Attenuated Salmonella Typhimurium Expressed Eimeria Tenella EtMIC3 And EtAMA1 As Oral Vaccine In Protecting Chicken Against Coccidiosis

Coccidiosis is a kind of protozoiasis caused by Eimeria species, which was parasite in the enterocyte of chickens and caused tremendous economic losses in industry worldwide.Current methods to control coccidiosis are the anti-coccidial drugs and the virulent or attenuated live vaccines. However, these methods have some disadvantages, so, the coccidiosis has not been controlled well. Then, to find new ways, such as live oral vaccine, to control coccidiosis became one of the most urgent jobs. Live oral vaccine has high immune effective and good security, and it is operated easily. On the other way, live oral vaccine does not need an adjuvant when immune, and it is easy to construct a multivalence vaccine with a low cost. Attenuated Salmonella typhimurium expressed exogenous antigens has been proved to have good protective efficient.An important part for constructing an attenuated S.typhimurium vaccine is to select an antigen that has high antigennicity. Microneme organelles is located at the apical tip of invading stage of coccidian, proteins secreted by microneme organelles were almost involved in the invading stage. Studies have showed that microneme protein have high antigennicity.So, we select two kind of microneme protein, EtMIC3 and Et AMA1, as antigens to construct attenuated S.typhimurium vaccines.In present study, the two proteins were analysised with softwares, and then part of EtMIC3 and the extracellular region of the EtAMA1 were selected as the antigen to construct the vaccine. Then, total RNA were extracted from merozoite of E.tenella SD-01 strain, and the gene of EtMIC3 and Et AMA1 were obtained using reverse transcriptase. The two genes were cloned into S.typhimurium expression vector pYA3342 to form the recombinant plasmid pYA3342-EtMIC3 and pYA3342-EtAMA1. The two completed plasmids were transformed into X4550, S.typhimurium vaccine strain, respectively. The results of SDS-PAGE and western blot showed that the recombinant proteins could be expressed in vitro. The protectiveefficiency of the two vaccines was detected by animal experiment. 120 chickens were randomly divided into 6 groups at 4d, group I and II were implemented with PBS for control,group III, IV and V were implemented with X4550(pYA3342), X4550(pYA3342-EtMIC3)and X4550(pYA3342-Et AMA1), respectively. Group VI was implemented with both X4550(pYA3342-EtMIC3) and X4550(pYA3342-EtAMA1). All the vaccines were orally immunized with a dose of 109 CFU. At 25 d, chickens in group II, III, IV, V and VI were infected with12000 sporulated E.tenella oocysts.At 35 d, chickens in all groups were killed. The protective efficiency of the vaccinea was evaluated via the body weight gain, the relative growth rate, the oocysts output, the lesion score, the oocysts decrease and the anti-coccidian index. The result showed that when compared with the control group(group I), the growth rate of chickens in group III, IV, V and VI was 80.49%, 75.21% and 87.82%, respectively. And the oocysts decrease was 61.9%,46.03% and 63.49%, respectively. The lesion score of the three vaccine groups was 2.0, 2.3,1.7, respectively. All the data showed that attenuated S.typhimurium vaccines carried E.tenella antigen could provide partial protection against homologous infection. Different antigen could provide different protection, so, select a high antigennicity antigen which would provide more protection needs more studies.