Anguillarum Bacterin Research Articles

Biocompatibility of intraperitoneally implanted TEMPO-oxidized cellulose nanofiber hydrogels for antigen delivery in Atlantic salmon (Salmo salar L.) vaccines

Disease outbreaks are a major impediment to aquaculture production, and vaccines are integral for disease management. Vaccines can be expensive, vary in effectiveness, and come with adjuvant-induced adverse effects, causing fish welfare issues and negative economic impacts. Three-dimensional biopolymer hydrogels are an appealing new technology for vaccine delivery in aquaculture, with the potential for controlled release of multiple immunomodulators and antigens simultaneously, action as local depots, and tunable surface properties. This research examined the intraperitoneal implantation of a cross-linked TEMPO cellulose nanofiber (TOCNF) hydrogel formulated with a Vibrio anguillarum bacterin in Atlantic salmon with macroscopic and microscopic monitoring to 600-degree days post-implantation. Results demonstrated a modified passive integrated transponder tagging (PITT) device allowed for implantation of the hydrogel. However, the Atlantic salmon implanted with TOCNF hydrogels exhibited a significant foreign body response (FBR) compared to sham-injected negative controls. The FBR was characterized by gross and microscopic external and visceral proliferative lesions, granulomas, adhesions, and fibrosis surrounding the hydrogel using Speilberg scoring of the peritoneum and histopathology of the body wall and coelom. Acutely, gross monitoring displayed rapid coagulation of blood in response to the implantation wound with development of fibrinous adhesions surrounding the hydrogel by 72 h post-implantation consistent with early stage FBR. While these results were undesirable for aquaculture vaccines, this work informs on the innate immune response to an implanted biopolymer hydrogel in Atlantic salmon and directs future research using cellulose nanomaterial formulations in Atlantic salmon for a new generation of aquaculture vaccine technology.

Oral Immunization of Larvae and Juvenile of Lumpfish (Cyclopterus lumpus) against Vibrio anguillarum Does Not Influence Systemic Immunity.

Vibrio anguillarum, a marine bacterial pathogen that causes vibriosis, is a recurrent pathogen of lumpfish (Cyclopterus lumpus). Lumpfish is utilized as a cleaner fish in the Atlantic salmon (Salmo salar) aquaculture in the North Atlantic region because of its ability to visualize and prey on the ectoparasite sea lice (Lepeophtheirus salmonis) on the skin of Atlantic salmon, and its performance in cold environments. Lumpfish immunity is critical for optimal performance and sea lice removal. Oral vaccine delivery at a young age is the desired method for fish immunization because is easy to use, reduces fish stress during immunization, and can be applied on a large scale while the fish are at a young age. However, the efficacy of orally delivered inactivated vaccines is controversial. In this study, we evaluated the effectiveness of a V. anguillarum bacterin orally delivered to cultured lumpfish and contrasted it to an intraperitoneal (i.p.) boost delivery. We bio-encapsulated V. anguillarum bacterin in Artemia salina live-feed and orally immunized lumpfish larvae. Vaccine intake and immune response were evaluated by microscopy and quantitative polymerase chain reaction (qPCR) analysis, respectively. qPCR analyses showed that the oral immunization of lumpfish larvae resulted in a subtle stimulation of canonical immune transcripts such as il8b, il10, igha, ighmc, ighb, ccl19, ccl20, cd8a, cd74, ifng, and lgp2. Nine months after oral immunization, one group was orally boosted, and a second group was both orally and i.p. boosted. Two months after boost immunization, lumpfish were challenged with V. anguillarum (7.8 × 105 CFU dose−1). Orally boosted fish showed a relative percentage of survival (RPS) of 2%. In contrast, the oral and i.p. boosted group showed a RPS of 75.5% (p < 0.0001). V. anguillarum bacterin that had been orally delivered was not effective in lumpfish, which is in contrast to the i.p. delivered bacterin that protected the lumpfish against vibriosis. This suggests that orally administered V. anguillarum bacterin did not reach the deep lymphoid tissues, either in the larvae or juvenile fish, therefore oral immunization was not effective. Oral vaccines that are capable of crossing the epithelium and reach deep lymphoid tissues are required to confer an effective protection to lumpfish against V. anguillarum

Brain and Pituitary Response to Vaccination in Gilthead Seabream (Sparus aurata L.).

