Concomitant exposure of rainbow trout fry to Gyrodactylus derjavini and Flavobacterium psychrophilum: effects on infection and mortality of host

Although rainbow trout fry mortality syndrome caused by the bacterium Flavobacterium psychrophilum is widespread in fish farms it is difficult to reproduce infection of rainbow trout in the laboratory using immersion exposure with bacterial suspensions. It has therefore been speculated that ectoparasites could act as enhancers of bacterial infections under natural conditions. In the present study rainbow trout fry were exposed to infections with F. psychrophilum (immersion for 30 min or 10 h) alone, exposed to the ectoparasitic monogenean Gyrodactylus derjavini alone or exposed to both pathogens in combination. Infection levels and host mortality were subsequently monitored to elucidate if the ectoparasitic monogeneans could enhance infection of fish with the bacterium. Immersion of fish in bacterial suspensions alone did not result in infection. Only one fish became infected with the bacterium and this fish belonged to the combination exposure group. The parasite populations increased differently in the various groups and it was found that host mortality was correlated to gyrodactylid infection levels (r=0.94) but not to bacterial exposure. The results emphasise the pathogenicity of the parasite G. derjavini, the relative resistance of intact fish to direct exposure to F. psychrophilum but provide only a weak indication of a possible enhancement of bacterial invasion due to ectoparasitic infections. It cannot be excluded that higher parasite burdens and/or prolonged immersion (more than 10 h) in bacterial suspensions may result in bacterial invasion.     

The immune response during proliferative kidney disease in rainbow trout: a case history

Young-of-the-year rainbow trout clinically affected by proliferative kidney disease (PKD), a systemic protozoan infection, exhibit a massive proliferation of small lymphocytes in the posterior kidney and spleen, a decrease in erythrocyte packed cell volume, hemoglobin, and serum albumins and an increase in beta-globulins. Electronphotomicrographs of the causative agent indicate that it is engulfed within a macrophage which is surrounded by small lymphocytes. Fish having survived a clinical episode are completely refractory to subsequent infection. Their serum electrophoretic patterns are quite unremarkable.     

Effect of florfenicol on the immune response of rainbow trout (Oncorhynchus mykiss)

Florfenicol, a drug effective against several bacterial diseases of fish, was tested for possible immunomodulatory effects. The aim of the study was to follow the kinetics of the immune response after vaccination with simultaneous oral antibiotic treatment. The fish were immunised with a commercial oil-based divalent (furunculosis/vibriosis) vaccine and were simultaneously given oral antibiotic treatment. The specific immune response was monitored by analysing the levels of specific antibodies with ELISA. As an indicator of the non-specific immune response the phagocytic activity of circulating leucocytes was measured by a chemiluminescence assay. Total circulating leucocyte counts and differentials were also monitored. The disease resistance was evaluated by challenge tests at the end of the experiment. The results showed that florfenicol did not have any significant effect on antibody production and circulating leucocyte levels but caused a suppression in chemiluminescence response/phagocytic cell 5-6 weeks after vaccination. The survival after challenge was slightly suppressed by the florfenicol treatment. The RPS-value for the vaccinated group was 98% and for the florfenicol-treated group was 88%.     

Effect of sulphadiazine and trimethoprim on the immune response of rainbow trout (Oncorhynchus mykiss)

A combination of sulphadiazine and trimethoprim at a ratio of 5:1 (SDZ/TMP) was tested for possible immunomodulatory effects. The aim of the study was to follow the immune response after vaccination with simultaneous drug treatment. The fish were immunised with a commercial oil-based divalent (furunculosis/vibriosis) vaccine and were simultaneously given oral drug treatment. The specific immune response was monitored by analysing the levels of specific antibodies with ELISA. As indicators of the nonspecific immune response, the lysozyme activity of serum was measured and the phagocytic activity of circulating leucocytes was monitored by a chemiluminescence assay. Total circulating leucocyte counts and differentials were also monitored. The disease resistance was evaluated by challenge tests at the end of the experiment.The results indicate that SDZ/TMP at a ratio of 5:1 does not interfere negatively with the immune response in rainbow trout after vaccination. A slight stimulation in the antibody response as well as in the chemiluminescence response of circulating granulocytes was recorded in fish treated with the drugs in connection with vaccination. The drugs did not significantly affect the survival after challenge.     

