Partial cross protection against Ichthyophthirius multifiliis in Gyrodactylus derjavini immunized rainbow trout.

Partial cross protection against a skin-parasitic ciliate has been recorded in rainbow trout previously immunized with an ectoparasitic platyhelminth. The susceptibility to infection by Ichthyophthirius multifiliis differed significantly between naive and Gyrodactylus derjavini immunized rainbow trout. Fish partly immune to the ectoparasitic monogenean G. derjavini became less infected and experienced lower mortality than naive fish when exposed to I. multifiliis infections. In vitro studies on immobilization of theronts using decomplemented (heat-inactivated) serum from G. derjavini immune or non-immune hosts showed no immobilization. However, untreated serum from both immune and non-immune fish containing intact complement immobilized theronts (titre 128-256). In addition, non-specific priming of the host response with interleukin (IL-1), bacterial lipopolysaccharide (LPS), concanavalin A (Con A) or mannan did confer a partial resistance to I. multifiliis infection. This will suggest that non-specific factors including complement could be partly responsible for the host response against infections with this ciliate.     

Effect of Gyrodactylus derjavini infections on cortisol production in rainbow trout fry

Cortisol production in fry of rainbow trout (367-456 mg body weight) infected with the ectoparasitic monogenean Gyrodactylus derjavini (mean intensities 4.7 and 4.9 parasites per fish) was studied at two temperature levels, 4-6 degrees C and 11-12 degrees C, respectively. Due to difficulties in obtaining an adequate amount of plasma from the small sized fish, the corticosteroid concentration was measured in the body fluid recovered (as supernatant) after sonication and centrifugation of whole fry. Infected fry at 11-12 degrees C showed an elevated level of cortisol compared to uninfected fry. However, the cortisol concentration was lower than in fish exposed to handling stress. At 4 degrees C, the cortisol level in infected fish compared to uninfected was insignificantly increased. The findings are discussed in relation to the role of monogeneans as inducers of secondary infections.     

Rainbow trout leucocyte activity: influence on the ectoparasitic monogenean Gyrodactylus derjavini

The ectoparasitic monogenean Gyrodactylus derjavini from rainbow trout Oncorhynchus mykiss was exposed in vitro to macrophages isolated as peritoneal exudate cells or as pronephros cells from the host. Cells colonized the parasite especially in the mannose-rich regions in the cephalic ducts where ciliated structures were abundant. Opsonization with fresh serum, in contrast to heat-inactivated serum, enhanced colonization also on other body parts. The adverse effect of the activated macrophages towards G. derjavini was associated with a heat-labile component released from these cells to the culture medium. Analysis of substances released from the cells showed reactivity for a number of enzymes, complement factor C3, interleukin (Il-1) and reactive oxygen metabolites. Chemotaxis assays with pronephric leucocytes showed chemoattractants in G. derjavini, and the respiratory burst level of macrophages was slightly elevated due to parasite exposure. It is suggested that skin leucocytes contribute to an increased level of complement factors in the trout skin during the host response, whereby a hostile microenvironment for the parasites is created. In addition, the IL-1 production could affect mucous cell secretion and hyperplasia and add to the antiparasitic action of the epithelium. Likewise, reactive oxygen metabolites and various enzymes are likely to be involved in the skin response.     

Characterisation of a low pathogenic form of Gyrodactylus salaris from rainbow trout

Gyrodactylus salaris was isolated from rainbow trout in a Danish freshwater trout farm, and a laboratory population of this particular parasite form was established on rainbow trout. Challenge infections were performed using different salmonid strains and species, including East Atlantic salmon Salmo salar (from the Danish River Skjern?), Baltic salmon S. salar (from the Swedish River Ume Alv) and rainbow trout Oncorhynchus mykiss (from the Danish rainbow trout farm Fousing). These were compared to infection studies on the Norwegian Laerdalselva parasite form kept under exactly the same conditions in the laboratory. The Danish G. salaris form had low virulence towards both Atlantic and Baltic salmon, whereas rainbow trout proved susceptible to the parasite. The Danish G. salaris form was able to maintain a very low infection on East Atlantic salmon, but not on the Baltic salmon, which eliminated the infection within 2 wk. Rainbow trout developed infection intensities ranging up to several hundred parasites per host. The host colonization patterns of the parasite differed clearly from those of previous studies on microhabitats of the Norwegian form of G. salaris. A comparative study on morphological characters (opisthaptoral hard parts) from the Danish parasite form and Norwegian G. salaris showed no significant differences. Selected genes comprising internal transcribed spacers 1 and 2 (ITS), ribosomal RNA intergenic spacer (IGS) and cytochrome c oxidase subunit I (COI) regions were cloned and sequenced. Five sequenced ITS clones from 5 individuals of the Danish strain consistently revealed a single base substitution compared to ITS sequences from all other known species and strains of Gyrodactylus. Mitochondrial COI gene sequences demonstrated that the Danish G. salaris form is closely similar to the Laerdalselva parasite form found in Norway. The IGS sequences were highly variable, but very similar to those obtained from German isolates of G. salaris.     

