Yishan Lu

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.

Loop-mediated isothermal amplification method for rapid detection of Vibrio alginolyticus, the causative agent of vibriosis in mariculture fish

Aims:  The purpose of this study was to develop a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish.

Cloning and expression of gene encoding the thermostable direct hemolysin from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis of crimson snapper (Lutjanus erythopterus)

Aims:  The main aims of this study were to clone and express complete open reading frame (ORF) of thermostable direct haemolysin gene (tdh) from Vibrio alginolyticus strain HY9901 in Escherichia coli, and further evaluate the virulence of expressed TDH on mouse and crimson snapper.

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.

Evaluation of a loop-mediated isothermal amplification method for the rapid detection of Vibrio harveyi in cultured marine shellfish.

Aims:  The purpose of this study was to establish a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive detection of Vibrio harveyi in mariculture shellfish.

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.

Immunoproteomic analysis and identification of novel immunogenic proteins from Vibrio harveyi.

Aims:  The main aim of this study was to screen novel immunogenic proteins of Vibrio harveyi, which could be vaccine candidates.

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.