophilum showed that serotype Th was the dominant serotype found. The serotypes Th and Fd were involved in disease outbreaks of fry and larger fish. All isolates of F. psychrophilum showed proteolytic activities; however, a few isolates, belonging to serotype FpT did not degrade elastin and were not associated with mortality. Increasing resistance problems to oxytetracycline were demonstrated. More than half of the F. psychrophilum isolates showed resistance to oxolinic acid and oxytetracycline. No antibiotic resistant isolates were found among Y. ruckeri and A. salmonicida .     

Dietary formic acid enhances apparent digestibility of minerals in rainbow trout, Oncorhynchus mykiss (Walbaum)

The effect of dietary formic acid on the availability of phosphorus (P) from a fishmeal-based diet by rainbow trout, Oncorhynchus mykiss (Walbaum), reared in brackish water (5–6 g L⁻¹) was determined. Diets containing a low amount of P (6.0 mg P g⁻¹ dry matter) were acidified with 0, 4 and 10 mL kg⁻¹ formic acid and fed to trout (average weight, 520 g) for 4 weeks. The measured pH of the diets were 6.3, 5.8 and 5.3, respectively. The intestinal solubility of P and the digestibility of P were measured by stripping the faecal matter from the fish. The apparent digestibility coefficient (ADC) of P significantly ( P < 0.05) increased from 69.5% to 75.0% of the basal diet in fish fed diets containing 10 mL kg⁻¹ formic acid. The solubility of P in the intestine was highly variable within each treatment, and the differences were not significant. The pH of intestinal content increased with the increase in dietary formic acid concentration. The ADC of magnesium and calcium also showed a significant ( P < 0.05) increase with the acidification of diet by formic acid.     

Immunogenicity of amoebic antigens in rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Rainbow trout developed a humoral immune response against numerous antigens of sonicated amoebae which were emulsified with Freund's complete adjuvant and injected into the peritoneum. The amoebae were cultured from the gills of Atlantic salmon, Salmo salar L., affected by amoebic gill disease. Antibodies in fish sera were detected by both enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Non-spedfie reactivity in fish serum against Escherichia coli, the bacterium used in co-cultivation of amoebae in vitro, was removed by immunoadsorption. Results obtained using ELISA and immunoblotting were comparable and indicated no significant difference in response to immunization with 10, 50 or 250 μg of sonieatcd amoebic protein. Amoebae contained immunogenie components of > 100, 100, 89, 49, 37 and 34kDa.     

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.