Molecular And Serological Epidemiology Of Bovine Piroplasm Species From Selected Areas Of China And Pakistan

Piroplasmosis in the large ruminants is considered as the summer nightmare.Theileria and Babesia are haemoprotozoans(Apicomplexa: Piroplasmida),which are the etiological agents of theileriosis and babesiosis,respectively.Ticks(Acari: Ixodidae)have been incriminated as vicious blood feeders and efficient vectors for these mentioned piroplasms.These diseases have been reported to cause annual economic losses of several billion US dollars in terms of production losses and cost of control measures worldwide.Thus,a rapid and specific detection method for piroplasms and the data of prevalence of piroplasms for some areas are needed.In the present study,a recombinase polymerase amplification(RPA)test method for T.annulata was established.A 281 bp fragment of enolase gene of T.annulata was amplified and the test was utilized for 274 field samples while comparing with the conventional PCR.The RPA results were positive for 48 of 274(17.51%)samples.All 274 samples were screened using conventional PCR and 21(7.66%)samples were positive for T.annulata.All the samples that were RPA positive but PCR negative were sequenced,which confirmed the results of RPA.Theileria annulata and T.orientalis are usually co-infected in field samples.A multiplex RPA detection method for simultaneous detection of T.annulata as well as T.orientalis was established.The multiplex RPA specifically amplified 282 bp and 229 bp fragments of the enolase gene from T.annulata and T.orientalis respectively and had no cross-reaction with other piroplasm species.The detection limit of the multiplex RPA was 100 copies of enolase gene plasmid DNA.Aim to evaluate the application of the established multiplex RPA method.188 field samples were detected with multiplex RPA and a published multiplex PCR method.It was found that 45(23.9%)and 5(2.6%)out of 188 blood samples were positive for T.annulata and T.orientalis,respectively through RPA.The multiplex PCR detection showed that 32(17.0%)and 3(1.6%)blood samples were positive for T.annulata and T.orientalis,respectively.In order to study the prevalence of piroplasms in Pakistan,450 cattle severely infested with ticks but asymptomatic,were selected for collection of blood from various regions of Pakistan including Chakwal,Jhang and Faisalabad.A published semi-nested PCR which targets V4 hypervariable region of 18 S rRNA gene,was used for detection the DNA of piroplasms.Depending upon the short amplified sequence,T.annulata,T.orientalis and B.bigemina were found to be prevalent in the study areas.And the positive rate for piroplasms of the selected samples from Pakistan was 128/450(28.44%).Aim to know the genetic relationship of piroplasms between Pakistan and China,120 DNA samples from Inner Mongolia were used to screen the piroplasm positive samples showing 16/120(13.33%)as positive.Large fragment of 18 S rRNA gene was amplified for the confirmation of Theileria species;while rhoptry associated protein(RAP-1c)gene was amplified for confirmation of B.bigemina.Aiming to study the prevalence of the bovine theileriosis from three provinces in China,an ELISA method based on recombinant major piroplasm surface protein(rMPSP)was used for detection of the selected field sera samples.Total 840 field bovine sera samples were collected from 44 prefectures of 3 provinces in China including Tibet,Xinjiang and Inner Mongolia.From the detection results,the overall positive rate was 18.57%.Highest prevalence was found in Xinjiang province 105/389(27%),followed by Inner Mongolia 7/50(14%)and Tibet 44/401(10.94%).The described iELISA might be an appropriate tool to identify the bovine theileriosis,and the results also jot down the important data regarding the current occurrence of theileriosis in the study areas of China.