The humoral immune response of rainbow trout (Salmo gairdneri) immunised by various regimes and preparations of Aeromonas salmonicida antigens

Antibody production in rainbow trout to extracellular antigens was investigated. The following antigen preparations and immunisation regimes were used: native extracellular products (ECP) in Freund's complete adjuvant (FCA), intraperitoneally (i.p.) with and without booster; formalinized ECP in FCA, i.p. with and without booster; washed, formalinized A. salmonicida cells in FCA, i.p., with booster; native ECP in saline, i.m., four weekly injections at two different doses, 45 micrograms and 6 micrograms each injection (after the protocol of Shieh, 1985). Using crossed normal rainbow trout serum, i.p., single injection (after the protocol of Sakai, 1985). Using crossed immunoelectrophoresis all antisera contained precipitating antibodies to three to five ECP components except that from fish immunised i.m. with 6 micrograms protein where antibodies were undetectable. In no case were specific antibodies to ECP protease or haemolysin detected. In a rabbit immunised with formalinized ECP in FCA under a similar regime to the rainbow trout, antibodies to at least 15 ECP components, including protease and haemolysin, were detected. The assumption of a specific immune response to the protease, at least in respect of antibody production, in recent reports of protection afforded by vaccines composed of either crude ECP or partially purified protease (Shieh, 1985) or partially purified protease inactivated by normal serum (Sakai, 1985) is not supported by the present findings.     

Identification of differentially expressed protective genes in liver of two rainbow trout strains

Since 1975, the rainbow trout strain BORN (Germany) has been bred in brackish water from a coastal form imported from Denmark. Accompanying phenotypic monitoring of the adapted BORN trout until now revealed that this selection strain manifested a generally elevated resistance towards high stress and pathogenic challenge including lower susceptibility towards Aeromonas salmonicida infections in comparison to other trout strains in local aqua farms. We focus on the elucidation of both, genetic background and immunological basis for the increased survivorship to infections. A first comparison of gene expression profiles in liver tissue of healthy rainbow trout from the local selection strain BORN and imported trout using a GRASP 16K cDNA microarray revealed six differentially expressed genes evoking pathogen and wounding responses, LEAP2A (encoding for liver-expressed antimicrobial peptide), SERPINA1 (alpha-1 antitrypsin), FTH1 (middle subunit of ferritin), FGL2 (fibroleukin), CLEC4E (macrophage-inducible C-type lectin), and SERPINF2 (alpha-2 antiplasmin). Since the latter gene is not described in salmonid species so far, our first aim was to characterize the respective sequence in rainbow trout. Two trout SERPINF2 genes were identified, which share only 48% identical amino acid residues and a characteristic SERPIN domain. Second, we aimed to analyse the expression of those genes after temperature challenge (8 °C and 23 °C). Only FTH1 was upregulated in BORN and import trout after increase of temperature, while SERPINA1 and FGL2 were only elevated in import trout. Third, the expression of all named genes was analyzed after pathogen challenge with A. salmonicida subsp. salmonicida. As a main finding, we detected a comparably faster regeneration of LEAP2A mRNA abundance in BORN trout following bacterial infection. Ingenuity Pathways Analysis suggested a functional interplay among the mentioned factors and the pro-inflammatory cytokine TNF, whose stronger expression was validated in liver of BORN trout. This data indicate that the examined genes contribute to an improved first barrier against invading pathogens in BORN trout.     

Activation of rainbow trout complement by C-reactive protein

The activation of the complement system of rainbow trout by trout C-reactive protein (CRP) was investigated. Complement fixation tests were performed by using rabbit hemolysin-sensitized sheep erythrocytes and rainbow trout complement. Purified CRP increased the consumption of complement in the presence of Streptococcus pneumoniae C-polysaccharide (CPS), indicating the activation of the complement system. In contrast to this, acute phase serum activated the complement in the absence of CPS. Consumption of the complement by acute-phase serum was depressed when CRP was removed from acute-phase serum by CPS-sepharose 4B affinity chromatography. The acute-phase serum, as well as CRP plus CPS, suppressed in vitro growth of Vibrio anguillarum in the presence of complement, and enhanced the phagocytosis of the bacteria by glass-adherent peritoneal exudate cells. These results indicated that CRP has a role in host defense during acute-phase response through the activation of the complement system, enhancement of phagocytosis, and suppression of bacterial growth.     