In vitro interactions between epithelial cells and Gyrodactylus derjavini

Skin responses of fish to various parasites have been shown to involve various immunologically competent cells producing factors which guide the reactions of epithelial cells. However, the present study has demonstrated that a monoculture of epithelial cells has the ability to encapsulate and partially degrade ectoparasites without involvement of leukocytes. The ectoparasitic monogeneanGyrodactylus derjavini was kept on a monolayer of Epithelioma Papulosum Cyprini (EPC) cells in 24-well multidishes supplied with tissue culture medium. Gyrodactylus derjavini did not reproduce but survived an incubation period of up to139h in the system. Due to sterile conditions, dead gyrodactylids were not subjected to microbial degradation and remained intact for several weeks. However, at 40 days G. derjavini was overgrown by EPC-cells and became partly degraded during the following 15 days. Analysis of enzyme reactivity in EPC-cells showed reactions for ten enzymes including esterases, amidases, phosphatases and phosphohydrolases. No marked differences for the ten enzymes between cell cultures with and without the ectoparasites were found but it cannot be excluded that some of these enzymes took part in parasite degradation. The study showed the in vitro capability of epithelial cells to interact, encapsulate and degrade G. derjavini without the involvement of leukocytes. This response probably is non-specific and will not exclude that various immunocompetent cells and their products normally optimize and accelerate elimination of invading parasites in vivo.     

Dexamethasone treatment affects skin mucous cell density in Gyrodactylus derjavini infected Salmo salar

Atlantic salmon, Salmo salar, is normally rather refractive to infection with the ectoparasitic monogenean Gyrodactylus derjavini but dexamethasone treatment of the host increases the susceptibility. The causative mechanisms were elucidated in this work. Groups of Atlantic salmon were treated by intra-peritoneal dexamethasone injections and subsequently infected with G. derjavini. It was shown that both the infection level and the mucous cell density of caudal and pelvic fins were affected by the treatment. Significantly higher mucous cell densities were found on infected and treated fish whereas non-infected and treated fish showed no significant elevation of cell density. This suggests that mucous cell discharge elicited by infection is inhibited by the drug. The association with elevated parasite counts in these fish can be explained either by decreased anti-parasitic mucus action or by parasite predilection for intact mucous cells.     

Demonstrating freedom from Gyrodactylus salaris (Monogenea: Gyrodactylidae) in farmed rainbow trout Oncorhynchus mykiss

This paper describes an approach to demonstrate freedom of individual rainbow trout farms from Gyrodactylus salaris Malmberg, 1957. The infection status of individual farms is relevant should G. salaris be introduced into a country or zone previously known to be free of the parasite. Trade from farms where G. salaris may have been introduced would be restricted until freedom had been demonstrated. Cage, fish and parasite sample sizes were calculated based on the minimum detectable prevalence (P*), test characteristics, population size, and Type I and II errors. Between 5 and 23 cages per farm would need to be sampled to demonstrate freedom at a cage level P* of 10%. The number of fish sampled per cage depended mainly on the test sensitivity (probability of correctly identifying an infected fish). Assuming a test sensitivity of 99% at the fish level, 59 fish per cage are needed (P* = 5%). Since G. salaris may exist in mixed infection with G. derjavini, testing a sample of gyrodactylid parasites may not result in the parasite being detected when present. Test sensitivity at the fish level depends on the number of gyrodactylids on the fish, the proportion of which are G. salaris and the number examined. Assuming a P* of 5% (i.e. G. salaris are at least 5% of the gyrodactylid population), between 20 and 73 parasites per fish would need to be sampled (depending on abundance) to maintain the Type I error at 0.01 (thus a fish level test sensitivity of 99%). This work identifies the critical information, and further research, needed to assess freedom from G. salaris with a known level of confidence; this is essential to provide a sound scientific basis for decision-making about disease control measures.     

