| Coccidiosis is a protozoan disease caused by Eimeria Coccidiosis parasitic in chicken intestinal epithelial cells.It seriously damages the growth and feed utilization rate of chickens and causes huge economic losses to chicken breeding industry.It is one of the most serious poultry parasitic diseases in the world.The prevention and treatment of the disease mainly depend on chemical drugs and vaccines.However,with the widespread and long-term use of chemical drugs,the continuous emergence of drug-resistant coccidia strains has dramatically reduced the therapeutic effect of traditional drugs.The use of the attenuated vaccine to prevent coccidiosis also risks returning and dispersing the virus.Therefore,it is imperative to develop a new vac-cine.Genetic engineering vaccine has the advantages of safety,stability,and easy preparation,so it usually immunizes the body with protective antigen and molecular adjuvant to enhance the immune effect of the vaccine.Lactic acid bacteria are recog-nized as GRAS,generally recognized as safe microorganisms,and can be used as car-riers to transmit specific protective antigens by oral delivery of antigen genes into the cells of the body,thus triggering local or systemic immune responses.E.tenella pro-filin can affect parasite movement,invasion,and host immune response by regulating the polymerization and depolymerization of actin.It has good immunogenicity and can be used as a new target of the coccidia vaccine.Salmonella typhimurium flagellin(FliC)is a natural ligand of Toll-like receptor 5,which can assist in inducing antigen-specific responses against pathogens such as viruses and bacteria.In this study,the recombinant Lactobacillus Plantarum was constructed with pSIP-409-pgs A’surface anchored expression vector,profilin as protective antigen and FliC as an adjuvant,and the protective effect of recombinant bacteria against coccidiosis was evaluated.The main research contents and results are as follows:Concerning the profilin and FliC sequences logged in by Gen Bank,the gene pro-filin-FliC was optimized and routinely synthesized.The profilin and profilin-FliC gene fragments were amplified by PCR and ligated to the anchored expression vector pSIP409-pgs A’.The results of double endonuclease digestion and base sequencing showed that the recombinant plasmids pSIP409-pgs A’-profilin-FliC and pSIP409-pgs A’-Profilin were constructed successfully.The co-anchored expression plasmids were electro transformed into NC8 competent cells,and the recombinant Lactobacil-lus Plantarum strains NC8-pSIP409-pgs A’-profilin-FliC and NC8-pSIP409-pgs A’-profilin were obtained after resistance screening.Western blot and indirect immuno-fluorescence showed that the successful expression of profilin and profilin-FliC pro-teins anchored on the surface of the bacterial wall had good reactivity.Healthy 1-day-old broilers were randomly divided into 6 groups with 20 chick-ens in each group:PBS group,PBS-challenge(challenge)group,empty vector NC8-pSIP409-pgs A’(409A’),NC8-pSIP409-pgs A’-profilin-FliC group(profilin-FliC),NC8-pSIP409-pgs A’-profilin group(profilin)and commercial live anti-coccidial vac-cine group(Vaccine).Oral immunization was carried out at 3,4,5,17,18,and 19days old,and each chicken was immunized with 1×10~9 CFU/200μL.At the age of 30days,5×10~4 oocysts were inoculated with E.tenella.Before challenge,the secretory antibody SIgA in intestinal contents and the specific antibody IgG in serum were de-tected by the ELISA method.The transcription level of spleen cytokines was detected by the q PCR method.Before and after worming,the proliferation of splenocytes was detected by the CCK-8 method,the levels of CD3~+CD4~+and CD3~+CD8~+T lympho-cytes in the spleen were detected by flow cytometry,and the contents of cytokines IFN-γand IL-2 in serum were detected by ELISA kit.After the challenge,the relative weight gain rate,oocyst excretion,cecal lesion score,and other protective indexes were counted and analyzed.The results of ELISA showed that the specific IgG detected by expression profil-in-FliC genome and single expression profilin genome was significantly higher than that in Challenge group(P<0.001)and 409p’group(P<0.001),and that in profilin-FliC group was significantly higher than that in profilin group(P<0.001).The spe-cific SIgA level of recombinant lactic acid bacteria were also significantly higher than that in the Challenge group and 409p’group(P<0.001),and that in the profilin-FliC group was significantly higher than that in the profilin group(P<0.001).The content of IFN-γin the profilin-FliC genome before and after the challenge was significantly higher than that in the challenge group(P<0.001)and 409p’group(P<0.0l).The con-tent of IL-2 in the profilin-FliC group was significantly higher than that in the Chal-lenge group(P<0.001),but it was significantly higher than that in the Challenge group and 409p’group after worming(P<0.001).The proliferation ability of spleen lymphocytes in the profilin-FliC group was significantly higher than that in the Chal-lenge group(P<0.001).The m RNA transcription levels of IL-2,IL-4,and IFN-γin spleen lymphocytes in the profilin-FliC group were significantly higher than those in the Challenge group(P<0.001,P<0.001).The flow cytometry results showed that immunized with recombinant lactic acid bacteria could increase the level of CD4~+CD8~+T lymphocytes in the chicken spleen.The level of CD4~+CD8~+in the profil-in-FliC group before and after the challenge was significantly higher than that in the challenge group and empty vector 409p’group(P<0.001,P<0.01).After the challenge,it was found that immunized recombinant lactic acid bacteria could increase the body weight of chickens,and the weight gain rate was significantly higher than that of the challenge and vaccine group(P<0.001).The relative weight gain rate of the profilin-FliC group was 82%.The excretion of oocysts in chicks’feces in the profilin-FliC group,profilin group,and Vaccine group decreased significantly.The oocyst reduc-tion rates in the PBS group were 42%,54%,and 75%,respectively.The results of the pathological section showed that the pathological intestinal damage,oocyst,intestinal villus injury,bleeding point,and inflammatory cells in the oral immune recombinant bacteria group were significantly less than those in the challenge group.The Vaccine,profilin,and profilin-FliC groups ACI index were 149,136,160,respectively.Results Oral administration of recombinant lactic acid bacteria vaccine containing the expres-sion of profilin-FliC protein on the surface had an excellent anti-coccidias effect.The protein was successfully anchored and expressed on the surface of Lactoba-cillus Plantarum NC8 by pgs A’,and the recombinant Lactobacillus Plantarum ex-pressing profilin and FliC adjuvant was constructed.Recombinant bacteria can stimu-late the production of specific IgG and s IgA antibodies,improve cellular and humoral immunity against coccidiosis,and have an excellent immune protection effect against coccidiosis,which provides a new strategy for immune prevention and treatment of coccidiosis in chickens.In addition,it was also found that Flic adjuvant could effec-tively enhance the anti-coccidias immune response of recombinant vaccine,which proved its potential as a mucosal vaccine adjuvant. |
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