Yucong Huang

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.

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.

Identification of DLD, by immunoproteomic analysis and evaluation as a potential vaccine antigen against three Vibrio species in Epinephelus coioides.

Vibrio spp. represent a serious threat to the culture of Epinephelus coioides (Orange-spotted Grouper) in Southeast Asia. In this study we used two-dimensional electrophoresis (2-DE) and Western blotting to identify common immunogenic proteins of Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus. Membranes were probed with orange-spotted grouper anti-V. alginolyticus sera and accordingly 60, 58 and 48 immunogenic protein spots were detected. By matching analysis for the three Western blotting membranes, 6 cross immunogenic spots for the three Vibrio species were identified. They were Outer membrane protein W (OmpW), dihydrolipoamide dehydrogenase (DLD), succinate dehydrogenase flavoprotein subunit(SDHA), elongation factor Ts(Ts), peptide ABC transporter periplasmic peptide-binding protein and phosphoenolpyruvate carboxykinase(PEPCK). One of the proteins, DLD, was used to evaluate the cross protective function for E. coioides with a bacterial immunization and challenge method. The relative percent survival rate of E. coioides against V. alginolyticus, V. harveyi and V. parahaemolyticus was 90%, 86% and 80%, respectively. This work may provide potential cross protective vaccine candidate antigens for three Vibrio species, and DLD may be considered as an effective cross-protective immunogen against three Vibrio species.

Protection of red snapper (Lutjanus sanguineus) against Vibrio alginolyticus with a DNA vaccine containing flagellin flaA gene

Aims:  The main aims of this study were to construct a DNA vaccine containing flagellin flaA gene from Vibrio alginolyticus strain HY9901 and to explore the potential application of pcDNA‐flaA as a DNA vaccine candidate for red snapper (Lutjanus sanguineus).

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.

Expression and immunogenicity analysis of accessory colonization factor A from Vibrio alginolyticus strain HY9901.

The accessory colonization factor A (ACFA) of Vibrio alginolyticus plays an important role in the efficient colonization of the bacterium and is potential candidates for vaccine development. In present study, the acfA gene was cloned, expressed and purified. Western blot analysis revealed protein recognition with the native ACFA in different V. alginolyticus strains. To analyze the immunogenicity of the recombinant ACFA, Lutjanus erythropterus Bloch were immunized by intraperitoneal injection, and the results demonstrated that the recombinant ACFA produced an observable antibody response in all sera of the vaccinated fish. The differential expressions of RAG1 gene in various tissues of L. erythropterus were analyzed by fluorescent quantitative real-time PCR, and the results showed the RAG1 mRNA expression was significantly up-regulated in thymus, head kidney and spleen tissue. Furthermore, the protective property of recombinant ACFA was evaluated through challenge with six heterogeneous virulent V. alginolyticus strains, and the immunohistochemical analysis in different tissues after challenge with V. alginolyticus. The results showed L. erythropterus vaccinated with recombinant ACFA were more tolerant of the infection by virulent V. alginolyticus strains. The data indicate that the recombinant ACFA could provide heterologous protection for the different virulent V. alginolyticus strains.

Draft Genome Sequence of the Fish Pathogen Vibrio harveyi Strain ZJ0603

Vibrio harveyi is an important pathogen that causes vibriosis in various aquatic organisms. Here, we announce the draft genome sequence of V. harveyi strain ZJ0603, which was isolated from diseased Orange-spotted grouper (Epinephelus coioides) in Guangdong, China.

VscO, a putative T3SS chaperone escort of Vibrio alginolyticus, contributes to virulence in fish and is a target for vaccine development.

Type III secretion system (T3SS) in Vibrio alginolyticus is essential for its pathogenesis. VscOs homologous proteins FliJ, InvI and YscO have been suggested to be putative chaperone escorts although its function in V. alginolyticus is unclear. To investigate the physiological role of VscO, a mutant strain of V. alginolyticus with an in-frame deletion of the vscO gene was constructed in the present study. One finding was that the mRNA expression levels of SycD, VopB and VopD proteins decreased in the ΔvscO mutant. In addition, the ΔvscO mutant showed an attenuated swarming ability and a ten-fold decrease in the virulence to fish. However, the ΔvscO mutant showed no difference in the biofilm formation and ECPase activity. Complementation of the mutant strain with the vscO gene could restore the phenotypes of the wild-type strain. Finally, the recombinant VscO protein caused a high antibody titer and an effective protection against lethal challenge with the wild-type strain V. alginolyticus. These results indicated that VscO protein has a specific role in the pathogenesis of V. alginolyticus and it may be a candidate antigen for development of a subunit vaccine against vibriosis.

Characterization and identification of streptococci from golden pompano in China

Streptococcal infections cause significant mortality and high economic losses in the fish farm industry worldwide, including in the culture of golden pompano Trachinotus ovatus L., a species gaining popularity in China. A total of 9 streptococcal strains were isolated from cage-cultured diseased golden pompano in Beihai, Zhanjing, and Shenzhen, China, between 2012 and 2014. Conventional and rapid identification systems were used to determine that the isolates were Streptococcus agalactiae, S. iniae, and S. dysgalactiae subsp. dysgalactiae. All isolates were gram-positive cocci cells in pairs or short-chain, non-motile, catalase negative, α or β hemolytic cocci. The results of multiplex PCR assays and 16S rRNA BLAST analysis also showed that the β hemolytic strains were S. agalactiae and S. iniae and the α hemolytic strain was S. dysgalactiae subsp. dysgalactiae, respectively. Pathogenicity assays revealed that S. agalactiae (lethal dose [LD50]: 6.38 × 10(4) CFU ml(-1)) was more virulent for golden pompano than S. iniae (LD50: 1.47 × 10(7) CFU ml(-1)) and S. dysgalactiae subsp. dysgalactiae (LD50: 2.57 × 10(6) CFU ml(-1)) when they were challenged by intraperiotoneal (i.p.) injection. The results of antibiotic susceptibility showed that all strains were extremely susceptible to cefradine, erythromycin, and cefotaxime but resistant to gentamicin, penicillin G, novobiocin, neomycin, ciprofloxacin, roxithromycin, furazolidone, enrofloxacin, norfloxacin, kanamycin, ampicillin, tetracycline, and vancomycin This is the first report of a phenomenon of golden pompano coinfection with S. agalactiae and S. iniae, which will contribute to the diagnosis and prevention of streptococcicosis.