Experimental induction of nocardiosis in yellowtail, Seriola quinqueradiata Temminck and Schlegel by artificial challenge

Challenge methods for inducing nocardiosis, caused by Nocardia seriolae in yellowtail, Seriola quinqueradiata, were evaluated. The first method involved intraperitoneal injection of 1.1 x 10(2), x 10(3) and x 10(4) cfu of N. seriolae; the second was by bath immersion with three different concentrations of bacterial suspension for 10 min; the third was by intradermal injection using a multipuncture device; the fourth was by oral administration using a tube; the fifth was based on the co-habitation of uninfected fish with others that had been artificially infected, i.e. intraperitoneally or by immersion for 10 min. The LD(50) values for the infection trials were 1.9 x 10(2) (intraperitoneal injection), 1.5 x 10(4) mL(-1) (immersion), 4.3 x 10(6) (intradermal injection) and 1.7 x 10(7) (oral administration). In the co-habitation challenge, mortalities were 70% and 50% in groups of non-infected fish mixed with fish infected by the i.p. injection and immersion methods, respectively. Fish challenged by intraperitoneal injection developed adhesions. Fish challenged by other methods did not show any gross clinical signs and moribund fish were similar to natural infection.     

Some factors affecting the potency of Yersinia ruckeri bacterins

Abstract. Several factors affecting the potency of Yersinia ruckeri bacterins were evaluated by vaccinating rainbow trout, Salmo gairdneri Richardson, with various bacterins using the immersion method and determining the level of protective immunity after a virulent challenge. The potency of bacterins prepared with tryptic soya broth at room temperature was not affected by growth at pH from 6.5 to 7.7 or by a culture age from 9 to 96 h. Chloroform and formalin inactivated (0.3%) bacterins gave comparable results and no enhancement of potency occurred by prior extraction of bacterial cells with either butanol or phenol. Cell lysis, as measured by reduced optical density, occurred when cells were held at pH 9–8 for 60 to 120 min. Bacterins prepared from pH-lysed cells resulted in a significant increase in protective immunity. Bacterins prepared at pH 7.2 for 48 h, pH-lysed and inactivated with 0.3 % formalin could be diluted up to 1:100 without loss of efficacy when applied to rainbow trout by a single 20 s immersion. However, with bacterin diluted 1:10 loss of potency occurred after 20 consecutive immersions (100 kg of fish) in the same bacterin at a rate of 0.5 kg/1 of diluted bacterin for each immersion. Factors affecting optimum duration of immunity are discussed.     

Potential for immersion vaccination against Aeromonas salmonicida

Abstract. The efficacy of the immersion method of vaccination was evaluated using Aeromonas salmonicida bacterin. In general a 2 min immersion vaccination was not as effective as vaccination by intraperitoneal injection; however, the level of immunity was improved by giving multiple vaccinations several days apart. A primary immersion vaccination with bacterin diluted 1:4 followed by a secondary vaccination diluted 1:2 gave good results. The most concentrated secondary booster was more effective than boosting with more dilute bacterin.     

Development,distribution and expression of a DNA vaccine against nodavirus in Asian Seabass,Lates calcarifier (Bloch, 1790)

Fish nodavirus (betanodavirus), a viral pathogen responsible for viral nervous necrosis (VNN) was isolated from infected Asian sea bass (Lates calcarifer). The distribution, clearance and expression of nodavirus vaccine, on the basis of DNA vaccine (pFNCPE42 DNA‐pcDNA3.1) construction, were analysed in tissues of the Asian seabass by PCR, RT‐PCR, ELISA and Immunohistochemistry. Fish immunized with a single intramuscular injection of 20 μg of the pFNCPE42‐DNA vaccine showed a significant increase in the serum antibody level in the 3rd week after vaccination, compared to control eukaryotic expression vector pcDNA3.1 vaccinated fish. Results from PCR studies indicated that the vaccine‐containing plasmids were distributed in heart, intestine, gill, muscle and liver 10 days after vaccination. Clearance of pFNCPE42‐DNA vaccine was studied at 10, 25, 50, 75 and 100 days of post vaccination (d p.v). At 100 days p.v. pFNCPE42‐DNA was cleared from muscle of vaccinated sea bass. In vitro and in vivo expression of fish nodavirus capsid protein gene (FNCP) was determined by fluorescent microscopy. Asian seabass was immunized with pFNCPE42‐DNA vaccine at a dose of 20 μg per fish and were challenged with betanodavirus by intramuscular injection. The vaccinated seabass was protected from nodaviral infection and 77.33% of relative percent survival (RPS) was recorded.     

