A nanocarrier immersion vaccine encoding surface immunogenic protein confers cross-immunoprotection against Streptococcus agalactiae and Streptococcus iniae infection in tilapia.

Streptococcosis is a highly contagious aquatic bacterial disease that poses a significant threat to tilapia. Vaccination is a well-known effective measure to prevent and control fish bacterial diseases. Among the various immunization methods, immersion vaccination is simple and can be widely used in aquaculture. Besides, nanocarrier delivery technology has been reported as an effective solution to improve the immune effect of immersion vaccine. In this study, the surface immunogenic protein (Sip) was proved to be conserved and potential to provide cross-immunoprotection for both Streptococcus agalactiae (S. agalactiae) and Streptococcus iniae (S. iniae) by multiple sequences alignment and Western blotting analysis. On this basis, we expressed and obtained the recombinant protein rSip and connected it with functionalized carbon nanotubes (CNT) to construct the nanocarrier vaccine system CNT-rSip. After immersion immunization, the immune effect of CNT-rSip against above two streptococcus infections was evaluated in tilapia based on some aspects including the serum specific antibody level, non-specific enzyme activities, immune-related genes expression and relative percent survival (RPS) after bacteria challenge. The results showed that compared with control group, CNT-rSip significantly (P < 0.05) increased the serum antibody levels, related enzyme activities including acid phosphatase, alkaline phosphatase, lysozyme and total antioxidant capacity activities, as well as the expression levels of immune-related genes from 2 to 4 weeks post immunization (wpi), and all these indexes peaked at 3 wpi. Besides, the above indexes of CNT-rSip were higher than those of rSip group with different extend during the experiment. Furthermore, the challenge test indicated that CNT-rSip provided cross-immunoprotection against S. agalactiae and S. iniae infection with RPS of 75 % and 72.41 %, respectively, which were much higher than those of other groups. Our study indicated that the nanocarrier immersion vaccine CNT-rSip could significantly improve the antibody titer and confer cross-immuneprotection against S. agalactiae and S. iniae infection in tilapia.

Nocardia seriolae mediates liver granulomatous chronic inflammation in Micropterus salmoides through pyroptosis.

Granulomatous diseases caused by Nocardia seriously endanger the health of cultured fish. These bacteria are widely distributed, but prevention and treatment methods are very limited. Chronic granulomatous inflammation is an important pathological feature of Nocardia infection. However, the molecular mechanisms of granuloma formation and chronic inflammation are still unclear. Constructing a granuloma infection model of Nocardia is the key to exploring the pathogenesis of the disease. In this study, we established a granuloma model in the liver of largemouth bass (Micropterus salmoides) and assessed the infection process of Nocardia seriolae at different concentrations by analysing relevant pathological features. By measuring the expression of pro-inflammatory cytokines, transcription factors and a pyroptosis-related protein, we revealed the close relationship between pyroptosis and chronic inflammation of granulomas. We further analysed the immunofluorescence results and the expression of pyroptosis-related protein of macrophage infected by N. seriolae and found that N. seriolae infection induced macrophage pyroptosis in vitro. These results were proved by flow cytometry analysis of infection experiment in vivo. Our results indicated that the pyroptosis effect may be the key to inducing chronic inflammation in the fish liver and further mediating granuloma formation. In this study, we explored the molecular mechanism underlying chronic inflammation of granulomas and developed research ideas for understanding the occurrence and development of granulomatous diseases in fish.

Effect of dietary Ficus carica polysaccharides on the growth performance, innate immune response and survival of crucian carp against Aeromonas hydrophila infection.

Ficus carica polysaccharides (FCPS), one of the most effective and important compo-nents in Ficus carica L., had been considered to be a beneficial immunostimulant and may be used in immunotherapy for animals and human. However, studies were little about the effect of FCPS used as immunomodulatory and the suitable dosage in fish. The present study investigated the effect of four different dietary levels of FCPS (0.1%, 0.2%, 0.4%, 0.8%) on the growth performance, innate immune responses and survival of crucian carp against Aeromonas hydrophila infection. The results showed that compared with control group, dietary FCPS had positive effects the growth performance (final weight, feed conversion ratio and survival rate) of crucian carp. FCPS induced significant higher (p < 0.05) leukocyte phagocytosis activity, serum bactericidal activity, lysozyme activity, com-plement C3, SOD activity and total protein level in the serum of crucian carp. Moreover, innate immune response of fish in FCPS groups increased first and then decreased with increasing dietary FCPS from 0.1% to 0.8%, and reached up to the peak in 0.4% dietary FCPS groups. Besides, the cumulative mortalities in FCPS groups were remarkably lower than that of control group when challenged with A. hydrophila, the relative percent survivals were 22.67%, 55.56%, 62.22% and 17.78% in 0.1% group, 0.2% group, 0.4% group and 0.8% group, respectively. These results suggested that dietary FCPS could improve the growth performance, innate immune response and disease resistance against A. hydrophila in fish, and the suitable dietary dose of FCPS was 0.4% in crucian carp.

