Immune response of largemouth bass, <Emphasis Type="Italic">Micropterus salmoides</Emphasis>, to whole cells of different <Emphasis Type="Italic">Nocardia seriolae</Emphasis> strains

Largemouth bass Micropterus salmoides were immunized with four different N. seriolae strains—two α-glucosidase-positive (961113, KU040801) and two α-glucosidase-negative (94260, OTTS) strains—along with Freund’s incomplete adjuvant. After primary immunization (week 0), a booster was administered at weeks 4 and 8. Nonspecific immune responses to multiple immunizations with the different N. seriolae strains were determined based on serum lysozyme activity and nitroblue tetrazolium (NBT)-positive cells in peripheral blood. The serum lysozyme activity and NBT-positive cells in peripheral blood were not significantly increased even after the two booster immunizations. Specific antibody responses against N. seriolae cells were investigated by enzyme-linked immunosorbent assay. At 4 weeks after immunization, all groups immunized with N. seriolae antigens showed significant increases in their specific antibody levels. The sera from fish immunized with different N. seriolae strains exhibited reactivity with N. seriolae sonicated antigens of 28, 30, 36 and 84 kDa by western blot analysis. After two boosters, fish were challenged with live N. seriolae to assess the vaccine’s efficacy; however, multiple injections of the N. seriolae strains did not reduce mortality, irrespective of the bacterin.     

Amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> interleukin-10 negatively suppresses host cell-mediated immunity

Interleukin-10 (IL-10) is an anti-inflammatory cytokine and negatively regulates cell-mediated immunity (CMI) induction by inhibiting cytokine production in type 1 T helper cells. IL-10 genes have been isolated from several fish, and inflammatory cytokine inhibition by IL-10 has been well examined. However, a CMI regulator of IL-10 in fish has not yet been identified. In this study, we cloned the IL-10 gene in amberjack Seriola dumerili and analyzed its function using its recombinant protein (rIL-10). In an in vitro culture experiment, gene expression of inflammatory cytokines was suppressed in leukocytes incubated with rIL-10 compared with cells that only received Nocardia seriolae stimulation. This result suggests amberjack IL-10 has conserved function as an inflammatory cytokine inhibitor. Bactericidal activity of amberjack cells against intracellular pathogen stimulation was decreased in a rIL-10 dose-dependent manner. Furthermore, a significant reduction in the T-bet/GATA-3 ratio was observed in N. seriolae living cell (LC)?+?rIL-10-injected fish. Taken together, these results suggest amberjack rIL-10 suppresses CMI induction both in vitro and in vivo. In addition, the number of IgM⁺ cells among spleen leukocytes in N. seriolae?+?rIL-10-injected fish was higher than in only N. seriolae LC, suggesting that Th2-dominant immunity was induced by adding rIL-10.     

An experimental vaccine against Aeromonas hydrophila can induce protection in rainbow trout,Oncorhynchus mykiss (Walbaum)

A candidate vaccine against Aeromonas hydrophila in rainbow trout, Oncorhynchus mykiss, was developed using a bacterial lysate. To test the strength of protection, A. hydrophila challenge models were compared using injection into both the intraperitoneal (IP) cavity and the dorsal sinus (DS) with selected doses of live bacteria washed in saline or left untreated. Unlike the IP route, injection into the DS with either saline washed or unwashed cells resulted in consistent cumulative mortality and a dose response that could be used to establish a standard challenge having an LD₅₀ of approximately 3 × 10⁷ colony forming units per fish. Survivors of the challenge suffered significantly lower mortality upon re‐challenge than naïve fish, suggesting a high level of acquired resistance was elicited by infection. Passive immunization using serum from hyper‐immunized fish also resulted in significantly reduced mortality indicating protection can be transferred and that some portion of resistance may be antibody mediated. Vaccination of groups of rainbow trout with A. hydrophila lysate resulted in significant protection against a high challenge dose but only when injected along with Freund’s complete adjuvant. At a low challenge dose, mortality in all groups was low, but the bacterial lysate alone appeared to offer some protection.     

