Development Of The PCR For Detection Of Piroplasms Infective To Cattle And Sheep In Haemaphysalis Qinghaiensis And Its Application

Haemaphysalis qinghaiensis is one species of hard ticks distributed only in China.Previous studies suggested that it was the vector of four species of piroplasms, which includes Theileria uilenbergi,T.luwenshuni,T.sinensis and Babesia motasi. These pathogens seriously affected the animal industry.The classical method for detection of piroplasms in ticks is based on microscopic examination,which is time consuming and not specific,and cannot differentiate the species of piroplasms in ticks. In this study,one pair of universal primer,which could amplify all of the four species of piroplsama in H.qinghaiensis,and four pairs of primers,which were specific to the four species of piroplsama respectively,were designed based on the sequences 18S ribosomal RNA gene of the pathogens,as it was demonstrated that the gene had some conservative and variable regions.A nested PCR tool for detection of these four pathogens in ticks was developed.It was demonstrated that the sensitivities of the method to the genomic DNA of T.uillenbergi,T.luwenshuni,T.sinensis and B.motasi were 0.1pg,1ng,10pg and 100pg respectively.Using the tool,136 H.qinghaiensis tick samples(55 females and 81 males)from Lintan County,Gannan Tibetan Rigeion, Gansu Provine,were examined.The results showed that 62 out of 136 samples were positive for piroplasms,and the infection rate was 45.59%.Twelve of them were infected by more than one pathogen and covered 19.35%.Of the 55 females,27 were positive and infection rate was 49.1%.Of the 81 males,35 were positive and infection rate was 43.2%.It was found that 62 samples were infected with T.uilenbergi and infection rate was 45.59%,including 27 females and 35 male.Nine of the 136 samples were infected with T.luwenshuni and infection rate was 6.62%.Five of the 9 ticks were females and covered 55.6%,and 4 were males and covered 44.4%.Two of the 136 samples were infected by T.sinensis and infection rate was 1.47%.Both of the ticks were females.Only one tick of these 136 ticks found to be infected by B. motasi and infection rate was 0.74%.From above results,it could be concluded that the dominant tick borne pathogen in H.qinghaiensis was T.uilenbergi,and the next one was T.luwenshuni,the infection rate of females was slightly higher than that of males.In addition,the examination also showed that the T.luwenshuni and T. uilenbergi occupied the majority of the mixture infection of four piroplasma species, and it implies that the animals in these region were maily suffering from T. luwenshuni and T.uilenbergi,which the result coincided with the former epidemicological survey completely.