Screening the potential part of the G protein antigen is an achievable strategy to improve the immune effect of DNA vaccine against MSRV infection.

DNA vaccines, as an effective prophylactic technology to induce both humoral and cellular immune responses, have already been widely studied to prevent and control viral and bacterial infections in aquaculture. To find a more effective and safer way to control Micropterus salmoides rhabdovirus (MSRV) infection in largemouth bass, two different DNA vaccines expressing partial (pcDNA3.1-G2) and full-length (pcDNA3.1-G) of the MSRV G protein were developed and injected intramuscularly with different doses. The immune effect was comprehensively compared and evaluated by detecting immune-related parameters including serum antibody levels, immune-related physiological indexes, immune-related gene expression and relative survival rates in this study. The results showed that compared with the pcDNA3.1-G vaccine, the pcDNA3.1-G2 vaccine induced higher serum antibody levels, a lower nonspecific immune response in serum (ACP, SOD and T-AOC activities), higher immune-related gene expression and a higher relative survival rate. Moreover, the immune effect of pcDNA3.1-G2-vaccinated fish showed gradually higher with the increasing pcDNA3.1-G2 concentration, especially in pcDNA3.1-G2 (10µg/per fish) group, the relative survival rate reached to 82.5%, which was significant higher (p < 0.05) than pcDNA3.1-G (10µg/per fish) group. This study indicated that screening the potential core part of an antigen is an achievable strategy to improve the immunogenicity and immunoprotective effect of DNA vaccine.

Yersinia ruckeri strain SC09 disrupts proinflammatory activation via Toll/IL-1 receptor-containing protein STIR-3.

Virulent pathogenic microorganisms often enhance their infectivity through immune evasion mechanisms. Our research on the integrative and conjugative element (ICE(r2)) of the virulent fish pathogen Yersinia ruckeri SC09 led to the identification of genes related to immune evasion (designated stir-1, stir-2, stir-3 and stir-4), among which stir-1 and stir-2 were determined as the key contributors to bacterial toxicity and immune evasion. Here, we further examined the ability of stir-3 to mediate immune evasion based on detailed bioinformatic analysis of ICE(r2) from Y. ruckeri SC09. Interactions among the translated STIR-1, STIR-2, STIR-3 and STIR-4 proteins in the secretory process were additionally explored. STIR-3 was positively correlated with bacterial toxicity and inhibited host toll-like receptor (TLR) signaling by interacting with MyD88, thereby facilitating bacterial survival in host cells. Importantly, our data showed co-secretion of STIR-1, STIR-2 and STIR-3 as a complex, with secretion failure occurring in the absence of any one of these proteins. While stir-1, stir-2, stir-3 and stir-4 genes werespecific to Y. ruckeri SC09, the ICE(r2) region where these genes were located is a mobile component widely distributed in bacteria. Therefore, the potential transmission risk of these immune evasion genes requires further research attention.

Toll/IL-1 receptor-containing proteins STIR-1, STIR-2 and STIR-3 synergistically assist Yersinia ruckeri SC09 immune escape.

Immune escape is a common feature of bacteria, viruses, parasites and even cancer cells. Our earlier work on an integrative and conjugative element (ICEr2) of Yersinia ruckeri SC09 demonstrated contributory roles of stir-1, stir-2 and stir-3 in bacterial toxicity and ability to code for immune evasion. Here, we further examined the ability of stir-4 in ICE (r2) and its encoded STIR-4 protein to mediate immune evasion using comparative genomic analysis. Additionally, the mechanisms underlying the synergistic activities of STIR-1, STIR-2, STIR-3 and STIR-4 in immune evasion were examined. Our results showed that STIR-4 did not contribute to bacterial toxicity, either in vivo nor in vitro, or show the ability to assist in bacterial immune escape. STIR-1, STIR-2, and STIR-3 formed heterotrimers in bacteria while facilitating immune evasion, which we speculate may be essential to maintain their stability. This discovery also partially explains the previous finding that a single gene can mediate immune evasion. Our data provide further knowledge on the distribution of ICE (r2)-like elements in bacteria, validating the prevalence of large-scale gene transfer in pathogens and its potential for enhancing virulence levels. Further studies are necessary to establish the biological significance of the ICE (r2) component.

