Specific and natural antibody response of cod juveniles vaccinated against Vibrio anguillarum

The purpose of the present study was to study specific and natural antibody levels in individual cod juveniles before and after being vaccinated against Vibrio anguillarum. Different vaccine preparations and vaccination regimes, i.e. bathing, dipping, i.p. injection or combination of treatments were employed and the performance of different groups to bath challenge by the bacterium tested. Antibody responses to V. anguillarum antigens in groups vaccinated by bathing and/or dipping were negligible, while responses were observed in i.p. injected fish. Fish receiving i.p. injection in addition to bathing, showed significant antibody response. Both groups showed increased levels of natural antibodies while levels were low in other groups. Fish bathed or dipped showed higher mortality when challenged than untreated fish, while fish that received a second vaccination showed the best protection. It was not ascertained whether there is a long term difference between the effects of immersion versus i.p. injection as a booster method. Levels of antibodies against V. anguillarum antigens or natural antibodies in groups with the lowest mortalities show that neither could have been used to predict protection given by the vaccines tested.     

The immune responses of dace Leuciscus leuciscm (L.) to injected antigenic materials

The fundamental features of the primary responses of dace Leuciscus leuciscus (L.), a freshwater cyprinid, to a number of standard antigens—haemocyanin, bovine serum albumin, horse serum, a bacterial antigen and an erythrocyte suspension, were examined. The fish were capable of producing either precipitating or agglutinating antibody in response to each of these antigens. Determinations of latent periods of antibody production by dace over a range of temperatures −18, 10, 5 and 2°C, showed that although the latent periods of the primary response increased with decreasing temperature, fish were able to produce antibodies to the erythrocyte antigen at 2°C and to the Salmonella and horse serum protein antigens at 5°C. The demonstration of antibodies at 5 and 2°C suggests that the fish are capable of producing antibodies over the complete range of their normal environmental temperatures.     

Susceptibility of three different strains of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at two different temperatures to Vibrio anguillarum and temperature effect on antibody response

Three geographically distinct-reared strains (Canadian, Icelandic, Norwegian) of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at optimal and super-optimal growth temperatures (12 and 18 degrees C respectively), were challenged with a virulent isolate of Vibrio anguillarum by injection. The halibut were injected intraperitoneally with 100 microl of the bacterial suspension (1 x 10(6) cells per fish). After challenge, temperature and strain-related differences in survival were observed. Canadian and Icelandic halibut cultured at the super-optimal temperature of 18 degrees C were significantly more susceptible to infection than those strains cultured at 12 degrees C. Total mortality at 18 degrees C for the Canadian and Icelandic strains was 56.4 and 61.85% respectively, compared to 32 and 26.6% respectively at 12 degrees C. Norwegian halibut were significantly more resistant to infection with V. anguillarum at 18 degrees C compared to the other strains, with total mortality of 13.3%. There was no significant difference in total mortality of Norwegian halibut at 18 or 12 degrees C (13.3, 25% respectively). The specificity of the antibodies in sera from challenged halibut cultured at 18 degrees C was primarily to LPS. Immunoblots showed the presence of antibodies against O-side chain antigens. This reaction was strongest in sera from the Norwegian halibut strain compared with the Canadian and Icelandic halibut, which suggests that the difference in resistance to challenge may be ascribable to the presence of antibodies to LPS. Specific antibody levels, as measured by ELISA, increased with increasing temperature and strain differences were apparent, however these did not relate to disease resistance.     

First report on antibody response of Seriola dumerilii (Risso 1810) challenged with Listonella anguillarum

The greater amberjack, Seriola dumerilii (Risso 1810) is a semi-pelagic fish and a worldwide species; it is considered a promising candidate for the diversification of Mediterranean aquaculture. In this paper an experimental injection with Listonella (Vibrio) anguillarum was performed to study the immune response of S. dumerilii. Antibody titres to L. anguillarum O1 were determined with indirect-ELISA at different times over a period of 42 days. Results showed that the antibody levels against L. anguillarum were significantly higher in the challenged fish compared to the control. They started developing since the 5th day reaching the highest peak on day 20 after injection, indicating a fast response of the immune system. The observed antibody titre was very high versus L. anguillarum if compared to other fish species.     

