Non-immunized Fish Research Articles

Protective effect of cutaneous antibody produced by channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius multifiliis Fouquet on cohabited non-immune catfish.

Fish which survive a sublethal ichthyophthiriasis acquire protective immunity against Ichthyophthirius multifiliis Fouquet (Ich). This study evaluated the protective effect of cutaneous antibody secreted by channel catfish, Ictalurus punctatus (Rafinesque), immune to Ich on cohabited non-immune catfish. Non-immune and immune fish controls were separately maintained and infected with theronts. The Ich infection was assessed by scoring 0, < 50, 50-100, and > 100 trophonts fish(-1) at 5 days post-infection. The results of infection showed that cohabited fish at the ratio of 15 non-immune to two immune fish had < 50 trophonts fish(-1). Eighty per cent of the cohabited fish at the ratio of 10 non-immune to two immune fish showed 0 or < 50 trophonts fish(-1). The 76% of control non-immune fish had more than 100 trophonts fish(-1). The control immune fish had 0 trophonts fish(-1). Anti-Ich antibody was detected using enzyme-linked immunosorbent assay in water samples taken from tanks containing immune fish after the water samples were concentrated 40-fold. The study suggests that immune fish cohabited with non-immune fish may protect non-immune fish against Ich infection.

Host site of activity and cytological effects of histone-like proteins on the parasitic dinoflagellate Amyloodinium ocellatum.

Histone-like proteins (HLPs) are broad-spectrum, endogenously produced antibiotics which we have isolated from tissues of rainbow trout Oncorhynchus mykiss and hybrid striped bass (Morone saxatilis male x M. chrysops female). Here, we show that HLP-1, which has high sequence homology to histone H2B, equally inhibited both young and mature trophonts of the important ectoparasite Amyloodinium ocellatum. In addition to direct killing of Amyloodinium trophonts, there was evidence that HLP-1 from both rainbow trout and hybrid striped bass caused severe developmental abnormalities, including delayed development, in both the parasitic trophont stage as well as the reproductive tomont stage. The deleterious effects of HLP-1 also were manifested in what appeared to be 'delayed mortality', where parasites of normal appearance would die later in development. Similar serious damage was also seen with calf histone H2B and the unrelated peptide antibiotic magainin 2. A comparison of the antibiotic activity in mucus versus epidermis compartments of the skin of hybrid striped bass suggested that the majority of antibiotic (including HLP-1) activity resided in the epidermis, although some activity was present in the mucus. These data suggest that normal, nonimmune fish skin contains potent defenses against protozoan ectoparasites and that the effects of these defenses may extend beyond their transient interactions with the parasites, which has important implications for this host-parasite relationship.

Isolation of salmon pancreas disease virus (SPDV) in cell culture and its ability to protect against infection by the ‘wild-type’ agent

A Scottish salmon pancreas disease virus (SPDV) has been isolated and its optimum growth conditions determined. Although several fish cell lines have been tested, successful culture was achieved only with CHSE-214 cells. Cytopathic effects were observed after 5 days. The highest virus titres, calculated by microtitration assay, were reached at 15°C. After 7–9 days post-inoculation, CHSE-214 cell supernatants contained between 107–108TCID50ml−1. The cultured isolate is chloroform- and pH3·0-sensitive, and virions are 50–60nm in diameter. These characteristics are similar to the Irish SPDV isolates. The culture isolate induced typical pancreas disease (PD) lesions in experimentally infected Atlantic salmon and convalescent fish were resistant to experimental infection with PD-infective kidney homogenates obtained by serial in vivo passages from a PD-infected farmed salmon (termed wild-type SPDV). Furthermore, fish immunised with the inactivated cultured virus were protected against a cohabitation challenge with the wild-type virus. Immunised fish sera showed virus-neutralising activity before challenge (7 weeks post-immunisation) and from 3–6 weeks post-challenge, when sera from non-immunised fish did not neutralise the virus. At 6 weeks post-cohabitation challenge, previously immunised fish had neutralising titres of up to 1:65. Following intraperitoneal (i.p.) challenge, immunised fish showed neutralising titres as high as 1:226 at 8 weeks post-challenge. Non-immunised fish injected i.p. with the wild-type virus developed serum-neutralising activity against the cultured isolate when sampled 8 weeks after infection, confirming an antigenic relationship between the wild-type and cultured virus. The results demonstrate that the tissue culture-adapted isolate of SPDV could be successfully used to protect against challenge by the wild-type virus and could therefore have potential use as an inactivated vaccine against PD.