Single-walled carbon nanotubes enhance the immune protective effect of a bath subunit vaccine for pearl gentian grouper against Iridovirus of Taiwan.

Iridovirus of Taiwan (TGIV) has been threatening the grouper farming since 1997, effective prophylaxis method is urgently needed. Subunit vaccine was proved to be useful to against the virus. Bath is the simplest method of vaccination and easy to be administrated without any stress to fish. In this research, we constructed a prokaryotic expression vector of TGIV's major capsid protein (MCP) to acquire the vaccine. Single-walled carbon nanotubes (SWCNTs) were used as the carrier to enhance the protective effect of bath vaccination for juvenile pearl gentian grouper (bath with concentrations of 5, 10, 20 mg/L for 6 h). Virus challenge was done after 28 days. Survival rates were calculated after 14 days. The level of antibody, activities of related enzymes in serums and expression of immune-related genes in kidneys and spleens were test. The results showed that vaccine with SWCNTs as carrier induced a higher level of antibody than that without. In addition, the activities of related enzymes (acid phosphatase, alkaline phosphatase, superoxide dismutase) and the expression of immune-related genes (Mx1, IgM, TNFαF, Lysozyme, CC chemokine 1, IL1-ß, IL-8) had a significantly increase. What's more, higher survival rates (42.10%, 77.77%, 89.47%) were provided by vaccine with SWCNTs than vaccine without SWCNTs (29.41%, 38.09%, 43.75%). This study suggests that the protective effect of vaccine that against TGIV with the method of bath vaccination could be enhanced by SWCNTs and SWCNTs could be a potential carrier for other subunit vaccines.

TcpA, a novel Yersinia ruckeri TIR-containing virulent protein mediates immune evasion by targeting MyD88 adaptors.

TIR domain-containing protein is an important member for some bacterial pathogens to subvert host defenses. Here we described a fish virulent Yersinia ruckeri SC09 strain that interfered directly with Toll-like receptor (TLR) function by a TIR-containing protein. Firstly, the novel TIR-containing protein was identified by bioinformatics analysis and named as TcpA. Secondly, the toxic effects of TcpA in fish was demonstrated in vivo challenge experiments through knockout mutant and complement mutant of tcpA gene. Thirdly, The study in vitro revealed that TcpA could down-regulate the expression and secretion of IL-6, IL-1ß and TNF-α. Finally, we demonstrated that TcpA could inhibit the TLR signaling pathway through interaction with myeloid differentiation factor 88 (MyD88) in experiments such as NF-κB dependent luciferase reporter system, co-immunoprecipitation, GST pull-down and yeast two-hybrid. The study revealed that TcpA was essential for virulence and was able to interact with the TIR adaptor protein MyD88 and inhibit the pre-inflammatory signal of immune cells and promote the intracellular survival of pathogenic Yersinia ruckeri SC09 strain. In conclusion, our results showed that TcpA acted as a new virulence factor in Y. ruckeri could suppress innate immune response and increase virulence by inhibiting TLR and MyD88-mediated specific signaling, highlighting a novel strategy for innate immune evasion in bacteria.

Molecular characterization, phylogenetic analysis and adjuvant effect of channel catfish interleukin-1ßs against Streptococcus iniae.

