Establishment Of Xinjiang Epidemic Strain In Vivo Test Of Theileria Annulata And Indirect Immunofluorescent Antibody Test

Bovine babesiosis is the general name of a kind of tick-borne hemozoon disease that is caused bytheileria and babesia (old for babesiosis). In China (including our region), theileria annulata has the mostserious damage and the highest pathogenicity rate,therefore, OIE has listed it as Class B disease. It mostlyoccurs suddenly and is featured by high fever, anemia, bleeding, emaciation and body swollen lymph node,with a high death rate among acute cases. It is populated in some provinces and municipalities in thewestnorth, north and eastnorth of China. The infected giardias include theileria, babesia and etc. Theileriaannulata has the most severe risk and the highest pathogenicity rate in China. In Xinjiang, the infection ratecan be as high as60%, which has greatly threatened the healthy development of cattle breeding industry.Theileria annulata epidemic strains are inoculated on experimental animals and are carried throughexpanding propagation. Establish acute, sensitive and fast indirect immunofluorescent antibody test on thebasis of developing slide merozoite antigen, and this has important significance on the early diagnose,epidemiologic study and integrated control of this disease.(Ⅰ) During this experimental study, give splenectomized operation to40Kunming whitemice at samelitter, with the weight of20g and male and female in half. Put them in a warm and clean place afteroperation and feed them warm water and dedicated fodder. Take the splenectomized Kunming white miceas the experimental animals and inoculate Xinjiang epidemic strain of Theileria annulata. Conductexpanding propagation experiment of theileria annulata in vivo on the experiment animals. Observe theirpercentage of parasitemia and the expanding propagation condition in the body of Kunming white mice inevery8h,12h,24h,48h and3d~10d. Gather the whole blood and their merozoite with the highestpercentage (over15%) of parasitemia through the way of heart and eyeball blood sampling and thencryopreserve it, and grope its storage and anabiosis method. The experimental results indicate that37micesurvive after operation and their breath, heart beat, appetite and mental state are good, which proves thatthe splenectomized operation is successful. Merozoite can be observed in the mice’s blood on2d and thepercentages of parasitemia reach the higher peak between5d and9d. The average percentage of parasitemiaof every group can reach15%~20%, and the percentages begin to decrease after10d and agamont can notbe observed in blood film, which indicates that theileria annulata can be replaced and proliferated inside thebody of the splenectomized Kunming white mice. The research has found that the expanding propagationrates of male and female had an evident difference (P=0.007＜0.01), while the difference of the percentageof parasitemia of the splenectomized Kunming white mice and the Kunming white mice are evident (P=0.0002＜0.01). The merozoite in the blood of Kunming white mice with a percentage of parasitemiaover15%can be stored using cryoapplication (it can also be made into slide merozoite antigen), and therecovery activity of agamont is best in half a year. This study can lay a foundation of establishing theileriaannulata models and providing rich strains for teaching and research.(Ⅱ) This study takes the blood source sex merozoite of positive theileria annulata case or themerozoite (make slide antigen using test I method and store it in-80°C refrigerator) after expandingpropagation of Kunming white mice as antigen, and gradient dilutes primary antibody and second antibody1:20~1:640,1:20~1:1600in the way of cross interior extrapolation method, assuring the best workingconcentration. Establish fast indirect immunofluorescent antibody test of Theileria annulata. Test itsspecificity, sensitivity and repeatability (observe its cross reaction with bovine babesiosis and babesia) andtest the suspected antibody samples of Theileria annulata by applying the established method. The testresults indicate that the laboratory self-made slide merozoite antigen of Theileria annulata (this antigen hasbeen made by the blood source sex merozoite of positive Theileria annulata case and the merozoite afterexpanding propagation of Kunming white mice, which has been stored in-80℃) still has good antigenicityafter being stored in refrigerator for half a year. When the dilution of primary antibody (the tested serum) is1:80and the dilution of secondary antibody (marked by fluorescence as Rabbit Anti-Bovine IgG) is1:100,it will be the most proper working concentration of indirect immunofluorescent antibody test of Theileriaannulata.Duplicate detection of the internal and external ones and the variable coefficient are both lowerthan10%; The established method has successfully detected the antibody level in the tested serum samples(N=290) of the cow from suspected region. Its seroprevalence of Theileria annulata is72.4%(210/290),which has a higher relevance ratio than dyeing microscopic examination of blood smear (the positive rate is27.6％). This study can provide slide merozoite antigen resources for the diagnosis of Theileria annulata;the established IFAT method, with a relatively high sensitivity, specificity and repeatability, can be used tothe early diagnosis of this disease and lay a foundation for the comprehensive control.