Identification of a mitochondrial‐targeting secretory protein from Nocardia seriolae which induces apoptosis in fathead minnow cells

Nocardia seriolae is the main pathogen responsible for fish nocardiosis. A mitochondrial‐targeting secretory protein (MTSP) 3141 with an N‐terminal transit peptide (TP) from N. seriolae was predicted by bioinformatic analysis based on the genomic sequence of the N. seriolae strain ZJ0503. However, the function of the MTSP3141 and its homologs remains totally unknown. In this study, mass spectrometry analysis of the extracellular products from N. seriolae proved that MTSP3141 was a secretory protein, subcellular localization research showed the MTSP3141‐GFP fusion protein co‐localized with mitochondria in fathead minnow (FHM) cells, the TP played an important role in mitochondria targeting, and only the TP located at N‐terminus but not C‐terminus can lead to mitochondria directing. Moreover, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase‐3 activity and apoptosis‐related gene (Bcl‐2, Bax, Bad, Bid and p53) mRNA expression suggested that cell apoptosis was induced in FHM cells by the overexpression of both MTSP3141 and MTSP3141ΔTP (with the N‐terminal TP deleted) proteins. Taken together, the results of this study indicated that the MTSP3141 of N. seriolae was a secretory protein, might target mitochondria, induce apoptosis in host cells and function as a virulence factor.     

Modified resazurin microtiter assay for in vitro and in vivo assessment of sulfamonomethoxine activity against the fish pathogen <Emphasis Type="Italic">Nocardia seriolae</Emphasis>

Resazurin microtiter assay (REMA) was carried out using four sulfonamides, three culture media, and four inoculum sizes as a first screening step to establish an easy-to-interpret sulfonamides susceptibility testing method for Nocardia seriolae. The in vitro activity of sulfamonomethoxine (SMM) against 190 clinical N. seriolae isolates was then examined, and in vivo experimental treatment was performed. When the culture medium and the inoculum size were considered in tandem, a 0.5× the original concentration of cation-adjusted Mueller–Hinton broth and an inoculum size of 10² CFU/well showed the clearest endpoint reading for all tested drugs, and the REMA-generated data were in excellent agreement with those generated by the reference Etest method. SMM activity showed minimum inhibitory concentration (MIC) values of 4–32 μg/ml against all tested N. seriolae isolates. Treatment of amberjack groups experimentally infected with N. seriolae isolates having SMM MICs of 4 and 32 μg/ml, resulted in survival rates of 100% and 87.5% in the two groups, respectively. In this study, we developed a simple visual method to test SMM activity against N. seriolae.     

一种(鱼师)鱼诺卡氏菌酪氨酸蛋白磷酸酶基因的克隆及功能初步研究

這鱼诺卡氏菌是鱼类诺卡氏菌病的主要病原,可导致鱼类慢性系统性肉芽肿疾病.這鱼诺卡氏菌全基因组序列分析发现了一个酪氨酸蛋白磷酸酶(protein tyrosine phosphtase,PTP)基因,生物信息学分析显示该基因很可能编码一个靶向定位于宿主细胞线粒体的分泌蛋白.本实验对這鱼诺卡氏菌PTP进行了基因克隆、分泌蛋白鉴定、亚细胞定位、过表达和线粒体膜电位检测,结果显示,在這鱼诺卡氏菌胞外产物中质谱鉴定到了PTP肽段,证实其为分泌蛋白.亚细胞定位研究观察到PTP-GFP融合蛋白均匀地分布在FHM细胞中,与线粒体分布不重合,说明這鱼诺卡氏菌PTP蛋白并未靶向定位于线粒体.亚细胞定位和过表达研究都显示PTP蛋白在FHM细胞中表达后,细胞核出现固缩浓染、凋亡小体等明显的细胞凋亡特征.通过线粒体膜电位检测表明,在pcDNA-PTP转染后48 h,线粒体跨膜电位被明显破坏,说明這鱼诺卡氏菌PTP很可能是一种可诱导细胞凋亡的细菌蛋白.通过对這鱼诺卡氏菌PTP开展基因克隆和功能初步研究,为进一步揭示该基因的功能和深入了解這鱼诺卡氏菌的分子致病机理奠定了基础.     