Characterisation of mucosal and systemic immune responses in rainbow trout (Oncorhynchus mykiss) using surface plasmon resonance

Mucosal and systemic antibody production in rainbow trout, Oncorhynchus mykiss (Walbaum), was evaluated following different antigen delivery routes. A BIAcore instrument (Pharmacia) allowed direct detection of antibody–antigen interactions by surface plasmon resonance changes. These interactions were measured in real-time without secondary reagents or extraneous labels. Groups of rainbow trout were immunised with a hapten-carrier antigen consisting of fluorescein isothiocyanate (FITC) conjugated to keyhole limpet haemocyanin (KLH) or phosphate buffered saline (PBS) pH7·2. Antigens were administered intraperitoneally (i.p.) with or without Freund's complete adjuvant (FCA) or peranally (p.a.) directly to the gastrointestinal (GI) tract. Serum and mucosal anti-FITC responses were significantly (P< 0·05) higher in FITC–KLH/FCA groups, clearly showing that adjuvant incorporation enhances mucosal as well as sytemic immunity. Antigen uptake and processing in fish immunised p.a. and i.p. without adjuvant was much less efficient and resulted in relatively low levels of serum and mucosal antibody production. Interestingly, mucosal responses in these groups peaked prior to serum responses suggesting possible early stimulation of mucosal defences. Mucosal antibody production in fish receiving FITC–KLH/FCA correlated more closely with serum responses, indicating possible transfer of serum derived antibody to mucosal sites. Mucosal and serum responses were confirmed as immunoglobulin (Ig) by subsequent reactivity with an anti-trout serum IgM monoclonal antibody (1·14) passed over flow cells containing anti-FITC antibodies. Further analysis showed significantly lower (P< 0·05) reactivity of early mucus anti-FITC components (4 weeks post-immunisation) to 1·14. Purified serum and mucus Ig from non-immunised fish showed different protein banding patterns by SDS–PAGE under reducing conditions. Immunoblotting with 1·14 also showed weak reactivity to mucus Ig in control fish while reacting strongly to mucus Ig from immunised fish. These data suggest that early mucosal responses in trout may consist of heterogeneous forms of Ig differing in characteristics to serum Ig. BIAcore analysis in this context and as a means of measuring antibody response proved useful, and has the potential to become a valuable new tool in the study of fish immunology.

Antibacterial proteins in rainbow trout, Oncorhynchus mykiss

Antibacterial proteins are an important part of the innate immune system for all animals. They have been extensively studied in mammals, amphibians and invertebrates, but have received only scant attention in fish. Their expression and processing, however, provide a way of monitoring defence vigour during development or with seasonal changes in physiology. The aim of the present work was to identify and characterise antibacterial proteins in rainbow trout. In vitro analyses of extracts of the peripheral blood leucocytes, head kidney leucocytes and mucus from adult unstimulated (non-immune) fish showed marked antibacterial activity against Gram positive bacteria. Fractionation by ion exchange chromatography and RP-HPLC of head kidney extracts showed the presence of two forms of lysozyme but no constitutively expressed antimicrobial proteins of <10kDa. By contrast, chromatographic analyses of mucus revealed at least four antibacterial proteins. Two are conventional lysozymes, a third is an unusual lysozyme-like protein with a low isoelectric point, and the fourth is a highly hydrophobic, cationic peptide of c. 3kDa.

Evaluation of immune function in juvenile turbot Scophthalmus maximus (L.) exposed to sediments contaminated with polychlorinated biphenyls

Turbot of approximately 2·3g body weight were held in the laboratory on sediment which had been taken from a clean marine site, then either spiked with a set dose of polychlorinated biphenyls (PCBs) or left unspiked as the reference sediment. Serum antibody levels were measured at week 10 of exposure to the sediment (8 weeks after primary immunisation with Keyhole Limpet Haemocyanin [KLH] in solute) and again at week 18 of exposure (8 weeks after secondary immunisation with KLH in Freund's Complete Adjuvant [FCA]). All fish grew well and blood leucocyte counts were similar in all groups. There was no difference in anti-KLH antibody levels between the fish on the PCB-spiked sediment and those on the reference sediment. Assays for non-specific immune responses also failed to detect differences between fish on the PCB-spiked sediments and their respective controls on the reference sediment, whether immunised or non-immunised, with one exception, i.e. it was found that in non-immunised fish, adherent leucocytes from the PCB-exposed fish at week 10 (but not at week 18) showed significantly decreased production of intracellular superoxide anion (O2−) compared with their non-exposed controls. A more marked difference in the non-specific immune parameters occurred as a result of immunisation, irrespective of the type of sediment. Thus at week 10, intracellular O2−production was lower in the immunised fish than in their non-immunised controls. Extracellular hydrogen peroxide (H2O2) was also affected, the levels being higher in immunised fish than in non-immunised fish on the reference sediment. By week 18 the total serum protein and lysozyme levels were considerably lower in the KLH-immunised groups than in their controls. The results are discussed in relation to their relevance to fish in the wild; also to the complexity of the interactions which occur during the course of an immune reaction.

