| Avian coccidiosis is a parasitic disease caused by Eimeria spp.in chickens’intestinal epithelial cells,which seriously endangers the growth and development of chicks and is one of the most serious parasitic diseases of chickens in the world.The control of the disease is most commonly used chemical drugs mainly antibiotics,but the widespread use of antibiotic drugs may lead to drug residues in animals or animal products and other problems,thus endangering human health.Moreover,prolonged and continuous use of certain chemical drugs may increase the likelihood of drug-resistant strains,thus affecting the therapeutic effects of traditional commonly used drugs.On the other hand,using attenuated live vaccines for prevention carries risks of reversion to virulence and dissemination.Therefore,developing new,safe,and environmentally friendly vaccines is essential.Nowadays,emerging genetic engineering vaccines have a shorter production time,lower costs,and are more accessible,making the development of new vaccines the primary research direction against coccidiosis.In recent years,lactic acid bacteria have gained recognition and attention as a safe level of microorganisms.Lactobacillus plantarum(L.plantarum)can effectively colonize the host and play a crucial role in maintaining the balance of the natural microbial community.Studies have found that it can also act as an antigen carrier,transporting specific antigens into cells and exhibiting immune regulation effects on humans and animals.The RON2 of the tachyzoite stage of the Eimeria tenella(E.tenella)microneme can induce the host to produce specific antibodies.It is an important invasion-related molecule expressed during the invasion phase of the parasite.The formed Moving Junction(MJ)is a vital structure that connects the parasite’s body and the host cell,affecting parasite movement and invasion as well as the host’s immune response.Therefore,it has immunogenicity and can be used as a new target for researching coccidiosis vaccines.This study constructed a recombinant L.plantarum vaccine using p SIP-409-pgsA’as the expression vector,RON2 protein as the protective antigen,and dendritic cell peptide(Dcpep)as the adjuvant,and evaluated the vaccine’s protective effect against E.tenella.The main research contents and results are as follows:Firstly,gene recombinant technology was used to construct NC8-p SIP-409-pgsA’-RON2-Dcpep and NC8-p SIP-409-pgsA’-RON2 L.plantarum strains.The target fragment of RON2 gene was amplified by PCR,and RON2-Dcpep and RON2 were connected to p SIP-409-pgsA’,respectively,followed by double enzyme digestion and sequencing identification.The results showed that the recombinant plasmids of p SIP-409-pgsA’-RON2-Dcpep and p SIP-409-pgsA’-RON2 were successfully constru-cted.The recombinant plasmids were electro-transformed into NC8 competent cells to obtain the recombinant L.plantarum strains NC8-p SIP-409-pgsA’-RON2-Dcpep and NC8-p SIP-409-pgsA’-RON2.The successful expression of RON2-Dcpep and RON2proteins was confirmed by Western-blotting.Animal experiments were conducted using healthy 1-day-old chicks,Experimental groups:Empty control group(PBS),PBS-Challenge group(Challenge),NC8-p SIP-409-pgsA’-RON2 group(RON2),NC8-p SIP-409-pgsA’-RON2-Dcpep group(RON2-Dcpep),empty vector NC8-p SIP-409-pgsA’group(Vector),and commercialized live anticoccidial vaccine group(Vaccine),20 animals per group.Immunization procedure:divided into two immunizations,and all of them by oral immunization,first immunization period:3,4,5 days of age;second immunization period;17,18,19 days of age;Coccidial challenge:30 days of age.The spleen cell proliferation was determined by the CCK-8 assay,The levels of IL-2,IgG,IFN-γin serum,and secretory IgA levels in the intestine were detected by ELISA before and after coccidial challenge(at 29 and 38 days of age);The levels of CD3~+CD4~+and CD3~+CD8~+T lymphocytes in the spleen after immunization were detected by flow cytometry.The weight of the experimental animals was recorded daily after coccidial challenge,and the weight gain rate,anti-coccidial index(ACI),and intestinal pathological changes were calculated using the H&E.The results of ELISA analysis of cytokine levels in serum showed that both IFN-γand IL-2 levels in the RON2-Dcpep group were significantly higher than those in the Challenge group before and after infection(P<0.001).Furthermore,after infection,the RON2-Dcpep group exhibited significantly higher levels of IFN-γthan the Vaccine group(P<0.001).In addition,the RON2-Dcpep group had significantly higher levels of IgG and SIgA antibodies before and after infection compared to the Challenge group(P<0.001),and the SIgA antibody levels in the RON2-Dcpep group were significantly higher than those in the Vaccine group after infection(P<0.001).The spleen lymphocytes exhibited significantly higher specific proliferation in the RON2-Dcpep and RON2 groups than in the Challenge group(P<0.001)after RON2protein stimulation.Flow cytometry analysis showed that the levels of CD4~+CD8~+T lymphocytes increased after two immunizations with recombinant lactobacilli,and the RON2-Dcpep group had significantly higher levels of CD4~+CD8~+T lymphocytes than the Challenge group(P<0.001).Analysis of experimental data after infection revealed that the relative weight gain and weight gain rate of chickens in the immunized lactobacilli group were higher than those in the Challenge group(P<0.001),with a relative weight gain rate of 93.89%in the RON2-Dcpep group,93.49%in the RON2group,and 84.14%in the Vaccine group.The oocyst output in the RON2-Dcpep,RON2,and Vaccine groups were significantly reduced,with oocyst shedding reduction rates of 60.61%,54.55%,and 42.42%,respectively.Pathological tissue sections of chickens after immunization with recombinant L.plantarum showed reduced cecal lesions,a significant decrease in oocyst shedding in the intestinal tissues,reduced damage to the intestinal villi,and a significant reduction in the numbers of red blood cells and inflammatory cells in the intestinal tissues.The ACI indices of the RON2-Dcpep,RON2,and Vaccine groups were 170.89,170.49,and150.14,respectively,indicating that the recombinant L.plantarum carrying RON2protein had good immune protection effects when taken orally.These results indicate that this study successfully anchored the target protein RON2 to the surface of NC8 plant lactobacilli using the pgsA’-anchored expression vector and constructed recombinant L.plantarum capable of correctly expressing the RON2 protein.Furthermore,immunization with this recombinant L.plantarum improved the immune protection of chickens against E.tenella,stimulating the enhancement of SIgA and IgG antibody levels in the body,and increasing the levels of IFN-γand IL-2.The recombinant L.plantarum also exhibited good immune protection effects against E.tenella infection,providing a new strategy for the prevention and control of chicken coccidiosis. |
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