Temperature-dependent protection against Ichthyophthirius multifiliis following immunisation of rainbow trout using live theronts

Rainbow trout Oncorhynchus mykiss Walbaum, 1792 fingerlings were vaccinated by intraperitoneal (i.p.) injection using live theronts of the skin parasitic ciliate Ichthyophthirius multifiliis Fouquet, 1876 at 2 temperatures (12 and 20 degrees C), and protection against challenge infections was subsequently evaluated by bath exposure to live theronts. Vaccination conferred a relative protection (evaluated as the decrease in the number of established theronts) at 12 degrees C and almost complete immunity at 20 degrees C. Significantly increased immobilisation titers (using plasma immobilisation of live theronts) were found in immunised fish at Week 2 and 4 post-vaccination. Lysozyme activity of plasma from vaccinated fish increased from Week 1 to 4. Both immobilisation titers and lysozyme activity were significantly higher at 20 degrees C. This study demonstrated that live theronts are good candidates for an antigen source for development of effective vaccines against white spot disease in this fish host, and further indicated that the protection of rainbow trout against I. multifiliis infection is highly temperature dependent and may be associated with both adaptive and innate response mechanisms.     

Acquired resistance in rainbow trout against Gyrodactylus derjavini

Investigations were conducted on the host response in rainbow trout and the associated changes in mucous cell density during infection with the skin monogenean Gyrodactylus derjavini. Parasite populations increased on all naive hosts and peaked 4-5 weeks p.i. after which infection levels decreased. Introduction of naive fish into responding host populations resulted in heavy infections of the naive fish, whereas parasite expulsion continued in the responding host groups showing an acquired, non-sterile immunity. This non-sterile immunity lasted at least a month as these hosts were refractory to reinfection despite being exposed to a high infection pressure. Mucous cell hyperplasia was seen in some groups during the intermediary phase of infection, but at the termination of the study a significant depletion was evident. Passive immunization of naive host (with sera from immune hosts) did not confer protection. This indicates differences between host responses to G. derjavini compared to responses against other pathogens where such a passive immunity has been described.     

Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: molecular and immunohistochemical studies

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish induces a protective immune response in rainbow trout (Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response and indicating interactions between the systemic and local responses. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IL-8, IL-22 and IFN-γ were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, IgM, CD4, CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of gill filaments and in hyperplastic gill tissue but following infection a clear efflux of these cells was detected. MHC II positive cells were distributed across the gill filaments and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in gill arterioles and the lamellar capillaries. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) and humoral elements such as immunoglobulins IgT and IgM which are orchestrated by cytokines in gill tissue reacting against I. multifiliis.     

Protective immunity of Nile tilapia against Ichthyophthirius multifiliis post-immunization with live theronts and sonicated trophonts

Two immunization trials were conducted to evaluate host protection of Nile tilapia, Oreochromis niloticus against Ichthyophthirius multifiliis (Ich). Immunizations were done with live theronts or sonicated trophonts by bath immersion and intraperitoneal (IP) injection. The immunized fish were challenged with theronts 21 days post-immunization in trial I and 180 days post-immunization in trial II. The serum anti-Ich antibody and cumulative mortalities of tilapia were determined after theront challenge. Serum anti-Ich antibody was significantly higher (P<0.05) in tilapia immunized with live theronts by immersion or IP injection or with sonicated trophonts administered by IP injection than tilapia immunized with sonicated trophonts by immersion, with bovine serum albumin by IP injection, or non-immunized controls. Host protection was acquired in fish immunized with live theronts by immersion or IP injection. Tilapia immunized with sonicated trophonts by IP injection were partially protected with a 57-77% survival in both trials. At 180 days post-immunization, serum antibody titers had declined in immunized fish yet they were still able to survive challenge. The protection appears not to be solely depending on serum antibody response against Ich.     

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.     

