| Bovine piroplasmosis is a tick-borne protozoan disease caused by Theileria annulata which infect bovine erythrocytes and reticuloendothelial system.It is reported by FAO that the world’s annual economic losses caused by tick-borne piroplasmosis up to 7 billion dollars.Xinjiang,as largest province in China,with all sorts of hard tick distributed.However,there is no effective method for early diagnosis,biological characteristics of dominant media tick and comprehensive prevention and control of ticks and tick borne diseases,which hampered the development of bovine production in Xinjiang.Therefore,it is urgent to study the diagnosis,culture and control methods for the tick-borne protozoan diseases in Xinjiang.Current studies were carried out as followings.(ⅰ)In order to establish a rapid,sensitive and reliable method for quantitative detection of bovine Theileria,two sets of primers and TadMan probes were designed according to Tams1 gene and 18S rRNA gene of bovine Theileria,respectively,and after optimization of the reaction parameters,specific PCR and qPCR were established to detect bovine Theileria with positive DNA and plasmid of bovine Taylor as template.The results showed that the PCR and qPCR could detect at least 1.26×10~2 copies/μL and 1.26×10~1copies/μL bovine Theileria DNA,respectively.The variation coefficients within group and between group were 0.26%,0.26%,0.52%,0.85%;and 0.30%,0.21%,0.56%,0.36%,all less than 0.85%,0.56%,indicating the PCR and qPCR have has good repeatability.The both methods have no cross-reaction with bovine babesiosis,bovine bigemina,and Tyrpanosoma.As compared with OIE reference test,the positive coincidence rate for the PCR was 93.8%.Total of 47 clinical samples were detected by the PCR and qPCR established in this study as well as by the OIE reference test,and positive rate were 29.79%,36.17%and 31,90%,respectively,indicating that the PCR and qPCR have high sensitivity,specificity and and repeatability,which can be used for early diagnosis and epidemiological investigation of clinical bovine Taylor disease.These study provided technical support for the prevention and control of ticks of Theileria annulata.(ⅱ)In order to understand the situation of cattle infected with annular Taylor’s disease and the prevalence regularity of the disease in Xinjiang,blood samples of local cattle(N=3511)and hard ticks(N=130926)were collected from four local areas,and the blood samples were subjected to etiology and serology analysis including in vitro and in vivo culture of Theileria annulata,and the tick samples were subjected to parasite detection,species identification and classification.The results showed that 68,167ticks were identified as Hyalomma tick,including hyalomma anatolicum,hyalomma dromedary,hyalomma dromedarii and hyalomma asiaticum.The positive rate for Theileria annulata were 17.03%,19.08%,25.21%from the total of 3511 blood samples.One Theileria annulata strain named Turpan strain Ta-t was isolated and propagated in Kunming splenectomy mice.The results showed that the merozites of Theileria annulata could be observed in the blood of inoculated splenectomy mice 48 h.p.i,and the parasitemia reach10%~20%5-9 d.p.i.The merozites from mice with 15%parasitemia could be stored at-80℃for future use.Current study provide basic data for understanding of the epidemiology of bovine piroplasmosis in Xinjiang.(ⅲ)In order to understand the biological characteristics,population structure,and the diversity and genetic evolution of the vector ticks transmitted Theileria annulata in Xinjiang,the identified hyalomma ticks(N=68167)were subjected to further analysis.Among the hyalomma ticks,the dominant species is hyalomma anatolicum(40.48%).The carrying rate of Theileria annulata in hyalomma anatolicum,hyalomma dromedary,hyalomma dromedarii and hyalomma asiaticum were 100%,81.94%,70.65%,30.00%,respectively.Gene sequence analysis showed that the above four tick species were clustered into corresponding classes with 97%,100%,98%and 98%,supporting rates,respectively.Base on COI gene sequence analysis,the support rates were 100%,99%,100%and 98%,respectively for above four tick species.(ⅳ)To find out the efficient treatment of the disease,60 cattle which were confirmed to infected with Theileria annulata were divided into 4 groups including Western medicine treatment group 1,Western medicine treatment group 2,Western medicine treatment group 1,at random-Western medicine treatment group,traditional Chinese medicine treatment group and blank control group.On the 1,2,3,4,5 and 7days post treatment,the clinical symptoms,body temperature,blood smear,infection rate and treatment effect of treated animal were recorded individually in the four groups.The results showed that the recovery rate of the infected animals treated with Western medicine group 1 was 86.67%(13/15),that of the animals treated with Western medicine group 2 was 93.33%(14/15),and that of the animal treated with traditional Chinese medicine was 73.33%(11/15).Uranidin is the efficient medicine for initial and intermediate infection of Bovine piroplasmosis.Finally,according to the treatments,the disease prevention and control scheme is proposed.In this study,the specific and sensitive PCR and real-time PCR early for diagnosis of Bovine piroplasmosis were development.With the developed methods,the epidemiology of Bovine piroplasmosis in Xinjiang was investigated.Field strains of Theileria annula was obtained and cultured in mice model.The study also revealed the tick population structure and genetic diversity of the dominant species,explored the treatment options.The works in this study provide the foundation for the prevention and control of Bovine piroplasmosis. |
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