Studies on delayed hypersensitivity to protein antigen. Induction of delayed hypersensitivity by chemically modified antigen.

it was shown in our previous paper that mice primed with chemically modified bacterial alpha-amylase (BaA), which was neither cross-reactive with anti-BaA antibody nor able to induce a humoral anti-BaA response, developed enhanced responses to a subsequent challenge with native BaA and that the magnitude of the immunological memory was closely related to the priming dose of modified BaA. This paper describes the experimental conditions for induction of delayed hypersensitivity (DH) by modified BaA in relation to the development of immunological memory for antibody response to native BaA. Mice primed with either an intraperitoneal (i.p.) or subcutaneous (s.c.) injection of modified BaA in complete Freunds adjuvant (CFA) developed enhanced anti-BaA as the immunogen and modified BaA as the eliciting antigen, the relationship of anti-BaA responses to a subsequent challenge with BaA. In contrast, when mice were immunized with an s.c. injection of the modified BaA only, a significant level of DH to native BaA could be induced, as measured by the footpad reaction (FPR). The highest degree of DH was observed in mice given 50 micrograms of modified BaA. DH was detectable within 5 days and persisted for 25 days after immunization. In the reciprocal combination of native BaA as the immunogen and modified BaA as the eliciting antigen, the relationship of anti-BaA responses to DH was examined. The primary anti-BaA responses induced by an i.p. injection of large doses of BaA was markedly higher than those induced by an s.c. injection, while DH was exhibited only in mice given s.c. injection of BaA in CFA. With respect to DH to native BaA induced by the modified BaA, it was shown that C3H/He mice were high and C57BL/6 mice were low responders.     

Temperature effects on immune response and hematological parameters of channel catfish Ictalurus punctatus vaccinated with live theronts of Ichthyophthirius multifiliis

This study evaluated the influence of temperature on the immune responses and hematological parameters in channel catfish Ictalurus punctatus immunized via intraperitoneal injection with live theronts of Ichthyophthirius multifiliis. Fish were distributed in 18 aquaria and received 9 treatments: 4 groups of fish were vaccinated with live theronts and maintained at constant temperature 15 °C, 20 °C, 25 °C and 30 °C; 3 groups of fish vaccinated and subjected to cycling temperature regime from 15-25 °C, 20-25 °C and 20-30 °C, changed 5 °C each day; 2 groups of fish were not vaccinated and served as controls at 25 °C, one with Ich challenge and the other without challenge. Non vaccinated fish and those vaccinated at 15 °C or 15-25 °C did not show anti-Ich antibodies in the serum 14 and 21 days post-immunization. The antibody levels were significantly higher from fish vaccinated at 25 °C, 30 °C, 20-25 °C and 20-30 °C compared to fish at 15 °C, 20 °C and 15-25 °C both 14 and 21 days post-immunization. At constant water temperature, fish vaccinated at 15 °C showed significantly higher mortality rate (67.8%, P < 0.05) than those vaccinated at 20 °C, 25 °C, and 30 °C (0-10.7% mortalities). At cycling water temperature, fish vaccinated at 15-25 °C showed significantly higher mortality rate (67.8%) than those vaccinated at 20-25 °C and 20-30 °C (P < 0.05). Twenty days after immunization fish vaccinated at 30 °C and 20-30 °C showed significant increase in the red blood cells, white blood cells, thrombocytes and monocytes. Six days after challenge with I. multifiliis theronts the fish showed decreased white blood cells, thrombocytes and monocytes. This study suggests that vaccinated catfish were severely impacted by low temperature, either at 15 °C constant temperature or at 15-25 °C cycling temperature. The fish showed no anti-Ich antibodies and suffered high mortality similar to non vaccinated control fish.     

