Immunization of channel catfish, Ictalurus punctatus Rafinesque, against Ichthyophthirius multifiliis (Fouquet): killed versus live vaccines

Abstract. Fish surviving infection with the pathogenic ciliated protozoan, Ichthyophthirius multifiliis (Fouquet, 1876), become resistant to subsequent infection by the parasite. The acquired immunity suggests that development of a vaccine against the parasite may be possible. Because of the advantages of immunoprophylaxis for treatment of the disease, an effort has been made to determine whether fish exposed to killed parasite preparations can resist subsequent lethal challenge. Both the route of administration and the effects of stage specific antigens have been examined. Channel catfish vaccinated by intraperitoneal (i.p.) injection or bath immersion with killed I multifiliis tomites show 100% mortality following a standard challenge protocol. Similarly, 100% mortality was observed in test groups injected with tomite cilia. In both cases, a consistent difference in days to death between control and test group animals was observed. Although complete mortality was seen with fish injected with tomite preparations, fish vaccinated with killed trophonts (the feeding stage of the parasite) had a much greater degree of protection with approximately 50% of fish surviving an otherwise lethal challenge. Finally, animals injected intraperitoneally with live tomites showed nearly complete immunity and were identical in their response to fish which survive natural infection. The response of fish vaccinated with live parasites indicates that animals injected intraperitoneally can develop surface immunity and that i.p. injection is a suitable route of administration for potential I. multifiliis vaccines.     

Experimental Cryptobia salmositica (Kinetoplastida) infections in Atlantic salmon, Salmo salar L.: cell-mediated and humoral immune responses against the pathogenic and vaccine strains of the parasite

Hatchery-reared Atlantic salmon, Salmo salar L., were vaccinated intraperitoneally (i.p.) with a live attenuated Cryptobia salmositica vaccine (either 100 000 or 5000 parasites fish⁻¹) and 4 weeks later were challenged with the parasite (either 100 000 or 5000 parasites fish⁻¹). Unvaccinated, infected salmon had high parasitaemias and were anaemic. Fish given a high dose (100 000 parasites fish⁻¹) had higher parasitaemias than fish given the lower dose. Vaccinated fish had low parasitaemias and a mild anaemia, but recovered quickly after challenge. Complement-fixing antibody increased in vaccinated fish after challenge and was highest at 2 weeks post-challenge. The cell-mediated response (both T cells and B cells) was depressed in infected fish until 4 weeks after infection. In vaccinated fish, the humoral response (i.e. B-lymphocytes) was greater than the cell-mediated response (i.e. T-lymphocytes). In contrast, infected fish had a greater cell-mediated than humoral immune response.     

Vaccination trials of sea bass,Dicentrarchus labrax (L.), against Photobacterium damsela subsp. piscicida,using novel vaccine mixtures

Bacterial cells of the marine fish pathogen Photobacterium damsela subsp. piscicida were grown in novel culture media. A mixture of whole cells and extracellular components was inactivated and used in bath, intraperitoneal (i.p.) and oral vaccination of sea bass, Dicentrarchus labrax, employing two sizes of fish. A commercial vaccine was used for comparative purposes. Control and immunized fish were either bath or intraperitoneally challenged 6 and 12 weeks post-vaccination. Small fish had significantly higher relative percentage survival with the novel vaccine mixture both at 6 and 12 weeks post-vaccination by bath, in comparison with the commercial vaccine. No protection was afforded at 6 or 12 weeks post-immunization by either vaccine after challenge via i.p. injection. Sea bass (1.5-2 g) intraperitoneally vaccinated with various adjuvanted vaccine mixtures were not protected against pasteurellosis. In contrast, larger sea bass (20 g) benefited from vaccination with the novel vaccine mixtures. Intraperitoneal challenge with the pathogen resulted in protection in both fish groups vaccinated with novel vaccine mixtures, whereas control fish suffered high mortalities (> 80%). Orally vaccinated fish were immersion challenged with the pathogen. At 6 and 12 weeks post-vaccination the control fish had a high mortality and the fish vaccinated with the novel vaccine mixture achieved good protection.     

