Jichang Jian

Complete genome sequence of Streptococcus agalactiae ZQ0910, a pathogen causing meningoencephalitis in the GIFT strain of Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae (group B streptococcus [GBS]) is a pathogen that causes meningoencephalitis in Nile tilapia (Oreochromis niloticus). Here, we reported the complete genome sequence of S. agalactiae strain ZQ0910, which was isolated from the GIFT strain of Nile tilapia in Guangdong, China.

Immunostimulatory effects of artificial feed supplemented with a Chinese herbal mixture on Oreochromis niloticus against Aeromonas hydrophila

The effects of a Chinese herbal mixture (CHM) composed of astragalus, angelica, hawthorn, Licorice root and honeysuckle on immune responses and disease resistant of Nile tilapia (Oreochromis niloticus GIFT strain) were investigated in present study. Fish were fed diets containing 0 (control), 0.5%, 1.0%, 1.5% or 2.0% CHM (w/w) for 4 weeks. And series of immune parameters including lysozyme, cytokine genes TNF-α and IL-1β, superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA) were measured during test period. After four weeks of feeding, fish were infected with Aeromonas hydrophila and mortalities were recorded. Results of this study showed that feeding Nile tilapia with CHM-supplementation diet stimulated lysozyme activity, SOD activity and POD activity in serum, induced TNF-α and IL-1β mRNA expression in head kidney and spleen, but decreased serum MDA content. All CHM-supplemental groups showed reduced mortalities following A. hydrophila infection compared with the group fed the control diet. These results suggested that this CHM can be applied as a tilapia feed supplement to elevate fish immunity and disease resistance against A. hydrophila.

Expression, characterization and immunogenicity of flagellin FlaC from Vibrio alginolyticus strain HY9901

Vibrio alginolyticus is one of ubiquitous pathogens infecting human and marine animals. Flagellins of bacteria play an important role in infecting animals and inducing host immune response. In the present research, flagellin flaC gene of V. alginolyticus strain HY9901 was cloned and expressed. The open reading frame of flaC gene contains 1155 bp and the putative protein consists of 384 amino acid residues. Polyclonal antibodies were raised in mouse against the purified recombinant FlaC protein and the reaction of the antibody was confirmed by western blot analysis using the FlaC protein and crude protein extracts of V. alginolyticus. Red snapper (Lutjanus sanguineus) vaccinated with recombinant FlaC produced specific antibodies, and were highly resistant to infection by virulent V. alginolyticus. This study indicates that the conserved FlaC is an effective vaccine candidate against V. alginolyticus infection.

Molecular and functional characterization of CD59 from Nile tilapia (Oreochromis niloticus) involved in the immune response to Streptococcus agalactiae.

CD59, the major inhibitor of membrane attack complex, plays a crucial role in regulation of complement activation. In this paper, a CD59 gene of Nile tilapia, Oreochromis niloticus (designated as On-CD59) was cloned and its expression pattern under the stimulation of Streptococcus agalactiae was investigated. Sequence analysis showed main structural features required for complement-inhibitory activity were detected in the deduced amino acid sequence of On-CD59. In healthy Nile tilapia, the On-CD59 transcripts could be detected in all the examined tissues, with the most abundant expression in the brain. When immunized with inactivated S. agalactiae, there was a clear time-dependent expression pattern of On-CD59 in the skin, brain, head kidney, thymus and spleen, with quite different kinetic expressions. The assays for the complement-inhibitory activity suggested that recombinant On-CD59 protein had a species-selective inhibition of complement. Moreover, our works showed that recombinant On-CD59 protein may possess both binding activities to PGN and LTA and inhibiting activity of S. agalactiae. These findings indicated that On-CD59 may play important roles in the immune response to S. agalactiae in Nile tilapia.

Immune response in Lutjanus erythropterus induced by the major outer membrane protein (OmpU) of Vibrio alginolyticus

The outer membrane proteins (OMPs) of the marine aquatic animal pathogen Vibrio alginolyticus play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In this study, the major 35.6 kDa OMP of V. alginolyticus was isolated by gel excision from the crude OMP fraction from V. alginolyticus. The sequence of the first 27 amino acid residues from the N-terminal end of the protein is ATV YKD GGT ELL VGG RVE FRG DFI GSD, which has high homology with OmpU proteins from other Vibrio spp. (92%). Lutjanus erythropterus were vaccinated with OmpU, and immunogenicity was confirmed by subsequent western blotting. Enzyme-linked immunosorbent assay (ELISA) analysis demonstrated that OmpU produced an observable antibody response in all sera of the vaccinated fish. L. erythropterus vaccinated with OmpU produced specific antibodies, and were highly resistant to infection with virulent V. alginolyticus. These results indicate that OmpU is an effective vaccine candidate against V. alginolyticus for L. erythropterus.

