Evaluation Of Novel Attenuated Salmonella Formulation Against Avian Necrotic Enteritis

Necrotic enteritis is one of the most common diseases in broiler chickens,usually secondary to coccidiosis,with the decreased weight gain rate,the increased mortality rate,resulting in direct and indirect economic losses to poultry industry.Clostridium perfringen A and its secrete toxins are mainly responsible for the outbreak of NE,whose main symptoms are small intestine mucosal necrosis.This research utilized novel Salmonella vectors with delayed attenuation and delayed protein expression to construct recombinant attenuated Salmonella vaccines to deliver a carboxyl-terminal fragment of ? toxin(PlcC)and a GST-NetB fusion protein(gst NetB).Then expression and immunogenicity of the target antigens were detected in vitro,evaluation of the immune protective effects against NE in vivo were also conducted.The specific contents and results are as follows:?The construction and immunogenicity analysis of recombinant Salmonella ?9959(pYA3681-PlcC),?9959(pYA3681-gstNetB)This experiment utilized Salmonella shuttle vector pYA3681 to construct recombinant plasmids pYA3681-PlcC and pYA3681-gstNetB,then electroporated the plasmids into Escherichia coli ?6212 peptide.After identification by double enzyme digestions and successful plasmid sequencing,another electroporation to final host bacteria ?9959 were performed.After the induction of IPTG,SDS-PAGE assay and Western Blotting assay could prove that the recombinant bacteria did express the target proteins.The relative molecular size of the recombinant PlcC was about 14 kD,the recombinant NetB fused with GST was about 65 kD.?Evaluation and the immune protection effects of the recombinant SalmonellasThis research also evaluated immune protections in broiler chicken against NE through animal experiments.The specific experimental procedures were as follows: PBS group,Empty vector group(pYA3681 group)and Plc group(pYA3681-PlcC group),NetB group(pYA3681-gstNetB),Mixture group(pYA3681-PlcC+pYA3681-gstNetB)were arranged.One-day-old broiler chickens were bought and then,orally inoculated the recombinant bacteria 109 CFU /200?l /per chicken for three times,one time a week.After three immunizations,flow cytometry was performed to detect the differentiations of CD3+CD4+T and CD3+CD8+T cells in the spleen.The specific antibody titers were detected by Elisa in the serum we separated from blood and in the intestinal lavage fluid.After the first immunization,on day28~day32,challenged all the chickens for five consecutive days,twice a day,109 CFU /1ml/per chicken.On day33,sacrificed all the experimental animals and removed the small intestines to calculate intestinal disease scores.Body weight changes rate were calculated too.The results indicated that,compared with PBS group and empty vector group,anti-PlcC and anti-gstNetB specific antibodies in serum and in the intestinal lavage fluid of the Mixture Group had significant increase;Flow cytometry results showed that compared with PBS group and empty vector group,CD3+CD4+T cells of the Experiment Group increased significantly,while the CD3+CD8+T cells did not,which indicated the recombinant Salmonellas could induce the proliferation of CD3+CD4+T cells;Weight gain rate and intestinal lesions scores also demonstrated ?9959(pYA3681-PlcC),?9959(pYA3681-gstNetB)could offer immune protection against NE in some extent and among them the mixture group could offer a better protection effects.In summary,all the results obtained indicate the multiple antigens could be exploited as a new orientation to develop NE vaccine.