Protection against atypical furunculosis in Atlantic halibut,Hippoglossus hippoglossus (L.); comparison of a commercial furunculosis vaccine and an autogenous vaccine

Atlantic halibut, Hippoglossus hippoglossus (L.), was shown to be sensitive to infection by three different isolates of Aeromonas salmonicida ssp. achromogenes in pre-challenge tests using intraperitoneal (i.p.) and intramuscular (i.m.) injections as well as bath challenges. A commercial furunculosis vaccine, Alphaject 1200, and an autogenous vaccine, AAS, based on the challenge strain, induced immune protection as shown in challenge tests 8 weeks post-immunization. The survival rate of vaccinated fish after i.p. challenge was 100%, whereas mortality of control fish was 61%. Employing i.m. challenge, relative percentage survival induced by the furunculosis vaccine and the AAS vaccine was 47 and 44, respectively. Mortality of i.m. injected controls was 68%. Vaccinated fish behaved normally following vaccination but the weight gain was significantly reduced in vaccinated fish 8 weeks post-vaccination compared with control fish receiving phosphate-buffered saline. At the same time, intra-abdominal adhesions were observed in fish injected with either of the two vaccines or adjuvant alone. Antibody response against A. salmonicida ssp. achromogenes was detected in sera from fish receiving either vaccine.     

Immunosuppression in rainbow trout, Salmo gairdneri Richardson, caused by the haemoflagellate Cryptobia salmositica Katz, 1951

Abstract. An experimental Cryptobia salmositica infection in rainbow trout, Salmo gairdneri Richardson, produced suppression of the humoral response against sheep red blood cells as measured by direct haemagglutination. Two-month and 5-month infections produced equal suppression. The parasite also produced suppression of the humoral response against a bacterial pathogen, Yersinia ruckeri . Anti- Y. ritckeri titres were significantly lower in most fish infected with C. salmositica than in non-infected fish. Immunosuppression became evident when C. salmositica first appeared in the blood (first 2 weeks of infection), Immunosuppression was confirmed by challenge with Y. ruckeri . Mortality at challenge occurred in 64·3% to 83·3% of the fish already infected with C. salmositica at the time of initial Y. ruckeri exposure. There was no mortality at challenge if fish were not infected with C. salmositica at initial bacterial exposure, nor in those concurrently infected with both pathogens. Antigenic competition may have caused the immunosuppression.     

Onset of immunity in salmonid fry vaccinated by direct immersion in Vibrio anguillarum and Yersinia ruckeri bacterins

Abstract. The fry of several salmonid species were vaccinated by direct immersion in either Yersinia ruckeri or Vibrio anguillarum bacterin and the level of protective immunity was determined by the survival of fish after bath challenge with virulent organisms. The immersion time for effective vaccination was obtained within 5 s and protective immunity was demonstrated within 5 days at 18°C and within 10 days at 10°C. The minimum size at which maximum protective immunity occurred was between 1.0 and 2.5 g. Immunity appeared to be a function of size and not age, but differences in response among several species were indicated. In fish under 1.0 g, the level of protective immunity could be increased using a more concentrated bacterin. The results were similar with both bacterins.     

Efficacy of Vibrio anguillarum antigen administered by intraperitoneal injection route in Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel)

Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel), were immunized by the intraperitoneal (i. p.) injection route with formalin‐killed whole cells of Vibrio anguillarum that originated from a diseased fish. Fifty days later, a booster vaccination was given by the same route. Control fish were similarly treated with sterile phosphate‐buffered saline. The efficacy of vaccination was evaluated based on protection against two bacterial challenges and immune responses (both specific and non‐specific). The challenges were performed by i. p. injection with V. anguillarum or V. parahaemolyticus. The results indicated that the vaccinated fish showed higher non‐specific immune activity than the unvaccinated fish. The effects of vaccinations on the phagocytic activity of phagocyte, bactericidal and lysozyme activities were notable, especially on bactericidal activity. Determined by ELISA, antiserum of vaccinated fish displayed high antibody titres. The vaccination conferred protection against V. anguillarum challenge (81.25–93.75% relative percentage survival (RPS)). The RPS was 46.15–53.85% against V. parahaemolyticus challenge, indicating some degree of cross‐protective immunity.     

