Relative Percent Survival Values Research Articles

Oral feed-based administration of phage cocktail protects rohu fish (Labeo rohita) against Aeromonas hydrophila infection.

Aeromonas hydrophila is one of the major freshwater fish pathogens. In the current study, a cocktail of D6 and CF7 phages was given orally to Labeo rohita to assess phage survival in fish organs as well as to determine the therapeutic efficacy of phage treatment against fish mortality caused by A. hydrophila. In the phage-coated feed, prepared by simple spraying method, phage counts were quite stable for up to 2 months with a decline of ≤ 0.23 log10 and ≤ 1.66 log10 PFU/g feed during 4 oC and room temperature storage. Throughout the experimental period of 7 days, both phages could be detected in the gut of fish fed with phage-coated feed. Besides, both CF7 and D6 phages were also detected in fish kidneys indicating the ability of both the phage to cross the intestinal barrier. During challenge studies with LD50 dose of A. hydrophila, phage cocktail doses of 1 × 106 - 1 × 108 PFU/g feed prevented the mortality in L. rohita with relative percentage survival (RPS) of 8.7-65.2. When challenged with LD90 dose of A. hydrophila, an RPS value of 28.6 was obtained at a phage cocktail dose of 1 × 108 PFU/g feed. The RPS data showed that orally-fed phage cocktail protected the fish against the mortality caused by A. hydrophila in a dose-dependent manner. Simple practical approaches for phage cocktail development, medicated feed preparation and oral administration along with phage survival and protection data make the current study useful for farmer-level application.

Efficacy of the Whole Cell Vaccine Streptococcus agalactiae with Vector Artemia sp. for Streptococcosis Prevention in Nila Fish (Oreochromis niloticus)

This study aims to examine the efficacy of the oral whole-cell vaccine of S. agalactiae with Artemia sp as a vaccine vector for prevention against Streptococcosis in tilapia fish. The fish seeds used in the study were 2-3 cm in size which did not carry S. agalactiae bacteria and were reared in jars with a volume of 3 liters as many as ten birds/jar. Giving the vaccine to fish orally by first soaking the vaccine in Artemia sp. The soaking times for the vaccine were 15, 30, 60, 90, 120 and 150 minutes. Parameters observed were relative percent survival (RPS), antibody titer value, total leukocytes, phagocytic index, and leukocyte differential. The results showed that the treatment between vaccines had no significant effect, while the treatment with the control vaccine had a significant effect (P<0.05). The highest RPS value was obtained in the vaccine soaking treatment in Artemia sp. for 30 minutes and 120 minutes, with a value of 93.33%. Furthermore, the RPS was 86.67% at 15 and 150 minutes of soaking and 80% RPS at 30 minutes of soaking. This shows that the level of vaccine protection against streptococcosis in tilapia fish is very high.

The protective effect of inactivated Flavobacterium columnare vaccine in grass carp (Ctenopharyngodon idellus).

Flavobacterium columnare, which causes columnaris disease, is responsible for significant mortality in grass carp. Vaccination is a safe and effective measure to combat this disease, and this study aimed to investigate the immune protective effects of different treatments using an inactivated F. columnare vaccine. The vaccine was prepared by inactivating the bacteria with 0.05% formaldehyde at 4°C for 24 hours. The experiments involving grass carp were divided into two parts. In Experiment 1, the immune effects of two isolates, JX-01 (genomovar I) and MU-04 (genomovar II), were compared, along with the impact of white oil adjuvant and the number of immunizations. The results showed that when the white oil adjuvant was used as a booster, the relative percent survival (RPS) of the JW2 group and MW2 group after 8 weeks of the first immunization was 34% and 61%, respectively. In Experiment 2, only the MU-04 (genomovar II) isolate was used as an antigen, with the white oil adjuvant as a booster. The effects of different doses (CFU=108, 107, and 106 bacteria/mL) on immune responses were compared, and the RPS values in the MW6, MW7, and MW8 groups after 4 weeks of the first immunization were found to be 38%, 57%, and 71%, respectively. Furthermore, in the cross-antigen protection experiment, the MW2 group exhibited an RPS of 55% against the JX-01 isolate, which was significantly higher than the control group (33%). These findings suggest that an inactivated vaccine comprising an appropriate antigen isolate when administered with a white oil adjuvant as a booster, can provide effective protection in grass carp.

