Analysis Of AMA1 And RON2 Genes And Developmemt Of Detection Techniques Of Chinese Ovine Babesia Species

Babesiosis is a zoonotic disease caused by Babesia spp.,which is mainly transmitted by hard ticks.The disease is clinically characterized by fever,anemia,jaundice and hemoglobinuria,even leads organ failure and death in acutely infected animals.The disease is epidemic,causing huge economic losses to the breeding industry in China.In present study,the CDS regions of movement junctions(MJs)of AMA1 and RON2 were obtained using PCR amplification from 6 strains of 2 ovine Babesia species,which are widely distributed in China.Then the structure characteristics of the genes were analyzed and the taxonomic status of the Babesia strains was studied using these genes.Finally,we developed a molecular and serological techniques for detecting the Babesia spp.using AMA1 as target gene and antigen,respectively.This study provides the detection techniques for performing epidemiological investigation of the disease,as well essential data for studing the classification relationship of Babesia spp.and the role of MJs during the process of Babesia invading host cells.The main research contents and results are as follows:1.The full-length sequences of RON2 and AMA1 genes were successfully amplified from cDNAs and genomic DNAs of the 6 Babesia strains infective to small ruminants.Bioinformatics analysis showed that there were no introns both in RON2 and AMA1 genes of the ovine Babesia.The protein encoded by RON2 gene contained a signal peptide and three trans-membrane domains.Like the other protozoa,AMA1 was a typical type I trans-membrane protein,composing of three regions(extracellular region,trans-membrane region and cytoplasmic region).The results of nucleotide and amino acid sequence alignment of the two genes and the MP phylogenetic tree construction indicated that there were two Babesia species infective to small ruminant in China,Babesia sp.and B.motasi.B.motasi strains were further divided into two branches,BmLT/BmTZ and BmNX/BmHB.2.The universal and species-specific primers were designed targeting on the conserved and interspecies hyper-variable regions of the ovine Babesia AMA1 genes,respectively.A nested-multiplex PCR assay was developed for detecting the 2 ovine Babesia species on the basis of optimizing reaction conditions.Two target fragments with different sizes were amplified from the two Babesia species by two reaction processes in a single step and the detection limitation reached to 5pg/μL.There was no cross-reactivity with the genomic DNAs of T.uilenbergi,T.luwenshuni,T.annulata,T.sinensis,A.ovis,A.marginale,B.bigemina,B.bovis and B.major.The 232 field-collected sheep samples from Wuwei city of Gansu province were tested using the nested-multiplex PCR method.The results showed that the positive rates of Babesia sp.and B.motasi was 1.72% and 6.03%,respectively.3.The truncated AMA1 gene was successfully obtained from Babesia sp.and B.motasi cDNA,and the prokaryotic expression vectors pET30a-XJ-AMA1 and pET30a-LT-AMA1 were constructed.The recombinant proteins rBspAMA1 and rBmAMA1 were expressed,identified,purified.And then their reactivity was analysed.The results of ELISA showed that both rBspAMA1 and rBmAMA1 had good reactivity with positive sera from the sheep infected by the 6 ovine Babesia strains,while there were no reactivities with positive sera of T.uilenbergi,T.luwenshuni and A.ovis.Therefore,a universal ELISA assay was developed using rBspAMA1 as antigen for detecting the two ovine Babesia species infections.The antibody kinetics against the Babesia was evaluated using the sera collected from experimentally infected sheep by B.motasi and Babesia sp.using the ELISA assay.The results demonstrated that the antibodies against AMA1 of the 2 Babesia species could be detected in 7th days post infection.The Babesia sp.antibody level reached the peak in 28-42 days post infection and gradually descended to the level of pre-infection after 170 days.The B.motasi antibody level reached the peak in 12-28 days post infection and gradually descended to the level of pre-infection after 230 days.Field-collected sera from 554 sheep in Gulang,Tianzhu,Liangzhou and Jingtai counties of Gansu Province were tested using the ELISA.The result showed that the average positive rate was 51.98%.