An Attenuated Salmonella Typhimurium DNA Vaccine Against Eimeria Stiedai

Rabbit coccidiosis is a kind of sporozoan disease caused by Eimeriidae species, which jeopardizes rabbit breeding industry in our country. Among rabbit coccidian, Eimeria stiedai is the most virulent and hazardous coccidian, which parasitizes in the bile duct epithelial cells of rabbit, causing serious liver coccidiosis. Although chemotherapy is the conventional method to control rabbit coccidiosis, more and more scholars have sought immunological means to control the disease due to the increasing problems such as resistant strains and drug residue. In the study, E. stiedai LZ strain was isolated with coccidia-free rabbits by a single oocyst technique, then microneme protein 5 (Es MIC-5) gene was cloned from sporozoites of the E. stiedai LZ strain. Es MIC-5 was successfully expressed in E. coli by use of prokaryotic expression vector pETMIC-5. The expressed recombinant protein MIC-5 was purified for detection of specific antibody against E. stiedai. Rabbit IL-15 gene was amplified by RT-PCR, and MIC-5 and IL-15 were cloned into two multiple cloning sites (MCS) of eukaryotic expression vector pBudCE4.1 (the positive named as pBMIC-5-IL-15), respectively. The expression of MIC-5 and IL-15 in BHK-21 transfected by the recombinant plasmid pBMIC-5-IL-15 was confirmed by indirect immunofluorescent test. Moreover, asd gene from S. typhimtwium was introduced into DNA vaccine vector pBMIC-5-IL-15 and the Zeocin resistance gene was then fragmented and a new DNA vaccine vector pBMIC-5-IL-15-asd without an antibiotic resistance gene was constructed according to the principle of the balance-lethal system. Then pBMIC-5-IL-15-asd was transformed into ?asd S. typhimtmium X3730 and X4550 strains one after another. The results of genetic stability test showed DNA vaccine vector pBMIC-5-IL-15-asd stablely existed in the ?asd S. typhimtwium X4550 strain, which puts light on antibiotic resistance and instability of DNA vaccines in hosts. Based on the immunization of 45 coccidia-free rabbits, the results showed that the recombinant S. typhimtwium X4550 strain carrying DNA vaccine vector pBMIC-5-IL-15-asd could efficiently induce specific humoral and cellular immune responses to E. stiedai, supplying immune protection to some extent, which lays a solid foundation for further studies on oral vaccines to prevent liver coccidiosis in rabbits.