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In July 2010, heavy mortalities were seen in cultured swimming crabs, Portunus trituberculatus, from Jiangsu Province in China. Two isolates, XA1 and XA2, were isolated from cardiac muscle and somatic muscle of the moribund crabs on ZoBell's marine 2216E agar and were confirmed as the causative agent for the mass mortalities seen on that farm. The morphological and physiological characteristics as well as the biochemical tests of the two isolates were identical and were similar to Vibrio natriegens. Both were gram-negative, oxidase positive, produced acid when fermenting glucose, and failed to grow in the media containing 1% NaCl. Furthermore, the sequencing of the 16S rRNA, gapA, ftsZ, mreB, and topA genes revealed that the two isolates were identical and had a 0.99 homology coefficient with V. natriegens. The Neighbor-Joining tree based on the concatenated sequences of four housekeeping genes (gapA, ftsZ, mreB, and topA) strongly supported that XA1 was most closely related to V. natriegens within the Vibrio harveyi–related clade. In addition, pathogenicity testing using immersion challenge confirmed that strain XA1 was virulent to juvenile crabs, and the LD50 value was 1.09 × 106. This is the first report on V. natriegens as a virulent pathogen for Portunus trituberculatus.

Outbreaks of mass mortality among cultured juvenile Chinese shrimp Fenneropenaeus chinensis occurred during summer 2010 in Jiangsu Province, PR China. Gross signs of disease included lethargy, swimming near the water surface and close to pond edges, pale red shell and appendages, breaking of antenna, and reduction in food consumption. Green and yellow bacterial colonies were recovered on thiosulfate citrate bile salt sucrose agar plates from the diseased shrimp. The predominant observed colonies were purified (VP1 and VR1) and their virulence toward postlarvae of Fenneropenaeus chinensis confirmed by bath experimental challenges. The phenotypic characteristics of the isolates, including morphological, physiological, and biochemical traits, were determined. Three housekeeping genes (16S rRNA, RNA polymerase a-chain (rpoA), and DNA gyrase B subunit (gyrB)) were sequenced, and phylogenetic trees based on rpoA and gyrB sequences were constructed. Phenotypic and genetic results confirmed the diseased ...

Outbreaks of mass mortalities among cultured megalopa of swimming crab (Portunus trituberculatus) occurred in a commercial hatchery during the spring of 2012 in Jiangsu province, P. R. China. Dominant bacteria were isolated and characterized by biochemical reactions, enzyme production, and sequencing analysis of the 16S rRNA, gyrB (DNA gyrase B subunit) genes, and two strains (DY1 and DY2) were selected as representative strains for virulence tests by immersion. The results showed that the characteristics of identified strains consist with Vibrio harveyi; the 16S rRNA and gyrB genes of the tested strains exhibited high similarity with V. harveyi; and the phylogenetic trees constructed using the neighbor-joining method based on 16S rRNA and gyrB genes. Two strains (DY1 and DY2) clustered with the V. harveyi and were supported by a higher bootstrap value, and two strains (DY1 and DY2) were found lethal to the healthy megalopa and juvenile crab. LD 50 of DY1 and DY2 to megalopa and juvenile crab were 2.4 × 106, 3.0 × 106, 1.9 × 106 and 2.5 × 106 CFU/ml, respectively. The results confirmed that the diseased megalopa were infected by V. harveyi. In addition, we analyzed the tested strains (DY1 and DY2) by PCR for the presence of virulence genes that were known in V. harveyi, and the results showed that five virulence genes (luxR, toxR, vhhA, vhhB and pap6) were detected by a specific PCR assay, further supporting the assignment of isolates to V. harveyi and its pathogenicity. To our knowledge, this is the first report of V. harveyi as pathogenic bacteria of megalopa of swimming crab.