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.     

Protection against enteric redmouth disease in rainbow trout, Salmo gairdneri Richardson, after vaccination with Yersinia ruckeri bacterin

Abstract. Rainbow trout, Salmo gairdneri Richardson, (average weight 100g) were vaccinated intraperitoneally (i.p.) with Yersinia ruckeri bacterin in saline or in oily adjuvant. Agglutinating antibody kinetics were followed during 445 days before challenge (1.2×10⁷ bacteria i.p.). Fourteen days after challenge 88.5% of the control fish had died while few mortalities were observed with vaccinated fish regardless of their agglutinating titre. Protection against Y. ruckeri does not seem to be due to antibodies.     

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.     

Modification of the hyperosmotic infiltration method of vaccination against vibriosis in cultured ayu, Plecoglossus altivelis Temminck and Schlegel

Abstract. A vaccine solution of a formalin-killed culture of Vibrio anguillarum cells was observed to be toxic to young ayu when administered by the hyperosmotic infiltration method. The toxin was present in the culture broth. After the toxin was removed from the broth by centrifugation, the fish were dipped in 5.32% NaCl solution for 2 min and then in a solution containing precipitated cells for 3 min. The immunized fish were protected against vibriosis when challenged one month after immersion. The bacterin was administered to ayu by a further two methods, both using lyophilized whole cells of formalin-killed V. anguillarum. In one method, the fish were placed in a 5.32% NaCl solution for 2 min and then in a solution containing lyophilized cells at 2 g/l of well water for 3 min (two-step immersion). In the other method, the fish were placed in a 5.32% NaCl solution containing lyophilized cells also at 2 g/l for 3 min (one-step immersion). A high level of protection against artificial challenge was achieved with either method. No agglutinating antibodies to V. anguillarum were detected in either the serum or mucus of fish dipped in a vaccine solution, a supernatant, or a precipitated solution, one month after immersion. On the other hand, serum titres were detected in fish vaccinated by injection, although no titres were detected in mucus. LD₅₀ values are presented for the virulence of the V. anguillarum strain. Compared to the original strain, virulence increased after the third passage in ayu, but decreased after the thirteenth passage in medium.     

Aeromonas salmonicida: determination of an antigen associated with protective immunity and evaluation of an experimental bacterin

