Qian Yang

A recombinant truncated surface immunogenic protein (tSip) plus adjuvant FIA confers active protection against Group B streptococcus infection in tilapia.

PURPOSEnTilapia is an important agricultural fish that has been plagued by Group B streptococcus (GBS) infections in recent years, some of them severe. It is well-known that surface immunogenicity protein (Sip) is an effective vaccine against GBS.nnnEXPERIMENTAL DESIGNnSince Sip was not expressed in either E. coli BL21 or E. coli Rosetta, we removed the N-terminal signal peptide and LysM of the virus to produce purified truncated Sip (tSip(1)), which multiplied easily in an E. coli host. The antibodys ability to recognize and combine with GBS was determined by Western-blot and specific staining in vitro. The relative percentage of survival (RPS), antibody titers, bacterial recovery, and pathologic morphology were monitored in vivo to evaluate the immune effects. Freunds incomplete adjuvant (FIA) plus tSip and aluminum hydroxide gel (AH) plus tSip were also evaluated.nnnRESULTSnIt revealed that tSip mixed with FIA was an effective vaccine against GBS in tilapia, while AH is toxic to tilapia.

Molecular cloning, expression and the adjuvant effects of interleukin-8 of channel catfish ( Ictalurus Punctatus ) against Streptococcus iniae

Interleukin-8 (IL-8) as an important cytokine involving in inflammatory and immune response, has been studied as effective adjuvants for vaccines in mammals. However, there are fewer reports about the characterization and adjuvant effects of IL-8 in fish. In this study, cloning and sequence analysis of IL-8 coding region of channel catfish (Ictalurus punctatus) were conducted, mature IL-8(rtIL-8) was expressed and evaluated for its adjuvant effects on the immunoprotection of subunit vaccine encoding α-enolase (rENO) of Streptococcus iniae from several aspects in channel catfish. The results showed co-vaccination of rENO with rtIL-8 enhanced immune responses including humoral and cellular immunity, with higher relative percent survival(RPS,71.4%) compared with the moderate RPS of rENO alone(50%) against S. iniae infection at 4 week post vaccination. While rtIL-8 failed to maintain long-lasting immune protection, only with RPS of 26.67% in rENOu2009+u2009rtIL-8-vaccinated fish compared with that of rENO alone(20%) at 8 week, signifying that IL-8 hold promise for use as potential immunopotentiator in vaccines against bacterial infections in fish, whereas it is insufficient to extend the immunoprotection for long time, and further studies are required to understand the mechanisms of IL-8 used as an adjuvant and seek for more effective way to strengthen the adjuvanticity of IL-8.

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.

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.

OmpN, outer membrane proteins of Edwardsiella ictaluri are potential vaccine candidates for channel catfish (Ictalurus punctatus).

Outer membrane proteins (OMPs) are a class of proteins that reside in the outer membrane of Gram-negative bacteria. OMPs act as epitopes and are potential vaccine candidates. Outer membrane protein N (OmpN) is a component of the outer membrane of Edwardsiella ictaluri (E. ictaluri). In a previous study, the OmpN1-, OmpN2-, OmpN3-encoding genes of E. ictaluri were cloned, and here they were expressed in Escherichia coli. Western blotting showed that these three proteins had molecular weights of ∼60kDa. Channel catfish were immunized with recombinant OmpNs (rOmpNs) and then challenged with E. ictaluri. The results showed that rOmpN1, rOmpN2, and rOmpN3, as well as a mixture of all three proteins (in a ratio of 1:1:1) generated moderate immune protection (relative percentage of survival=62.5, 62.5, 67.5, and 75%, respectively). In an agglutination antibody titer assay, fish antisera showed an antibody titer of 1:128. Furthermore, each of the proteins stimulated high levels of lysozyme activity. In addition, a real-time polymerase chain reaction analysis revealed significant up-regulation of immune-related genes encoding major histocompatibility complex class I (MHC I), MHC II, CD4L, tumor necrosis factor-α, and interferon-γ after 24 and 48h of challenge, compared with the levels stimulated by phosphate-buffered saline. Taken together, we conclude that rOmpNs may elicit immune responses and generate protection against E. ictaluri in channel catfish. Thus, rOmpNs could be promising vaccine candidates against E. ictaluri.

Outbreak of infectious pancreatic necrosis virus (IPNV) in farmed rainbow trout in China.

Infectious pancreatic necrosis virus (IPNV) is a member of the Aquabirnavirus genus, which caused mass mortality (nearly 100%) in farmed rainbow trout (Oncorhynchus mykiss) in aquaculture farms in 2016, China. Major clinical signs included decreased appetite, mucous-like stools, and darkened pigmentation. Pathological changes in moribund fish were observed, such as marked vacuolar degeneration of the pancreatic cells with pyknotic nucleus and decreased zymogen granules, severe hemorrhage in the liver, and tumidness of respiratory epithelium in gills. In addition, the tissue fluid of diseased fish could produce a cytopathic effect (CPE) in RTG-2 cells. The presence of specific 206bp fragments by the reverse transcription-polymerase chain reaction (RT-PCR) using tissue homogenate of diseased fish and supernatant of infected cells revealed that IPNV could be confirmed. The pathogenicity test of cell culture supernatant detected cumulative mortality of 80%, and the clinical symptoms observed in the moribund and dead fish were similar to the naturally infected fish. Furthermore, the sequence analysis of VP2 gene showed that the isolated virus strain belonged to genogroup 1, and 97% homology with the Mexican IPNV isolate was found. To our knowledge, this is the first report on IPNV natural infection in the southwest of China.

