Outer membrane protein U (OmpU) mediates adhesion of Vibrio mimicus to host cells via two novel N-terminal motifs.

Vibrio mimicus (V.mimicus) is a causative agent of ascites disease in aquatic animals. Our previous studies have demonstrated that the outer membrane protein U (OmpU) from V.mimicus is an immunoprotective antigen with six immunodominant linear B-cell epitopes. Although the N-terminus of OmpU contains potential binding motifs, it remained unclear whether OmpU possesses adhesion function. Here, the adhesive capacity of recombinant OmpU and V.mimicus to epithelioma papulosum cyprinid (EPC) cells was determined by immunofluorescence and adherence assay. The results showed that after co-incubated with rOmpU, an obvious visible green fluorescence could be observed on the EPC cell surface and the nuclei exhibited blue fluorescence; while the control cell surface did not show any signal, only nuclei exhibited blue fluorescence. The average number of wild-type strain adhered to each cell was 32.3 ± 4.5. The average adhesion number of OmpU gene deletion mutant was significantly reduced to 10.8 ± 0.5 (P < 0.01) and restored to 31.3 ± 2.8 by complement strain (P >0.05). Pretreatment of cells with rOmpU reduced the average adhesion number of wild-type strain to 9.7 ± 2.9 (P < 0.01). Likewise, binding was significantly decreased to 8.8 ± 3.2 (P < 0.01) due to blocking role of OmpU antibodies. To determine binding motifs of OmpU, six immunodominant B-cell epitope peptides labeled with FITC were employed in flow cytometry-based binding assay. Two FITC-labeled epitope peptides (aa90-101 and aa173-192) showed strong binding to EPC cells (the fluorescence positive cell rate was 99 ± 0.6% and 98 ± 0.3%, respectively), which could be specifically competed by excess corresponding unlabeled peptides, whereas the remaining four showed a low level of background binding. This is the first demonstration that OmpU possesses adhesion function and its N terminal 90-101 and 173-192 amino acid regions are critical sites for cell surface binding.

Identification of capsule, biofilm, lateral flagellum, and type IV pili in Vibrio mimicus strains.

Vibrio mimicus is a bacterium that causes gastroenteritis; it is closely related to Vibrio cholerae, and can cause acute diarrhea like cholera- or dysentery-type diarrhea. It is distributed worldwide. Factors associated with virulence (such as hemolysins, enterotoxins, proteases, phospholipases, aerobactin, and hemagglutinin) have been identified; however, its pathogenicity mechanism is still unknown. In pathogenic Vibrio species such as V. cholerae, Vibrio. parahaemolyticus and Vibrio vulnificus, capsule, biofilms, lateral flagellum, and type IV pili are structures described as essential for pathogenicity. These structures had not been described in V. mimicus until this work. We used 20 V. mimicus strains isolated from water (6), oyster (9), and fish (5) samples and we were able to identify the capsule, biofilm, lateral flagellum, and type IV pili through phenotypic tests, electron microscopy, PCR, and sequencing. In all tested strains, we observed and identified the presence of capsular exopolysaccharide, biofilm formation in an in vitro model, as well as swarming, multiple flagellation, and pili. In addition, we identified homologous genes to those described in other bacteria of the genus in which these structures have been found. Identification of these structures in V. mimicus is a contribution to the biology of this organism and can help to reveal its pathogenic behavior.

Immunological responses of customised probiotics-fed marron, Cherax tenuimanus, (Smith 1912) when challenged with Vibrio mimicus.

A two-phased experiment was conducted to investigate the effects of dietary supplementation of customised probiotics on marron physiology. During the first phase marron were fed probiotic supplemented feed for 70 days, while in phase two the same marron were challenged with Vibrio mimicus and their physiological responses were investigated for 4 days post-challenged. The experiment was carried out in a purpose-built room, designed for aquaculture research, using 18 of 250 L cylindrical plastic tanks. Five species of isolated probiotic bacteria from commercial probiotic products and marron's intestine were tested in this experiment. The probiotic bacteria were (Bacillus sp.); A10 (Bacillus mycoides); A12 (Shewanella sp.); PM3 (Bacillus subtilis); and PM4 (Bacillus sp.), which were added to the formulated basal marron diet (34% crude protein, 8% crude lipid, 6% ash) at a concentration of 10(8) cfu/g of feed. Immune responses of marron fed probiotics were evaluated by investigating organosomatic indices, growth rate, survival, intermoult period, total haemocytes counts (THC), proportion of granular cells (GC), bacteraemia, bacteria load in the intestine and water quality. The results showed that dietary supplementation of probiotics in marron had no significant impact on growth, intermoult period and survival of the marron. However, their supplementation improved the physiological condition of marron in terms of significantly higher tail muscle indices, THC and proportion of granular cells (GC) and reduced bacterial load in the haemolymph. The addition of probiotics in marron diets also increased the bacteria load in the marron intestine. In addition, dietary supplementation of the customised probiotics was effective in improving the resistance of marron against V. mimicus as they had higher THC, higher proportion of GC and lower presence of bacteria in their haemolymph, after marron were challenged with V. mimicus. The results also showed that probiotic Bacillus mycoides (A10) and PM4 are the most beneficial dietary probiotics for marron health.

Dietary organic selenium improves growth, survival and resistance to Vibrio mimicus in cultured marron, Cherax cainii (Austin, 2002).