Abstract. Protection against Aeromonas salmonicida was determined by passive immunization and with various bacterin preparations. Rabbit antiserum was prepared against a rough, virulent strain of A. salmonicida (AS-1R), the same strain boiled (AS-1R, boiled), and an avirulent, smooth strain of this same isolate (AS-1S). Cross-absorption, cross-passive protection and analysis by counter immunoelectrophoresis of various extraction methods were studied. It was shown that AS-1R cells contained an additional antigen not present in AS-1R (boiled) and AS-1S cells. Antiserum to the AS-1R antigen passively protected sockeye salmon, Oncorhynchus nerka (Walbaum), against a virulent challenge, and antisera to AS-1R (boiled) and AS-1S were not protective. The antigen was not destroyed by formalin or heat at 5°C for 60 min, but it appeared to be partially inactivated with proteolytic enzymes. The antigen was produced in casein yeast beef (CYB) broth up to 32 h but not thereafter, and low yields were obtained in tryptic soy or brain heart infusion (BHI) broth. It was extracted from cells with ethylenediamine tetraacetic acid (EDTA) and especially alkaline hydrolysis, but not with proteolytic enzymes or detergents. The detergents appeared to destroy the antigen. We concluded that the antigen was protein and is most likely the external A-protein (AP) reported for rough, virulent strains of A, salmonicida. Various methods of preparing A. salmonicida bacterins were evaluated by determining the level of protective immunity induced in intraperitoneally (i.p.) vaccinated fish. Growth of cells in CYB or BHI broth resulted in production of only rough (autoagglutinated in saline) variants of A. salmonicida. Although only rough variants were associated with protective immunity, one strain was not protective, it was avirulent by bath challenge. Bacterins prepared in CYB were more efficacious than those grown in BHI, but inactivation with formalin, iodine, or glutaraldehyde worked equally well. However, boiling the bacterin or filtering the cells from the bacterin removed its efficacy. Methods of releasing the AP were evaluated by sonification, pH-lysis, disaggregation and treatment with EDTA, and all treatments worked equally well. Also, precipitation on to aluminium or use of Freund's complete adjuvant did not significantly improve the protection. In parenterally vaccinated fish, protection was demonstrated by challenging the fish at various levels by bath, injection or cohabitation with infected fish. The best protection was demonstrated using the cohabitation challenge method. The potency and field efficacy of an A. salmonicida bacterin prepared in CYB broth and extracted with 5 mM EDTA was evaluated. Fish were vaccinated by i.p, injection and potency was determined in the laboratory by experimental challenge and in the field by natural challenge. Chinook salmon, O.ishawytschu (Walbaum), developed immunity within seven days at 10°C. The bacterin could be diluted up to 1:2000 without loss of potency. The field tests results were equivocal; however, (he prevalence of infection was lower in vaccinated fish.     

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.     

A histological study of the response to challenge with vibriosis in ayu, Plecoglossus altivelis Temminck and Schlegel, vaccinated by immersion and injection with Vibrio anguillarum bacterin

Abstract. The histological responses of ayu, Plecoglossus altivelis , given an intramuscular injection of a formalin-killed bacterin of Vibrio anguillarum are described. Lesions at the site of injection showed muscular necrosis and infiltration of inflammatory cells by the second day after injection, and production of granulation tissue from the fifth to the fourteenth day. Protective responses against vibriosis were studied histopathologically in ayu that were vaccinated by intramuscular injection with formalin-killed Vibrio bacterin and by immersion in sonicated Vibrio bacterin, and challenged by a subcutaneous injection with viable Vibrio on the fourteenth day after the vaccination.
Efficacy of both methods was confirmed by the survival of vaccinated fish after the challenge. There was slight bacterial multiplication in the fish, and bacterial phagocytosis by infiltrated neutrophils and tissue necrosis in the injected area on the third day after the challenge. In contrast, non-vaccinated fish died within 24h of challenge, with extensive bacterial multiplication and tissue necrosis in the injected area and visceral organs.     

Comparison of passive and active immunization of fish against streptococcosis (enterococcosis)

Passive immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), was carried out to determine the persistence of anti-Streptococcus sp. antibodies (ASA) raised in sheep, rabbits or rainbow trout. The protection afforded by passive immunization was compared with the protection obtained from active immunization by immersion in or intraperitoneal (i.p.) injection with formalin-killed cells. Assessments were undertaken concurrently for up to 3 months post-immunization (PI) to evaluate the practical potential of passive immunization. Passively administered sheep and rabbit antibodies were detected in fish sera by enzyme-linked immunosorbcnt assay for more than 60 days after i.p. injection. Fish responded immunologically to these antibodies and the highest humoral responses to sheep and rabbit ASA occurred at 2 months PI. The relative per cent survival (RPS) of rainbow trout challenged with virulent Streptococcus sp. after an i.p. injection (0.1 ml 100 g^?1 fish body weight) of sheep, rabbit or fish ASA was: 88.8, 50 and 0% after 1 month; 33.3, 6.8 and 6.8% after 2 months; and 13.3, 0 and 6.6% after 3 months PI, respectively. Fish immunized actively had an RPS of 88.8 and 11.1% after 1 month, 38.1 and 4.7% after 2 months, and 36 and 0% after 3 months PI for the i.p. injection and immersion routes, respectively.     