Thrombocyte aggregation in rainbow trout

• 1.1. Thrombocytes from mature rainbow trout (Sahmo gairdneri) aggregated in vitro after addition of ADP, ATP, collagen, epinephrine, 5-hydroxytryptamine or thrombin to thrombocyte-rich plasma.
• 2.2. Thrombocyte aggregation was dose dependent and could be inhibited by preincubating the thrombocyte-rich plasma with adenosine, acetylsalicylic acid or prostaglandin E₁.
• 3.3. Thrombocytes released ADP and ATP when aggregated by 10 μM epinephrine. This release was blocked by 1 mM adenosine.
• 4.4. Electron microscopic observations of thrombocytes revealed them to contain numerous microtubules and electron-dense vesicles. In addition a possible shape change associated with thrombocyte aggregation was noted.

     

Overwinter feeding in rainbow trout

The contents of the stomachs of 38 rainbow trout stocked in Llyn Alaw, Anglesey, in August 1969 and caught between October 1969 and February 1970 were analysed. The fish were actively feeding on the bottom fauna throughout the winter and 21 of the stomachs were full or distended. The mean volume of the contents of the stomachs was 2–8 times greater than that of the contents of stomachs of similarly sized brown trout caught at the same time.     

Spleen size predicts resistance of rainbow trout to Flavobacterium psychrophilum challenge

Selective breeding of animals for increased innate resistance offers an attractive strategy to control disease in agriculture. However, this approach is limited by an incomplete knowledge of the heritability, duration, and mechanism(s) of resistance, as well as the impact of selection on the immune response to unrelated pathogens. Herein, as part of a rainbow trout broodstock improvement program, we evaluated factors involved in resistance against a bacterial disease agent, Flavobacterium psychrophilum. In 2005, 71 full-sibling crosses, weighing an average of 2.4 g, were screened, and resistant and susceptible crosses were identified. Naive cohorts were evaluated at 10 and 800 g in size, and most maintained their original relative resistant or susceptible phenotypes, indicating that these traits were stable as size increased >300-fold. During the course of these studies, we observed that the normalized spleen weights of the resistant fish crosses were greater than those of the susceptible fish crosses. To test for direct association, we determined the spleen-somatic index of 103 fish crosses; created high, medium, and low spleen-index groups; and determined survival following challenge with F. psychrophilum or Yersinia ruckeri. Consistent with our previous observations, trout with larger spleen indices were significantly more resistant to F. psychrophilum challenge; however, this result was pathogen-specific, as there was no correlation of spleen size with survival following Y. ruckeri challenge. To our knowledge, this is the first report of a positive association between spleen size and disease resistance in a teleost fish. Further evaluation of spleen index as an indirect measure of disease resistance is warranted.     

Plasmid coding for transferable drug resistance in bacteria isolated from cultured rainbow trout

The occurrence of drug resistance and plasmid-mediated transferability was investigated in 170 strains belonging to eight bacterial groups isolated from cultured rainbow trout. It was found that 87.6% of the strains were resistant to at least one drug, with the highest percentages of resistance being detected for ampicillin (54.7%), sulfadiazine (46.5%), nitrofurantoin (38.2%), and chloramphenicol (37.0%). Six enterobacteria, two Vibrio, and one Aeromonas isolate transferred resistance factors to Escherichia coli K-12. The most common transmissible R factor determined resistance to chloramphenicol and sulfadiazine, demonstrating an association between a specific plasmid and the resistance pattern transferred. The presence of chloramphenicol in fish food was detected by bioassay. In general, transfer frequencies were similar in primary and secondary matings, which indicate the potential water-borne dissemination of these R plasmids.     

Increased susceptibility of Atlantic salmon Salmo salar to infections with Gyrodactylus derjavini induced by dexamethasone bath treatment

Dexamethasone, a known immunosuppressant, was administered by bath or injection to Atlantic salmon Salmo salar (Conon stock) to study if this treatment could affect the susceptibility of fish to infections with a Danish strain of Gyrodactylus derjavini (Monogenea). Three groups of S. salar (Conon stock) were immersion treated either with 10, 60 or 240 microg dexamethasone l-1 water, respectively. In addition, one group (positive control) was treated intraperitoneally with 200 microg dexamethasone per fish and one negative control group was kept untreated. A single G. derjavini parasite was placed on the anal fin of each fish and the infection was subsequently monitored weekly for 6 weeks. An increase in parasite populations on the salmon was positively correlated with the amount of immunosuppressant used. Infection levels in the group immersion treated with dexamethasone (240 microg l-1 water) and in the i.p. treated positive control group were significantly higher compared to the untreated control group.     