Identification and functional characterization of Histone‐like DNA‐binding protein in Nocardia seriolae (NsHLP) involved in cell apoptosis

Nocardia seriolae, a facultative intracellular bacterium, is the main pathogen of fish nocardiosis. Bioinformatic analysis showed that the histone‐like DNA‐binding protein (HLP) gene of N. seriolae (nshlp) encoded a secreted protein and might target the mitochondria in the host cell. To further study the preliminary function of HLP in N. seriolae (NsHLP), the gene cloning, extracellular products identification, subcellular localization, overexpression and apoptosis detection assay were carried out in this study. Mass spectrometry analysis of the extracellular products from N. seriolae showed that NsHLP was a secreted protein. Subcellular localization of HLP‐GFP fusion proteins mainly assembled in the nucleus, which indicated that the NsHLP was co‐located with the nucleus rather than mitochondria in fathead minnow (FHM) cells. Notably, the expression of NsHLP had changed the distribution of mitochondria into lumps in the FHM cell. In addition, apoptotic features were found in the transfected FHM cells by overexpression of NsHLP. Quantitative assays of mitochondrial membrane potential value, caspase‐3 activity and pro‐apoptotic genes mRNA (Bad, Bid and Bax) expression level demonstrated that the cell apoptosis was induced in the transfected FHM cells. All the results presented in this study provided insight on the function of NsHLP, which suggested that it may participate in the cell apoptosis regulation and play an important role in the pathogenesis of N. seriolae.     

Preliminary investigation into the killing effect of kingfish (Seriola lalandi) serum and mucus against the monogenean parasites Benedenia seriolae and Zeuxapta seriolae

The survival of two economically important monogenean parasites, Benedenia seriolae and Zeuxapta seriolae, exposed to either naïve or infected yellowtail kingfish Seriola lalandi serum and mucus was observed over an 8-h period. Infection status of the host had no effect on survival of monogeneans in either serum or mucus therefore data were grouped according to each parasite. Serum exposure at the highest concentration of 1:5 did not have any effect on Z. seriolae, however, 50% killing activity against B. seriolae was observed within 3 h at concentrations greater than 1:100. This killing activity was completely abolished following heat treatment and inactivation of the serum. Prior incubation of parasites with heat-treated serum for 2 h in order to potentially coat the parasites with antibodies did not enhance the activity of fresh serum against either species. Addition of 5 mM EDTA, but not 5 mM EGTA-Mg, inhibited the killing ability of fresh serum against B. seriolae, suggesting that previously observed activity against this parasite was most likely mediated via the alternative complement pathway. Cutaneous mucus was not found to have any effect on B. seriolae or Z. seriolae survival. Overall these results suggest that the blood-feeding gill parasite Z. seriolae is considerably more resistant to host immune responses compared with skin- and mucus-dwelling B. seriolae.     

Vaccination of Full‐sib Channel Catfish Families Against Enteric Septicemia of Catfish with an Oral Live Attenuated Edwardsiella ictaluri Vaccine

This study evaluated the efficacy of an oral live attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish in 20 full‐sib fingerling channel catfish families. The vaccine was delivered orally by feeding fish a diet coated with an attenuated E. ictaluri isolate. Sixty‐nine days postvaccination, control and vaccinated fish were challenged with virulent E. ictaluri and mortality was examined for 21 d postchallenge. Vaccinated fish had significantly lower mortality than nonvaccinated fish following challenge (P < 0.001). Mortality of vaccinated fish was 1.7 ± 1.4% as opposed to 47.8 ± 28.7% in nonvaccinated fish. Relative percent survival ranged from 87.7 to 100% with an average of 95.2 ± 4.0% (±SE) among the 20 families of fish. There were significant differences in mortality among families in nonvaccinated fish (P < 0.01) while there were no differences among vaccinated families of fish. Results indicate that the live attenuated E. ictaluri vaccine is effective at reducing mortality in channel catfish exposed to virulent E. ictaluri. These data demonstrate that genetic differences among healthy families of channel catfish are not major considerations in developing an effective vaccination program utilizing the oral vaccination platform described in this study.     