Immunization of rainbow trout, Salmo gairdneri Richardson, against bacterial kidney disease: preliminary efficacy evaluation

Abstract. A bacterin for immunization against bacterial kidney disease of salmonid fishes caused by Renibacterium salmoninarum is described. Cultures were grown in Evelyn's KDM2 medium containing 10% calf serum in a fermenter under the following conditions: pH 7.2, 15°C, 800ml/min air, 200 rev/min agitation and 5–15 days of incubation. Possible substitutes for calf serum were 10% horse serum 0.15% starch and leptospira medium. The bacterins were inactivated with 0.3% formalin and no adjuvants were used. Other tests evaluated pH-lysed bacterin, 50% concentrated bacterin and 50% concentrated pH-lysed bacterin. Juvenile rainbow trout, salmo gairdneri Richardson, were vaccinated either by intraperiotoneal (i.p.) injection, 2 min immersion or 2-step hyperosmotic infiltration. Fish were held from four to six weeks at 11°C, then challenged by i.p. injection with the homologous virulent bacterium. Fish died from days 19 to 40 after challenge. The best preparation was pH-lysed bacterin given by a single i.p. injection; hyperosmotic and immersion vaccination were not effective. Typically when 80% or more of unvaccinated controls were infected as detected by Gram stain, 10% or less of the vaccinated fish were infected.     

Protection, immune response and side-effects in European whitefish (Coregonus lavaretus L.) vaccinated against vibriosis and furunculosis

As indicated by market demand and cultivation prospects, the whitefish (Coregonus lavaretus L.) has the potential to become an important alternative to rainbow trout in fish farming in Finland. The fish processing industry has called for a fish species having non-pigmented flesh, and there is a long tradition of whitefish farming for stocking purposes in Finland. However, cultivation in net cages in the brackish water of the coastal area exposes fish to vibriosis (Listonella (Vibrio) anguillarum) and furunculosis (Aeromonas salmonicida salmonicida). Hence, profitable whitefish farming requires the efficient control of both of these diseases. The efficacy of vaccination (Apoject 1800®) was studied in the laboratory using challenge tests and by monitoring specific antibody production (ELISA) and the blood leucocyte pattern. The fish (22 g) were vaccinated at 15°C and the immunity developed during 8 weeks prior to challenge with vibriosis (1.8×10⁵ cfu) and 12 weeks prior to challenge with furunculosis (4.9×10² cfu). The challenge was performed by i.p. injection. Side-effects around the injection site were studied 16 weeks post-vaccination. The immune system of the whitefish responded well and vaccination gave good protection against vibriosis and furunculosis. The RPS was 100% following the Vibrio challenge and 99% following the Aeromonas challenge. The antibody levels continued increasing in the vaccinated group throughout the study period. Lymphocyte and neutrophil counts were elevated in vaccinated compared to non-vaccinated fish at 8 weeks post-vaccination, but not after 16 weeks. Vaccination produced weak adhesions around the injection site in 42% and minor pigmentation in 9% of the 65 fish examined.     

