Vibrio Alginolyticus Infection Research Articles

Development of monoclonal antibodies for simple identification of Vibrio alginolyticus

The present study was aimed to produce monoclonal antibodies (MAbs) for simple and specific identification of Vibrio alginolyticus infection in shrimp. Mice were immunized with heat killed V. alginolyticus four times at 2-week intervals. The best response mouse was used for spleen donor in hybridoma production. Screening of hybridoma clones producing desired antibodies was performed by dot blotting against V. alginolyticus and other bacterial species, Western blotting and immunohistochemistry of infected shrimp tissues. Four groups of MAbs were obtained; the first group of MAbs demonstrated their limited specificity only to V. alginolyticus used for immunization, while the second and the third groups recognized all three isolates of V. alginolyticus used for testing. The fourth group of MAbs bound to all three isolates of V. alginolyticus and also recognized Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis and Vibrio vulnificus but did not bind to Vibrio mimicus, Vibrio cholerae, Vibrio penaeicida and other bacterial species tested. MAbs in groups 1, 2 and 3 were able to use for the detection of bacterial infection in the tissues by means of immunohistochemistry. MAbs specific to V. alginolyticus was produced. These MAbs can be used for specific identification of the bacteria by simple 'dot blotting' method and immunohistochemistry. This study demonstrated an immunological tool that can be used for simple and accurate identification of V. alginolyticus as well as for the diagnosis of V. alginolyticus infection in animals. This immunological tool can replace costly and laborious biochemical tests.

Molecular cloning of growth hormone from silver sea bream: Effects of abiotic and biotic stress on transcriptional and translational expression

The pituitary growth hormone (GH) gene of silver sea bream ( Sparus sarba) was cloned and characterized and found to be 615 base pairs encoding a protein of 204 amino acids. Using a bacterial expression system, recombinant protein was prepared and rabbit polyclonal antibody was raised. Transcript and protein amounts of GH were measured in fish that were adapted to a range of salinities, acclimated to different temperatures, or undergoing a natural time course of Vibrio alginolyticus infection. Isoosmotic salinity (12 ppt) adaptation resulted in increased GH transcript and protein in comparison to freshwater (0 ppt) and seawater (33 ppt) adapted fish. It was also found that cold temperature (12 °C) acclimated sea bream had higher amounts of pituitary GH transcript and protein when compared to warm temperature (25 °C) acclimated fish. Finally, the amounts of GH transcript and protein were found to be rapidly downregulated from an early stage of disease. The results from the present study demonstrate how GH can be modulated during both abiotic and biotic stress in fish.

The peroxinectin of white shrimp Litopenaeus vannamei is synthesised in the semi-granular and granular cells, and its transcription is up-regulated with Vibrio alginolyticus infection

Peroxinectin mRNA expression in the different types of haemocytes of white shrimp Litopenaeus vannamei was studied by in situ hybridisation using digoxigenin-UTP-labelled riboprobes and reverse transcription-polymerase chain reaction (RT-PCR). Granular cells (GC) and a mixture of semi-granular cells (SGC) and hyaline cells (HC) were separated by 70% Percoll gradient centrifugation. Peroxinectin was synthesised in both GC and the mixture of SGC-HC. An in situ hybridisation assay indicated that peroxinectin mRNA expression occurred in GC and SGC, but not in HC. Peroxinectin transcript up-regulated significantly, whereas haemocyte count decreased significantly at 6, 12 and 24 h post Vibrio alginolyticus-injection with slower restoration as compared to that of saline-injected shrimp. The RT-PCR assay indicated that peroxinectin exists extensively in several species of decapod crustaceans including L. vannamei, freshwater prawn Macrobrachium rosenbergii, common caridina Caridina pseudodenticulata, stone crab Thalamita crenata and mud crab Scylla serrata suggesting that this protein plays an important role in defence against pathogens.

Vibrio alginolyticus infection in the white shrimp Litopenaeus vannamei confirmed by polymerase chain reaction and 16S rDNA sequencing

A gram-negative, rod-shaped bacterium identified as Vibrio alginolyticus was isolated from diseased Litopenaeus vannamei (also called Penaeus vannamei) in Taiwanese culture ponds. The diseased shrimp displayed poor growth, anorexia, inactivity, reddish pleural borders of antennae, uropods and telson, opaque and whitish musculature, and mortality. In histological preparations, melanized hemocytic granulomas were observed in the connective tissue around hemal sinuses together with hemocytic aggregation in necrotic musculature. Six isolates of Vibrio were collected from diseased shrimp at 3 farms, and these were evaluated for characteristics including morphology, physiology, biochemistry and sensitivity to antibiotics. The results indicated that the isolates belonged to a single species that grew in 1 to 8% NaCl, at 10 to 40 degrees C and on TCBS (thiosulfatecitrate-bile sucrose) agar, and that gave positive catalase, O/F (Oxidation/Fermentation), lysine decarboxylase, gelatinase and cytochrome-oxidase tests. Identification of CH003 (1 of 6 isolates) was confirmed by PCR assay for V. alginolyticus (expected amplicon 1486 bp). The 16S rDNA sequence (GenBank accession number AY373027) gave 99.9% sequence identity to V. alginolyticus (GenBank accession number X74690). The calculated 96 h LD50 dose of the isolated strain was 3.0 x 10(5) colony forming units (CFU) shrimp(-1) (6.6 x 10(4) CFU g(-1)).

Vibrio alginolyticus infections in Hawaii.

Clinical and bacteriological features of eight cases of Vibrio alginolyticus infections in Hawaii are presented. These isolates occurred in superficial sites, primarily related to infections caused by swimming.