Decreased resistance to bacterial cold-water disease and excessive inflammatory response in ayu (Plecoglossus altivelis) reared at high water temperature.

Temporal elevation of water temperature positively affects immune activity and disease resistance in poikilothermic teleost fish. The ayu, Plecoglossus altivelis, an important fish species for Japanese freshwater fisheries, is usually produced under higher water temperatures than the natural conditions to facilitate rapid growth. However, it has been reported that rearing fish at higher water temperatures inhibits the development of the thymus, suggesting that resistance to infectious diseases is reduced in ayu reared at higher water temperatures. Here, we show that decreased resistance to bacterial cold-water disease and excessive inflammatory responses occurred in ayu reared at 22°C compared with those reared at lower temperatures. Ayu larvae were reared at 12°C, 15°C and 22°C for 77 days and fed 3% of their body weight. Thymus index and condition factor was calculated after the fish rearing. Then, ayu reared at the different temperatures were challenged with Flavobacterium psychrophilum and the fish were sampled for histopathology and gene expression analyses. Further, the fish were vaccinated with formalin-killed F. psychrophilum and continuously reared at the three different water temperatures. Serum antibody titer was determined by ELISA and cumulative mortality in each group was recorded after the bacterial challenge. Ayu reared at 22°C showed a significantly lower thymus index and higher condition factor than those reared at lower temperatures. Infiltrated leukocytes and many melanin pigments were frequently observed in the adipose tissues and spleens of ayu reared at 22°C, respectively, but not in those reared at 12°C. The gene expression levels of inflammatory cytokines such as IL-1β, IL-8 and TNFα in the spleen were significantly higher in the 22°C group than in the 12°C group. The cumulative survival rate after challenge with Flavobacterium psychrophilum was 51.7%, 40.0% and 13.3% in the 12°C, 15°C and 22°C groups, respectively. The relative percent survival values of vaccinated fish reared at 15°C and 22°C groups were lower than those reared at 12°C. Moreover, the specific antibody titer of the vaccinated fish was the lowest in the 22°C group and the highest in the 12°C group. These results suggest that rearing the fish under high water temperature causes excessive inflammatory responses similar to metabolic inflammation in human obesity, resulting in a decrease of disease resistance. In addition, thymic involution induced by higher water temperature probably leads the poor response to vaccination. The present study provides insights into the physiological and immunological changes of fish under global warming.

Saponin and chitosan-based oral vaccine against viral haemorrhagic septicaemia virus (VHSV) provides protective immunity in olive flounder (Paralichthys olivaceus)

Production losses of olive flounder (Paralichthys olivaceus) have increased owing to viral haemorrhagic septicaemia virus (VHSV) infection. In this study, we determined safe concentrations of orally administered saponin and chitosan by analysing serum enzyme (AST/ALT) levels as biochemical markers of hepatic injury. Furthermore, we demonstrated the efficacy, duration of protection, and safety of saponin and chitosan-based vaccines with inactivated VHSV (IV). Oral administration of saponin, chitosan, and their combination did not induce fish mortality at all tested concentrations (0.29, 1.45, and 2.9 mg/g of fish body weight/day) 10 days after administration. However, AST level was high at a dose >0.29 mg/g of fish body weight/day. Both saponin and chitosan were found to be safe and acceptable for vaccination studies at a dose of 0.29 mg/g of fish body weight/day. Administration of IV alone did not induce protection at 2 and 4 weeks post vaccination (wpv). Olive flounders administered saponin + IV and chitosan + IV vaccines had higher immunity against VHSV with relative percentage survival (RPS) of 12.5–7.5% and 0–20.1%, respectively; however, additional immunisation with combination of saponin + chitosan + IV clearly enhanced the protection with RPS values of 10–15%, 26.7%, 42.9%, and 37.5% at 4, 8, 12, and 20 wpv, respectively. Although the RPS value of oral immunisation was not comparable to that of injectable vaccines, the manufacturing process is simple and oral administration causes less stress to juvenile fish. To investigate the development of a protective immune response, olive flounder were re-challenged with VHSV (107.8 TCID50/fish) at 70 days postinfection; 100% of the previously unexposed fish died, whereas 80–100% of the previously immunised fish survived. Our results showed the possibility of developing preventive measures against VHSV using saponin and chitosan-based oral vaccines with inactivated virus.