Efficacy of injection vaccines against Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum)

Efficacy of mineral oil-based experimental injection vaccines against Flavobacterium psychrophilum were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), under laboratory and field conditions. The vaccines consisted of formalin- or heat-inactivated whole bacterium cell preparations of two different serotypes (Fd and Th) or a combination of serologically different F. psychrophilum (Fd and/or Th and/or Fp(T);Th). Specific antibody responses against the bacterium in plasma and skin mucus were evaluated post-vaccination with enzyme-linked immunosorbent assay. Efficacy of the vaccinations was determined by challenge trials to F. psychrophilum with the vaccinated rainbow trout. Significantly higher antibody levels in plasma were detected in vaccinated fish compared with mock-vaccinated fish. Injection vaccination did not trigger specific antibody production in the skin mucus. Significantly higher survival of i.p. vaccinated fish compared with non-vaccinated fish was observed during the challenge. The results suggest that mineral oil-based injectable vaccines containing formalin- or heat-inactivated virulent cells of F. psychrophilum effectively triggered specific antibody production and protected the fish against bacterial cold water disease.     

Improved recapture rate of vaccinated sea-ranched Atlantic salmon, Salmo salar L.

Vaccination of sea-ranched Atlantic salmon was conducted in order to investigate if immuno-prophylactic measures could improve their survival. Fish were either vaccinated by bath or injection. A total of 66 000 fish were reared in fresh water at a hatchery on the island of Bornholm and at the presmolt stage were separated in three groups each comprising of 22 000 fish. One group was vaccinated intraperitoneally with a polyvalent vaccine (containing killed Vibrio anguillarum serotype O1 and O2, Yersinia ruckeri and Aeromonas salmonicida ). A second group was bath vaccinated with the corresponding vaccine-components and the third group was used as a non-vaccinated control. One month after vaccination these groups were allocated to three separate net-pens located 500 m from the coastline of the island. After 4 months in the net-pens, 1000 fish from each cage were tagged with Carlin-tags below the dorsal fin. The fish were then released for a migration period in the Baltic Sea. Following a sea period of 40 months (45 months post-vaccination), the recapture rates of the groups were calculated from the returned tags from fishermen. Recapture of the injection vaccinated group was significantly higher (25%) compared with the bath vaccinated fish (14.7%) and the control group (16.8%).     

Efficacy of furunculosis vaccines in turbot, Scophthalmus maximus (L.): evaluation of immersion, oral and injection delivery

The commercial furunculosis vaccine Aquavac Furovac 5 and an autogenous vaccine, based on the challenge strain, induced immune protection in turbot, Scophthalmus maximus (L.), as shown in challenge tests 120 days post-immunization by injection (relative percentage of survival, RPS = 72-99%). This protective effect lasted for at least 6 months post-immunization at appreciable levels (RPS = 50-52%). Neither the autogenous vaccine nor the commercial vaccine was able to induce significant levels of protection against Aeromonas salmonicida in turbot when administered by immersion. Antibody levels were high or moderate in fish vaccinated by injection with the different vaccines and very low in fish vaccinated by immersion. The field results show that delivering an oral boost after the primary vaccination by injection did not enhance protection of turbot against furunculosis and that water-based (autogenous vaccine) and oil adjuvanted (Alpha Ject 1200) vaccines administered by injection conferred similar levels of protection (RPS > 80%) in turbot.     

Characterization of susceptibility and carrier status of burbot,Lota lota (L.), to IHNV,IPNV, Flavobacterium psychrophilum,Aeromonas salmonicida and Renibacterium salmoninarum

In this study, susceptibility and potential carrier status of burbot, Lota lota, were assessed for five important fish pathogens. Burbot demonstrated susceptibility and elevated mortality following challenge with infectious haematopoietic necrosis virus (IHNV) by immersion and to Aeromonas salmonicida by intraperitoneal (i.p.) injection. IHNV persisted in fish for at least 28 days, whereas A. salmonicida was not re-isolated beyond 17 days post-challenge. In contrast, burbot appeared refractory to Flavobacterium psychrophilum following intramuscular (i.m.) injection and to infectious pancreatic necrosis virus (IPNV) by immersion. However, i.p injection of IPNV resulted in re-isolation of virus from fish for the duration of the 28 day challenge. Renibacterium salmoninarum appeared to induce an asymptomatic carrier state in burbot following i.p. injection, but overt manifestation of disease was not apparent. Viable bacteria persisted in fish for at least 41 days, and bacterial DNA isolated by diagnostic polymerase chain reaction was detected from burbot kidney tissue 90 days after initial exposure. This study is the first to investigate susceptibility of burbot to selected fish pathogens, and this information will aid in efforts to culture and manage this species.     

