Evaluation Of The Immune Protective Effects Of REmMIC2 And REmMIC3 From Eimeria Magna In Rabbits

Eimeria magna was widely distributed in our country.Animal infection mainly causes symptoms such as loss of appetite and diarrhea.Drug control of rabbit coccidiosis is the primary method for clinical use.However,the long-term irrational use of drugs has led to a sharp increase in the number of drug-resistant strains and drug-resistant residues.There are precedents for vaccines to prevent and control coccidiosis,and the effect on chicken coccidiosis is good.In this study,two genes(EmMIC2 and EmMIC3)were screened from the transcriptome data of Eimeria magna and expressed in prokaryotes.Recombinant microneme proteins were evaluated by immune protection for developing rabbit coccidia genetic engineering vaccines.The screening was performed on Eimeria magna transcriptome data to obtain EmMIC2and EmMIC3 genes.After the TA cloning,real-time fluorescent quantitative PCR was used to analyze the transcription levels of EmMIC2 and EmMIC3 genes in different stages of Eimeria magna(unsporulated oocysts,sporulated oocysts,merozoites,and gametophytes).Both genes were found to have higher relative transcript levels in sporulated oocysts.The pET-32a(+)-EmMIC2 and pET-32a(+)-EmMIC3 expression vectors were constructed after induction for 8 hours at 18°C and 0.5M concentration of IPTG,it was found that rEmMIC2and rEmMIC3 were expressed in inclusion urea and supernatant,respectively.Both rEmMIC2 and rEmMIC3 could be recognized by the serum of rabbits infected with Eimeria magna in the western blot,and the two recombinant proteins had good immunoreactivity.Forty-eight New Zealand young rabbits(861.11±96.45 g)were divided into six groups(unimmunized and unchallenged group,unimmunized and challenged group,saponin adjuvant group,pET-32a(+)vector group,rEmMIC2 immunization group,and rEmMIC3immunization group),eight rats in each group.The first immunization was carried out at 35days,and the second immunization was carried out two weeks later.The unimmunized and unchallenged group was excluded at the age of 63 days,and the remaining groups were orally inoculated with 1×10~5 sporulated oocysts of Eimeria magna.The animal experiment was completed after the coccidia challenge,and the protection of rEmMIC2 and rEmMIC3was jointly evaluated according to the animal’s clinical symptoms,intestinal lesions,relative weight gain ratio,oocyst decrease rate,anticoccidial index,feed conversion ratio,specific Ig G antibody and cytokine levels.The control groups showed anorexia and soft poop,whereas the experimental group showed few anorexia symptoms.Significantly different from the control group,the relative weight gain ratio of the immunized rEmMIC2 and rEmMIC3 groups were 78.37%and 75.29%,respectively,and the oocyst reduction was77.95%and 76.09%,respectively,and the anticoccidial index were 171.12 and 169.29,respectively.Ig G antibodies,IFN-γ,IL-4,IL-10,and IL-17 levels were significantly increased in the experimental group.The results showed that rEmMIC2 and rEmMIC3 have the potential as vaccine-candidate antigens.