Evaluation of an oral DNA nanovaccine against photobacteriosis in Solea senegalensis

Infectious diseases are one of the main causes of social and economical losses in world aquaculture. Senegalese sole (Solea senegalensis) is an important species for aquaculture in southern Europe, whose production is affected by the appearance of bacterial diseases such as photobacteriosis, a septicemia caused by Photobacterium damselae subsp. piscicida (Phdp). The aim of this study was to obtain an oral DNA nanovaccine and to evaluate its efficacy against Phdp in S. senegalensis juveniles. For this purpose, the amplified product corresponding to the protein inosine-5′-monophophate dehydrogenase (IMPDH) from Phdp, was cloned into the expression vector pcDNA™6.2/C-EmGFP-GW obtaining the DNA vaccine named as pPDPimpdh. The correct transcription and protein expression was verified at 48 h post tansfection in HEK293 cells. Chitosan nanoparticles (CS-TPP NPs) were prepared by ionotropic gelation and their features were appropriate for use as oral delivery system. Therefore, pPDPimpdh was protected with chitosan CS-TPP NPs throughout complex coacervation method giving as a result a DNA nanovaccine referred as CS-TPP+pPDPimpdh NPs. Sole juveniles were vaccinated orally with CS-TPP NPs, pPDPimpdh and CS-TPP+pPDPimpdh NPs followed by a challenge with Phdp at 30 days post vaccination (dpv). The relative percentage survival (RPS) for pPDPimpdh vaccinated groups was 6.25%, probably due to its degradation in the digestive tract. RPS value obtained for CS-TPP NPs and CS-TPP+pPDPimpdh NPs was 40% and antibodies were observed in both cases. However, a delay in mortality was observed in sole juveniles vaccinated orally with CS-TPP+pPDPimpdh NPs. In fact, an upregulation of tf, mhcII, cd8a and igm in the posterior gut and c3, hamp1, tf and cd4 in spleen was observed in juveniles vaccinated with CS-TPP+pPDPimpdh NPs. After challenge, a modulation of cd8a and cd4 expression levels in the posterior gut and c3, tf, lyg, cd4, igm and igt expression levels in spleen was observed. Moreover, the concentration of lysozyme in skin mucus significantly increased in fish vaccinated orally with CS-TPP+pPDPimpdh NPs at 11 dpc. These data indicate that oral vaccination with CS-TPP+pPDPimpdh NPs could be acting through the non-specific immune responses as well as the specific humoral and cell mediated immunity and provide the first step toward a development of an oral DNA nanovaccine against Phdp in sole.

Effects of dietary hydrolyzable tannins on growth performance, antioxidant capacity, intestinal microflora and resistance against Vibrio parahaemolyticus of juvenile Pacific white shrimp, Litopenaeus vannamei (Boone, 1931)