Duration of immunity in salmonids vaccinated by direct immersion with Yersinia ruckeri and Vibrio anguillarum bacterins

Abstract. The level of protective immunity was determined for several salmonid species following vaccination by the direct immersion method with commercial Vibrio anguillarum (two serotypes) and Yersinia ruckeri (Hagerman strain) bacterins. The duration of protective immunity varied with the bacterin concentration, size and species of fish, but the duration between the two bacterins was comparable. In fish under 1 g duration of protective immunity was longest when the most concentrated bacterin was used. Generally, immunity lasted in 1-g fish for about 120 days, in 2-g fish for about 180 days, but in 4-g fish and larger immunity lasted for a year or longer. Coho salmon ( Oncorhynchus kisutch ) and sockeye salmon ( O. nerka ) retained immunity for a longer time and pink salmon ( O. gorbuscha ) the shortest time. Chinook salmon ( O. tshawytscha ) and rainbow trout ( Salmo gairdneri ) were intermediate. Field data generally followed the laboratory tests, but the duration appeared somewhat shortened. In one test 20-g rainbow trout were vaccinated by the shower method and no loss of immunity occurred after 311 days.     

Vaccination strategies with oral booster for surubim hybrid (Pseudoplatystoma corruscans × P. reticulatum) against haemorrhagic septicaemia

This study evaluated the efficiency of differently prepared vaccines against Aeromonas hydrophila in the hybrid surubim (Pseudoplatystoma corruscans × P. reticulatum). Survival and haemato‐immunological parameters were compared between the treatments: non‐vaccinated fish (C); bacterin‐vaccinated fish (B); bacterin plus oral booster vaccinated fish (B+O); bacterin and toxoid‐vaccinated fish (B+T) and bacterin, toxoid and oral booster‐vaccinated fish (B+T+O). Fourteen‐days vaccinated fish from B+O and B+T+O were fed with an oral booster for 4 days. After 1 week, the fish were intraperitoneally challenged with 2 × 10⁸ CFU mL^?1 of A. hydrophila. Fish from the treatment B+T+O showed the lowest cumulative mortality (11.36%) 96 h after challenge, compared with other treatments (22.72–44.04%), and a relative survival of 74%. Serum immunoglobulin in B+T+O fish was higher than in other treatments. All vaccinated fish showed an increased agglutination titre when compared with non‐vaccinated fish, both before and after challenge. Fish fed with oral booster showed an increase in phagocytic percentage before and after challenge. It can be inferred that the oral booster vaccination was efficient in reducing mortality in hybrid surubim by enhancing the response against haemorrhagic septicaemia due to A. hydrophila infection.     

The use of a real-time PCR primer/probe set to observe infectivity of Yersinia ruckeri in Chinook salmon, Oncorhynchus tshawytscha (Walbaum), and steelhead trout, Oncorhynchus mykiss (Walbaum)

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a common pathogen affecting aquaculture facilities and implicated in large losses of cultured fish. Fisheries scientists continue to gain a greater understanding of the disease and the pathogen by investigating methods of identification and pre- and post-infection treatment. In this study, a real-time PCR probe set for Y. ruckeri was developed to detect daily changes in the bacterial load during pathogen challenges. Two species of fish, Chinook salmon, Oncorhynchus tshawytscha, and steelhead trout, Oncorhynchus mykiss, were exposed to two strains of Y. ruckeri (Hag and SC) during bath challenges. A subset of fish was killed daily for 14 days, and the kidney tissue was biopsied to enumerate copies of pathogen DNA per gram of tissue. While Chinook exposed to either the Hag or SC strains exhibited similar pathogen loads, those exposed to the Hag strain displayed higher mortality (～66%) than fish exposed to the SC strain (～24% mortality). Steelhead exposed to the Hag strain exhibited a greater pathogen load and higher mortality (～42%) than those exposed to the SC strain (<1% mortality). Steelhead challenged with either strain showed lower pathogen loads than Chinook. The study illustrates the efficacy of the probe set to enumerate Y. ruckeri bacterial growth in the kidneys of fish. Also, strains of Y. ruckeri display species-specific growth patterns that result in differential mortality and pathogen load.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the pathogenic haemoflagellate, Cryptobia salmositica Katz, and protection against cryptobiosis in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), inoculated with a live vaccine