2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein that exerts antiviral effects through the RNase L- or retinoic acid-inducible gene I (RIG-I)-dependent signalling pathway. In this study, we identified and cloned the OASL gene (named duOASL) from healthy adult Cherry Valley ducks. Full-length duOASL cDNA (1630bp) encoded a 504-amino acid polypeptide containing three conserved domains, namely, nucleotidyltransferase domain, 2'-5'-oligoadenylate synthetase domain, and two ubiquitin-like repeats. DuOASL mRNA expression was quantified by performing quantitative reverse transcription-PCR (qRT-PCR). Results of qRT-PCR showed that duOASL was broadly expressed in all examined tissues, with the highest mRNA expression in the large intestine. Antiviral activity of duOASL was measured by determining its effect on Duck Tembusu virus (DTMUV) replication in vitro. We found that duOASL overexpression slightly inhibited DTMUV replication, whereas duOASL knockdown by using a specific small interfering RNA increased DTMUV replication in DF-1 cells. Thus, we successfully cloned and characterized the antiviral protein duOASL from Cherry Valley ducks and found that it exerted antiviral effects against DTMUV. These results provide a solid foundation for performing further studies to determine the mechanism underlying the antiviral effect of duOASL at the cellular level.

This study aimed to evaluate the survival and gene expression of Vibrio harveyi under starvation conditions. The microcosms V. harveyi were incubated in sterilized seawater for four weeks at room temperature. Overall, the cell numeration declined rapidly about 103 CFU/ ml during starvation, with a tiny rebound at day twenty-one. Scanning electron microscopy revealed rod-shaped cells became sphere with a rippled cell surface. By polymerase chain reaction (PCR) assay, nine genes, named lux R, tox R, vhh B, fla A, top A, fur , rpo S, mre B, fts Z were detected in the non-starvedi cells. In the starved, the expression levels of the detected genes declined substantially ranging from 0.005-fold to 0.028-fold compared to the non-starved performed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) with 16s rRNA as the internal control. In the recovering cells, the expression levels of the detected genes, except lux R and mre B, were upregulated dramatically compared to the wild, especially top A (23.720-fold), fur (39.400-fold) and tox R (9.837-fold), validating that the expressions of both the metabolism and virulence genes were important for growth and survival of V. harveyi. The results may shed a new light on understanding of stress adaptation in bacteria.

A bacterial disease was reported from cultured loach Misgurnus anguillicaudatus in China. Symptoms included haemorrhages at the body surface (including base of fin, head, mouth, and abdomen), ulceration of muscle, and high-level mortalities. The dominant bacterial colonies were isolated in nutrient agar from different infected body parts and confirmed the pathogenicity by challenge experiments. The 3 strains (NQ1-3) were randomly selected and characterized phenotypically and genotypically. Biochemically, the 3 strains showed properties and biochemical characteristics similar toVibrio cholerae. Phylogenetic analysis of the 16S rRNA gene (GenBank accession No. JF939043) and 3 housekeeping genes, RNA polymerase a-chain (rpoA, GenBank accession No. JF939042), recombination repair protein (recA, GenBank accession No. JF939045) and DNA gyrase B subunit (gyrB, GenBank accession No. JF909344) of strain (NQ1) exhibited high similarity to those of V. cholerae from GenBank. The 3 strains (NQ1-3) were positive for toxR, lolB (previously called hemM) and rtxA (presumptive cytotoxin) genes, potential virulence factors. To sum up, the isolated strains were confirmed as V. cholerae on the basis of phenotypic characteristics, phylogenetic analysis of the 16S rRNA gene and three housekeeping genes, as well as potential virulence factors studies. The susceptibility of isolates to 28 antimicrobial agents was determined, NQ1 were found to be sensitive to some of the drugs tested, including carbenicillin, azlocillin, cefoperazone, ceftazidime, ceftriaxone, cephradine, azithromycin, lomefloxacin, levofloxacin, enoxacin, norfloxacin, neomycin, streptomycin, doxycycline, tetraecycline, and minomycin. Key words: Misgurnus anguillicaudatus, Vibrio cholerae, housekeeping genes, ulcer disease.