Vaccination is a widely used therapeutical strategy in aquaculture, but whether vaccination elicits stress responses in the central neuroendocrine system and enhances the crosstalk between the immune and endocrine systems in the brain or pituitary after vaccination is unclear. To answer this question two experiments using two different vaccine exposure routes, i.e., bath or intraperitoneal (i.p.) injection, were carried out on gilthead seabream (Sparus aurata L.). In the first one, the stress responses of fish subjected to waterborne Vibrio anguillarum bacterin were compared with responses after air exposure or their combination. In the second experiment, fish were subjected to an intraperitoneal injection of Lactococcus garvieae bacterin and we assessed the central stress response and also whether or not a significant immune response was induced in brain and pituitary. In both experiments, blood, brain and pituitary tissues were collected at 1, 6, and 24 h post stress for plasma hormone determination and gene expression analysis, respectively. Results indicated that bath vaccination induced a decreased central stress response compared to air exposure which stimulated both brain and pituitary stress genes. In the second experiment, injection vaccination kept unchanged plasma stress hormones except cortisol that raised at 6 and 24 h. In agreement, non-significant or slight changes on the transcription of stress-related genes were recorded, including the hormone genes of the hypothalamic pituitary interrenal (HPI) axis and other stress markers such as hsp70, hsp90, and mt genes in either brain or pituitary. Significant changes were observed, however, in crhbp and gr. In this second experiment the immune genes il1β, cox2, and lys, showed a strong expression in both brain and pituitary after vaccination, notably il1β which showed more than 10 fold raise. Overall, vaccination procedures, although showing a cortisol response, did not induce other major stress response in brain or pituitary, regardless the administration route. Other than main changes, the alteration of crhbp and gr suggests that these genes could play a relevant role in the feedback regulation of HPI axis after vaccination. In addition, from the results obtained in this work, it is also demonstrated that the immune system maintains a high activity in both brain and pituitary after vaccine injection.

Comparative study of stress and immune-related transcript outcomes triggered by Vibrio anguillarum bacterin and air exposure stress in liver and spleen of gilthead seabream (Sparus aurata), zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss)

The stress and immune-related effects of short-term (1, 6 and 24 h) air exposure stress (1 min), bath vaccination with Vibrio anguillarum bacterin, and both stressors combined were evaluated in liver and spleen of Sparus aurata, Danio rerio and Onchorhynchus mykiss. Expression profiles of immune (interleukin 1 beta: il1β; tumor necrosis factor alpha: tnfα; interleukin 10: il10; tumor growth factor beta: tgfβ1; immunoglobulin M: igm; lysozyme: lys; complement protein c3: c3) and stress-related genes (glucocorticoid receptor: gr; heat shock protein 70: hsp70; and enolase) were analysed by RT-qPCR. Cortisol level was assessed by radioimmunoassay. The gene expression patterns in liver and spleen were found to be differentially regulated in a time- and organ-dependent manner among species. In seabream, a higher il1β-driven inflammatory response was recorded. In zebrafish, air exposure stress but not bath vaccination alone modulated most of the changes in liver and spleen immune transcripts. Stressed and vaccinated trout showed an intermediate pattern of gene expression, with a lower upregulation of immune-related genes in liver and the absence of changes in the expression of hsp70 and enolase in spleen (as it was observed in seabream but not in zebrafish). Following air exposure, cortisol levels increased in plasma 1 h post-stress (hps) and then decreased at 6 hps in O. mykiss and D. rerio. By contrast, in S.aurata the cortisol level remained higher at 6 hps suggesting a greater degree of responsiveness to this stressor. When fish were exposed to combined air exposure plus bath vaccination cortisol levels were also augmented at 1 and 6 hps in O. mykiss and S.aurata and restored to basal level at 24 hps, whereas in D. rerio the response was higher in response to the combination of both stressors. In addition, V. anguillarum bacterin vaccination triggered cortisol secretion only in D. rerio, suggesting a greater responsiveness of D. rerio hypothalamic-pituitary-interrenal axis. Overall, comparing the tissue transcription responsiveness, liver was found to be more implicated in the response to handling stress compared to spleen. These results also indicate that a species-specific response accounts for the deviations of stress and immune onset in the liver and spleen in these fish species.