Detection of Vibrio anguillarum antigen by the dot blot assay

The dot blot assay, modified and adapted for detection of antigens from Vibrio anguillarum in fish tissues, was specific for V. anguillarum and did not react with antigens of V. ordalii, Pseudomonas sp., or Yersinia ruckeri. The blot assay enabled detection of as little as 2.3 ng of a mixture of protein antigens obtained from cell-free extracts of V. anguillarum; it was about 100 times more sensitive than either the indirect fluorescent antibody technique or bacterial isolation for detecting V. anguillarum in fish tissues.     

Inhibition of vibrio anguillarum by Pseudomonas fluorescens AH2, a possible probiotic treatment of fish

To study the possible use of probiotics in fish farming, we evaluated the in vitro and in vivo antagonism of antibacterial strain Pseudomonas fluorescens strain AH2 against the fish-pathogenic bacterium Vibrio anguillarum. As iron is important in virulence and bacterial interactions, the effect of P. fluorescens AH2 was studied under iron-rich and iron-limited conditions. Sterile-filtered culture supernatants from iron-limited P. fluorescens AH2 inhibited the growth of V. anguillarum, whereas sterile-filtered supernatants from iron-replete cultures of P. fluorescens AH2 did not. P. fluorescens AH2 inhibited the growth of V. anguillarum during coculture, independently of the iron concentration, when the initial count of the antagonist was 100 to 1, 000 times greater that of the fish pathogen. These in vitro results were successfully repeated in vivo. A probiotic effect in vivo was tested by exposing rainbow trout (Oncorynchus mykiss Walbaum) to P. fluorescens AH2 at a density of 10(5) CFU/ml for 5 days before a challenge with V. anguillarum at 10(4) to 10(5) CFU/ml for 1 h. Some fish were also exposed to P. fluorescens AH2 at 10(7) CFU/ml during the 1-h infection. The combined probiotic treatment resulted in a 46% reduction of calculated accumulated mortality; accumulated mortality was 25% after 7 days at 12 degrees C in the probiotic-treated fish, whereas mortality was 47% in fish not treated with the probiont.     

Opposite effects of total lymphoid irradiation on T cell-dependent and T cell-independent antibody responses

The effect of total lymphoid irradiation (TLI) on the primary antibody response to the dinitrophenylated heterologous protein, keyhole limpet hemocyanin (DNP-KLH), in complete Freund's adjuvant (CFA), and to the trinitrophenylated polysaccharide antigen, Brucella abortus (TNP-BA), was studied in BALB/c mice. The antibody response to both antigens was diminished in comparison with nonirradiated mice when antigens were injected within 3 days after TLI. When the mice were immunized 30 days after completion of TLI the antibody response to DNP-KLH in CFA was still diminished, but the antibody response to TNP-BA was enhanced 5- to 10-fold as compared with that of control animals. The opposite effect of TLI on the two antibody responses was also observed in a syngeneic primary adoptive transfer system.     

Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum produces a 38-kDa major outer membrane porin protein (OMP) for biofilm formation and bile resistant activity. The gene encoding the porin was used to construct DNA vaccine. The protective efficiency of such vaccine against V. anguillarum causing acute vibrio haemorrhagic septicaemia was evaluated in Asian seabass (Lates calcarifer Bloch), a common species of the Indian coast and a potential resource for the aquaculture industry. In vitro protein expression of porin gene was determined by fluorescent microscopy after transfection of seabass kidney cell line (SISK). Fish immunized with a single intramuscular injection of 20 microg of the OMP38 DNA vaccine showed significant serum antibody levels in 5th and 7th weeks after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1. Asian seabass vaccinated with the OMP38 DNA vaccine was challenged with pathogenic V. anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 55.6% was recorded. Bacterial agglutination and serum complement activity was analysed by using DNA vaccinated seabass serum above 80% of analysed strain was killed at the highest agglutination titre. Histopathological signs of V. anguillarum challenged fish were observed in around 45% of pVAOMP38, 90% of PBS and 87% of pcDNA3.1-vaccinated control fish. The results indicate that L. calcarifer vaccinated with a single dose of DNA plasmid encoding the major outer membrane protein shows moderate protection against acute haemorrhagic septicaemia and mortality by V. anguillarum experimental infection.     