Channel catfish is one of the most extensively cultured species worldwide, which is widely used as a classical model for comparative immunology. Interleukin-1ß (IL1ß) is an immunoregulatory cytokine with the potential to enhance the immune response induced by vaccines in many animals. To characterize the molecular characterization and identify the immunoadjuvant role of channel catfish IL1ß, molecular cloning, phylogenetic analysis, and expression of two IL1ß genes were performed, the bioactivity of their recombinant proteins (rIL1ß1 and rIL1ß2) were detected in vitro and their adjuvant effects on a subunit vaccine encoding C5a peptidase (pSCPI) of Streptococcus iniae were evaluated. The results indicated that two IL1ßs remained highly conserved possessing five conserved motifs compared with other fish IL1ßs, although there were 28 nucleotide differences and 16 amino acid differences between channel catfish IL1ß1 and IL1ß2. Analysis of the ratios of nonsynonymous (dN) and synonymous (dS) substitutions revealed that fish IL1ß genes were subjected to negative/purifying selection with global dN/dS ratios value 0.425. The results of adjuvant effect showed that compared with injection of pSCPI alone, co-injecting pSCPI with both rIL1ß1 and rIL1ß2 significantly enhanced antibody levels, serum bactericidal activity, lysozyme activity, alternative complement hemolytic activity, and the expression of endogenous IL1ß and TNF-α in head kidney and spleen. Although vaccination with rIL1ß1 or rIL1ß2 failed to offer immunoprotection against S. iniae infection, the RPS (relative percent survival) of pSCPI+rIL1ß1 and pSCPI+rIL1ß2 groups were both higher than pSCPI alone (RPS, 50%), with 64.26% and 60.71%, respectively. Moreover, pSCPI+rIL1ß1+rIL1ß2 offered significantly higher (P < 0.05) immunoprotection (RPS, 75%) against S. iniae infection than pSCPI alone. Our present results not only enrich the molecular structure study of fish IL1ßs but also signify that two recombinant channel catfish IL1ßs can be used as potential adjuvants in a subunit vaccine model against bacterial infection, which are of profound importance to prevent and control bacterial disease in channel catfish.

Multiplex genome editing by natural transformation in Vibrio mimicus with potential application in attenuated vaccine development.

Vibrio mimicus (V. mimicus) is a significant pathogen in freshwater catfish, though knowledge of virulence determinants and effective vaccine is lacking. Multiplex genome editing by natural transformation (MuGENT) is an easy knockout method, which has successfully used in various bacteria except for V. mimicus. Here, we found V. mimicus strain SCCF01 can uptake exogenous DNA and insert it into genome by natural transformation assay. Subsequently, we exploited this property to make five mutants (△Hem, △TS1, △TS2, △TS1△TS2, and △II), and removed the antibiotic resistance marker by Flp-recombination. Finally, all of the mutants were identified by PCR and RT-PCR. The results showed that combination of natural transformation and FLP-recombination can be applied successfully to generate targeted gene disruptions without the antibiotic resistance marker in V. mimicus. In addition, the five mutants showed mutant could be inherited after several subcultures and a 668-fold decrease in the virulence to yellow catfish (Pelteobagrus fulvidraco). This study provides a convenient method for the genetic manipulation of V. mimicus. It will facilitate the identification and characterization of V. mimicus virulence factors and eventually contribute to a better understanding of V. mimicus pathogenicity and development of attenuated vaccine.

A Yersinia ruckeri TIR Domain-Containing Protein (STIR-2) Mediates Immune Evasion by Targeting the MyD88 Adaptor.

TIR domain-containing proteins are essential for bacterial pathogens to subvert host defenses. This study describes a fish pathogen, Yersinia ruckeri SC09 strain, with a novel TIR domain-containing protein (STIR-2) that affects Toll-like receptor (TLR) function. STIR-2 was identified in Y. ruckeri by bioinformatics analysis. The toxic effects of this gene on fish were determined by in vivo challenge experiments in knockout mutants and complement mutants of the stir-2 gene. In vitro, STIR-2 downregulated the expression and secretion of IL-6, IL-1ß, and TNF-α. Furthermore, the results of NF-κB-dependent luciferase reporter system, co-immunoprecipitation, GST pull-down assays, and yeast two-hybrid assay indicated that STIR-2 inhibited the TLR signaling pathway by interacting with myeloid differentiation factor 88 (MyD88). In addition, STIR-2 promoted the intracellular survival of pathogenic Yersinia ruckeri SC09 strain by binding to the TIR adaptor protein MyD88 and inhibiting the pre-inflammatory signal of immune cells. These results showed that STIR-2 increased virulence in Y. ruckeri and suppressed the innate immune response by inhibiting TLR and MyD88-mediated signaling, serving as a novel strategy for innate immune evasion.

Preparation, characterization and evaluation of the immune effect of alginate/chitosan composite microspheres encapsulating recombinant protein of Streptococcus iniae designed for fish oral vaccination.