Cross-immunity and antibody responses to different immobilisation serotypes of Ichthyophthirius multifiliis

Vaccination of channel catfish with either of two serotypes of the parasitic ciliate Ichthyophthirius multifiliis conferred protection against challenge infection by either serotype. Fish were vaccinated by intracoelomic injection with live theronts of isolate G5 (serotype D) or isolate G12 (a new serotype), which express different surface immobilisation antigens. Vaccination with live G12 theronts conferred complete protection against subsequent challenge by both serotypes while vaccination with G5 theronts elicited only partial protection against both serotypes. Vaccination with trophont lysates did not protect against challenge infection. Sera from vaccinated fish were tested in immobilisation assays, ELISAs, and Western blots. Serum antibodies recognised only immobilisation antigens of the serotype used for vaccination in immobilisation assays or on Western blots. No antigens common to both serotypes were identified by Western blots. In contrast, serum antibodies bound antigens in cell lysates from both serotypes by ELISA, demonstrating that antibodies recognising both serotypes are produced in response to infection, which presumably confer observed cross-serotype protection.     

卵形鲳鯵对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫(Cryptocaryon irritans)的幼虫对卵形鲳鯵(Trachinotus ovatus)进行腹腔注射和体表感染,然后每隔一周用阻动试验(Immobilization assay)检测免疫鱼的抗血清和皮肤培养液对刺激隐核虫幼虫的阻动效价,在第14周中,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示:两种免疫方法都能让卵形鲳鯵的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异:两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤黏膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是黏膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。       

卵形鲳鲹对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫（Cryptocaryon irritans）的幼虫对卵形鲳鲹（Trachinotus ovatus）进行腹腔注射和体表感染,然后每隔1周用阻动试验（Immobilization assay）检测免疫鱼的抗血清和皮肤培养液对激刺隐核虫幼虫的阻动效价,在第14周,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示：两种免疫方法都能让卵形鲳鲹的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异：两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤粘膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是粘膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。     

Lectins in fish skin: do they play a role in host-monogenean interactions?

Mucus samples from rainbow trout skin with or without infections by Gyrodactylus derjavini were tested for the presence of lectins reacting with mannose, galactose and lactose. The samples inhibited the binding of biotinylated lectins (from Canavalia ensiformis, Artocarpus integrifolia and Erythrina corallodendron, respectively) to microtitre plates with covalently bound carbohydrates (mannopyranoside, galactopyranoside and lactose, respectively). However, the inhibition of C. ensiformis and A. integrifolia lectins was slightly greater when mucus from infected (but recovering) fish was used, suggesting an increase of mannose and galactose binding lectins in fish skin exposed to parasites. As mannose, galactose and lactose are present on the glycocalyx of Gyrodactylus derjavini, it is suggested that lectins could play a dual role in interactions between fish hosts and their monogenean parasites. Thus, recognition between parasite and host and also host responses towards parasite infections could both, at least partly, involve carbohydrate-lectin binding.     

Putative immune response of rainbow trout, Salmo gairdneri, to Diplostomum spathaceum infections

The immune response of rainbow trout to infection with the cercariae of the digenean parasite Diplostomum spathaceum was investigated. Rainbow trout infected with cercariae at weekly intervals, or injected with a suspension of dead cercariae, did not produce a specific humoral response against D. spathaceum cercariae, as tested using immunoperoxidase, agglutination and cytotoxicity assays. However, a significant difference occurred in the rate of infection of rainbow trout given weekly exposure to cercariae of D. spathaceum in winter and summer. Rainbow trout injected with a suspension of dead cercariae acquired significantly fewer metacercariae than controls when exposed to a challenge infection. This suggested a specific immune response by the host and is the first example of a reduction in the infection rate of rainbow trout immunized against a digenean parasite, when exposed to a challenge infection.     

Comparison of serum antibody responses and host protection against parasite Ichthyophthirius multifiliis between channel catfish and channel × blue hybrid catfish

There is limited information available on the immune protection of channel catfish Ictalurus punctatus × blue catfish I. furcatus (CB) hybrid against the fish parasite Ichthyophthirius multifiliis (Ich). The objective of this study was to compare serum antibody response and host protection between channel catfish and CB hybrid catfish using a cohabitation model. Channel catfish and CB hybrid catfish were immunized with live theronts by immersion or by IP injection at the dose of 10,000–20,000 theronts per fish in two trials. The fish were then challenged with theronts to compare serum antibody response and protection against the parasite between channel catfish and CB hybrid catfish. The immunized channel catfish and CB hybrid catfish showed a significantly higher (p < 0.05) serum anti-Ich antibody (titer > 1120) compared to non-immunized controls (titer = 0). After being challenged with live theronts, the immunized channel catfish and CB hybrid catfish had none or a low number of the parasites (<50 trophonts per fish) and showed a significantly higher (p < 0.05) survival (90–100%) than non-immunized controls (0%). Overall results indicated that there was no statistical (p > 0.05) difference on serum anti-Ich antibody, parasite infection and fish survival between immunized channel catfish and CB hybrid catfish.     