The modulation of catecholamines to the immune response against bacteria Vibrio anguillarum challenge in scallop Chlamys farreri

Catecholamines are pivotal signal molecules in the neuroendocrine-immune regulatory network, and implicated in the modulation of immune response. In the present study, the activities of some immune-related enzymes and the concentration of catecholamines were determined in circulating haemolymph of scallops Chlamys farreri after bacteria Vibrio anguillarum challenge. The activities of superoxide dismutase (SOD), catalase (CAT) and lysozyme (LYZ) increased significantly and reached 610 U mg(-1) at 12 h, 37.6 U mg(-1) at 6 h and 261.5 U mg(-1) at 6 h after bacteria challenge, respectively. The concentration of norepinephrine, epinephrine and dopamine also increased significantly and reached 114.9 ng mL(-1) at 12 h, 86.9 ng mL(-1) at 24 h and 480.4 pg mL(-1) at 12 h after bacteria challenge, respectively. Meanwhile, the activities of these immune-related enzymes in haemolymph were monitored in those scallops which were challenged by bacteria V. anguillarum and stimulated simultaneously with norepinephrine, epinephrine and adrenoceptor antagonist. The injection of norepinephrine and epinephrine repressed significantly the induction of bacteria challenge on the activities of immune-related enzymes, and they were reduced to about half of that in the control groups. The blocking of α and β-adrenoceptor by antagonist only repressed the increase of CAT and LYZ activities significantly, while no significant effect was observed on the increase of SOD activities. The collective results indicated that scallop catecholaminergic neuroendocrine system could be activated by bacteria challenge to release catecholamines after the immune response had been triggered, and the immune response against bacteria challenge could been negatively modulated by norepinephrine, epinephrine, and adrenoceptor antagonist. This information is helpful to further understand the immunomodulation of catecholamines in scallops.     

The humoral immune response of Salmo trutta L. and Cyprinus carpio L. exposed to heavy metals

Salmo trutta L. and Cyprinus carpio L. were exposed to low levels of waterborne heavy metals, 0.75 mg Ni dm^-3, 1.06mg Zn dm^-3, 0.29 mg Cu dm^-3 and 1.01 mg Cr dm^-3, at pH 7.83, water hardness 206.9 mg CaCO, dm^-3, and water temperature 15.5° C. During a 38 week exposure period, the humoral antibody response to MS2 bacteriophage was followed using a 50% viral neutralization assay (SD₅₀) method. A suppression of the immune response was observed in fish exposed to the four heavy metals. Total suppression of the humoral antibody response was found only in C. carpio exposed to Cu or Cr, and these fish exhibited symptoms of acute toxicosis. The time for the primary blood clearance of live bacteriophage was increased in S. trutta exposed to the heavy metals, with the exception of Zn-exposed fish, and in C. carpio exposed to Cu. Following the suppressed primary responses, the Ni-exposed S. trutta and Zn-exposed C. carpio exhibited an adjuvant-like response to the second bacteriophage challenge.     

Subclinical Listonella anguillarum infection does not impair recovery of swimming performance in rainbow trout Oncorhynchus mykiss

This study examines whether injections of the commonly used bacterial-challenge pathogen Listonella anguillarum (formerly Vibrio anguillarum) negatively impact the ability of rainbow trout Oncorhynchus mykiss Walbaum to perform repeat swimming trials. Fish were given intraperitoneal injections of either a sub-lethal (10(5) colony forming units; CFUs) or a lethal (10(7) CFUs) dose of L. anguillarum, held for 48 h, and then given 2 successive ramp critical swimming speed (Ucrit) tests separated by 45 min. Compared with saline-injected control fish, the low-dose injection did not significantly impair swimming performance and recovery. Similarly, Ucrit and re-performance for fish surviving the high-dose injection were comparable to control (2 of 6 fish died after injection and before testing). In contrast, a positive control test of seawater challenge did impair recovery of swimming performance. In view of these results and common use of L. anguillarum as a challenge pathogen for toxicological studies, it seems unlikely that the consequences of pathogenesis impact the important cardiorespiratory changes associated with exercise.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum.

