Preparation Of UV-attenuated Chicken Eimeria Tenella Vaccine And Evaluation Of Its Immune Effect

Chicken coccidiosis is a protozoal disease that do serious harmness to the development of poultry industry and reduces economic benefits worldwide.It can lead to reduced feed intake,depression,morbidity and even death to chickens.In order to reduce the occurrence of chicken coccidiosis,the attenuated vaccine has become the most wide usage to control coccidiosis.In this experiment,the physical and chemical weakening method was used to attenuate E.tenella by ultraviolet(Ultraviolet,UV)irradiation,and evaluate its effect.The test results are as follows:1.Isolation and identification of chicken coccidia oocysts.E.tenella stored in the laboratory was rejuvenated and proliferated,and high-purity oocysts of E.tenella was obtained by isolation and purification technique,the species was identified by morphological and molecular biology measure.The result showed that the amplified ITS-1 gene size was about 551 bp,which was consistent with the expected one.Through homology analysis and alignment,the homology arrived at 99.47 % compared with the reported one from Gen Bank E.tenella(GQ856310.1).2.To carry out the UV-irradiated chicken coccidiocyst oocyst test.The relative sporulation rate of irradiated oocysts was observed by irradiating high-purity E.tenella at UV wavelength of 365 nm at different times.The results showed that short-term UV radiation had no obvious effect on the viability of oocysts,but with the prolongation of irradiation time,the relative sporulation rate of oocysts significantly decreased,and the time should be controlled within 30-40 min.Secondly,2.5 % K2Cr2O7,PBS buffer solution and 50 % glycerol physiological saline were selected as the protective solution of E.tenella respectively,and placed under UV radiation.Among them,the PBS protective solution had the best effect on the development of coccidial oocysts,and the relative sporulation rate could reach 70.03 %.Combined with the previous results,E.tenella was placed in the optimal irradiation time and optimal solution,and subjected to UV irradiation with different intensities.It was found that with the increase of irradiation intensity,the relative sporulation rate of oocysts decreased,and the irradiation intensity was at 1174 uw/cm2.After the development of oocysts is seriously affected,and the radiation intensity should be controlled at 562 uw/cm2 ~ 785 uw/cm2.3.To carry out the pathogenicity study of E.tenella after UV weakened.After UVweakened,E.tenella oocysts at the dosage of 1×104 were inoculated into to every chicken,the virulence of attenuated E.tenella oocysts was determined by measuring the chicken survival rate,the relative weight gain rate and average cecal lesion score.The relative weight gain rate decreased and the average lesion score also increased.The result indicated that the stronger UV radiation intensity was the weaker the pathogenicity,and under the radiation intensity was 2140 uw/cm2,there was almost no pathogenicity.After UV irradiated,it showed that the sporocysts were clearly visible,and there were four sporocysts in the oocysts,and each sporocyst contained two sporozoites,and most of the sporozoites were unstained by staining with ethidium bromide,the survival rate of E.tenella oocysts after UV irradiated was higher than that of the group of tempertur treatment.4.Carrying out the immune protection test of irradiation-attenuated chicken coccidial vaccine.The 3-day age chicks were immunized with the oocysts after irradiation for the first time,and the second immunization was performed 7 days later.The challenge experiment was made at the 7th day after the second immunization.The relative weight gain rate and oocyst discharge numbers of the chicks were determined,the anti-coccidial index(ACI)also was calculated;the intestinal damage was analyzed by immunohistochemistry,and the expression level of cytokines was detected to evaluate the immune protection effect after immunization with the vaccine.The results showed that the detection indexes of the immunized group were higher than that of the group of nonimmunized with discharged.Among them,amount of oocysts decreased significantly in the UV intensity of 785 uw/cm2 group compared with the trial groups(P<0.05);the relative expression of each cytokine was significantly increased in the two groups of UV intensity of 562 uw/cm2 and 785 uw/cm2(P<0.05);In ACI index indicated,the attenuated vaccine by UV intensity of 785 uw/cm2 had strong immune protection(173.73),followed by 562 uw/cm2 and 1174 uw/cm2(163.82 and 167.35).In summary,the attenuated UV vaccine has good immune protection,especially the 562 uw/cm2 and 785 uw/cm2 attenuated groups have the best immune effect.