Tannins have great potential as an alternative to in-feed antibiotics. However, no study has investigated the effects of tannins on shrimp. The present study assessed the eff ;ects of hydrolyzable tannins (HTs) fed at different inclusion levels (F0: control, F0.5: 0.05 %, F1: 0.1 %, F1.5: 0.15 % and F2: 0.2 % (g−1 feed) on the growth performance, antioxidant capacity, immune response, intestinal microflora and resistance to Vibrio parahaemolyticus of juvenile Pacific white shrimp (Litopenaeus vannamei). After 60 days of the feeding trial, a significant increase (P < 0.05) of weight gain rate (WGR), specific growth rate (SGR), activities of alkaline phosphatase (ALP), acid phosphatase (ACP), superoxide dismutase (SOD) and trypsin in serum and hepatopancreas, and the expression level of catalase (CAT), Cu/Zn superoxide dismutase (Cu/Zn-SOD) and glutathione peroxidase (GPx) gene in the hepatopancreas of 0.15 % HT treated groups were achieved. The dietary supplementation of HTs also changed the composition of intestinal microflora. At the phylum level, Proteobacteria accounted for the majority of bacteria, followed by Firmicutes, Bacteroidetes and Actinobacteria. At the genus level, Stenotrophomonas, Ruegeria, and Photobacterium were dominant in the F0.5, F1, and F1.5 groups, respectively. The relative abundance of Vibrio and Carboxylicivirga decreased in the intestines of shrimp fed with HTs diets. Furthermore, F1.5 group showed the highest relative percent survival value (80 %) (P < 0.05), which indicates that the supplementation of 0.15 % HTs in challenge test significantly enhanced the resistance of shrimp against V. parahaemolyticus infection after 24 h. These results indicated that 0.15 % HTs could remarkably improve the growth performance, antioxidant capacity, intestinal microflora, and resistance against V. parahaemolyticus of L. vannamei, and can be a potential feed additive for shrimp in aquaculture. According to our study, the optimal dietary supplementation dosage of HTs is 0.15 % feed in diets.

Stimulatory effect of dietary chasteberry (Vitex agnus-castus) extract on immunity, some immune-related gene expression, and resistance against Aeromonas hydrophila infection in goldfish (Carassius auratus)

Chasteberry is highly recommended as an herbal medicine across the globe for treating of many gynaecological disorders. In this study, chasteberry extract (CBE) was supplemented in goldfish diet to evaluate the immunity responses at the cellular and molecular levels. Moreover, after the feeding trial, the fish were challenged with Aeromonas hydrophila. The fish (300 individuals, 2.4 ± 0.12 g initial weight) were randomly distributed in 12 tanks and were fed with 0 (control), 5, 10, and 15 g CBE per kg of feed for 8 weeks. Based on the results, lysozyme activity, alkaline phosphatase, and total immunoglobulin (Ig) in the skin mucus samples were significantly enhanced in the fish fed with 15 g/kg CBE (P < 0.05). Moreover, dietary CBE positively affected lysozyme activity, complement components, and IgM content of the serum samples compared to the control group. Also, the number of monocytes and lymphocytes were increased significantly with increasing CBE in the diet (P < 0.05). The highest mRNA levels of tumor necrosis factors (TNF-α, TNF-2α) and Lysozyme were observed in 15 g/kg CBE treatment. After the challenge test, the highest relative percentage survival value (60%) was observed in the fish fed with 15 g/kg CBE. We concluded that dietary CBE especially at 15 g/kg has an immunomodulatory effect in goldfish by stimulating the innate immunity and some inflammatory cytokines as well as disease resistance against A. hydrophila.

Carvacrol essential oil stimulates growth performance, immune response, and tolerance of Nile tilapia to Cryptococcus uniguttulatus infection.

This study investigated the antifungal activity of 5 essential oils (EOs) towards yeasts recovered from diseased fishes; and focused on the efficacy of one EO (carvacrol) on growth performance, non-specific immunity, and disease resistance of Nile tilapia Oreochromis niloticus against Cryptococcus uniguttulatus challenge. Thymoquinone, thymol, carvacrol, eugenol, and cinnamon were first tested in vitro against 20 clinical yeast strains in comparison with antifungal drugs (fluconazole, ketoconazole, itraconazole, amphotericin B, nystatin, and clotrimazole) using disc diffusion and broth microdilution methods. For the in vivo challenge, fish (n = 150) were divided into 5 groups (carvacrol prophylaxis, carvacrol treatment, itraconazole treatment, unchallenged control, and positive control; 30 fish group-1) with 3 replicates. Phagocytic activity, reactive oxygen species production, reactive nitrogen species production, myeloperoxidase, lysozyme activity, and total immunoglobulins were tested before and after challenge. Relative percent survival (RPS) and mortality percent were determined as indicators for functional immunity. EOs displayed divergent degrees of antifungal activity, and carvacrol was the most effective against the tested yeasts. The dietary additive of carvacrol significantly enhanced growth performance, all immunological parameters, and the RPS values (90%) compared to other treatments. This unique experimental model indicates that carvacrol seems promising not only for enhancing immunity and promoting fish growth, but also for controlling emerging fungal infections. Future studies should investigate different concentrations of carvacrol as well as its antifungal activity in different fish species.