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. multifiliisinfections. 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.

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.

Acquired immunity to amyloodiniosis is associated with an antibody response.

The dinoflagellate Amyloodinium ocellatum, which causes amyloodiniosis or 'marine velvet disease', is one of the most serious ectoparasitic diseases plaguing warmwater marine fish culture worldwide. We report that tomato clownfish Amphiprion frenatus develop strong immunity to Amyloodinium ocellatum infection following repeated nonlethal challenges and that specific antibodies are associated with this response. Reaction of immune fish antisera against dinospore and trophont-derived antigens in Western blots indicated both shared and stage-specific antibody-antigen reactions. A mannan-binding-protein affinity column was used to isolate IgM-like antibody from A. frenatus serum. The reduced Ig consisted of one 70 kD heavy chain and one 32 kD light chain with an estimated molecular weight of 816 kD for the native molecule. Immunoglobulin (Ig) isolated from immune but not non-immune fish serum significantly inhibited parasite infectivity in vitro. An enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal rabbit antibody produced against affinity-purified A. frenatus Ig. Anti-Amyloodinium serum antibody was not always detectable in immune fish, although serum antibody titers in immune fish increased after repeated exposure to the parasite. These results suggest that there may be a localized antibody response in skin/gill epithelial tissue, although antibody was rarely detected in skin mucus.

Ｅｎｔｅｒｏｃｏｃｃｕｓ ｓｅｒｉｏｌｉｃｉｄａホルマリン不活化菌体の投与がブリの生体防御系に与える影響

In the present study, immunobiological activities in the serum and phagocytes of yellowtail Seriola quinqueradiata immunized with formalin-killed E. seriolicida (FKC) by intraperitoneal injection were investigated to elucidate the induced protective mechanisms. Immunized fish showed 66.7% survival, while no survivors were observed in the control group 4 weeks after the challenge. Agglutinating antibody levels, alternative complement pathway activity, antibacterial activity, opsonic activity of serum were also higher in immunized than in non-immunized fish. Phagocytic activity and intracellular bactericidal activity of macrophages did not show differences between the immunized and non-immunized fish.The results suggest that non-cellular defense activities could be important mechanisms of yellowtail in protection to E. seriolicida infection.

Immune-complex trapping in the splenic ellipsoids of rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss), immunised with horseradish peroxidase, were given horseradish peroxidase intravenously, and the trapping of antigen in the spleen was followed 1, 24, and 48 h after injection. After 1 h, the localisation of horseradish peroxidase indicated that the antigen had been extensively trapped in the walls of the splenic ellipsoids. The colocalization of horseradish peroxidase with rainbow trout immunoglobulin M and complement factor 3 was shown with a double immunofluorescence technique and suggested that horseradish peroxidase was trapped in the form of immune complexes. After 24 and 48 h, very little horseradish peroxidase was detected in the ellipsoids, and horseradish peroxidase was mainly found in association with large cells with prominent cytoplasmic extensions. In non-immunized fish given horseradish peroxidase intravenously, antigen was not detected in ellipsoids. Thus, the observed difference between immunised and non-immunized trout suggests a specific role for the splenic ellipsoids in rapid immune-complex trapping and invites speculation on its significance in a secondary immune response.