Interactions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with immune responses of rainbow trout

Chlorinated dioxins, as typified by the most potent isomer, TCDD, are immunosuppressive in mammalian species and can enhance the susceptibility to a number of diseases. In recent years chlorinated dioxins have been detected in fish in many freshwater and marine habitats. Thus far, the effects of these chemicals on the immune responses of fish have not been examined. We studied the influence of TCDD on the defense mechanisms of rainbow trout. Yearling trout were injected intraperitoneally with the vehicle, 0.1, 1.0 or 10 micrograms/kg of TCDD. Interactions with the humoral immune response to sheep red blood cells (SRBC) were assessed by the Jerne plaque assay using head kidney and spleen leukocytes. Serum antibody was measured by complement-mediated lysis of SRBC in a chromium release assay. Effects of TCDD on the cellular immune responses were evaluated by the response of thymic and splenic lymphocytes to Con A and PWM. In addition, the phagocytic activity of peritoneal macrophages was examined in vitro. Trout which received 0.1 or 1.0 micrograms/kg TCDD remained clinically normal, and defense mechanisms were unaltered in these fish. Trout which received 10 micrograms/kg of TCDD became hypophagic and exhibited fin necrosis, ascites and suppression of hematopoiesis. In this treatment group, Con A-induced blastogenesis of thymic and splenic lymphocytes was not significantly changed, however, suppression of the PWM-induced response of splenic lymphocytes occurred. No statistically significant alterations occurred in humoral immune responses, and phagocytic activity of peritoneal macrophages was not decreased. The dose-response curve for various biologic effects of TCDD in the rainbow trout appears different from that in sensitive mouse strains. The 30-day, single-dose, parenteral LD50 for TCDD in the C57BL mouse is 100 micrograms/kg, and TCDD suppresses both cell-mediated and humoral immune responses at 1-2 micrograms/kg in this mouse strain. In the rainbow trout, however, immunosuppression was evident only at doses of TCDD approaching the 80-day, single-dose, parenteral LD50 of 20 micrograms/kg.     

Contribution of food deprivation to the immune response in rainbow trout (Oncorhynchus mykiss) vaccinated against Cryptobia salmositica and Aeromonas salmonicida

The aims of the present study were to determine (a) the effectiveness of an attenuated live Cryptobia salmositica vaccine; (b) the effects of food deprivation on the immune response and its duration in rainbow trout (Oncorhynchus mykiss) immunised with a live C. salmositica vaccine or with a killed Aeromonas salmonicida vaccine. The fish were divided into three groups (I, II and III; 14 fish per group), those in Groups I and II were under food deprivation (0.40% of body weight), while Group III fish were fed to satiety. The study showed that the attenuated strain of C. salmositica did not cause anaemia and disease, and the fish were protected from clinical disease when they were challenged with virulent parasites. Parasitaemia in all fish vaccinated and challenged with virulent C. salmositica fluctuated and was relatively low; however, fish in Group III had higher parasitaemia than those in Groups I and II between weeks 8 and 14. The numbers of activated neutrophils increased [nitroblue tetrazolium (NBT) assay] after immunisation with both Cryptobia and Aeromonas vaccines and they remained high throughout the experiment. Antibody production (ELISA values) increased after vaccination and were slightly higher in Group III. ELISA titres against A. salmonicida increased after vaccination and decreased after 5 weeks. The titres increased again after the vaccinated fish were given booster, and they were higher than those in the first vaccinated fish.     

Immune responses in rainbow trout Oncorhynchus mykiss induced by a potential probiotic bacteria Lactobacillus rhamnosus JCM 1136

This study was undertaken to examine the effect of supplementing a suggested probiotic bacteria Lactobacillus rhamnosus JCM 1136 in feed on immune response and gut flora composition of rainbow trout Oncorhynchus mykiss. The probiotic bacteria were incorporated into a commercial feed to constitute two experimental diets containing either 10(9) or 10(11) colony forming unit of live bacteria/g of feed while a third diet without the bacterial supplement served as the control diet. The diets were offered to rainbow trout (75g average weight) in triplicate tanks for 30 days. Fish were sampled at 10, 20 and 30 days after commencement of the feeding trial to determine the proportion of the given probiont in the gut microflora composition and the nonspecific humoral and cellular immune responses on the 30th day. The relative proportion of the probiont increased with the feeding duration in the intestine, but not in the stomach. The proportion of L. rhamnosus in the stomach corresponded to the intake levels while no such relation existed in the intestine. The serum lysozyme and complement activities were significantly greater in fish fed the higher level of probiont compared with the control fish. The phagocytic activity of head kidney leucocytes also showed similar tendencies. These observations indicate the potential immuno-regulatory role of probiotic organisms in rainbow trout.     

