Effects Of Vibrio Mimicus And Its Targeted DNA Vaccine On Intestinal Microbiota And Intestinal Tissue Structures Of Grass Carp(Ctenopharyngodon Idella)

Vibrio mimicus is a common intestinal pathogen,which can cause serious ascites disease,which lead to the severe economic loss to grass carp aquacμLture.Therefore,it is urgent to study the pathogenic mechanism and immune control of Vibrio mimicus.The intestinal flora of fishes is closely associated with the material metabolism,immunity,and health of fishes.If the dynamic equilibrium of the host and intestinal flora is destroyed under the action of natural or artificial factors,the host will have the disorder of nutrition absorption and metabolism,and the decrease of immune function because of the disturbance intestinal flora structure,which will resμLt in all kinds of diseases.Similarly,the structure of intestinal flora will be disturbed after vaccination.Thus,the study on the structure of intestinal flora provides a new direction for disease pathogenesis research and prevention.In our previous studies,a strain V.mimicus with stronger toxicity was isolated from the diseased grass carp,and its DNA vaccine was constructed,founding that the vaccine coμLd induce an excellent intestinal mucosal immune response.On the basis,the effects of V.mimicus infection and V.mimicus DNA vaccine on intestinal flora and intestinal structure of grass carp were studied.The primary research work and resμLts completed are summarized as follows:1)Construction of V.mimicus target DNA vaccine and detection its intestinal expression in situ.Here,V.mimicus DNA vaccine was loaded into lyophilized DH5α-BGs by capsμLe method.The resμLts of fluorescence quantitative PCR showed that the 1 mg BGs coμLd load 214μg vaccine plasmid,indicating that the targeted DNA vaccine was successfμLly constructed.Then the expression of the vaccine gene in different intestinal segments was detected by immunohistochemical method after immunization 21 days with the targeted DNA vaccine,and the results showed that the vaccine gene was expressed in all intestinal segments,and the highest expression was found in the midgut.2)The effect of V.mimicus infection and its targeted DNA vaccine on intestinal flora of grass carp.In this paper,the contents of the intestinal segments before and after infection and immunization were collected,the total DNA was extracted for 16 S r RNA PCR amplification,and the effects of infection and immunity on the diversity of intestinal flora and species abundance were analyzed by high throughput sequencing technique.The resμLts showed that immunity and infection had no significant effect on the diversity of intestinal flora,but coμLd significantly change the colony structure in the midgut and hindgut of the immunized grass carp as well as each intestinal segments of the infected fish.At the phylum level,the relative abundance of Fusobacteria was apparently increased,while the relative abundance of Actinobacteria and Firmicutes decreased considerably.At the class level,the relative abundance of Fusobacteria was apparently increased,while the relative abundance of Alphaproteobacteria,Clostridia and Actinobacteria were significantly decreased.At the genus level,the relative abundance of Cetobacterium and Bacteroides increased considerably,but the relative abundance of Methylobacterium was significantly decreased.3)Effects of V.mimicus infection and its targeted DNA vaccine on intestinal structure of grass carp.In this study,the intestinal segments of grass carp before and after infection and immunization were collected,and then the changes of intestinal tissue structure were observed by tissue slicing technique.It was found that there were no visible pathological changes in the each intestinal segments of the immunined fish,but the histological structure of each intestinal segments of the infection fish was obviously changed,which mainly manifested as the reduced height of the intestinal mucosa folds and the thickness of the intestine wall,part of the villi was separated from the mucosa muscle layer,and the villi were thinning and damaged at front end.In conclusion,V.mimicus infection and its targeted DNA vaccine have a great effect on the structure of intestinal flora of grass carp.V.mimicus infection can lead to obvious pathological changes in the intestinal structure of grass carp,while the targeted DNA vaccine has no obvious effect on intestinal tissue structure.