Differences in virulence between two serotypes of Ichthyophthirius multifiliis

Naive channel catfish Ictalurus punctatus were infected by 2 isolates of the parasitic ciliate Ichthyophthirius multifiliis that differed in virulence. The isolates, NY1 and G5, Serotypes A and D, respectively, express different surface immobilization-antigens. The virulence of the 2 isolates was compared using tail-fin infections to quantitate parasite numbers and by analysis of the survival of infected fish. Although NY1 infected fish at a lower level than G5, all NY1-infected fish died, but 51% of G5-infected fish survived. The greater virulence of NY1 is apparently a consequence of its shorter life cycle, which results in overwhelming reinfection of fish before they can develop a protective immune response. This report represents the first experimental evidence for differences in virulence between serotypes of I. multifiliis.     

Ichthyophthiriasis in carp Cyprinus carpio: infectivity of trophonts prematurely exiting both the immune and non-immune host.

Ichthyophthirius multifiliis exposed to naturally immunised carp established short-term infections, the majority of parasites actively emerging within 2 h of entering the epidermis. A small, but significant, number of these expelled parasites were shown to retain theront-like properties with the capacity to directly re-invade a further fish host. Infectivity fell rapidly with time in the host and was comparable to that of trophonts of a similar age artificially induced to emerge from non-immune hosts with the aid of MEM (minimal essential medium). Trophonts recovered with MEM from immune carp 2 to 8 h post infection rarely established infections upon exposure to susceptible new hosts and no infections resulted from older trophonts recovered after 8 to 24 h exposure; older trophonts, however, represented only a small percentage of the original parasite population. A low level of infectivity was recorded in trophonts collected with the aid of MEM from non-immune carp after up to 24 h of infection. The results are discussed in relation to theront transformation and evasion of the host immune response.     

Evaluation of the protective immunogenicity of the N, P, M, NV and G proteins of infectious hematopoietic necrosis virus in rainbow trout oncorhynchus mykiss using DNA vaccines.

The protective immunogenicity of the nucleoprotein (N), phosphoprotein (P), matrix protein (M), non-virion protein (NV) and glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV) was assessed in rainbow trout using DNA vaccine technology. DNA vaccines were produced by amplifying and cloning the viral genes in the plasmid pCDNA 3.1. The protective immunity elicited by each vaccine was evaluated through survival of immunized fry after challenge with live virus. Neutralizing antibody titers were also determined in vaccinated rainbow trout Oncorhynchus mykiss fry (mean weight 2 g) and 150 g sockeye salmon Oncorhynchus nerka. The serum from the 150 g fish was also used in passive immunization studies with naive fry. Our results showed that neither the internal structural proteins (N, P and M) nor the NV protein of IHNV induced protective immunity in fry or neutralizing antibodies in fry and 150 g fish when expressed by a DNA vaccine construct. The G protein, however, did confer significant protection in fry up to 80 d post-immunization and induced protective neutralizing antibodies. We are currently investigating the role of different arms of the fish immune system that contribute to the high level of protection against IHNV seen in vaccinated fish.     

The auxotrophic aroA mutant of Aeromonas hydrophila as a live attenuated vaccine against A. salmonicida infections in rainbow trout (Oncorhynchus mykiss)

An auxotrophic aroA mutant of the Aeromonas hydrophila AG2 strain is a live attenuated vaccine against A. hydrophila infection in rainbow trout (Oncorhynchus mykiss). The protection conferred by the live attenuated vaccine against A. salmonicida strains is reported here, and several parameters of the specific and non-specific immune response in vaccinated trout were characterised. Vaccination with a dose of 10(7)cells/fish of the aroA mutant elicited significant protection against the Hooke and DK30 strains of A. salmonicida (relative percent survival RPS >60%). This cross-protection correlated moderately with the activation of the humoral and cellular specific immune responses, which show cross-reactivity against antigens shared by the two bacterial species, and a moderate increase in the lysozyme and antiprotease activities in the serum of vaccinated trout.     