Comparative Immune- and Stress-Related Transcript Response Induced by Air Exposure and Vibrio anguillarum Bacterin in Rainbow Trout (Oncorhynchus mykiss) and Gilthead Seabream (Sparus aurata) Mucosal Surfaces.

Fish have to face various environmental challenges that may compromise the efficacy of the immune response in mucosal surfaces. Since the effect of acute stress on mucosal barriers in fish has still not been fully elucidated, we aimed to compare the short-term mucosal stress and immune transcriptomic responses in a freshwater (rainbow trout, Oncorhynchus mykiss) and a marine fish (gilthead seabream, Sparus aurata) to bacterial immersion (Vibrio anguillarum bacterin vaccine) and air exposure stress in skin, gills, and intestine. Air exposure and combined (vaccine + air) stressors exposure were found to be inducers of the cortisol secretion in plasma and skin mucus on both species in a time-dependent manner, while V. anguillarum bacterin exposure induced cortisol release in trout skin mucus only. This was coincident with a marked differential increase in transcriptomic patterns of stress- and immune-related gene expression profiles. Particularly in seabream skin, the expression of cytokines was markedly enhanced, whereas in gills the response was mainly suppressed. In rainbow trout gut, both air exposure and vaccine stimulated the transcriptomic response, whereas in seabream, stress and immune responses were mainly induced by air exposure. Therefore, our comparative survey on the transcriptomic mucosal responses demonstrates that skin and gut were generally more reactive in both species. However, the upregulation of immune transcripts was more pronounced in gills and gut of vaccinated trout, whereas seabream appeared to be more stress-prone and less responsive to V. anguillarum bacterin in gills and gut. When fish were subjected to both treatments no definite pattern was observed. Overall, the results indicate that (1) the immune response was not homogeneous among mucosae (2), it was greatly influenced by the specific traits of each stressor in each surface and (3) was highly species-specific, probably as a result of the adaptive life story of each species to the microbial load and environmental characteristics of their respective natural habitats.

Modulatory in vitro effect of stress hormones on the cytokine response of rainbow trout and gilthead sea bream head kidney stimulated with Vibrio anguillarum bacterin

In fish, the stress response and their consequences in the immune system have been widely described. Recently, a differential cytokine regulation between rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) was reported after treatment with stress hormones together with their receptor antagonists. Nevertheless, there is no evidence of whether antagonists for stress hormone receptors may influence the interaction between hormones and cytokines after bacterial administration. Thus, the aim of our study was to evaluate the cytokine expression in the presence of stress hormones (cortisol, ACTH, adrenaline), hormone receptor antagonists and inactivated Vibrio anguillarum bacterin in rainbow trout and gilthead sea bream head kidney primary cell culture (HKPCC). Mifepristone, spironolactone, propranolol and phentolamine were used to block GR, MR, MC2R, and β-/α-adrenoreceptors.Our results showed an expected increase of the pro-inflammatory and anti-inflammatory response after inactivated V. anguillarum bacterin treatment in both species. Cortisol, ACTH and adrenaline did not modulate the expression of immune-related genes in rainbow trout, while in sea bream cortisol was able to reduce the stimulated gene expression of all cytokines. This effect was only restored to basal expression level in IL-1β and TNF-α by mifepristone. ACTH reduced both pro-inflammatory and anti-inflammatory cytokine expression, excluding IL-1β, only in sea bream. Adrenaline enhanced the expression of IL-1β and TGF-β1 stimulated by inactivated V. anguillarum in sea bream, and the effect was diminished by propranolol. In sum, our results confirm that the immunoendocrine differences reported at gene expression profile between two teleost species are also observed after exposure to inactivated V. anguillarum bacterin, suggesting that stress hormones would differentially modulate the immune response against pathogens in teleost species.