Protective Immunity of Goldfish Against Ichthyophthirius multifiliis Infection Induced by Different Trophont Vaccine Preparations

Ichthyophthirius multifiliis, commonly called “Ich,” is a protozoan parasite that infects the epidermis and gills of freshwater fish. Here, we used goldfish (Carassius auratus) to determine whether the four vaccine preparations (live trophonts, formalin‐fixed trophonts, freeze‐thawed trophont lysates, and trophont cilial and cell cortical fractions) of I. multifiliis (Fouquet) can elicit resistance of immunized fish against subsequent theront challenge, and whether a relationship exists between in vitro immobilization of theronts in sera or mucus from immunized fish and host protective immunity against the parasite. Experimental goldfish were randomly divided into five groups with five parallel controls, with each group or subgroup consisting of 20 individuals. Each test goldfish was immunized with one of the four Ich vaccine preparations administered by one of the three routes: live trophonts by gavage (Group 1), formalin‐fixed trophonts by injection (Group 2) or by immersion (Group 3), freeze‐thawed trophont lysates by injection (Group 4), and trophont cilial and cell cortical fractions by injection (Group 5). The fish were then challenged by a standard Ich theront challenge on Day 21 postimmunization. For every 10 d following immunization, we tested the in vitro immobilization of Ich theronts by sera or mucus from immunized and control fish. We found that although all control (nonimmunized) and Group 3‐immunized goldfish died following theront challenge, more than half (55–65%) of the immunized fish from Groups 1, 2, 4, and 5 survived. Furthermore, except for the fish from Groups 3 and 4, the number of mean days to death in each test group was significantly higher than that in the respective control. These results indicate that goldfish immunized by injection or by gavage with any of the four vaccine preparations gained resistance to Ich infection. Further analysis indicated that this protective immunity was closely associated with the in vitro immobilization of Ich theronts by fish sera or mucus.     

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.     

Antibacterial effect of Lactococcus lactis subsp. lactis on Artemia franciscana nauplii and Dicentrarchus labrax larvae against the fish pathogen Vibrio anguillarum

The antibacterial effect of Lactococcus lactis subsp. lactis against the fish pathogen Vibrio anguillarum was determined in Artemia franciscana nauplii and in European sea bass (Dicentrarchus labrax) larvae fed with ten pulses of nauplii enriched with L. lactis. The evaluation of the bactericidal activity of the extracellular products of L. lactis in vitro showed inhibition of growth of Vibrio (Listonella) anguillarum and Photobacterium damselae subsp. piscicida. The incorporation of L. lactis in Artemia nauplii did not affect their survival and offered protection in a challenge with V. anguillarum, significantly increasing LD₅₀. The administration of Artemia nauplii enriched with L. lactis for 48 h to sea bass larvae for five consecutive days had no adverse effect on survival of fish. In an in vivo challenge test with V. anguillarum using sea bass larvae, fish treated with nauplii enriched with the probiotic L. lactis showed significantly (P < 0.001) increased survival rates of 81 % compared with the untreated group of challenged fish (24 %). Our results indicate that L. lactis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.     

Current knowledge of nocardiosis in teleost fish

Nocardia sp. is the causative agent of nocardiosis, a lethal granulomatous disease of the skin, muscle, and various inner tissues affecting various teleost and shellfish. Four species of Nocardia have been isolated from diseased fish and shellfish, namely Nocardia asteroides, Nocardia seriolae, Nocardia salmonicida and Nocardia crassostreae. Therefore, in fish aquaculture, nocardiosis has caused severe economic losses, especially in the Asian region. Considerable research has been performed, since the first report of identified Nocardia sp. in fish, to characterize Nocardia sp. and identify rapid detection techniques, immune response against infection and prophylactic approaches. In this review, the current state of knowledge about nocardiosis in fish has been presented, including the pathogenesis, diagnosis, host immune response and vaccine development.     

Identification of a cell-wall peptidase (NlpC/P60) from Nocardia seriolae which induces apoptosis in fathead minnow cells

Nocardia seriolae, a Gram-positive bacterium, is the main pathogen of fish nocardiosis. Protein NlpC/P60 is a cell-wall peptidase and a potential virulence factor of N. seriolae. Subcellular localization research revealed that both NlpC/P60-GFP and NlpC/P60Δsig-GFP fusion proteins were evenly distributed in the whole cell of fathead minnow (FHM) cells. Furthermore, typical apoptotic features, such as nuclear pyrosis and apoptotic bodies, were observed in the transfected FHM cells and grouper spleen cells by the overexpression of protein NlpC/P60. Then, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase-3 activity and apoptosis-related gene (Bax, BNIP3, TNF1 and TNF6) mRNA expression were conducted. The results showed that ΔΨm was decreased, caspase-3 was significantly activated, and the mRNA expression of pro-apoptotic genes (Bax and BNIP3) and tumour necrosis factors (TNF1 and TNF6) was up-regulated in NlpC/P60-overexpressed cells. Taken together, the results indicated that the protein NlpC/P60 of N. seriolae might involve in apoptosis regulation. This study may lay the foundation for further study on the function of N. seriolae NlpC/P60 and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae.     