Enteric coated microspheres as an oral method for antigen delivery to salmonids

A comparative study was conducted to determine the immunisation efficacy of enteric coated antigen microspheres (ECAMs) versus intraperitoneal (i.p.) and immersion antigen delivery. Anal intubation of antigen served as a positive control by directly delivering the antigen to the hindgut. Naive juvenile coho salmon Oncorhynchus kisutch were exposed to three different concentrations of trinitrophenylated lipopolysaccharide (TNP-LPS) or trinitrophenylated keyhole limpet haemocyanin (TNP-KLH) by the three routes, oral (ECAM), intraperitoneal, immersion or anal intubation. TNP-LPS or TNP-KLH were prepared for oral immunisation by coating onto 0·45 mm dextrose sugar beads followed by an application of Eurdragit LD-30 co-polymer. Serum was taken from immunised and non-immunised control fish fed diet containing nonantigen coated beads. The serum was assayed by ELISA at 4, 6 and 8 weeks post-immunisation to assess anti-TNP antibody titres. Statistical analysis of the serum showed that oral vaccination significantly increased anti-TNP titres at 4 and 6 weeks post-vaccination as compared with the controls (P<0·05). The anti-TNP titres after TNP-LPS i.p. immunisation were significantly higher than the controls (P<0·05) at 4, 6 and 8 weeks post immunisation, while the group that was immersed showed no significant difference from the control. There was no significant difference between TNP-LPS i.p. and ECAM immunisation methods.The TNP-KLH immunised ECAM group showed significantly elevated levels of anti-TNP antibodies compared with controls at 6 and 8 weeks, while the i.p. immunised group was significantly higher than the control throughout the 8-week period. The group immunised by anal intubation had significantly higher titres than controls at 6 and 8 weeks but not at 4 weeks.

Acquired protection in Atlantic salmon Salmo salar parr and post-smolts against pancreas disease

Experimental infection of Atlantic salmon Salrno salar parr and post-smolts with pancreas disease infective kidney homogenate induced a highly significant protection against the disease following exposure to challenge infections at either 3 , 4 , 7 or 9 mo after the initial infection. Protection was maintamed in fish infected as parr and exposed to a challenge infection following smolting and sea water transfer. A carrier status was not demonstrated in immune fish since homogenates of kidneys taken 1 and 2 wk post-challenge were not infective, unlike homogenates of kidneys from non-immune fish.

Antibacterial activity in the serum and mucus of rainbow trout, Oncorhynchus mykiss, following immunisation with Aeromonas salmonicida

Intraperitoneal injection of either sterile saline or formalin-inactivated Aeromonas salmonicida caused an increase in serum lysozyme in the rainbow trout, Oncorhynchus mykiss that peaked at c. 10 days post-challenge, Significantly larger amounts of lysozyme were found in fish challenged with A. salmonicida than saline alone. Both the serum and mucus from fish vaccinated 21 days previously with formalin-inactivated A. salmonicida showed significantly reduced growth of A. salmonicida, A. punctata and Serratia marcescens in vitro compared with that from saline-injected fish. There was no significant difference, however, between the growth rates of A. hydrophila and Escherichia coli following exposure to either serum or mucus from immunised or non-immunised fish. These data suggest that a number of antibacterial factors increase in concentration following immunisation and that these probably play a role in protection against microbial disease.

Ichthyophthirius multifiliis (Fouquet): survival within immune juvenile carp, Cyprinus carpio L.

Juvenile carp were immunised against Ichthyophthirius multifiliis by controlled exposure to theronts. Immediately following a potentially lethal challenge, immunised and non-immunised fish were injected intraperitoneally with either the corticosteroid triamcinolone acetonide (200μg g -1 body weight), saline or left untreated. More fish were similarly treated 1, 2, 3 or 4 days after challenge. Untreated immunised fish did not develop an infection, whereas 100% infection was recorded in untreated non-immunised fish and immunised and non-immunised fish treated with corticosteroid. Infection intensities decreased progressively in immunised fish treated with corticosteroid 1-4 days after challenge. Results indicate that theronts can enter immune fish which are immunosuppressed directly after challenge suggesting that pre-existing antibodies may only play a partial role in resistance. Immune suppression by corticosteroid 4 days after challenging immune fish did not lead to establishment of infection indicating that the cell mediated immune response effectively killed invading theronts within 4 days of entering the host.