Identification and characterization of tumor necrosis factor receptor (TNFR)-associated factor 3 from humphead snapper, Lutjanus sanguineus

Tumor necrosis factor receptor (TNFR)-associated factor 3(TRAF3) is a key regulator in TNFR and Toll-like receptor (TLRs)/RIG-I-like receptors (RLRs) signal pathway. Here, a TRAF3 gene (Ls-TRAF3, GenBank Accession No: KJ789921) is cloned from humphead snapper (Lutjanus sanguineus). The Ls-TRAF3 cDNA contains an open reading frame of 1788 bp, which encodes a polypeptide of 595 amino acids. The deduced amino acid of Ls-TRAF3 possesses a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain. Ls-TRAF3 protein shares high identities with other known TRAF3 proteins. In healthy fish, Ls-TRAF3 transcripts were broadly expressed in all examined tissues with highest expression levels in spleen, liver and head kidney. Quantitative real-time PCR (qRT-PCR) analysis revealed that Ls-TRAF3 could be induced by bacteria or viral PAMP poly I:C stimulation in vivo. Here, we also showed Ls-TRAF3 that, positively regulated IRF3 and Mx upon poly I:C stimuli, whereas prevented production of proinflammatory cytokine IL-6 after LPS injection. Moreover, over-expression of wide type (WT) Ls-TRAF3 and truncated forms, including ΔZinc finger 1, ΔZinc finger 2 and Δcoiled-coil suppressed NF-κB activity significantly, whereas the inhibitory effect of NF-κB was partially impaired when the RING finger or MATH domain deletion, suggesting the latter was more important for downstream signal transduction. Taken together, these results implicated that Ls-TRAF3 might play regulatory roles in immune response to pathogen invasion.

Phylogenetic analysis of newly isolated grass carp reovirus

Grass carp reovirus (GCRV) is a causative agent of haemorrhagic disease in grass carp that drastically affects grass carp aquaculture. Here we report a novel GCRV isolate isolated from sick grass carp that induces obvious cytopathic effect in CIK cells and name it as GCRV096. A large number of GCRV 096 viral particles were found in the infected CIK cells by electron microscope. The shape, size and the arrangement of this virus were similar to those of grass carp reovirus. With the primers designed according to GCRV 873 genome sequences, specific bands were amplified from sick grass carp and the infected CIK cells. The homology rates among vp4, vp6 and vp7 gene in GCRV 096 and those of some GCRV isolates were over 89%. In this study, the sequences of vp4, vp6 and vp7 were used to analyse sequence variation, phylogenetic relationships and genotypes in twenty five GCRV isolates. The results indicated these twenty five GCRV isolates should be attributed to four genotypes. And there were no obvious characteristics in the geographical distribution of GCRV genotype. The study should provide the exact foundation for developing more effective prevention strategies of grass carp haemorrhagic disease.

Integrated analysis neurimmiRs of tilapia (Oreochromis niloticus) involved in immune response to Streptococcus agalactiae, a pathogen causing meningoencephalitis in teleosts

Abstract MicroRNAs (miRNAs) are a class of noncoding RNA molecules and play important roles in a wide spectrum of biological processes, including in immune response. Recent years have witnessed considerable amount of research interest in studies on miRNA‐mediated modulation gene function during neuroinflammation. Here, we evaluated Streptococcus agalactiae infected tilapia (Oreochromis niloticus) brain for the expression profile of miRNAs, potential functions and their correlation with genes involved in inflammatory pathways. A total of 1981 miRNAs were identified, including in 486 miRNAs which have homologues in the currently available databases and 1945 novel miRNAs. The expression levels of 547 miRNAs were significantly altered at 6 h–48 h post‐bacterial infection, and these miRNAs were therefore classified as differentially expressed tilapia miRNAs. Real‐time PCR were implemented for 14 miRNAs co‐expressed in five samples, and agreement was confirmed between the high‐throughput sequencing and real‐time PCR data. For the 486 differentially expressed miRNAs target 41,820 genes. GO and KEGG enrichment analysis revealed that some target genes of miRNAs were grouped mainly into the categories of apoptotic, signal pathwayand immune response. This is the first report of comprehensive identification of teleost miRNAs being differentially regulated in brain in normal conditions relating to bacterial infection. HighlightsFive Oreochromis niloticus brain sRNA libraries were constructed and deep sequenced.A total of 486 conserved and 1954 novel miRNAs were identified.547 miRNAs were differentially expressed among in five libraries.Selected miRNA expression levels were consistent between sequencing and qPCR data.Annotation of targets revealed a broad range of signal pathways.

Draft Genome Sequence of Nocardia seriolae ZJ0503, a Fish Pathogen Isolated from Trachinotus ovatus in China

ABSTRACT Nocardia seriolae is a pathogen that causes nocardiosis in marine and freshwater fish. Here, we report the draft genome sequence of N. seriolae strain ZJ0503, which was isolated from Trachinotus ovatus in Guangdong, China.

Protection against Vibrio alginolyticus in crimson snapper Lutjanus erythropterus immunized with a DNA vaccine containing the ompW gene.

The outer membrane proteins of Vibrio alginolyticus play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In the present study, the ompW gene was cloned, expressed and purified. A DNA vaccine was constructed by inserting the ompW gene into a pcDNA plasmid. Crimson snapper Lutjanus erythropterus (Bloch) were injected intramuscularly with the recombinant plasmid pcDNA-ompW. The expression of the DNA vaccine was detected in gill, head kidney, heart, liver, spleen and injection site muscle of crimson snapper by RT-PCR 7 and 28 d post-vaccination. The ELISA results demonstrated that the DNA vaccine produced an observable antibody response in all sera of the vaccinated fish. In addition, crimson snapper immunized with the DNA vaccine showed a relative percentage survival (RPS) of 92.53%, indicating effective protection against V. alginolyticus infection.