Parasite Infections in Danish Trout Farms

Samples from 5 Danish freshwater trout farms rearing rainbow trout (Oncorhynchus my kiss) were examined for parasite infections from October 1993 until November 1994 and recorded parasites are listed. In addition, results from an examination of a mariculture net cage system are presented as well. A total of 10 metazoan and 10 protozoan parasites were recorded. The metazoans included Gyrodactylus derjavini, Gyrodactylus salaris, Eubothrium crassum, Triaenophorus nodulosus, Proteocephalus sp., Diplostomum spathaceum, Tylodelphus clavata and Argulus foliaceus from the freshwater farms. The protozoans Hexamita salmonis, Ichthyobodo necator, Ichthyophthirius multifiliis, Apiosoma sp., Epistylis sp., Trichodina nigra, T. mutabilis, T. fultoni, Trichodinella epizootica, and an Ichthyophonus like intestinal parasite were also detected in the freshwater trout farms. Based on lectin binding studies, few fish were found positive for the myxosporean parasite PKX although no clinical cases were reported. In the mariculture system, Lepeophtheirus salmonis and Caligus elongatus were found.     

Effects of dietary cholesterol on antioxidant capacity,non-specific immune response,and resistance to Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss) fed soybean meal-based diets

This study evaluated the effects of dietary cholesterol on antioxidant capacity, non-specific immune response and resistance to Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss) fed soybean meal-based diets. Fish were fed diets supplemented with graded cholesterol levels (0 [control], 0.3, 0.6, 0.9, 1.2, and 1.5%) for nine weeks. The fish were then challenged by A. hydrophila and their survival rate recorded for the next week. Dietary cholesterol supplementation generally increased the serum and hepatic superoxide dismutase (SOD), glutathione-peroxidase (GSH-Px), catalase (CAT), and total antioxidant capacity (TAC) activities, but decreased the serum and hepatic malondialdehyde (MDA) contents. Further, the hepatic CAT and serum SOD, CAT, and TAC activities were significantly higher in fish fed diets supplemented with 0.9 or 1.2% cholesterol compared to those fed the control diet, whereas the serum and hepatic MDA contents were significantly lower. The respiratory burst activity, alternative complement activity, and hepatic lysozyme activity increased steadily when the supplemental cholesterol was increased by up to 1.2% and then declined with further addition. The serum lysozyme activity and phagocytic activity increased steadily with increasing dietary supplemental cholesterol level up to 0.9% and then declined with further addition. Dietary cholesterol supplementation generally enhanced the protection against A. hydrophila infection, and fish fed diets supplemented with 0.9 or 1.2% cholesterol exhibited the highest post-challenge survival rate. The results indicated that cholesterol may be under-supplied in rainbow trout fed soybean meal-based diets, and dietary cholesterol supplementation (0.9–1.2%) contributed to improved immune response and disease resistance of rainbow trout against A. hydrophila.     

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.     

Histopathology of Ichthyophthirim multifiliis infections in fishes

Histopathological changes involved in Ichthyophthirius multifiliis infections are described from a wide range of host fishes—carp, crucian carp hydrids, Carassius carassius × C. auralus , goldfish, eels, African catfish, Glorias lazera , several species of cichlid fishes and rainbow trout. Fish were obtained from diverse geographical regions: Israel, South Africa, Portugal and Venezuela, as well as from laboratory experimental infections. Observed histopathological changes could not demonstrate any interspecific differences, but individual fish or different groups of fish of the same species demonstrated wide variability in susceptibility to infection and consequently in the nature and severity of the histopathological response. Low to moderate primary infection and secondary reinfestations did not induce significant histopathological changes in the integument. The proliferative response of the integumentary epithelium appeared only following subsequent infections. Repeated heavy reinfections induced massive cellular necrosis and in very heavy infections extensive lysis of the epithelial tissue. Host-parasite interactions in the light of the histological findings are discussed.