Analysis Of Bov57 Gene And Development Of High-resolution Melting Detection Method Of Ovine Babesia Species

Babesiosis,also known as tick fever,redwater,is a blood protozoal disease which transmitted by vector ticks.The main clinical symptoms of human and animal infection are high fever,jaundice,hemolytic anemia,hemoglobinuria,and even death in severe infection.The disease is widely distributed in China.It has caused huge economic losses to the development of animal husbandry in China and seriously restricted the development of breeding industry.The main results are as follows:1.Aim to investigate the prevalence of Ovine babesiosis in China,the genome DNAs of 302 sheep blood samples collected from 7 provinces in China were extracted,amplified by nested PCR,and sequenced.The results showed that only 2 of the 302 samples were positive for Babesia motasi from Guangdong province,with an average positive rate of 0.66%.Two positive samples for Babesia sp.were detected,including one from Chongqing and one from Hainan.The positive rates were 1.82%and 3.33%,respectively,and the average positive rate was 0.66%.2.The full-length bov57 gene were amplified from six Chinese ovine Babesia strains,and their gene structural feature and evolutionary relationship were analyzed.The results showed that there were no introns in bov57 genes of six Chinese ovine Babesia.The open reading frame of bov57 gene from Babesia sp.Dunhuang/Xinjiang(BspDH/XJ)and B.motasi Lintan/Tianzhu(BmLT/TZ)is 1608 bp and encoding 536 amino acids,However,the ORF of bov57 from B.motasi Hebei/Ningxian(BmHB/NX)is 1605 bp and encoding 535 amino acids,Sequences alignment has shown that nucleotide sequence similarities of B.motasi and Babesia sp.strains were ranged from 58.84%-61.71%,while those of the amino acid sequences were 45.93%-50.93%.The percentage identity of nucleotide sequence among B.motasi BmLT/TZ and BmHB/NX were 85.29%-85.60%and those of amino acids were 75.19%-75.74%.The results of phylogenetic analysis showed that the six chinese ovine Babesia strains were divided into two species and it’s possible that there were two sub-species in B.motasi.3.A high-resolution melting detection method based on bov57 gene,was established to distinguish Babesia sp.,B.motasi Lintan/Tianzhu and B.motasi Hebei/Ningxian.The minimum detection limit of this method was 2.06 copies/μL for Babesia sp.Xinjiang,4.93copies/μL for B motasi Hebei and 3.11 copies/μL for motasi Lintan in the standard curve.No cross reaction was found with T.luwenshuni by specific detection.