Sequencing of 16S−23S rRNA internal transcribed spacer and its application in the identification of Nocardia seriolae by polymerase chain reaction

A method for the diagnosis of nocardiosis in yellowtail (Seriola quinqueradiata), using polymerase chain reaction (PCR), was developed in this study. Primers specific for Nocardia seriolae were synthesized based on the alignment of 16S?23S rRNA internal transcribed spacer region sequences of N. seriolae. The primers did not amplify specific PCR product from other fish pathogens. However, two and three fishes could be diagnosed as infected with N. seriolae by clinical signs and bacterial isolation. PCR amplification of N. seriolae by specific primers detected six infected fishes. Thus, the primers used in this study are useful in detecting nocardiosis in 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.     

Development of attenuated erythromycin‐resistant Streptococcus agalactiae vaccine for tilapia (Oreochromis niloticus) culture

Streptococcus agalactiae is an important pathogen in fish, causing great losses of intensive tilapia farming. To develop a potential live attenuated vaccine, a re‐attenuated S. agalactiae (named TFJ‐ery) was developed from a natural low‐virulence S. agalactiae strain TFJ0901 through selection of resistance to erythromycin. The biological characteristics, virulence, stability and the immunization protective efficacy to tilapia of TFJ‐ery were determined. The results indicated that TFJ‐ery grew at a slower rate than TFJ0901. The capsule thickness of TFJ‐ery was significantly less (p < 0.05) than TFJ0901. When Nile tilapia were intraperitoneally (IP) injected with TFJ‐ery, the mortality of fish was decreased than that injected with TFJ0901. The RPS of fish immunized with TFJ‐ery at a dose of 5.0 × 10⁷ CFU was 95.00%, 93.02% and 100.00% at 4, 8 and 16 weeks post‐vaccination, respectively. ELISA results showed that the vaccinated fish produced significantly higher (p < 0.05) antibody titres compared to those of control at 2 or 4 weeks post‐vaccination. Taken together, our results suggest that erythromycin could be used to attenuate S. agalactiae, and TFJ‐ery is a potent attenuated vaccine candidate to protect tilapia against S. agalactiae infections.     

Therapeutic and prophylactic immunization against Streptococcus iniae infection in hybrid striped bass (Morone chrysops×Morone saxatilis)

Vaccination strategies have traditionally been used as preventative or prophylactic measures against disease (prophylactic immunization) in uninfected fish. Alternatively, therapeutic or remedial measures, such as antibiotic administration, are commonly employed to treat disease in infected fish. Vaccination as a therapeutic measure (therapeutic immunization), however, has not been adequately explored in sub‐clinically infected fish. Therapeutic and prophylactic immunization with three Streptococcus iniae vaccines, formalin‐killed whole S. iniae cells (FKC vaccine), concentrated S. iniae extracellular products (greater than 2 kDa) (ECP vaccine) and a combination of killed cells and extracellular products (FKC+ECP vaccine), were tested in hybrid striped bass, Morone chrysops×Morone saxatilis, previously naturally infected with S. iniae. Fish (mean weight 10.0 g) were injected intraperitoneally (IP) or intramuscularly (IM) with one of each of the vaccines, tryptic soy broth (TSB‐control) or non‐injected (non‐injected control) to evaluate therapeutic effects (Trial 1). Survivors of the natural infection and ECP and FKC+ECP vaccine immunization and another lot of non‐injected control fish were immersion challenged with 1.47 × 10⁶ CFU of S. iniae mL^?1 at 44 days post‐immunization to evaluate vaccine efficacy (Trial 2). Hybrid striped bass (1.0 g) were also IM injected with S. iniae ECP vaccine at an aquaculture facility and immersion challenged with 1.47 × 10⁶ CFU of S. iniae mL^?1 12 weeks post‐immunization (Trial 3). The ECP and FKC+ECP vaccines, regardless of injection route, significantly (P<0.001) increased survival in asymptomatic, sub‐clinically infected fish thereby providing therapeutic merit. Hybrid bass immunized IP or IM had mean per cent survival values ranging from 78 to 96 at 44 days post‐immunization (Trial 1) and 69–97 post challenge (Trial 2). Survival of fish injected with TSB or immunized with FKC vaccine was significantly lowered and ranged from 12 to 13 by IP injection and 40 to 50 by IM injection and thus, the FKC vaccine had no therapeutic effect. The survival of hybrid striped bass IM immunized with S. iniae ECP vaccine in field Trial 3 was 91 and the RPS was 83. These results demonstrate that therapeutic immunization using S. iniae ECP and FKC+ECP vaccines can control a natural S. iniae infection. Furthermore, S. iniae ECP or FKC+ECP vaccines can also be used prophylacticly to protect hybrid striped bass against subsequent pathogen challenge.     

