Rainbow trout (Oncorhynchus mykiss) immune response towards a recombinant vaccine targeting the parasitic ciliate Ichthyophthirius multifiliis

The protective effect in rainbow trout (Oncorhynchus mykiss) of an experimental subunit vaccine targeting antigens in the parasite Ichthyophthirius multifiliis has been evaluated and compared to effects elicited by a classical parasite homogenate vaccine. Three recombinant parasite proteins (two produced in E. coli and one in insect cells) were combined and injected i.p., and subsequently, protection and antibody responses were analysed. Both the experimental and the benchmark vaccine induced partial but significant protection against I. multifiliis when compared to control fish. Specific antibody responses of vaccinated trout (subunit vaccine) were raised against one neurohypophysial n‐terminal domain protein #10 of three recombinant proteins, whereas the benchmark vaccine group showed specific antibody production against all three recombinant proteins. The immunogenic parasite protein #10 may be a potential vaccine candidate supplementing the protective I‐antigen in future vaccine trials.     

Comparative effects of four feed types on white spot disease susceptibility and skin immune parameters in rainbow trout, Oncorhynchus mykiss (Walbaum)

The effects on rainbow trout, Oncorhynchus mykiss (Walbaum), immune parameters by differently formulated fish feed types containing immunostimulants have been tested in a double-blind, duplicated and controlled study performed over 50 days. A total of 800 rainbow trout (10-12 g) were kept in eight duplicate fish tanks (each containing 100 fish) and fed at a daily feeding rate of 1.5% of the biomass. The feed types were (1) control feed (C) without additives, (2) feed containing beta-glucan, nucleotides, manno-oligosaccharides (MOS), vitamins C and E (GNMCE), (3) feed containing probiotic bacteria and plant extracts (PP) and (4) feed with nucleotides, manno-oligosaccharides, vitamins C and E (NMCE). Plasma lysozyme activity was increased in fish fed two feed types (GNMCE and NMCE) but slightly depressed in fish fed PP. A non-significant trend for a higher mucous cell density at days 30 and 50 was shown in all fish receiving feeds with additives compared to the control group. All fish became infected with Ichthyophthirius multifiliis when exposed, but fish fed GNMCE showed a significantly lower infection both at days 30 and 50. Expression of genes encoding C3 and MHCII was significantly up-regulated in fish fed GNMCE for 50 days, and the expression of genes coding Hepcidin was significantly down-regulated in fish fed NMCE for 50 days. Beta-glucan was the single component, when used in combination with other feed ingredients, which was found associated with increased parasite resistance, increased lysozyme and immune gene up-regulation.     

Experimental evidence for direct in situ binding of IgM and IgT to early trophonts of Ichthyophthirius multifiliis (Fouquet) in the gills of rainbow trout, Oncorhynchus mykiss (Walbaum)

Freshwater fish are able to mount a protective immune response against the parasite Ichthyophthirius multifiliis (Ich) following a non‐lethal exposure. Factors involved in immunity comprise cellular and humoral factors, but antibodies have been suggested to play a prominent role in protection. However, host antibodies have not yet been demonstrated to bind to the parasite in situ. By the use of immunohistochemical techniques, this study demonstrated that IgT and IgM bind to surface structures, including cilia, on the early feeding stage of the parasite in the gills of immune rainbow trout, Oncorhynchus mykiss, shortly (2 h) after invasion. No binding of IgT and no or only a weak binding of IgM was observed on the parasites in the gills of similarly exposed but naïve rainbow trout. This study indicates that antibodies play an important part in the protection of immune fish against Ich although additional humoral and cellular factors may contribute to this reaction.     

The effectiveness of adenoviral vectors to deliver and express genes in rainbow trout,Oncorhynchus mykiss (Walbaum)

The efficacy of adenoviral vectors for gene delivery into fish cells, both in vitro and in vivo, was evaluated. Vectors utilized were of human adenovirus serotype 5 (Ad), which are commonly used in human clinical trials, but have not been assessed for gene delivery to fish. Because nothing is known about Ad receptors in fish, both an Ad (Ad5Luc1) with natural tropism for the coxsackie and adenovirus receptor (CAR), as well as an infectivity enhanced Ad (Ad5LucRGD) were included within this study. Gene expression was detected in cell lines using either vector. The levels seen with Ad5LucRGD were much higher than for Ad5Luc1 in most lines except CHSE-214. Transduction of CHSE-214 cells with Ad5Luc1 could be blocked with an excess of a competitive inhibitor, suggesting that these cells possess a CAR homologue thatmediates attachment of Ad, similar to that seen in mammalian cells. In vivo gene delivery was attempted by several methods, with significant expression seen only via intramuscular injection, although infection efficiency was low. Thus it was observed that several teleost cell lines are capable of being infected and one cell line expressed a human serotype adenoviral receptor homologue that aids in Ad infection. Additionally, in vivo studies indicated that muscle tissue of rainbow trout could be infected with Ad vectors, suggesting an alternative gene delivery strategy for this animal.     