Cloning and expression analyses of a unique IgT in ayu Plecoglossus altivelis

A unique heavy chain of immunoglobulin (Ig) T was cloned from ayu Plecoglossus altivelis, and its in vivo expression was compared with IgM and IgD expression using quantitative RT-PCR and in situ hybridization. Three immunoglobulin domains, corresponding to CH1, CH2, and CH4, in IgT of other teleost fish species were conserved in the heavy chain. IgT mRNA levels were higher in the spleen, head kidney, trunk kidney, and gill tissue than in the intestine, liver, and skin tissue. IgM transcripts were the most abundant transcripts in all tissues tested. In the in situ hybridization studies, IgT and IgM mRNA-positive cells were scattered across the trunk kidney of healthy fish, while IgD mRNA-positive cells were not detected. mRNA levels of IgT and IgM were significantly upregulated in the gills and trunk kidney after vaccination with Vibrio anguillarum bacterin, but the IgD mRNA level remained unchanged. These results suggest that this unique IgT is expressed in a small number of cells and that it plays a role in immune responses induced by immersion vaccination.

Vibrio anguillarum bacterin uptake via the gills of Japanese flounder and subsequent immune responses

The mucosal surfaces of fish allow for the introduction of foreign substances, including antigens, from the surrounding environment. In this study, uptake of Vibrio anguillarum J-O-3 serotype bacterin by Japanese flounder, and the subsequent immune responses were investigated. Immunohistochemistry revealed that the bacterin was taken up through the epithelial cells of gills. The transcription levels of inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor α were significantly up-regulated in the gills at 3 days following exposure to the bacterin. There was also a corresponding increase in IL-8 receptor, CD4-1, CD4-2 and CD8α transcript levels in the gills. Our findings suggest that the gills play a major role in the uptake of V. anguillarum bacterin and induction of inflammation, which results in an activation of the adaptive immune response in teleost fish.

Gene transcript changes in individual rainbow trout livers following an inflammatory stimulus

Inflammatory stimuli elicit liver synthesis and subsequent release into the plasma of several proteins (positive acute phase proteins, APP) with functions in innate immunity, tissue repair and restoration of homeostasis. To expand the basis for evaluating the degree of conservation of the APR in vertebrates and to assess the extent to which genes encoding both cellular and plasma proteins are affected, we profiled transcriptional changes in livers of individual rainbow trout ( Oncorhynchus mykiss) after intraperitoneal injection of Listonella ( Vibrio) anguillarum bacterin in Freund's Incomplete Adjuvant. Twenty genes were down-regulated, some unexpectedly such as complement component 3 and α2-macroglobulin. Sixteen up-regulated genes included three encoding proteins involved in iron metabolism (hepcidin, haptoglobin, and intelectin), from which we infer that sequestration of iron is likely to be a major component of the trout APR. Activated genes encoding proteins of unknown functions included precerebellin-like plasma protein, and differentially regulated trout protein which is predicted to be cell surface associated. The only complement component that increased was C7. Genes encoding proteins that are probably not released into plasma included two fatty acid binding proteins, two transport proteins (SEC61 and a Na–Ca exchanger), GAPDH, an amino transferase, and a hydrolase. When microarray data and quantitative RT-PCR analyses were used to evaluate specific transcripts, variations were notable between individual fish, possibly a basis for natural variation in susceptibility to infectious diseases. This study suggests novel hypotheses relating to NFκB, albumin-related protein, pentraxin, hypoferremia and the complement cascade. While the capacity to mount an APR is conserved throughout vertebrate evolution, the responding genes vary from species to species, and considerable variation is observed from individual to individual within a species.