Streptococcus iniae has caused serious harm to the fish farming industry in recent years. Vaccination is a potential approach for preventing and controlling disease, being oral vaccination the most suitable vaccination route in fish. Alginate and chitosan microspheres have been widely used as controlled release systems for oral vaccination in fish. In this study, we prepared and characterized alginate/chitosan composite microspheres encapsulating the recombinant protein serine-rich repeat (rSrr) of S. iniae. We evaluated effect of these microspheres on the immune system of channel catfish. The microsphere preparation conditions were optimized by Response Surface Method and target microspheres were obtained under 1.68% alginate (w/v), the W/O ratio 3.6:7.4 (liquid paraffin with 4% Span 80, v/v) with stirring at 1000 rpm, 9.64% CaCl2 (w/v) and 0.95% chitosan (w/v) with an encapsulation efficiency of 92.38%. The stability and safety of rSrr-microspheres were evaluated in vitro and in vivo, respectively. Furthermore, compared with control group, oral vaccination with rSrr-microspheres induced higher serum antibody titers, higher lysozyme activity, higher total protein and higher expression of immune-related genes, and resulted in higher relative percent survival (RPS) with the value of 60% for channel catfish against S.iniae infection. Our results thus indicate that alginate/chitosan microspheres encapsulating rSrr can be used as oral vaccine for channel catfish, providing efficient immunoprotection against S. iniae infection.

Subchronic toxicity and hepatocyte apoptosis of dietary olaquindox in common carp (Cyprinus carpio).

Olaquindox as one of the effective antimicrobial agents and growth-promoting feed additives, had been widely used in animal and fish production. However, few studies have been done to unveil its possible toxic effect and tissue injury on aquatic animal. In this study, the toxic effect and underlying mechanisms of olaquindox toxicity were investigated in common carp when feed with different doses of olaquindox for 90 days. The morbidity and mortality, pathological changes, hematology parameters, residue concentration in the tissues of common carp were assessed, hepatocyte apoptosis was detected through ultrastructural observation and flow cytometry methods. The results showed that the morbidity and mortality increased with the increasing dosages of dietary olaquindox, subchronic exposure to olaquindox caused remarkably pathological changes, including congestion and bleeding, intramuscular edema, vacuolar degeneration, degeneration and deformation in renal tubules architecture, respiratory epithelium fusion and intestinal epithelial microvilli disintegration. Besides, dietary olaquindox led to significant changes in blood biochemical parameters including red blood cell, hemoglobin, alanine aminotransferase and aspartate aminotransferase, an elevated residue concentration of olaquindox was detected in liver and kidney after exposure, hepatocyte apoptosis and necrosis were observed. Moreover, insulin-like growth factor I (IGF-I) mRNA level in liver was higher than normal level with the dose below 25 mg/kg olaquindox and was lower than normal level with the dose above 50 mg/kg. Our results demonstrated that dietary olaquindox may pose subchronic toxicity and residue in fish organs and provided scientific data for the safe application of olaquindox in fish.

Plant polysaccharides used as immunostimulants enhance innate immune response and disease resistance against Aeromonas hydrophila infection in fish.

Plant polysaccharides (PPS) are an important medicinal plant product, and play a major role in preventing and controlling infectious microbes in aquaculture. The present study investigated the effect of three PPS; Ficus carica polysaccharides (FCPS), Radix isatidis polysaccharides (RIPS), and Schisandra chinensis polysaccharides (SCPS), used as feed additives, on innate immune responses and disease resistance against Aeromonas hydrophila in crucian carp. Results show that crucian carp fed with these PPS showed significant (p < 0.05) enhancement of their innate immune response including leukocyte phagocytosis activity, serum bactericidal activity, lysozyme activity, total protein level, complement C3, and superoxide dismutase activity compared with the control group. Their degree of influence on these immune parameters was in the order of FCPS > RIPS > SCPS, except for lysozyme activity (RIPS > FCPS > SCPS). In addition, fish cumulative mortalities in the three treatment groups were remarkably lower than in the control group (95%) when challenged with A. hydrophila, relative percent survivals were 57.9%, 47.4%, and 42.1% in FCPS, RIPS, and SCPS groups, respectively. These results suggest that FCPS, RIPS, and SCPS used as immunostimulants are capable of enhancing immune responses and disease resistance against A. hydrophila in crucian carp, and that FCPS was the most effective. The findings from this study will help accelerate research of this topic, and promote the application and development of immunostimulants, such as Chinese herbs, in aquaculture.