Assessment of cross‐protection to heterologous strains of Flavobacterium psychrophilum following vaccination with a live‐attenuated coldwater disease immersion vaccine

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, remains one of the most significant bacterial diseases of salmonids worldwide. A previously developed and reported live‐attenuated immersion vaccine (F. psychrophilum; B.17‐ILM) has been shown to confer significant protection to salmonids. To further characterize this vaccine, a series of experiments were carried out to determine the cross‐protective efficacy of this B.17‐ILM vaccine against 9 F. psychrophilum isolates (representing seven sequence types/three clonal complexes as determined by multilocus sequence typing) in comparison with a wild‐type virulent strain, CSF‐259‐93. To assess protection, 28‐day experimental challenges of rainbow trout (Oncorhynchus mykiss) fry were conducted following immersion vaccinations with the B.17‐ILM vaccine. F. psychrophilum strains used in challenge trials were isolated from several fish species across the globe; however, all were found to be virulent in rainbow trout. The B.17‐ILM vaccine provided significant protection against all strains, with relative percent survival values ranging from 51% to 72%. All vaccinated fish developed an adaptive immune response (as measured by F. psychrophilum‐specific antibodies) that increased out to the time of challenge (8 weeks postimmunization). Previous studies have confirmed that antibody plays an important role in protection against F. psychrophilum challenge; therefore, specific antibodies to the B.17‐ILM vaccine strain appear to contribute to the cross‐protection observed to heterologous strain. The ability of such antibodies to bind to similar antigenic regions for all strains was confirmed by western blot analyses. Results presented here support the practical application of this live‐attenuated vaccine, and suggest that it will be efficacious even in aquaculture operations affected by diverse strains of F. psychrophilum.     

Identification of a secreted superoxide dismutase (SOD) from Nocardia seriolae which induces apoptosis in fathead minnow (FHM) cells

Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen. The pathogenesis and virulence factors of N. seriolae are not fully understood. Secreted superoxide dismutase (SOD) may be a virulence factor found by a comparative bioinformatics analysis of the whole genome sequence of N. seriolae and the virulence factor database (VFDB). In order to determine the subcellular localization and study the preliminary function of SOD from N. seriolae (NsSOD), gene cloning, secreted protein identification, subcellular localization in fish cells, and apoptosis detection of NsSOD were carried out in this study. Subcellular localization research revealed that NsSOD‐GFP fusion proteins were evenly distributed in the cytoplasm. Furthermore, apoptotic bodies were observed in the transfected FHM cells by the overexpression of protein NsSOD. Then, assays of mitochondrial membrane potential (ΔΨm) value, caspase‐3 activity and apoptosis‐related genes (Bax, Bid, Bad and Bcl‐2) mRNA expression were conducted. The results showed that ΔΨm was decreased, and caspase‐3 was significantly activated. The mRNA expression of the Bad gene showed significant up‐regulated expression at 24 h.p.t., while Bid and Bax genes showed significant up‐regulated expression at 72 and 96 h.p.t. and anti‐apoptotic gene (Bcl‐2) was down‐regulated in NsSOD overexpressed cells. Taken together, the results indicated that the protein NsSOD might be involved in apoptosis regulation. This study may lay the foundations for further studies on the function of NsSOD and promote the understanding of the virulence factors and the pathogenic mechanisms of N. seriolae.