Aeromonas salmonicida infection kinetics and protective immune response to vaccination in sablefish (Anoplopoma fimbria)

Effective vaccine programs against Aeromonas salmonicida have been identified as a high priority area for the sablefish (Anoplopoma fimbria) aquaculture. In this study, we established an A. salmonicida infection model in sablefish to evaluate the efficacy of commercial vaccines and an autogenous vaccine preparation. Groups of 40 fish were intraperitoneally (ip) injected with different doses of A. salmonicida J410 isolated from infected sablefish to calculate the median lethal dose (LD50). Samples of blood, head kidney, spleen, brain, and liver were also collected at different time points to determine the infection kinetics. The LD50 was estimated as ~3 × 105 CFU/dose. To evaluate the immune protection provided by an autogenous vaccine and two commercial vaccines in a common garden experimental design, 140 fish were PIT-tagged, vaccinated and distributed equally into 4 tanks (35 fish for each group, including a control group). Blood samples were taken every 2 weeks to evaluate IgM titers. At 10 weeks post-immunization, all groups were ip challenged with 100 times the calculated LD50 for A. salmonicida J410. A. salmonicida was detected after 5 days post-infection (dpi) in all collected tissues. At 30 days post-challenge the relative percentage survival (RPS) with respect to the control group was calculated for each vaccine. The RPS for the bacterin mix was 65.22%, for Forte Micro 4® vaccine was 56.52% and for Alpha Ject Micro 4® was 30.43%, and these RPS values were reflected by A. salmonicida tissue colonization levels at 10 days post-challenge. Total IgM titers peaked at 6–8 weeks post-immunization, where the autogenous vaccine group showed the highest IgM titers and these values were consistent with the RPS data. Also, we determined that the A. salmonicida A-layer binds to immunoglobulins F(ab)' in a non-specific fashion, interfering with immune assays and potentially vaccine efficacy. Our results indicate that vaccine design influences sablefish immunity and provide a guide for future sablefish vaccine programs.

Development of a live vector vaccine against infectious pancreatic necrosis virus in rainbow trout

Infectious pancreatic necrosis virus (IPNV) causes severe viral disease in salmonids, with major economic losses in the global rainbow trout aquaculture industry following from a high mortality rate. In the present study, we aimed to develop a safe and efficient vaccine to protect rainbow trout against IPNV infection. Administered via the immersion route, a live vector vaccine containing the coding region of the IPNV VP2 induced protective immune responses in rainbow trout. For best protection, juvenile rainbow trout should be immunized with a 1:100 dilution of the titer with 1 × 1010.0 ml−1 TCID50 of the live vector vaccine, via 10-min immersion. Expression of the IPNV VP2 gene was confirmed in spleens of vaccinated rainbow trout, and reached the highest level at 3 days post-vaccination (dpv) and thereafter gradually decreased between 3 and 15 dpv. The expression of TLR-3, TLR-7 and TLR-8 was upregulated after vaccination, and the highest expression levels of IFN-1, Mx-1, Mx-3, Vig-1 and Vig-2 were detected at 3 dpv. Four markers of the adaptive immune response (CD4, CD8, IgM and IgT) were continuously increased in the spleens of vaccinated fish as compared with controls, between 3 and 15 dpv. The cumulative percentage mortality differed significantly between the vaccinated fish and the controls (empty-plasmid-vaccinated). The results of the IPNV challenge showed that the live vector vaccine had a high protection rate against IPNV (relative percent survival value of 88.24), and the vaccinated rainbow trout displayed high levels of serum antibodies against IPNV infection. Taken together, our results demonstrate that this is a promising live vector vaccine that could be used to protect rainbow trout against IPNV infection.

