牛的巴贝斯虫18S rRNA基因序列比较研究

Studies on Comparison of 18S rRNA Gene Sequences of Babesia Species Infective to Cattle

对中国已报道的8株牛的巴贝斯虫(包括1株牛巴贝斯虫、1株双芽巴贝斯虫、1株大巴贝斯虫、3株卵形巴贝斯虫和2株巴贝斯虫未定种)的18S rRNA基因序列进行了测定与比较.自感染动物的血液中纯化虫体,提取基因组DNA,PCR扩增靶基因,然后将其连接到pGEM-T Easy载体上,进行克隆测序.研究结果显示:牛的巴贝斯虫18S rRNA基因大小在1 653～1 699 bp之间;用所测得的和自GenBank下载的各种动物的巴贝斯虫18SrRNA基因序列构建了系统发生树,发现由刻点血蜱传播的大巴贝斯虫伊犁株与由长角血蜱传播的3株卵形巴贝斯虫存在明显差别,应属于2个独立种;由小亚璃眼蜱传播的牛巴贝斯虫未定种不同于目前已报道的任何种类,在中国应为一个新种.因而,中国存在5种牛的巴贝斯虫,即:牛巴贝斯虫,双芽巴贝斯虫、大巴贝斯虫,卵形巴贝斯虫和巴贝斯虫未定种.

Ribosomal small-subunit RNA gene-sequence of eight Chinese Babesia stocks infective to cattle, including one Babesia bigemina isolate, one B. boris isolate, three Babesia ovata isolates, one Babesia major isolates and two unnamed Babesia sp. isolates （Babesia U sp. ）, were cloned and analyzed. The target DNA segments were amplified by PCR and the products were ligated into the pGEM-T Easy vector for sequencing. The length of the 18S rRNA gene of all Babesia species involved in this study varied between 1 653-1 699 bp. The phylogenetic trees were constructed based on 18S rRNA sequence of the Chinese isolates as well as other species of Babesia available in GenBank. The results showed that Chinese isolates of Babesia boris and Babesia bigemina were in the same branch with corresponding foreign isolates respectively; B. ovata transmitted by Haemaphysalis longicornis were locateded in the same group with B. ovata Korea, and homology among them exceed 96.5% ; B. major transmitted by H. punctata was situated in another branch, and the homology with other bovine Babesia species is less than 92. 5 %; Two isolates B. U sp. is dispensed on the same branch with B. caballi, and the homology with other bovine Babesia species is less than 91.8%. These results indicated that there are five Babesia species infective for cattle in China, including B. boris, B. bigemina, B. major, B. ovataand Babesia U sp.