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Temperature- and developmentally-induced variation in the histochemical profile of myofibrillar ATPase activity in carp

The histochemical profile of calcium activated acid stable myofibrillar ATPase (mATPase) activity in developing larval and juvenile carp was investigated. In the larval fish, differentiation of pink muscle fibres occurred after metamorphosis which was delayed by a week at 17° C compared to larvae grown at 27° C. After metamorphosis the 27° C group exhibited some small myofibres with acid stable mATPase activity in the deep white muscle. This was similar for the juvenile carp which were acclimated for more than a month at 25° C. In contrast, the cold (12° C) acclimated juvenile fish, contained very few small white muscle fibres with acid stable mATPase activity. It was also noted that the cold acclimated fish had lower background acid stable mATPase activity than the warm acclimated fish. Results indicate that after metamorphosis and more evidently in juveniles, temperature can influence the rate of myofibre hyperplasia.     

Mucosal response in African catfish after administration of Vibrio anguillarum O2 antigens via different routes

The aim of this study was to investigate the possibility of mucosal vaccination in African catfish (Clarias gariepinus) with Vibrio anguillarum O2 bacterins. The antigen was administered via different routes: anal intubation, oral administration, intraperitoneal injection and immersion. To monitor the antigen uptake, a competitive ELISA was used. The antibody response was measured using an indirect ELISA. Increased antibody levels were found in bile and mucus upon anal intubation, which was not the case after intraperitoneal injection. The data indicate that oral vaccination of fish may be possible when antigens can reach the second gut segment in sufficient quantities and in the right form as confirmed by the recorded substantial induction of systemic and mucosal immunity. The results obtained are a strong indication for mucosal immune response and the two compartmental models for immune response in fish.     

Kinetics of juvenile sea bass (Dicentrarchus labrax, L.) systemic and mucosal antibody secreting cell response to different antigens (Photobacterium damselae spp. piscicida, Vibrio anguillarum and DNP).

The ELISPOT assay was used to measure the number of specific antibody secreting cells (ASC) induced during the primary and secondary immune responses in the spleen, head kidney and gut of juvenile (5 g) sea bass (Dicentrarchus labrax) to bacterial (Vibrio anguillarum and Photobacterium damselae ssp. piscicida) and hapten dinitrophenyl-conjugated to keyhole limpet haemocyanin (DNP-KLH) antigens administered intraperitoneally. High variability among individuals was observed at each sampling day. All fish were bath vaccinated to V. anguillarum at an earlier stage (2 g) in the farm of origin prior to the development of the experiments, and therefore only secondary and tertiary responses were measured in the group immunised with this bacterium. Significant differences to the controls were observed in the primary responses of the head kidney and the spleen to P. damselae ssp. piscicida and DNP, respectively. Frequency analysis of the production of ASC suggests that significant responses in the gut might be masked by the high error variance. The peak of the primary response was observed 4 days earlier to DNP (18-20 days post-immunisation) and it was significantly higher than the response to P. damselae ssp. piscicida. Higher numbers of ASC were observed in the secondary responses of the head kidney and spleen, although they were not statistically significantly different from the primary levels, probably due to the high error variance as supported by the frequency analysis. Nevertheless, together with a faster response (peak at 7 days post-immunisation), the data suggest that memory formation had occurred. Additionally, the data suggest that some suppression of the secondary immune response in the gut might have occurred. The head kidney appears to produce the highest number of specific ASC of the organs tested. It appears that sea bass show a relatively fast but short duration antibody response.