Development of attenuated Vibrio parahaemolyticus mutant strains as potential live vaccines

Vibrio parahaemolyticus is a pathogenic Gram-negative bacterium that commonly infects numerous species of marine fishes. In this study, we obtained eight attenuated mutant strains by the selection of rifampicin-resistant V. parahaemolyticus from three wild-type strains. Among eight selected strains, four strains (L4650, A650, L4200 and A400) were widely attenuated by virulence analysis. Sequences of two genes, tlh and toxR, that related to virulence were analyzed. The results showed that seven attenuated strains were different in sequences of the tlh gene while there was no difference in sequence of toxR gene in comparison with that of wild-type strains. Three attenuated strains (L4650, L4200 and A650) had the nucleotide changes in tlh gene that resulted to the changes in the size of deduced amino acid sequences. In addition, rpoB gene coding for -subunit of RNA polymerase that related to Rifampicin resistance was analyzed for all selected strains. Two strains (A650 and L4650) had the nucleotide changes in ropB sequence. To assess the potential use of the L4650 strain as a live attenuated vaccine, the groupers (Epinephelus coioides) were intraperitoneally injected with the L4650 strain and later challenged with the virulent wild-type strain at three different doses. Results showed the protection for L4650 strain vaccinated-fish produced relatively earlier than usual at 15 days post-vaccination with relative percent survival (RPS) values from 96.91-100%. At 60 days post-vaccination, the RPS values of vaccinated fish were 96.27-100%. Our results suggest the L4650 strain could become a potential vaccine candidate against V. parahaemolyticus for future studies.

Maternal immunity of tilapia broodstock vaccinated with polyvalent vaccine and resistance of their offspring againstStreptococcus agalactiae

This study aimed to examine the use of Streptococcus agalactiae polyvalent vaccine in tilapia broodstock and the effect of maternal immunity and resistance on their offspring against S. agalactiae strain. The broodstock was injected with polyvalent vaccine of S. agalactiae at a dose of 108 CFU per fish at 2nd gonadal maturity until spawning. Challenge test was carried out on the offspring at the 5, 10, 15 and 20 days after hatching using NK1, N17O, N14G, N3M, N4M strain respectively and combination of them. We observed immunological parameters in broodstock, eggs and larvae and relative per cent survival (RPS) of larvae after challenged with pathogenic S. agalactiae. The results showed that the leukocytes, phagocytic activity, respiratory burst, lysozyme activity and antibody levels of vaccinated broodstock had higher level compared with unvaccinated broodstock. The high level of the lysozyme activity, antibody levels and recombination activating gene 1 (RAG1) were also observed in eggs and larvae from vaccinated broodstock. Larvae produced from vaccinated broodstock when challenged with variety strain of pathogenic S. agalactiae had RPS value more than 50% until 20 days after hatching. In conclusion, polyvalent vaccine of S. agalactiae administrated in the broodstock could enhance immunity in the broodstock and protect their offspring from pathogenic S. agalactiae.

Cross-immunity in Nile tilapia vaccinated with Streptococcus agalactiae and Streptococcus iniae vaccines

Streptococcus agalactiae and Streptococcus iniae are major bacterial pathogens of tilapia that can cause high mortality concomitant with large economic losses to aquaculture. Although development of vaccines using formalin-killed bacteria to control these diseases has been attempted, the mechanism of immunity against streptococcal infections and the cross-protective ability of these two bacteria remains unclear. To explore the immunological role of these vaccines, we compared the immune responses of tilapia after immunization with both vaccines and compared the relative percent survival (RPS) and cross-immunization protection of tilapia after separate infection with S. agalactiae and S. iniae. All results revealed that vaccinated fish had significantly higher (P < 0.05) levels of specific antibodies than control fish 14 days post secondary vaccination (PSV) and 7 days post challenge. In vaccinated fish, the mRNA expression of interleukin-8 (IL-8), interleukin-12 (IL-12), caspase-3 (C-3), tumour necrosis factor (TNF), and interferon (IFN) was significantly up regulated (P < 0.05) in the head kidney after immunized; similar results were found for IL-8, TNF and IFN in the posterior kidney, meanwhile the expression levels of C-3 and IFN were significantly increased (P < 0.05) in the spleen of vaccinated fish. Additionally, the levels of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) in vaccinated fish were improved at different degree when compared to the control fish. These results showed that vaccination with formalin-killed cells (FKCs) of either S. agalactiae or S. iniae conferred protection against infection by the corresponding pathogen in Nile tilapia, resulting in RPS values of 92.3% and 91.7%, respectively. Furthermore, cross-protection was observed, as the S. agalactiae FKC vaccine protected fish from S. iniae infection, and vice versa. These results suggested that the S. agalactiae and S. iniae FKC vaccines can induce immune responses and generate excellent protective effects in Nile tilapia.