Nutritional regulation of somatostatin expression in rainbow trout, Oncorhynchus mykiss

Previously we characterized three DNAs from the endocrine pancreas (Brockmann body) of rainbow trout (Oncorhynchus mykiss) that encode for distinct preprosomatostatin (PPSS) molecules: one containing somatostatin-14 and its C-terminus (PPSS I) and two containing [Tyr⁷, Gly¹⁰]-somatostatin-14 at their C-termini (PPSS II′ and PPSS II″). In the present study, the regulation of PPSS expression was studied in rainbow trout placed on varying nutritional regimes (fed continuously, fasted, fasted then refed). Fish that were fasted for one week displayed reduced growth compared to their fed counterparts, but no alteration in pancreatic PPSS expression was noted between the two groups. Fish fasted for 4 and 6 weeks also were growth retarded and displayed increased levels of PPSS I mRNA and PPSS II″ mRNA compared to fed animals; PPSS II′ mRNA levels were not affected by food deprivation. Refeeding fish for two weeks following 4 weeks of food deprivation restored growth and reduced PPSS I and PPSS II″ mRNA expression to levels similar to those displayed by continuously fed fish. Changes in PPSS expression were correlated with increases in plasma SS. These results indicate that nutritional state modulates differential expression of PPSSs. This revised version was published online in July 2006 with corrections to the Cover Date.     

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 probiotics to control furunculosis in rainbow trout, Oncorhynchus mykiss (Walbaum)

Aerobic heterotrophic bacteria were isolated from the intestinal contents of Atlantic salmon, Salmo salar , rainbow trout, Oncorhynchus mykiss , and turbot, Scophthalmus maximus , on tryptone soya agar and De Man Rogosa and Sharpe agar, of which 11 of 177 (6% of the total) of the isolates were antagonistic to Aeromonas salmonicida . Four of these cultures, which were identified tentatively as A. hydrophila , Vibrio fluvialis , Carnobacterium sp. and an unidentified Gram-positive coccus, were beneficial to fish when fed singly or as an equi-mixture. Feed supplemented with the putative probiotics indicated survival of the organisms in the gastrointestinal tract for 7 days. Feeding with the probiotics for 7 and 14 days led to better survival following challenge with A. salmonicida . There was no indication of serum or mucus antibodies to A. salmonicida , but there was an increased number of erythrocytes, macrophages, lymphocytes and leucocytes, and enhanced lysozyme activity in the fish.     

Relationships between growth and disease resistance in rainbow trout,Oncorhynchus mykiss (Walbaum)

Rainbow trout from 23 families were evaluated for growth and resistance to the bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum and infectious haematopoietic necrosis (IHN) caused by IHN virus. Average family weights were between 161 and 263 g with an average of 225 g at 213 days post‐fertilization with specific growth rates ranging from 2.37 to 2.88. Per cent survival of fish challenged with F. psychrophilum was between 18% and 100%, while for those challenged with IHNV, the range was between 12% and 93%. Significant positive correlations were found for end body weight and resistance to IHN (P < 0.05) and for early body weight and resistance to BCWD (P < 0.1). However, no significant correlations were detected between resistance to both pathogens or disease resistance and overall genetic diversity or diversity within the major histocompatibility locus.     

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.     

Immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), with a low molecular mass fraction isolated from Flavobacterium psychrophilum

Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome has become a widespread fish pathogen in freshwater aquaculture worldwide. In this study, a low molecular mass fraction (P25-33), with an approximate weight of 25-33 kDa, was identified among F. psychrophilum strains in an immunoblotting analysis with anti-F. psychrophilum sera. The immunogenic efficacy of the isolated and extracted P25-33 was investigated in two intraperitoneal immunization trials with rainbow trout, Oncorhynchus mykiss (Walbaum). The first trial included immunizations using P25-33 with Freund's complete adjuvant (FCA) and the second trial included immunizations using P25-33, formalin-inactivated whole and sonicated F. psychrophilum cell preparations without FCA. In both trials, antibody titres against F. psychrophilum were analysed with an enzyme-linked immunosorbent assay and the efficacy of the immunizations was determined by a challenge with F. psychrophilum. The P25-33 was shown to give rise to a protective immune response in rainbow trout after immunization with FCA, but not without FCA when a low concentration of P25-33 was used. Instead formalin-inactivated whole and sonicated cells of F. psychrophilum were able to protect the immunized fish more effectively when immunized without FCA. The results suggest that whole or sonicated F. psychrophilum cells could be better candidates for a cost-effective water-based injection vaccine than the immunogenic fraction.