The outer membrane proteins of Stenotrophomonas maltophilia are potential vaccine candidates for channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) is an important agricultural fish that has been plagued by Stenotrophomonas maltophilia (S. maltophilia) infections in recent years, some of them severe. The outer membrane proteins (OMPs) of S. maltophilia are one of the most immunogenic and highly conserved candidates for vaccine development in aquaculture. The present study investigated OMPs of S. maltophilia as vaccine on immune response and disease resistance against S. maltophilia of channel catfish and investigated the enhancement effect of natural adjuvants Propolis (Pro), FIG polysaccharide (Fcps), Glycyrrhizine (Gly) to OMPs of S. maltophilia for further study. The results indicated that channel catfish injected intraperitoneally with OMPs showed better immune response including leukocytes phagocytosis activity, serum bactericidal activity, complement C3, IgM level and an increasement of resistance against S. maltophilia compared to the control group. Moreover, Pro, Fcps and Gly could enhance the immune response of OMPs. The relative percent of survival (RPS) was 73.33%, 66.67%, 63.33%, 60%, 0% in fish injected OMPs + Pro, OMPs + Fcps, OMPs + Gly, OMPs and 0.65% normal saline, respectively. These results suggested that OMPs used as vaccine could induce and stimulate immune response and enhance disease resistance in channel catfish, especially for Pro as immunoenhancer. Results revealed that OMPs were an effective vaccine against S. maltophilia in channel catfish.

Adjuvant Immune Enhancement of Subunit Vaccine Encoding pSCPI of Streptococcus iniae in Channel Catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) is an important agricultural fish that has been plagued by Streptococcus iniae (S. iniae) infections in recent years, some of them severe. C5a peptidase is an important virulent factor of S. iniae. In this study, the subunit vaccine containing the truncated part of C5a peptidase (pSCPI) was mixed with aluminum hydroxide gel (AH), propolis adjuvant (PA), and Freund's Incomplete Adjuvant (FIA). The immunogenicity of the pSCPI was detected by Western-blot in vitro. The relative percent survival (RPS), lysozyme activity, antibody titers, and the expression of the related immune genes were monitored in vivo to evaluate the immune effects of the three different adjuvants. The results showed that pSCPI exerted moderate immune protection (RPS = 46.43%), whereas each of the three adjuvants improved the immune protection of pSCPI. The immunoprotection of pSCPI + AH, pSCPI + PA, and pSCPI + FIA was characterized by RPS values of 67.86%, 75.00% and, 85.71%, respectively. Further, each of the three different adjuvanted pSCPIs stimulated higher levels of lysozyme activity and antibody titers than the unadjuvanted pSCPI and/or PBS buffer. In addition, pSCPI + FIA and pSCPI + PA induced expression of the related immune genes under investigation, which was substantially higher than the levels stimulated by PBS. pSCPI + AH significantly stimulated the induction of MHC II ß, CD4-L2, and IFN-γ, while it induced slightly higher production of TNF-α and even led to a decrease in the levels of IL-1ß, MHC I α, and CD8 α. Therefore, we conclude that compared with the other two adjuvants, FIA combined with pSCPI is a more promising candidate adjuvant against S. iniae in channel catfish.

Cloning and Characterization of Surface-Localized α-Enolase of Streptococcus iniae, an Effective Protective Antigen in Mice.

Streptococcus iniae is a major fish pathogen that can also cause human bacteremia, cellulitis and meningitis. Screening for and identification of protective antigens plays an important role in developing therapies against S. iniae infections. In this study, we indicated that the α-enolase of S. iniae was not only distributed in the cytoplasm and associated to cell walls, but was also secreted to the bacterial cell surface. The functional identity of the purified recombinant α-enolase protein was verified by its ability to catalyze the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP), and both the recombinant and native proteins interacted with human plasminogen. The rabbit anti-rENO serum blockade assay shows that α-enolase participates in S. iniae adhesion to and invasion of BHK-21 cells. In addition, the recombinant α-enolase can confer effective protection against S. iniae infection in mice, which suggests that α-enolase has potential as a vaccine candidate in mammals. We conclude that S. iniae α-enolase is a moonlighting protein that also associates with the bacterial outer surface and functions as a protective antigen in mice.