Construction and evaluation of Vibrio alginolyticus ΔclpP mutant, as a safe live attenuated vibriosis vaccine

Vibrio alginolyticus is a common and serious pathogen threatening the progress of coastal aquaculture. ClpP protease has been proved to be closely associated with biofilm formation, stress tolerance, autolysis and virulence in several pathogens. Hence, targeting ClpP may be a potentially viable, attractive option for the preparation of vaccine in preventing vibriosis. In this study, an in-frame deleted mutant strain (ΔclpP) was constructed by allelic exchange mutagenesis to investigate physiological role of clpP in pathogenicity of V. alginolyticus and evaluate its potential as a live attenuated vaccine. The results exhibited that ΔclpP showed no differences in external morphology, growth, swarming motility and ECPase activity. However, ΔclpP represented an increment in biofilm formation, and a decrement in adherence to CIK cells. In addition, virulence of ΔclpP was examined in pearl gentian grouper and was found to be seriously attenuated. ΔclpP induced high antibody titers and provided a valid protection with a relative percent survival value of 83.8% without histopathologic abnormality. Our results indicated ΔclpP showed a great potential to be a live attenuated vaccine.

Immersion immunization with recombinant baculoviruses displaying cyprinid herpesvirus 2 membrane proteins induced protective immunity in gibel carp

Cyprinid herpesvirus 2 (CyHV-2) is the causative pathogen of herpesviral haematopoietic necrosis disease, which has caused huge economic losses to aquaculture industry in China. In this study, nine truncated CyHV-2 membrane glycoproteins (ORF25, ORF25C, ORF25D, ORF30, ORF124, ORF131, ORF136, ORF142A, ORF146) and a GFP reporter protein were respectively expressed using baculovirus surface displaying system. Western blot showed that the proteins were successfully packaged in the recombinant virus particles. In baculovirus transduced gibel carp kidney cells, the target proteins were expressed and displayed on the fish cell surface. Healthy gibel carp were immunized by immersion with the recombinant baculoviruses and the fish treated with phosphate-buffered saline (PBS) were served as mock group. The expression of interleukin-11 (IL-11), interferon α (IFNα) and a complement component gene C3 were significantly up-regulated in most experimental groups, and interferon γ (IFNγ) expression in some groups were also induced after immunization. Subsequently, the immunized gibel carp were challenged by intraperitoneal injection of CyHV-2 virus. All the immunized groups exhibited reduced mortality after CyHV-2 challenge. In the groups immunized with baculoviruses displaying and expressing ORF25, ORF25C and ORF146, the relative percentage survival values reached 83.3%, 87.5% and 70.8%, respectively. Our data suggested that baculovirus-displayed ORF25, ORF25C and ORF146 could be potential vaccine candidates for the prevention of CyHV-2 infection in gibel carp.

Anti-inflammatory mediators and appetite regulatory neuropeptides are affected by chronic stress in Salmo salar

Pseudomonas plecoglossicida is well-known as the cause of viscera granulomas disease in fish. In this study, a cspA1 knock-down strain was constructed and tested in Epinephelus coioides to observe the changes in virulence and evaluate its potential as an attenuated live vaccine. The results showed that the cspA1 knock-down strain caused a significant reduction in the ability of biofilm formation, motility, adhesion and virulence. E. coioides vaccinated with cspA1 knock-down strain were more tolerant of the infection by wild-type P. plecoglossicida. The relative percent survival value of E. coioides vaccinated with cspA1 knock-down strain reached 80% after challenging with wild-type P. plecoglossicida. In the meanwhile, the expression level of genes associated with immunity, including IL-1β, IgM, MHC-I and MHC-II, was up-regulated after vaccination, indicating that the cspA1 knock-down strain can induce effective and durable immune response in E. coioides and it may be an effective attenuated live vaccine candidate for the prevention of infections by P. plecoglossicida.

