Artificial Infection Experiment And Establishment Of The PCR Method For Detection Of Theileria Sp

Theileriosis is caused by genus Theileria, which is transmitted by ticks and parasitizes in macrophage, lymphblast and erythrocytes of a variety of domestic and wild animals.Ovine Theileriosis is due to Theileria infective to sheep and goats. Now the Theileria species which cause high-lethal Theileriosis of ruminants is described as a new Theileria species in China.The disease is widely distributed in northwest China:Gansu,Inner Mongolia,Xinjiang,Qinghai,Shanxi provine et al.And the disease is transmitted by Haemaphysalis qinghaiensis.The Theileriosis of sheep was finded firstly in China in late 1950's.At that time,Theileriosis of sheep was thought as Theileria ovis.It was described as Theileria hiric.Schnittager et al proved that Theileriosis of sheep was a new species different from Theileria lestoquardi and Theileria annulata by researching the gene sequences of small subunit ribosome.Now Theileriosis of sheep is named as Theileria sp.(China).The disease seriously affects sheep industry and results in many exotic sheep and local lambs to death each year in China. The disservice of ovine Theileriosis is very serious. However,there is little study on the disease. Most research work concentrated in Theileria annulata and Theileria parva.The artificial infection experiment The tested sheep were infected artificially by the Haemaphysalis qinghaiensis ticks collected from Gansu province. Then the changes about the clinical symptom,hematology,pathology and the time of the appearance of schizonts and gametocytes in lymph node and erythrocyte,immunity level and antibody titre,respectively were observed. The result expressed that the temperature of sheep showed two peaks obviously, and was all above40℃.The schizonts was observed about 18-22 day after infecting the tested sheep.The time of observing the gametocytes was the same as the time which the schizonts appeared or after one day.The observation of the hematology indicated the tested sheep showed anaemia.The harm to the sheep in the period of the schizonts was the seriousest.The RBC C3b receptor rosette rate,the RBC immune complet rosette rate and the lymphocyte transformation rate were determined.The results showed that all the data we determined in sheep-infecting were obviously lower than in healthy ones,and there were significant difference.This indicated that Theileria sp. restrained the function of immunity of sheep obviously.The antibody titre was highest when the temperature attained the second peak.Along with the condition of the tested sheep turned for the better,the antibody titre descended gradually.The antibody still exsited in a very long period of time with the disappearance of gametocytes.Study on the experimental animal model of Theileria sp The ovine red blood cell-substituted severe combined immune deficiency(O-RBC-SCID) mice infected with Theileria sp.developed a high level of parasitemia.The size of parasite grown in O-RBC-SCID mice was larger than that in sheep.Furthermore,multiple infections of erythrocytes containing 2,4,8,16 and 32 parasites were observed frequently.On 10-12 days after infection,the percentage of parasitized red blood cells from blood circulation ending was 20% at the miximum.The parasites could be detected for 20 to 28 days from blood circulation ending.This experiment successfully applied to the experimental animal model of Theileria sp.A PCR mothod was developed to detect Theileria sp Based on the sequence data of 18S rRNA gene of Theileria sp,a set of Theileria sp-specific primers were designed.These primers selectively amplified a 398bp DNA fragment of the 18S rRNA gene of Theileria sp.No PCR products were amplified from purified DNA of Babesia sp, Anaplasma ovis, Theileria annulata and Trypanosoma.It was estimated that as few as 0.12fg Theileria sp genomic DNA was detectable by PCR.Using this method ,the 61 blood samples from different area were successfully detected and 39 samples were positive with Theileria sp infection,the others were negative.The results showed that the PCR method developed was an effective tool for diagnosing Theileria sp infection in sheep and goats with highly sensitive and specific.It will be useful tools for detecting the Theileria sp and investigating the epidemiology of Theileria sp.