Uptake and processing of a Vibrio anguillarum bacterin in Artemia franciscana measured by ELISA and immunohistochemistry

Nauplii of Artemia franciscana were incubated in two different concentrations (undiluted and 1:9 in autoclaved sea water) of a divalent bacterin composed of two different serovars of Vibrio anguillarum. In order to investigate uptake and further processing of a bacterin in the live feed organism A. franciscana, immunohistochemistry was applied, visualising the presence of whole bacterial cells and antigens from the bacterin in individual nauplii. By using ELISA, it was shown that approximately 1·5–2·5×105cells were incorporated into each Artemia under the conditions used. Maximum incorporation of cells was measured after 30min, whereas after 60min there was a decline to levels of 0·9–1·6×105cells per Artemia. Immediately after incubation in the bacterin solution, the nauplii were transferred to a culture of the alga Isochrysis galbana, in order to simulate transfer of the nauplii to rearing tanks for fish larvae. From the ELISA, it could be concluded that the incorporated bacterial cells were excreted from the Artemia nauplii rapidly, however a large variation among different nauplii could be visualised by immunohistochemistry.

The Non-specific Immunostimulation and Adjuvant Effects of Vibrio anguillarum Bacterin, M-glucan, Chitin and Freund's Complete Adjuvant against Pasteurella piscicida Infection in Yellowtail.

The non-specific immunostimulation and adjuvant effects of Vibrio anguillarum bacterin (VAB), M-glucan, chitin and Freund's complete adjuvant (FCA) against pasteurellosis were examined in yellowtail, Seriola quinqueradiata. Control fish were injected intraperitoneally with 0.1 ml of sterile phosphate buffered saline (PBS). Experimental fish were injected similarly with either vaccine (lipopolysaccharide-mixed chloroform-killed Pasteurella piscicida, LPS-CKC), an immunostimulant or mixture of the vaccine and an immunostimulant.All fish were subsequently challenged by the same but live P. piscicida strain using an immersion method at 15, 25, 35 and 45 days post-inoculation. Immunostimulants alone were found to slightly increase the protectionagainst Pasteurella infection, but the effects were not statistically significant. The most effective adjuvant wasFCA, however the enhanced protection was not statistically different from that of fish inoculated with LPS-CKCalone. However, the relative percent survival (RPS) of fish vaccinated with M-glucan, chitin or Vibrio bacterinas adjuvant was lower than that of fish vaccinated with only LPS-CKC bacterin alone. Thus, M-glucan, chitin and Vibrio bacterin had no positive adjuvant effects on the P. piscicida vaccine.

An investigation of the biliary and intestinal immunoglobulin and the plasma cell distribution in the gall bladder and liver of the common dogfish, Scyliorhinus canicula L.

Local immune mechanisms were investigated in the gut and gall bladder of the common dogfish. Administration of sonicated Vibrio anguillarum bacterin and whole sheep red blood cells (SRBC) by the oral and anal routes elicited the production of low titres of specific antibodies in the bile but not in the serum. Parenteral administration of the V. anguillarum sonicate and SRBC elicited a strong systemic response and specific biliary antibodies were detected at higher titres than when antigens were introduced into the alimentary tract. Immunoglobulin was detected in the bile and intestine and was comparable in size to serum immunoglobulin. However, they had different electrophoretic mobilities and preliminary characterisation revealed some differences in the heavy and light chains. The origin of the bile and intestinal immunoglobulin was not elucidated although it is likely that the former contributes to the latter. A few plasma cells were found in the gall bladder and none in the liver.

Field testing of oral Vibrio anguillarum bacterin in pond-cultured ayu.