Abstract. An enzyme-linked immunosorbent assay using dried blood on filter paper, was developed for the detection of antibodies against the haemoflagellate Cryptobia salmositica in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum). Each fish (average weight about 5g) in three experimental groups was either inoculated with 20000 attenuated live C. salmositica vaccine, or inoculated with 2000 or 20000 pathogenic parasites per fish. The vaccine was effective in protecting juvenile trout 4 weeks after vaccination and antibody titers were higher in vaccinated and challenged fish than in unvaccinated and infected ones. Specific antibodies were detected one week post-infection (w.p.i.) with the pathogen and declined to low levels at 6 w.p.i. The high-dose group (20000 per fish) had antibody titres comparable to those of the vaccinated and challenged fish.     

Comparison of different challenge methods to evaluate the efficacy of furunculosis vaccines in Atlantic salmon, Salmo salar L.

Four challenge methods, intraperitoneal (i.p.) and intramuscular (i.m.) inoculation, bath and cohabitation exposure, were evaluated as methods for testing the efficacy of furunculosis vaccines in Atlantic salmon, Salmo salar L. Groups of fish vaccinated with one of two different vaccines containing aluminium phosphate or animal/vegetable oil as an adjuvant were challenged with Aeromonas salmonicida 6 and 12 weeks after vaccination. Relative per cent survival (RPS) was calculated daily during a 3-week observation period post-challenge. A large variation in protection measured by RPS, both between methods and between different time points for each method, was found. Towards the end of the observation period, RPS tended to be similar in the i.p. and cohabitation challenge groups. A high degree of protection was demonstrated for the oil adjuvanted vaccine. The i.m. challenge produced very low RPS numbers for both vaccines, but this was most marked for the aluminium-phosphate-adjuvanted vaccine. The bath exposure resulted in RPS values intermediate to the cohabitation and injection methods. The study also demonstrated that, after i.p. and i.m. challenges, the initial peak mortality caused by the inoculation was followed by a secondary increase in mortality, probably because of shedding of bacteria into the water during the first mortality phase and hence contributing to a superinfection state.     

溶藻弧菌鞭毛蛋白flaC基因DNA疫苗对红笛鲷的免疫保护研究

为研究溶藻弧菌鞭毛蛋白flaC基因DNA疫苗对红笛鲷的免疫保护作用,实验构建了重组真核表达质粒pcDNA-flaC并将该质粒肌肉注射红笛鲷,采用PCR、RT-PCR、ELISA和攻毒试验等方法检测了该真核表达质粒在红笛鲷组织内的分布、表达和对红笛鲷的免疫保护.PCR结果显示,免疫接种7和28 d,注射点周围肌肉、鳃、肾脏、肝脏和脾脏都存在质粒分布;RT-PCR结果显示,免疫接种后第7天、14天和28天,红笛鲷不同组织内均有目的基因表达.ELISA结果表明,鱼血清内产生了抗FlaC蛋白的抗体,表明DNA疫苗免疫后鱼体表达了目的蛋白,并诱导产生了相应抗体.攻毒实验表明,免疫后的红笛鲷能较好地抵抗致病性溶藻弧菌的感染.结果表明,质粒pcDNA-flaC可能是抵抗溶藻弧菌感染的有效的疫苗候选物.