The probiotic potential against vibriosis of the indigenous microflora of rainbow trout

The antibacterial properties of the indigenous microflora of rainbow trout ( Oncorhynchus mykiss Walbaum) and the potential use of inhibitory bacteria as fish probiotics were investigated. A total of 1018 bacteria and yeasts were isolated on tryptone soy agar (TSA) from skin, gills and intestine. Forty-five of these inhibited growth of the fish pathogenic bacterium Vibrio anguillarum in a well diffusion assay. The antagonism was most prominent among Pseudomonas spp., as 28 (66%) of the antagonistic bacteria belonged to this genus, despite constituting only 15% of the total tested flora. As pseudomonads are typically siderophore producers, chrome azurol S (CAS) agar was used as a semi-selective medium for isolation of antagonistic bacteria. On this medium, 75% of the iron-chelating strains were inhibitory to V. anguillarum . Eight strains out of a subset of 11 antagonists caused a 3–6 log unit reduction in the density of V. anguillarum [measured by polymerase chain reaction (PCR) detection in a most probable number (MPN) regimen] in a broth co-culture assay. Survival of rainbow trout infected with vibriosis was improved 13–43% by six out of nine antagonistic strains tested in vivo. All disease-protecting strains were pseudomonads, isolated from CAS plates, whereas two Carnobacterium spp. that were antagonistic in in vitro well diffusion assays did not alter the accumulated mortality of rainbow trout. The addition of live bacterial cultures to fish-rearing water may thus improve survival of the fish; however, in vitro antagonism could not completely predict an in vivo effect. Further studies on the underlying mechanism of activity are required to design appropriate selection criteria for fish probiotic bacteria.     

Immunization of rainbow trout Oncorhynchus mykiss against Discocotyle sagiffata (Monogenea)

Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with 2 different Discocotyle sagittata extracts dissolved in PBS and subsequently exposed to controlled infection. Immunization resulted in significantly reduced (p < 0.0001) worm intensities in > 50% of vaccinated fish (response arbitrarily defined as parasite burdens < mean control intensity - 1 SD), irrespective of the immunization regime (different parasite extracts, dosing and application schedules) employed. The protective effect of worm extract applied in Freund's complete adjuvant (FCA) did not differ significantly from extract given in PBS. Vaccination with embryonated parasite eggs extract and with FCA alone did not result in partial immunity, suggesting the observed protective effect is specific. Immunized fish had significantly higher specific antibody titres at the time of dissection (as determined by ELISA) than both naive and control fish. Overall, a significant negative correlation was found between antibody titres and worm burdens, suggesting immunoglobulins are implicated in mediating partial immunity. Western blot tests indicated the 2 different worm extracts used to immunize fish share antigens, but each one primarily induced recognition of a distinct band (30 and 38 kDa). Immunization seems to promote a shift between 2 equilibria, rather than progressively increasing protection. This would explain why boosting did not increase immunity, and why 2 different extracts primarily inducing recognition of 2 distinct antigens provide similar degrees of protection. Although several other non-specific and cellular factors are likely to be involved in controlling parasite numbers, it cannot be excluded that antibodies could be involved in mediating the observed partial immunity.     

Priming of host resistance to protect cultured rainbow trout Oncorhynchus mykiss against eye flukes and parasite‐induced cataracts

In the present study, immunologically naive rainbow trout Oncorhynchus mykiss were experimentally exposed to a low‐level Diplostomum spathaceum (Trematoda) infection to stimulate acquired resistance and, along with unexposed controls, were subsequently exposed to natural infection for 8 weeks. The priming of the host resistance, designed to simulate a procedure applicable in aquaculture, decreased the number of establishing parasites compared to untreated controls by the end of the experiment. This effect was slow and did not protect the fish against the parasite‐induced cataracts. The results suggest that this type of priming of host resistance is probably inefficient in preventing the deleterious effects of D. spathaceum infection in aquaculture conditions.