The efficacy of oral bacterin to control vibriosis was determined using pond cultured ayu (Plecoglossus altivelis). A formalin-killed whole cell bacterin of Vibrio anguillarum was fed to 322, 000 ayu in seven ponds eight to fifteen times at doses ranging from 0.08 to 0.16g bacterin/kg fish/day. Fish were exposed to natural infections due to vibriosis in the farm ponds. Experimental challenge was performed with fish lots where natural infection did not occur. Natural infection occurred in three ponds of vaccinated fish and three control ponds where bacterin was not fed. A decrease in the number of dead fish and the number of days of medication using oxolinic acid was shown in all of the vaccinated lots as compared to the non-vaccinated lots. Efficacy of the bacterin was assessed as the differences in the mortality profile and the need for medication during two months. The results of the experimental challenge showed the efficacy for more than 59 days. Combined use of oxolinic acid and bacterin did not reduce the efficacy of the bacterin. The smallest amount of bacterin yielding protection was eight feedings of 0.1 g/kg fish on alternate days. The total amount of bacterin required to vaccinate 322, 000 fish was approximately 1.4 kg equivalent to 4.3 mg/fish. The vibrio bacterin delivered by the oral route was shown to be highly effective when given to large commercial ayu populations showing ease in application and reduced stress for the fish.

Comparisons of the cellular immune response of fish vaccinated by immersion and injection of Vibrio anguillarum.

The cellular immune response of rainbow trout Salmo gairdneri immunized by immersion or injection with Vidrio anguillarum bacterin was investigated using the passive antibody producing cell assay and passive antidody rosette-forming cell assay. Antibody-producing cels (plaqueforming cell: PFC) were rarely detected in the anterior kidney and spleen of raindow trout immunized by immersion. PFCs were detected in fish immunized by injection and reached the maximum level on the 13th day after immunization. The number of antigen-bingding cells (rosette-forming cells: RFCs) increased rapidly in the anterior kidney and spleen of rainbow trout vaccinated by immersion or injection. The RFC reached the maximum level on the 11th day after immunization. After that the number of RFCs gradually decreased until it reached the same level as that in non-immunized fish on the 28th day. The hemagglutinating antibody of the serum against V. anguillarum of fish vaccinated by immersion tended to be as low as the serum of non-vaccinated fish. However, the hemagglutinating antibody in fish vaccinated by injection was detected first on the 13th day after immunization. The maximum value of the titer was reached on the 28th day after immunization.

Survival of Hatchery-reared Atlantic Salmon Smolts Vaccinated with a Vibrio anguillarum Bacterin

Abstract Atlantic salmon (Salmo salar) smolts were vaccinated with a single intraperitoneal injection of a trivalent Vibrio bacterin to evaluate the feasibility of increasing survival. Mortalities of the treated and untreated groups for the 4-5 week post-injection period at the hatchery were negligible. There was no significant difference in the rate of adult return of the vaccinated smolts compared to the controls, indicating that immunization did not enhance marine survival.

Effects of sodium dichromate exposure on the immune responses of juvenile coho salmon, Oncorhynchus kisutch, against Vibrio anguillarum.

Sublethal exposure of juvenile coho salmon (Oncorhynchus kisutch) to Cr (as sodium dichromate) in freshwater decreased their disease resistance and serum a-glutinin production against Vibrio anguillarum. Duplicate disease resistance experiments, in which groups of salmon were exposed to 0.0 or 0.5 mg Cr/L for two weeks and then injected in subgroups with one of four tenfold dilutions of live V. anguillarum (ca. 3-4 x 10(2-5) bacteria/.1 ml dose), showed that disease mortality increased with dose and was significantly greater in Cr-exposed fish. Serum agglutinin production, measured in fish which were injected with V. anguillarum bacterin and then exposed to 0.0 to 0.5 mg Cr/L for two weeks, was significantly inhibited in Cr-exposed salmon by six weeks after immunization. It is suggested that measurement of disease resistance and agglutinin production may be sensitive and ecologically realistic indicators of sublethal toxicity in aquatic animals.