Evaluation and Selection of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis of Gene Expression in Nile Tilapia (Oreochromis niloticus) during Vaccination and Infection.

qPCR as a powerful and attractive methodology has been widely applied to aquaculture researches for gene expression analyses. However, the suitable reference selection is critical for normalizing target genes expression in qPCR. In the present study, six commonly used endogenous controls were selected as candidate reference genes to evaluate and analyze their expression levels, stabilities and normalization to immune-related gene IgM expression during vaccination and infection in spleen of tilapia with RefFinder and GeNorm programs. The results showed that all of these candidate reference genes exhibited transcriptional variations to some extent at different periods. Among them, EF1A was the most stable reference with RefFinder, followed by 18S rRNA, ACTB, UBCE, TUBA and GAPDH respectively and the optimal number of reference genes for IgM normalization under different experiment sets was two with GeNorm. Meanwhile, combination the Cq (quantification cycle) value and the recommended comprehensive ranking of reference genes, EF1A and ACTB, the two optimal reference genes, were used together as reference genes for accurate analysis of immune-related gene expression during vaccination and infection in Nile tilapia with qPCR. Moreover, the highest IgM expression level was at two weeks post-vaccination when normalized to EF1A, 18S rRNA, ACTB, and EF1A together with ACTB compared to one week post-vaccination before normalizing, which was also consistent with the IgM antibody titers detection by ELISA.

A nanobody-mediated drug system against largemouth bass virus delivered by bacterial nanocellulose in Micropterus salmoides.

Diseases caused by pathogens severely hampered the development of aquaculture, especially largemouth bass virus (LMBV) has caused massive mortality and severe economic losses to the culture of largemouth bass (Micropterus salmoides). Considering the environmental hazards and human health, effective and environmentally friendly therapy strategy against LMBV is of vital importance and in pressing need. In the present study, a novel nanobody (NbE4) specific for LMBV was selected from a phage display nanobody library. Immunofluorescence and indirect ELISA showed that NbE4 could recognize LMBV virions and had strong binding capacity, but RT-qPCR evidenced that NBE4 did not render the virus uninfectious. Besides, antiviral drug ribavirin was used to construct a targeted drug system delivered by bacterial nanocellulose (BNC). RT-qPCR revealed that NbE4 could significantly enhance the antiviral activity of ribavirin in vitro and in vivo. The targeted drug delivery system (BNC-Ribavirin-NbE4, BRN) reduced the inflammatory response caused by LMBV infection and improved survival rate (BRN-L, 33.3 %; BRN-M, 46.7 %; BRN-H, 56.7 %)compared with control group (13.3 %), ribavirin group (RBV, 26.7 %) and BNC-ribavirin (BNC-R, 40.0 %), respectively. This research provided an effective antiviral strategy that improved the drug therapeutic effect and thus reduced the dosage.

Exploring the Immunoprotective Potential of a Nanocarrier Immersion Vaccine Encoding Sip against Streptococcus Infection in Tilapia (Oreochromis niloticus).

Tilapia, as one of the fish widely cultured around the world, is suffering severe impact from the streptococcus disease with the deterioration of the breeding environment and the increasing of breeding density, which brings serious economic loss to tilapia farming. In this study, the surface immunogenic protein (Sip) of Streptococcus agalactiae (S. agalactiae) was selected as the potential candidate antigen and connected with bacterial nano cellulose (BNC) to construct the nanocarrier subunit vaccine (BNC-rSip), and the immersion immune effects against S. agalactiae and Streptococcus iniae (S. iniae) in Nile tilapia were evaluated on the basis of the serum antibody level, non-specific enzyme activity, the immune-related gene expression and relative percent survival (RPS). The results indicated that Sip possessed the expected immunogenicity according to the immunoinformatic analysis. Compared with the rSip group, BNC-rSip significantly induced serum antibody production and improved the innate immunity level of tilapia. After challenge, the RPS of BNC-rSip groups were 78.95% (S. agalactiae) and 67.86% (S. iniae), which were both higher than those of rSip groups,31.58% (S. agalactiae) and 35.71% (S. iniae), respectively. Our study indicated that BNC-rSip can induce protective immunity for tilapia through immersion immunization and may be an ideal candidate vaccine for controlling tilapia streptococcal disease.