Passive immunisation of fish against vibriosis, comparison of intraperitoneal, oral and immersion routes

Passive immunisation of fish was conducted to determine whether anti-Vibrio anguillarum whole sera (AVA) and affinity-purified AVA raised in sheep, rabbits and rainbow trout (Oncorhynchus mykiss) were persistent when injected and orally administered into rainbow trout. These responses were compared with active immunisation by immersion in, and intraperitoneal (i.p.) injection with, formalin-killed V. anguillarum cells. Sheep and rabbit AVA were detected in rainbow trout sera for up to 70 days (half-life 21 days) after i.p. injection as determined by an enzyme-linked immunosorbent assay (ELISA). The relative percentage survival (RPS) of passively immunised rainbow trout challenged with virulent V. anguillarum after an injection was comparable to that of active immunisation by immersion after 1 month post-immunisation (p.i). Affinity-purified sheep and rabbit AVA exhibited the same protective potential as whole serum in rainbow trout. Rabbit and sheep immune sera diluted 1:8 and 1:50, respectively, provided equivalent protection as undiluted fish immune serum. An active immune response against passively acquired heterologous immunoglobulins was demonstrated by ELISA, with responses against sheep AVA being less than those against rabbit AVA. Rainbow trout given purified sheep AVA conjugated to LTB (the GM-1-binding subunit of Escherichia coli heat-labile toxin) and administered orally had an RPS of 37.5% at 15 days and 27% at 1 month p.i. In contrast, fish given sheep AVA conjugated to TraT (an internal membrane of E. coli) or in micellar form with Quil-A had RPSs of only 18.7 and 6.2%, respectively, after 15 days, and 13.3 and 0% after 1 month, respectively. The protection conferred by immune sera was shown to be due to the immunoglobulin component alone. Heat inactivation of the complement in sera had no effect on the potency of immune sera.     

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.     

Immune responses and host protection of channel catfish, Ictalurus punctatus (Rafinesque), against Ichthyophthirius multifiliis after immunization with live theronts and sonicated trophonts

The humoral immune responses and host protection of channel catfish, Ictalurus punctatus (Rafinesque), against Ichthyophthirius multifiliis (Ich) were determined after immunization with live theronts and sonicated trophonts. Immunizations with live theronts or sonicated trophonts were carried out by both bath immersion and intraperitoneal (i.p.) injection. Cutaneous and serum immunoglobulin (Ig) levels and anti-Ich antibodies were measured 12 and 21 days post-immunization. The level of Ich infection and survival of catfish were determined after theront challenge. Cutaneous and serum anti-Ich antibodies were significantly higher (P < 0.05) in fish immunized with live theronts by immersion or i.p. injection, or with sonicated trophonts administered by i.p. injection, than in fish immunized with sonicated trophonts by immersion, with bovine serum albumin by i.p. injection, or non-immunized controls. Host protection was noted only in fish immunized with live theronts by immersion or i.p. injection or with sonicated trophonts by i.p. injection. There was a positive correlation between higher levels of anti-Ich antibodies and host survival in the immunized fish.     

银鲫对嗜水气单胞菌灭活菌苗的免疫应答研究

以绵羊红细胞为载体,建立了检测银鲫血清抗体的间接血球凝集试验。用该方法研究了银鲫对嗜水气单胞菌灭活菌苗的免疫应答一般规律。研究结果表明,疫苗免疫组在免疫７天后可以测到血凝抗体,２１天达到高峰值４０９６,然后开始下降,３５天时抗体滴度降为１６。加弗氏不完全佐剂疫苗免疫组亦在７天时可以测到血凝抗体,２１天达到１０２４,３５天时为２０４８,抗体维持在较高的水平。对鱼体免疫后的保护力进行测定,结果表明,两个免疫组分别在７天左右和１４天左右开始产生保护力。免疫保护力在鱼体免疫初期随着抗体滴度的增加而加强,随后在一段时间内保持在较强的水平,并不随抗体滴度的下降而减弱。浸泡免疫试验的结果亦表明,银鲫可以通过浸泡免疫获得免疫保护力。     

Protective effect and antibody response of DNA vaccine against salmonid alphavirus 3 (SAV3) in Atlantic salmon