Effects of Dietary Zinc and Calcium on Select Immune Functions of Channel Catfish

Abstract The effects of dietary zinc and calcium on immunocompetence of channel catfish Ictalurus punctatus (mean initial weight = 3.1 g/fish) were determined after a 10-week feeding trial. Fish were fed daily amounts equal to 3% of their body weight of one of six purified diets that were deficient in zinc (2 mg/kg of diet), calcium (0.02% of diet), or both; replete with zinc (20 mg/kg of diet) and calcium (0.5% of diet); or excessive in zinc (200 mg/kg of diet) or calcium (2.5% of diet). Immunocompetence of immunized and nonimmunized fish fed the various diets was assessed by measuring serum immunoglobulin M levels and mortality after an intraperitoneal injection of live Aeromonas hydrophila. Channel catfish fed diets deficient in zinc, calcium, or both had significantly (P ≤ 0.05) less weight gain and survival as compared with those fed diets replete with or excessive in zinc or calcium. Tissue mineral concentrations of fish indicated that various levels of zinc and calcium nutriture existed when immune...

In vitro stimulation of salmonid leucocytes with mitogens and with Aeromonas salmonicida

Leucocytes from peripheral blood (PBL), spleen and head kidney of Atlantic salmon ( Salmo salar ) and rainbow trout ( Oncorhynchus mykiss ) responded well when stimulated with lipopolysaccharide from Escherichia coli and phytohaemagglutinin (PHA). Thymocytes responded to PHA only. PBL from Atlantic salmon vaccinated against furunculosis showed significantly higher responses as compared to non-vaccinated fish when stimulated with an antigen preparation of whole cells of Aeromonas salmonicida . In leucocyte cultures from rainbow trout vaccinated against furunculosis, specific antibodies to A. salmonicida were produced in vitro . None or very low antibody levels were detected in cultures from non-immunised fish during the first 7 days in culture. After 9 days, a primary antibody response to A. salmonicida could, however, be detected in these cultures. Good correlation ( r =0·88) between antibody production by blood leucocytes and splenocytes was seen.

Influence of Dietary Lipid and Temperature on Bactericidal Activity of Channel Catfish Macrophages

Abstract We fed juvenile channel catfish Ictalurus punctatus purified diets containing different lipid sources for 90–110 d to examine the influence of dietary lipids on intracellular killing of Edwardsiella ictaluri by pronephros macrophages. The diets contained either menhaden oil, soybean oil, beef tallow, or a combination ofall three lipids. We performed the study with nonimmune and immunized fish acclimated to optimal (28°C) and suboptimal (19°C) temperatures. In both temperature trials, bactericidal activity was positively correlated with the level of n-3 fatty acids in the diet (particularly long-chain highly unsaturated fatty acids). Vaccination resulted in substantially enhanced bactericidal activity, except at suboptimal temperature in fish fed the diet containing beef tallow (primarily saturated and monoenoic fatty acids). Overall results were similar at optimal and suboptimal temperatures. This study demonstrates the potential for nutritional manipulation of disease resistance in fish and illu...

Humoral Immune Response of White-Spotted Char Salvelinus leucomaenis to Aeromonas salmonicida Extracellular Products.

White-spotted char Salvelinus leucomaenis were immunized intramuscularly with Aeromonas salmonicida extracellular products (ECP). By immunization two or three times at four week intervals, the serum agglutinating antibody titers at twelve weeks after the first immunization were raised ranging from 1:128 to 1:512 or from 1:512 to 1:4, 096 respectively. Disc electrophoretic analysis did not show any difference in the protein composition between im-munized and non-immunized fish sera. By immunodiffusion analysis, char anti-ECP sera formed a single precipitating line against ECP and salmolysin. The component protein of the char antisera, which reacted to ECP, also formed a single precipitating line against ECP in β to γ-globulin region by immunoelectrophoresis. The antiserum was separated into three frac-tions by gel filtration on Sepharose 6B, and antibody activity was found in early eluate of the second peak with Kav of 0.4 to 0.6. The results indicate that salmolysin, which is included in ECP, possesses high immunogenicity to white-spotted char.

Dietary influences on disease resistance factors in channel catfish

The effects of diet on selected disease resistance factors were studied in channel catfish ( Ictalurus punctatus). Two commercial feeds and a “control”, laboratory-prepared diet were compared. Macrophage function (phagocytosis and intracellular killing) was used to assess nonspecific disease resistance and serum antibody was measured as an indication of specific immunity. We measured the immune response to Edwardsiella ictaluri, a common bacterial pathogen of catfish, as well as the phagocytosis and killing of the bacteria. In the initial experiment fish were maintained on the experimental diets for 116 days, vaccinated and responses assayed 14 days later. Significant differences among the groups were observed in the phagocytic index as well as in circulating antibody. An additional study showed that even when fed the experimental diets for only 42 days there were significant differences in the ability of macrophages from both immunized and nonimmunized fish to kill E. ictaluri.