Vibriosis Vaccination of Chum Salmon by Hyperosmotic Infiltration

Abstract When fry of chum salmon (Oncorhynchus keta) started to feed, they were vaccinated with Vibrio anguillarum bacterin by hyperosmotic infiltration. Mortalities of vaccinated and unvaccinated fish were compared in ambient and controlled challenge experiments. In the controlled challenge experiment, cumulative mortalities after 10 days were 6% in the vaccinated group and 63% in the unvaccinated group; in the ambient challenge experiment the respective cumulative mortalities after 70 days were 6% and 96%. The differences were statistically significant (P < 0.005), indicating that survival was highly contingent on vaccination under the conditions observed.

Vibriosis of Ayu

Two hundred fifty nine strains of Vibrio anguillarum were isolated from cultured ayu of various districts in Japan from 1974 to 1977. These strains were tested for their susceptibility to 6 chemotherapeutic agents : sulfamonomethoxine, chloramphenicol, tetracycline, nalidixic acid, nitrofurazone and trimethoprim. Nine of the 259 strains were sensitive to all drugs tested. The remaing 250 strains were resistant to various combinations of the six drugs tested. Transferable R plasmids were detected in 165 out of the 250 resistant strains, and strains from all districts turned out to have some transferable R plasmids. This high incidence of drug resistant strains of V. anguillarum was assumed to be due to the selective pressure exerted by chemotherapeutic agents used during culturing the fish. These strains fall into at least three different O sero-groups.The efficacy of hyperosmotic infiltration technique (HI) in controlling vibriosis of ayu was tested. Experimental fish were first placed in a solution containing 5.32% NaCl for 2 min. and then placed in a vaccine solution of V. anguillarum (5.0 × 108/ml) bacterin for 3 min. A month after immunization, the immunized fish were found to be resistant to V. anguillarum to intraperitoneally injected.In the second experiment, formalin-added culture of V. anguillarum was centrifuged and the precipitated bacterial cells were resuspended in saline solution of the same volume as the original culture. As in the first experiment, the fish were held in a solution of 5.32% NaCl for 2 min, and then placed either in the above suspension or the medium supernatetant for 3 min. Immersion of fish in the suspension provided adequate protection but immersion of fish in the supernatant was not effective to protect against intraperitoneally injected V. anguillarum a month after vaccination. Treatment with the supernatant caused high mortality to ayu presumably due to the exotoxin from bacteria but immersion in the Vibrio cell suspension did not cause higher mortality than a control group immersed only in 5.32% NaCl solution during are one month.In the third experiment, the fish were immersed in 5.32% NaCl solution containing lyophilized whole cells of V. anguillarum for 3 min. The vaccinated fish turned out to be well protected against V. anguillarum intraperitoneally injected a month after treatment. The HI method could be used most effectively to immunize ayu against vibriosis.

Field Testing of Injected Vibrio anguillarum Bacterins in Pen-Reared Pacific Salmon

Intraperitoneal injection of three types of Vibrio anguillarum bacterin (heat-killed, formalin-killed, and a combination of heat and formalin-killed) was tested in Chinook (Oncorhynchus tshawytscha) and coho salmon (O. kisutch) to assess the feasibility of preventing vibriosis by immunization in commercial aquaculture. A single injection of V. anguillarum bacterin gave substantial protection to chinook salmon for 6 mo postinoculation (32% mean mortality in all injected groups) in comparison to the control group (85% mortality). Chinook salmon showed the highest survival when treated with the heat-killed V. anguillarum bacterin. All groups of coho salmon had less than 7.0% mortality, and immunization was not beneficial to survival. Serum antibody titer of V. anguillarum agglutinins was increased by intraperitoneal injection. Vibrio anguillarum was the pathogen most frequently isolated from moribund chinook salmon; Aeromonas salmonicida was the predominant organism isolated from moribund coho salmon.