| Coccidiosis is one of the major avian infectious diseases in the world.Among all kinds of Eimeria,E.tenella is the most pathogenic,and E.maxima is one of the most popular strains.At present,the prevention and control of the disease mainly relies on drug prevention and live vaccine,but the production process of chemical drugs is complex,there are drug residues,drug-resistant strains and other problems after long-term use,and the virulence of live vaccine is strong,which seriously affects the prevention and control of the disease.Therefore,it is imperative to explore new,safe and efficient prevention and control means.Lactobacillus plantarum is an ideal carrier for oral vaccine because of its stable colonization in intestinal tract,natural adjuvant activity,simple culture conditions and high safety.EtMIC2 can induce the immune protection of E.tenella,and it can also partially protect E.maxima.AMA1 plays an important role in different developmental stages of chicken coccidia,and its immunogenicity has been confirmed.SO7 is a refractor protein in sporozoite and merozoite stages of E.tenella.SO7 has partial protective effect on E.maxima.In this study,recombinant Lactobacillus plantarum NC8-pSIP409-pgs A’SO7-EtMIC2-S and NC8-pSIP409-AMA1-S-pgs A’SO7 were constructed,and the immune protective effect of recombinant Lactobacillus plantarum was evaluated by E.tenella and E.maxima chicken infection models.Firstly,therecombinantLactobacillusplantarum NC8-pSIP409-pgs A’SO7-EtMIC2-S and NC8-pSIP409-AMA1-S-pgs A’SO7 were constructed.Based on the single anchored expression plasmids TOP10-pSIP409-EtMIC2-S、TOP10-pSIP409-pgs A’-SO7and TOP10-pSIP409-AMA1-Stheanchoredexpressionplasmids TOP10-pSIP409-pgs A’SO7-EtMIC2-S and TOP10-pSIP409-AMA1-S-pgs A’SO7were constructed.Using TOP10-pSIP409-EtMIC2-S and TOP10-pSIP409-pgs A’-SO7plasmids as templates,the expression cassettes SD-EtMIC2-S and SD-pgs A’SO7 with SD sequence were amplified respectively,and then the target fragments were cloned into TOP10-pSIP409-pgs A’SO7/HindⅢand TOP10-pSIP409-AMA1-S/HindⅢwhich were linearized by HindⅢsingle enzyme.HindⅢsingle enzyme digestion showed that the co-anchored expression plasmids were successfully constructed TOP10-pSIP409-pgs A’SO7-EtMIC2-S and TOP10-pSIP409-AMA1-S-pgs A’SO7.The recombinant Lactobacillus plantarum strains NC8-pSIP409-pgs A’SO7-EtMIC2-S and NC8-pSIP409-AMA1-S-pgs A’SO7 were obtained after resistance screening.The expression of the target protein was confirmed by Western blot.One day old chickens were randomly divided into seven groups:PBS group,PBS-challengegroup,emptybody409pgs A’(409p’),NC8-pSIP409-pgs A’SO7-EtMIC2-S group(SO7-EtMIC2),NC8-pSIP409-AMA1-S-pgs A’SO7 group(AMA1-SO7),Mixture group(MIX),and commercial live coccidiosis vaccine group(Vaccine).The mice were immunized orally at the age of 3,4,5,17,18 and 19 days with the dose of 2×10~9CFU/head.At the age of 30 days,5×10~4and 1×10~5sporulated oocysts of E.tenella and E.maxima were inoculated.ELISA and flow cytometry were used to detect the changes of anti-coccidiosis immune indexes before and after challenge.The weight gain rate,oocyst output,intestinal lesion score and anti-coccidiosis index(ACI)were recorded.The pathological changes of cecum were detected by H&E staining.The results of ELISA showed that the levels of IFN-γand IL-2 in chickens immunized with recombinant bacteria before challenge were higher than those in PBS group and empty body409pgs A’group(P<0.05);the levels of IFN-γand IL-2 in SO7-EtMIC2,AMA1-SO7and MIX groups after challenge were significantly higher than those in PBS challenge group(P<0.05).Compared with PBS group,the level of CD8~+was significantly increased in the immune recombinant Lactobacillus group(P<0.05).The proportion of CD8~+T lymphocytes in MIX group was the highest,but there was no significant difference in the level of CD4~+among the groups(P>0.05).Compared with PBS challenge group,the level of CD8~+in T lymphocytes of immune recombinant bacteria group increased significantly after challenge(P<0.05).The average weight gain and relative weight gain rate of chicken were calculated.The weight gain rate of MIX group was the highest.The weight gain rate of MIX group,SO7-EtMIC2 group and AMA1-SO7 group was significantly higher than that of PBS challenge group.The worm reduction rate of E.tenella in Vaccine group was the highest(59.65%),the worm reduction rate of SO7-EtMIC2 group was 49.81%,and that of AMA1-SO7group was 47.91%.The reduction rate of E.maxima was 66%in MIX group,55%in SO7-EtMIC2 group and 59%in AMA1-SO7 group.The results of pathological sections showed that compared with PBS challenge group,the intestinal lesions in oral recombinant bacteria group were significantly reduced,the number of oocysts,villi damage,bleeding points and inflammatory cells were significantly reduced.The ACI of E.tenella and E.maxima in MIX group were 151.5 and 161.5 respectively;The ACI of E.tenella and E.maxima in Vaccine group were 174 and 172 respectively.The results showed that recombinant Lactobacillus plantarum NC8-pSIP409-pgs A’SO7-EtMIC2-S and NC8-pSIP409-AMA1-S-pgs A’SO7 could improve the cellular immunity against coccidiosis in chickens,and had a good immune protection effect on mixed infection of coccidiosis,which provided a new idea for the immune control of chicken coccidiosis. |
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