Interleukin-8 holds promise to serve as a molecular adjuvant in DNA vaccination model against Streptococcus iniae infection in fish

DNA vaccines had been widely used in animal models against various viral infections, while it was not so convincing for many infectious diseases especially bacterial disease in aquaculture. Interleukin-8(IL-8) as one of the CXC chemokines, its immunological role and adjuvant potential which had been proved in mammals were rarely reported in fish species. In this study, recombination plasmid pcDNA3.1/IL-8(pcIL-8) was conducted and the capacity of IL-8 as molecular adjuvant was explored from several aspects by co-injecting with a DNA vaccine encoding a-enolase(pcENO) against Streptococcus iniae infection in channel catfish. The results suggested that co-injection of pcIL-8 with DNA vaccine increased the innate immunity and specific antibody levels, as well as increased the immune-related genes involving in pro-inflammatory response, humoral and cellular immunity. Moreover, pcIL-8 enhanced the immunoprotection of pcENO with the relative percent survival(RPS) of 60% to 80% against S.iniae infection at 4 week post vaccination(p.v.), with the significantly higher RPS of 73.33% in pcENO+pcIL-8 group compared with that of pcENO alone(53.33%) at challenge test of 8 weeks p.v. Taken together, these results indicate pcIL-8 as a molecular adjuvant co-injected with DNA vaccine not only improves the immunoprotection but also maintains long period of immunity for channel catfish against S.iniae infection. Our study signifies that IL-8 holds promise to serve as a potential adjuvant in DNA vaccines against bacterial infections for long time.

Effectivity of oral recombinant DNA vaccine against Streptococcus agalactiae in Nile tilapia

The effects of oral vaccination by two immune routes (gavage and mixed fodder administration) using a recombinant DNA vaccine SL7207-pVAX1-sip against Streptococcus agalactiae were assessed. SL7207-pVAX1-sip significantly enhanced various innate immune responses of tilapia, such as total serum protein, superoxide dismutase activity, lysozyme activity, complement C3 concentration in serum, serum antibacterial activity, and interleukin 1β and tumor necrosis factor-α. Vaccinated fish had higher relative percent survival values (57% and 63% for gavage group and mix fodder administration, respectively) at 10 weeks after S.xa0agalactiae infection. When administered mixed fodder, the DNA vaccine SL7207-pVAX1-sip against S.xa0agalactiae may produce more effective protection. These findings can promote the application and development of DNA vaccines in aquaculture.

Identification and screening of effective protective antigens for channel catfish against Streptococcus iniae

Vaccination is a potential approach for prevention and control of disease in fish. The use of genetically engineered vaccines is an effective method and a green intervention to control bacterial infection in aquaculture. However, efforts to develop these vaccines are limited by the lack of conserved protective antigens. In this study, three candidate immunogens (Srr, NeuA, and Hsp) of the pathogenic Streptococcus iniae strain DGX07 isolated from diseased channel catfish were identified and analyzed. Molecular cloning, expression, and purification of candidate antigen genes were carried out to obtain the candidate immunogens in the form of recombinant subunit vaccines. Western blotting was performed to evaluate immunogenicity in vitro and channel catfish were vaccinated by intraperitoneal injection and the specific antibody titers and relative percent of survival were determined to evaluate immune protection in vivo. The results showed that these three candidate immunogens were expressed correctly as recombinant proteins fused with His tags, with molecular weights of 70 kDa for Srr, 86 kDa for NeuA, and 51 kDa for Hsp, respectively. Moreover, each immunogen was predicted to be located either extracellularly or on the surface of S. iniae, and were able to offer protection against S. iniae infection in the form of recombinant subunit vaccines with adjuvant ISA763, especially Srr, with a relative percent of survival of 70% for Srr, 55% for NeuA, and 50% for Hsp, respectively.

Acute and Subacute toxicity study of Olaquindox by feeding to common carp (Cyprinus carpio L.)

Olaquindox, is a growth-promoting feed additive for food-producing animals. As the banned medicinal feed additive, olaquindox in animal feed and water must be concerned as an important hazard index. To improve studies of the toxicity of olaquindox, we provide a toxicological effects of olaquindox on a common freshwater fish, Cyprinus carpio L. The results of acute toxicity tests showed that the 7d-LD50 of olaquindox administered by feeding for common carp was determined to be 3746.3u202fmg/kg. We also found that the accumulation coefficient of olaquindox in carp was 1.45-1.9. Based on the studied hematological and blood biochemical parameters (RBCs count, hemoglobin content, ALT, AST and SOD activity), we found that olaquindox induced significant alterations in all studied parameters. Regarding bioaccumulation, the results showed that olaquindox had more efficiency to internalize fish tissues (liver, kidneys and muscle). The histopathological investigation of tissues from poisoning fish revealed various alterations that varied between adaptation responses and permanent tissue damage. Our results indicate that olaquindox are toxic to common carp and have obvious accumulation, and all the data from acute and subacute toxicity experiments in common carp may provide a useful tool for assessing the toxicity of olaquindox to aquatic organisms.