This work reports the effect of two DNA vaccines against salmonid alphavirus 3 (SAV3) in Atlantic salmon. Presmolts were vaccinated by intramuscular injection of plasmids encoding the SAV3 structural polyprotein C‐E3‐E2‐6K‐E2 (pCSP), E2 only (pE2), or plasmid without insert (pcDNA3.3). E2 is expressed at the surface of cells transfected with pCSP and internally in cells transfected with pE2. A commercial vaccine based on inactivated SAV (NCPD) was used for comparison. At 10 weeks post‐vaccination, only fish vaccinated with pCSP showed antibody against E2 and virus‐neutralizing activity. Vaccinated fish were infected with SAV3 to determine protection by virus quantitation in serum after 7 days and scoring of pathological changes after 21 days. Fish vaccinated with both pCSP and NCPD vaccines showed significant virus reduction in serum, while fish vaccinated with pE2 did not. All fish vaccinated with pcDNA3.3 and pE2 showed pathological changes in organs typical of PD, 60% of fish vaccinated with NCPD showed PD pathology, while fish vaccinated with pCSP did not show PD pathology. Taken together, DNA vaccination with pCSP provided strong protection for salmon against SAV3 infection, which in part may be due to production of virus‐neutralizing antibodies.     

Protective immunity against enteric septicaemia in channel catfish, Ictalurus punctatus (Rafinesque), following controlled exposure to Edwardsiella ictaluri

Protective immunity against enteric septicaemia of catfish (ESC) following immunization with Edwardsiella ictaluri bacterins and exposure to live E . ictaluri was investigated. Mean cumulative percentage survival was significantly higher ( P 0.05) in controlled live vaccinates (100%) than in immersion and oral bacterin vaccinates (68.3% and 50.0%, respectively). Bactericidal activity against E . ictaluri by peritoneal macrophages from controlled live vaccinates (85.9%) was significantly greater ( P 0.05) than bactericidal activity of macrophages from immersion bacterin vaccinates (71.4%) or non-vaccinates (68.1%). No significant ( P > 0.05) difference was found in the bactericidal activity of macrophages from oral bacterin vaccinates and macrophages from controlled live vaccinates. The E . ictaluri -specific antibody response of controlled live (0.08 OD) and immersion bacterin vaccinates (0.11 OD) was significantly higher ( P 0.05) than that of oral bacterin vaccinates and non-vaccinates (0.01 OD) 15 days post-vaccination. A significantly higher antibody response was seen in controlled live vaccinates (0.17 OD), when compared to other vaccinates or non-vaccinates 33 days after vaccination. Neither immersion nor oral bacterins protected the vaccinates against ESC. Controlled live E . ictaluri immunization of channel catfish resulted in production of specific antibodies, increased macrophage bactericidal activity and protection against ESC.     

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.     

Protection against heterologous Streptococcus iniae isolates using a modified bacterin vaccine in Nile tilapia,Oreochromis niloticus (L.)

Streptococcus iniae is a significant pathogen impacting aquaculture production worldwide. The objectives of this study were to determine whether a developed modified S. iniae (ARS-98-60) bacterin vaccine is efficacious in Nile tilapia, Oreochromis niloticus (L.), against challenge with heterologous isolates from diverse geographical locations and to evaluate protein and antigenic variability among the isolates tested. Two groups of tilapia (approximately 5 g) were intraperitoneally (IP) vaccinated with 100 μL of the vaccine or sham vaccinated with 100 μL of sterile tryptic soy broth and held for 28 days. Fish were challenged with each isolate by IP injection of 2–3 × 10⁷ CFU per fish using calcein to mark fish prior to cohabitation for challenge. The results demonstrated significant protection against all challenge isolates, and relative percent survivals ranged from 79% to 100%. SDS–PAGE analysis of whole-cell lysate proteins from the S. iniae isolates demonstrated similar protein profiles between 10 and 31 kDa and variation in profiles between 35 and 100 kDa. Western blot analysis using antiserum from vaccinated fish (ARS-98-60) demonstrated shared immunogenic proteins among all isolates in the molecular mass range of 22–35 kDa and high molecular mass material >150 kDa. The results suggest that the developed S. iniae vaccine has broad ranging protection among isolates exhibiting different protein profiles.