Primary in vitro stimulation of antibody production by rainbow trout lymphocytes

Trinitrophenylated (TNP) forms of E. coli lipopolysaccharide (LPS) and keyhole limpet hemocyanin (KLH) were used to produce antigen specific plaque-forming cell (PFC) responses with rainbow trout (Salmo gairdneri) splenocytes from unprimed fish in vitro. The culture system that was developed is described and characterized with respect to the kinetics and dose responses for both the haptenated and unhaptenated forms of the carriers. The induction of the PFC response to TNP-LPS was inhibited with TNP-lysine. Exposure to graded levels of gamma-radiation demonstrated a low dose augmentation of the PFC response with both antigens. Antigen addition experiments reveal that both antigens appear to stimulate the same population of antibody-producing B lymphocytes.     

Morphological and molecular characterisation of Gyrodactylus salmonis (Platyhelminthes, Monogenea) isolates collected in Mexico from rainbow trout (Oncorhynchus mykiss Walbaum)

Gyrodactylus salmonis (Yin et Sproston, 1948) isolates collected from feral rainbow trout, Oncorhynchus mykiss (Walbaum) in Veracruz, southeastern Mexico are described. Morphological and molecular variation of these isolates to G. salmonis collected in Canada and the U.S.A. is characterised. Morphologically, the marginal hook sickles of Mexican isolates of G. salmonis closely resemble those of Canadian specimens - their shaft and hook regions align closely with one another; only features of the sickle base and a prominent bridge to the toe permit their separation. The 18S sequence determined from the Mexican specimens was identical to two variable regions of SSU rDNA obtained from a Canadian population of G. salmonis. Internal transcribed spacer (ITS) regions (spanning ITS1, 5.8S and ITS2) of Mexican isolates of G. salmonis are identical to ITS sequences of an American population of G. salmonis and to Gyrodactylus salvelini Kuusela, Zi?tara et Lumme, 2008 from Finland. Analyses of the ribosomal RNA gene of Mexican isolates of G. salmonis show 98-99% similarity to those of Gyrodactylus gobiensis Gl?ser, 1974, Gyrodactylus salaris Malmberg, 1957, and Gyrodactylus rutilensis Gl?ser, 1974. Mexican and American isolates of G. salmonis are 98% identical, as assessed by sequencing the mitochondrial cox1 gene. Oncorhynchus mykiss is one of the most widely-dispersed fish species in the world and has been shown to be an important vector for parasite/disease transmission. Considering that Mexican isolates of G. salmonis were collected well outside the native distribution range of all salmonid fish, we discuss the possibility that the parasites were translocated with their host through the aquacultural trade. In addition, this study includes a morphological review of Gyrodactylus species collected from rainbow trout and from other salmonid fish of the genus Oncorhynchus which occur throughout North America.     

Immunological activation of rainbow trout macrophages induced in vitro by sperm autoantibodies and factors derived from testis sensitised leucocytes

Young cultures of rainbow trout macrophages were found to significantly increase in diameter following addition of muramyl dipeptide or of supernatants from testis/FCA sensitised leucocytes incubated with spermatozoa, or a testis extract. However, such supernatants caused little migration inhibition or increase in phagocytosis of spermatozoa by macrophages, whereas opsonization of spermatozoa with anti-sperm serum caused an almost six-fold increase in phagocytosis. These results suggest an interplay between humoral and cell-mediated immune reactions upon macrophages during experimental autoimmunity to the testis in trout.     

Evaluation of the cell-mediated immune response in cattle induced by 2,4-dinitrochlorobenzene

A technique has been evolved and standardized for evaluating the cell-mediated immune (CMI) response in cattle induced by 2,4-dinitrochlorobenzene (DNCB). Six healthy cross-bred Jersey cows were used. The dose and the number of applications for primary sensitization and the challenge dose after the 14th day have been standardized. The gross and histological pictures of the CMI response shown by these cattle are described in detail and their usefulness in assessing the CMI response in various disease conditions including tumour is indicated.