Jufen Tang

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.

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.

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.

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.

Molecular characterization and expression of CD2 in Nile tilapia (Oreochromis niloticus) in response to Streptococcus agalactiae stimulus

The cluster of differentiation 2 (CD2), functioning as a cell adhesion and costimulatory molecule, plays a crucial role in T-cell activation. In this paper, the CD2 gene of Nile tilapia, Oreochromis niloticus (designated as On-CD2) was cloned and its expression pattern under the stimulation of Streptococcus agalactiae was investigated. Sequence analysis showed On-CD2 protein consists of two extracellular Ig-like domains, a transmembrane region, and a long proline-rich cytoplasmic tail, which is a hallmark of CD2, and several important structural characteristics required for T-cell activation were detected in the deduced amino acid sequence of On-CD2. In healthy tilapia, the On-CD2 transcripts were mainly detected in the head kidney, spleen, blood and thymus. Moreover, there was a clear time-dependent expression pattern of On-CD2 after immunized by formalin-inactivated S. agalactiae and the expression reached the highest level at 12 h in the brain and head kidney, 48 h in the spleen, and 72 h in the thymus, respectively. This is the first report on the expression of CD2 induced by bacteria vaccination in teleosts. These findings indicated that On-CD2 may play an important role in the immune response to intracellular bacteria in Nile tilapia.

NK-lysin from Oreochromis niloticus improves antimicrobial defence against bacterial pathogens

ABSTRACT NK‐lysin, an effector of cytotoxic T cells and natural killer cells, is a potent antimicrobial peptide widely distributed in mammals. Homologues of NK‐lysin have been discovered in several teleost species, but only several of their natural functions was recorded so far. Here we identified an NK‐lysin from Nile tilapia (Oreochromis niloticus), On‐NKL, and analysed its expression model and biological effects on pathogen infection. The open reading frame of On‐NKL sequence spans 432 bp, codes for 143 amino acids and shares 27%–62% overall sequence identities with NK‐lysin of other species. The deduced mature peptide of On‐NKL possesses a saposin B domain and six well‐conserved cysteine residues that essential for antimicrobial activity by forming three intrachain disulphide bonds. The results of qRT‐PCR showed that On‐NKL expression was observed in multiple tissues and head kidney leucocytes and nonspecific cytotoxic cells (NCCs) and is most abundant in gills. After bacterial challenge, On‐NKL expression significantly varied in different tissues and NCCs. Following bacterial infection, On‐NKL‐overexpressing fish featured significantly lower pathogen loads in tissues than control fish. On‐NKL‐overexpressing fish also exhibited 33.3% relative percent survival compare with control groups. Findings suggested that On‐NKL could be the potential effector of NCCs and act as immune‐related gene that enhances antimicrobial defence. HighlightsHomologue of NK‐lysin gene (On‐NKL) was identified from Nile tilapia, Oreochromis niloticus.On‐NKL could respond to bacterial challenge in immune tissue and brain.Overexpression of On‐NKL apparently enhanced the survival rate after bacterial infection.On‐NKL could be a potential effector of NCCs.

Conservation of structural and interactional features of CD28 and CD80/86 molecules from Nile tilapia (Oreochromis niloticus)

ABSTRACT Interaction of CD28 with CD80 or CD86 molecules provides a costimulatory signals required in T cell activation. In this study, we cloned and analyzed a CD28 gene (On‐CD28) and a CD80/86 gene (On‐CD80/86) from Nile tilapia (Oreochromis niloticus). Sequence analysis revealed the typical characteristics of On‐CD28 protein; for instance, the proline‐based motif (117TYPPPL122) is essential in binding of CD28 to CD80/86 ligands. Moreover, an extracellular Ig domain was found in On‐CD80/86; this domain is responsible in binding of CD28 to CD80/86 receptors. Subcellular localization analysis showed that both On‐CD28 and On‐CD80/86 were distributed predominantly in the cytomembrane. Yeast two‐hybrid assay showed that On‐CD28 directly interacted with On‐CD80/86. On‐CD28 and On‐CD80/86 transcripts were detected in all the examined tissues of healthy Nile tilapia, and the highest expression levels of On‐CD28 and On‐CD80/86 were detected in the brain and heart, respectively. Following a bacterial challenge using Streptococcus agalactiae in vivo, On‐CD28 and On‐CD80/86 were upregulated in head kidney, spleen, intestines, and brain. However, they showed different expression profiles in response to stimulation with inactivated S. agalactiae in vitro. These findings indicated that the interaction of On‐CD28 with On‐CD80/86 provides a costimulatory signals that possibly play an important role in T cell activation during S. agalactiae infection. HighlightsCD28 and CD80/86 gene was identified from Nile tilapia (Oreochromis niloticus).On‐CD28 and On‐CD80/86 were distributed predominantly in the cytomembrane.On‐CD28 could directly interacted with On‐CD80/86.Induced transcriptional patterns were observed for both On‐CD28 and On‐CD80/86 after challenges with Streptococcus agalactiae.