Protection against Pseudomonas plecoglossicida in Epinephelus coioides immunized with a cspA1-knock-down live attenuated vaccine

Pseudomonas plecoglossicida is well-known as the cause of viscera granulomas disease in fish. In this study, a cspA1 knock-down strain was constructed and tested in Epinephelus coioides to observe the changes in virulence and evaluate its potential as an attenuated live vaccine. The results showed that the cspA1 knock-down strain caused a significant reduction in the ability of biofilm formation, motility, adhesion and virulence. E. coioides vaccinated with cspA1 knock-down strain were more tolerant of the infection by wild-type P. plecoglossicida. The relative percent survival value of E. coioides vaccinated with cspA1 knock-down strain reached 80% after challenging with wild-type P. plecoglossicida. In the meanwhile, the expression level of genes associated with immunity, including IL-1β, IgM, MHC-I and MHC-II, was up-regulated after vaccination, indicating that the cspA1 knock-down strain can induce effective and durable immune response in E. coioides and it may be an effective attenuated live vaccine candidate for the prevention of infections by P. plecoglossicida.

Superoxide dismutase B (sodB), an important virulence factor of Vibrio alginolyticus, contributes to antioxidative stress and its potential application for live attenuated vaccine

Vibrio alginolyticus is an opportunistic and halophilic Gram-negative pathogen in limiting the development of aquatic industry and affecting human health. SODs are oxidative enzymes that play a critical role in oxidative defense. In this study, an in-frame deleted mutant strain (ΔsodB) was constructed by allelic exchange mutagenesis to investigate physiological role of sodB in pathogenicity of V. alginolyticus. The results exhibited that ΔsodB showed no differences in growth compared with wild-type strain HY9901 (WT), but led to increasing in biofilm formation, ECPase activity and sensitivity to hydrogen peroxide, decreasing in swarming motility, adherence to CIK cells, SOD activity and virulence. In addition, ΔsodB induced a high antibody titer and provided a valid protection with a relative percent survival value of 86.5% without inducing clinical symptoms after challenging with WT. These results suggest that sodB is important for normal physiological function, oxidation resistance and virulence in V. alginolyticus, and ΔsodB may be considered as an effective live attenuated vaccine against V. alginolyticus.

Protection against Vibrio alginolyticus in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu) immunized with an acfA-deletion live attenuated vaccine

Vibrio alginolyticus is well-known as an opportunistic Gram-negative pathogen, which endangers the development of global aquaculture as well as human health. In this study, a ΔacfA mutant strain and complementation of the ΔacfA mutant (C-acfA) were constructed. The ΔacfA mutant was tested in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu) to observe the changes in virulence and evaluate its potential as an attenuated live vaccine. The results showed that the ΔacfA mutant caused a high antibody titer and a significant reduction in the ability to colonize the intestine of pearl gentian grouper. Grouper vaccinated with ΔacfA mutant were more tolerant of the infection by virulent V. alginolyticus HY9901 without inducing clinical symptoms and obvious pathological changes. The relative percent survival value of pearl gentian grouper vaccinated with ΔacfA mutant intraperitoneal injection reached 81.1% after challenging with V. alginolyticus HY9901. The specific antibody titers immunized with ΔacfA was significantly higher than that in the PBS group. The antibody titer of ΔacfA group displayed the tendency of rising up from the first to fourth week and declining from fifth to eighth week and reached the peak at the fourth week. In the meanwhile, the expression level of genes associated with immunity, including IL-1β, TNF-α, IL-16, IgM, CD8α and MHC-Iα, was up-regulated after vaccination, indicating that the ΔacfA can induce effective and durable immune response in pearl gentian grouper and it may be an effective attenuated live vaccine candidate for the prevention of infections by V. alginolyticus.