To determine the effects of dietary organic selenium (OS) supplementation on the growth performance and immune competence of marron, Cherax cainii (Austin, 2002), a group of marron were fed 0.2 g kg(-1) of Sel-Plex(®) supplemented basal diet and then compared with another group (control) of marron fed basal diet without any supplementation. After 90 days of feeding, final weight, average weekly gains (AWG), relative gain rate (RGR), specific growth rate (SGR), survival, total and differential haemocyte counts (THC and DHC), were compared between the two groups. Surviving marron from each group were then divided into three sub-groups (three tanks per sub-group with seven marron per tank); (1) first sub-group was injected with 20 µL of 3.24 × 10(6) cfu Vibrio mimicus; (2) the second sub-group was injected with 20 µL normal saline and (3) the third sub-group was not subjected to injection and became the control group. THC, DHC, neutral red retention time (NRRT) and Vibrio ranks of post-injected marron were evaluated for 96 h, at every 24-h interval. The results showed that after 90 days of feeding, final weight, AWG, RGR, SGR, survival, THC, proportion of hyaline cells of OS-fed marron were significantly higher (P < 0.05) than the control group, whereas proportion of granular and semigranular cells were not affected by dietary OS. After challenging with V. mimicus, survival rate of marron without dietary OS significantly decreased (P < 0.05) as compared to the control group of marron. THC of marron in all sub-groups were significantly reduced (P < 0.05) after the challenge. However, THC and granular cells of sub-groups fed OS were higher than other sub-groups. Vibrio ranks and NRRT of marron fed OS were significantly lower and slower, respectively, than marron fed without OS. These findings demonstrated the benefits of OS inclusion in the marron diet in terms of growth, health and disease resistance.

Dietary supplementation of mannan oligosaccharide improves the immune responses and survival of marron, Cherax tenuimanus (Smith, 1912) when challenged with different stressors.

Three trials were conducted to determine the effects of mannan oligosaccharide (Bio-Mos ) on the immune responses of marron. In the first trial marron were challenged with Vibrio mimicus infection, in the second with NH3 exposure and in the third, the marron were exposed to air during a simulated live transportation trial. For V. mimicus infection and live transportation trials, marron (10.44 +/- 0.20 g and 4.44 +/- 0.20 g initial weights, respectively) were fed three different diets containing 0% (control diet), 0.2% and 0.4% Bio-Mos for 30 days and 112 days respectively before challenge, whereas for the NH3 exposure trial, marron (94 +/- 2.17 g initial weight) were reared with the control diet and 0.4% Bio-Mos diet for 42 days before exposure to NH3. Marron were examined for survival and total haemocyte count (THC), differential haemocyte count (DHC), haemolymph clotting time, bacteraemia and lysosomal membrane stability as indicators of immune responses during the course of the challenge. Survival of marron infected with bacteria and exposed to NH3, were significantly improved when fed Bio-Mos. THCs were significantly reduced in marron fed the control diet when they were infected with bacteria and subjected to live transportation while it remained unchanged in the marron fed the Bio-Mos supplemented diets. THCs of marron fed any of the diets were reduced when they were exposed to NH3 but the THCs were higher (P < 0.05) in marron fed Bio-Mos diets. Vibrio spp. in haemolymph of marron fed the control diet significantly increased when they were infected with V. mimicus and challenged with NH3 but it remained unchanged in the marron fed the Bio-Mos diets. Haemolymph clotting time was higher in marron fed the control diets when subjected to live transportation and 3 days of exposure to NH3. After 96 h infection marron fed the Bio-Mos diets had longer NRR time than those fed the control diet. All the findings demonstrated the ability of Bio-Mos to improve the survival, health status and immunity of marron under the bacterial infection and stress conditions caused by air and NH3 exposures.

Proteolytic activation of Vibrio mimicus (Vm) major outer membrane protein haemagglutinin (HA) with Vm-HA/protease: Implication for understanding bacterial adherence.

Vibrio mimicus (Vm) haemagglutinins (HAs), such as an extracellular HA/protease (Vm-HA/protease) and a major outer membrane protein-HA (Vm-OMPHA), have been recognized as the putative adherence factors for the bacterium. However, the mechanism by which HAs coordinate the adherence function of the bacterium remains as yet unknown. We report herein the positive interaction between Vm-HA/protease and Vm-OMPHA resulting in significant enhancement of the haemagglutinating ability. In this interaction, no cleaved polypeptide was detected; however, limited proteolysis of Vm-OMPHA was confirmed by SDS-PAGE. The proteolytic activation of the native cell-associated Vm-OMPHA by limited proteolysis was also demonstrated in several V. mimicus strains. Proteolytic activation of OMPHA was also achieved with various proteases from bacterial and eukaryotic sources. These findings may indicate a novel coordination of V. mimicus HAs in the adherence of the bacterium.

Presence of LuxS/AI-2 based quorum-sensing system in Vibrio mimicus : luxO controls protease activity.

Presence of the quorum-sensing regulation system in Vibrio mimicus was investigated. The culture supernatants of V. mimicus strains were found to possess AI-2 autoinducer like activity, and the strains were found to harbor the genes which are homologous to luxS, luxO, and luxR of V. harveyi. These genes of V. harveyi have been shown to be important components of V. harveyi-like quorum-sensing system. The luxO gene homologue known to encode LuxO, the central component of the regulation system, was disrupted, and effects on protease and hemolysin activity were studied. Disruption of luxO gene resulted in the increased protease activity, but the hemolysin activity did not vary considerably.