Construction And Application Of Babesia Orientalis CDNA Library

Babesia orientalis is an intraerythrocytic protozoan parasite of the buffalo Bubalus bubalis transmitted by the tick Rhipicephalus haemaphysaloides haemaphysaloides. It is the cause of babesiosis, one of the most important diseases of buffalo in central and southern China and is characterized by apathy, fever, anemia, icterus, haemoglobinuria, and in some cases death. It is a new Babesia species which was isolated and named by our laborary.The purpose of this study was to investigate the molecular taxonomy of B. orientalis. A semi-nested PCR based on the 18S rRNA gene of B. orientalis was also developed and a cDNA library constructed. Based on the eDNA library, the ESTs were sequenced and analysed, and the ORF of the heat shock protein 70 (HSP70) gene of B. orientalis was cloned and expressed in prokaryotic cells.(1) The molecular taxonomy study of B. orientalisThe complete 18S rRNA gene sequence of B. orientalis was determined by PCR using the eukaryotic 18S rRNA universal primer. It was sequenced and BLASTed. The results indicated that the classification of the parasite belonged to the genus Babesia. The , 700 bp complete sequence was compared with those of 15 other Babesia spp. available on GenBank. The data was analyzed and a phylogenetic tree was established. The results indicated that the hereditary distance of the parasite was close to that of a Babesia sp. from South Africa and B. ovis, and far from B. bigemina and B. bovis. This result further proved that B. orientalis was a new species on molecular biology.(2) Development of a semi-nested PCR for the diagnosis of B. orientalisA semi-nested PCR was developed based on the V4 hypervariable region of the B. orientalis 18S rRNA gene and the specific 257 bp fragment was amplified. It was very sensitive and capable of detecting a parasitemia of approximately 0.00000012%. Blood samples collected from 121 asymptomatic buffaloes collected in 4 Babesia-epidemic counties and from 71 asymptomatic buffaloes in 3 Babesia-free counties in Hubei province, China, were examined for the presence of B. orientalis using both Wright-Giemsa stained blood smears and the semi-nested PCR. Microscopic examination revealed 5/121 animals were positive, compared to 24/121 for semi-nested PCR. All the samples from the 3 Babesia free counties were negative by semi-nested PCR, but 17/71 were positive microscopically for intraerythrocytic piroplasms. To identify the cause, the 18S rRNA gene of organisms in the 17 samples was amplified and BLASTed and the results indicated that a Theileria sp. was involved. A totle of 378 ticks (R.haemaphysaloides) collected from buffaloes of Babesia-epidemic counties were also examined by the semi-nested PCR and 35/378 were positive. The results showed that the semi-nested PCR was a useful method to investigate the epidemiology and enzootic potential of this parasite, and the buffalo babesiosis (B. orientalis) presented widely in the middle of China.(3) Construction of the B. orientalis cDNA libraryThe total RNA was obtained from infected blood using E. Z. N. A^TM Blood RNA Kit. The first-strand synthesis and cDNA amplification was done using LD-PCR and ligation of cDNA toλTriplEx2 vector was completed by SMARTTM cDNA Library Construction Kit. Then it was packaged using the Gigapack(?)â…¢Gold Packaging Kit. The cDNA library of B. orientalis was then constructed. The titers of the primary library and the mpified library were 2.0×10⁶ pfu/mL and 5.8×10⁸ pfu/mL respectively. The insert fragment length of the library ranged from 0.5 to 3.0 kb, and the recombination efficiency ccounted for 98.8%. This confirmed that the cDNA library was a successful one. The library was then screened by rabbit anti-B. orientalis serum and three strongly positive clones isolated. BLAST results showed that they were the heat shock protein 70 (HSP70) gene, nuclear movement protein gene and a hypothetical protein gene of B. orientalis.(4) Sequencing and analysis of the ESTs of B. orientalisThe positive clones were picked randomly from the library and those clones in which the insert fragment was over 300 bp as identified by PCR were sequenced. In total, 324 ESTs were sequenced and 305 effective ESTs were obtained. The effective ESTs were used to build 46 unigenes with a high level of similarity using phrap software. The results showed that they maybe the partial or complete genes of like-variant erythrocyte surface antigen protein, like-merozoite antigen protein, protein kinases/phosphates, ribosomal protein, heat shock protein 70, like-heat shock protein 90, actin protein, zinc finger protein or hypothetical protein of B. orientalis.(5) The clone and prokaryotic expression of the HSP70 gene of B. orientalisThe entired ORF of HSP70 gene of B. orientalis was amplified using the HSP70 complete sequence and cloned into a pGEX-KG vector, the recombinant plasmid pGEX-KG-HSP70 was then constructed. The plasmid was introduced into E. coli- BL₂1－CodonPlus. After inducement by IPTG, high expression was obtained. The expressed product of HSP70 could specifically bind to the antibody against B. orientalis by Western-blot.This study confirmed that B. orientalis was a new species based on molecular biological grounds. The results further showed that the semi-nested PCR was a valuable tool in diagnostic and epidemiological investigations of the disease caused by this parasite. It also showed the infection to be common in central China. During this study, a cDNA library was also constructed, the ESTs sequenced and the HSP70 gene of B. orientalis cloned and expressed. The findings will form the basis for further studies of the molecular biology and also of immunogenic and other genes of the parasite as well as of the immunity, pathogenesis, prevention and control of buffalo babesiosis.