Turtle Colonization And Phage Clearance Of Vibrio Cholerae

Vibrio cholerae is a Gram-negative aquatic bacterium which mainly transmitted through food and water.Toxigenic V cholerae could cause diarrhea,electrolyte disturbance and acidosis.In recent years,outbreaks caused by V.cholerae-contaminated soft-shelled turtles,mainly of toxigenic serogroup O139,have been frequently reported,posing a new foodborne disease public health problem.However,at present most studies were performed by using epidemiological method,little laboratory studies were used to study theirs interaction.In this study,the colonization of toxigenic V.cholerae on the body surface and intestine of the soft-shelled turtle was explored firstly.Preferred colonization sites on the turtle body surface,mainly the carapace and calipash of the dorsal side,were observed for the O139 strains.Intestinal colonization was also found.The colonization factors of V.cholerae played different roles in colonization of the soft-shelled turtle body surface and intestine.Mannose-sensitive hemagglutinin(MSHA)of V.cholerae was necessary for body surface colonization,but no roles were found for toxin-coregulated pili(TCP)and N-acetyl glucosamine-binding protein A(GBPA).Both TCP and GBPA play important colonization roles in the intestine,whereas the deletion of MSHA identified only a minor colonization-promoting role for this factor.Secondly,the preferred colonization sites and proliferation trend were studied by using bioluminescent imaging method.The results showed that the preferred colonization sites for O1 V.cholerae strains on body surface were similar to O139 serogroups.More V cholerae strains colonized on turtle's calipash and carapace on dorsal side and less strains colonized on ventral side.The ventral side colonization sites were mainly limbs.By constructing colonization gene mutants and colonization competition assays we make sure thay MSHA was one of the main colonization factor for O1 V.cholerae strains colonization.By colonization competition assays for different serogroups and different sources,we found that the O139 strains have stronger colonization ability than O1 strain.Besides,strains obtained from soft-shelled turtles tend to have stronger colonization ability than strains obtained from patients.At last,we tested the activities of virulent phage VP1 to VP5 on the surface of LB agar plate by using the toxigenic V.cholerae strains which isolated from soft-shelled turtles.The results showed that virulent phage VP2 could lyse all 01 serogroup toxigenic V.cholerae strains and the vast majority of O139 serogroup toxigenic V.cholerae strains.By using the colonization models in our first section,we tested the phage clearation effects for turtles' body surface,intestine and the aquatic environment.Gradually decreased phenomenon on turtle's surface was observed.Obvious clearing effects were also found in turtles' intestine and the aquatic environment.In summary,virulent phage VP2 can be effective at reducing oxigenic V.cholerae contamination on soft-shelled turtles and have the potential values to apply to soft-shelled turtles breeding.