云南临沧地区蜱种分子生物学鉴定及埃立克体基因序列分析

Molecular identification of tick species and sequence analysis of Ehrlichia in Lincang area of Yunnan Province,China

目的了解云南临沧地区蜱虫种类及其自然感染埃立克体的情况。方法采集耕牛体表寄生的蜱虫,经形态学鉴定和分组后,从蜱虫中提取DNA,应用PCR扩增蜱虫COI基因以及埃立克体16S rRNA和groEL基因,并测序分析。结果共采集到蜱虫42只,其中微小牛蜱38只占90.48%、血蜱4只占9.52%。将每种蜱的2只为1组,在21组样本中,检出COI片断21份(检出率100%);在相同的4组样本中均检出埃立克体16S rRNA片断和groEL片断4份(检出率19.05%)。基因序列分析,检出的COI片断序列中有2份与澳大利亚pava血蜱(JX573136)的同源性最高(89.3%),其余19份序列与马来西亚微小牛蜱B3的同源性最高(99.5%);检出的4份16S rRNA片断序列完全一致,与美国查菲埃立克体Arkansas和埃文埃立克体Aa2FT349及中国北京查菲埃立克体BY-YQ-HME-O18株的同源性均为100%;检出的4份groEL片断序列也完全一致,与日本埃立克体Yonaguni206株的同源性最高均为93.9%。结论经分子检测证实云南临沧地区耕牛体表微小牛蜱中存在一种类似日本Yonaguni206株的埃立克体感染,耕牛体表还可能寄生一种新的血蜱。

To investigate tick species and Ehrlichia infection among ticks in Lincang area of Yunnan province, 42 ticks were captured from body surface of farm cattle and divided into 21 groups by morphological identification. Partial COl segments of ticks and 16S rRNA and groEL segments of Ehrlichia were amplified separately by polymerase chain reaction （PCR）,and the homology were sequenced and analyzed with other known sequences. Among them, 90.48% （38/42） ticks were identified as Boophilus microplus and 9.52~ （4/42） ticks as Haernaphysalis by morphological identification. The 21 out of 21 groups sam- ples were positive for ticks CO1 fragments （detection rate, 100%）, 4 for Ehrlichia 16S rRNA fragments （detection rate, 19.05%） and 4 for Ehrlichia groEL segments （detection rate,19.05%）. Sequence analysis showed that 2 fragments of 21COI sequences and Haernaphysalis pava （JX573136） from Australia showed higher nucleotide homology,all reaching up to 89.3%. The 19 fragments of 21COI sequences and Boophilus m icroplus B3 from Malaysia showed higher nucleotide homology,all reaching up to 99.5%. Four Ehrlichia 16S rRNA fragments and E. chaffeensis Arkansas strains from USA,and E. chaffeensis BY-YQ HME-O18 stains from Beijing of China,and E. ewingii Aa2FT349 strains from USA showed higher nucleotide homology,all reaching up to 100~. Four Ehrlichia groEL fragments and E. ssp. Yonaguni206 stains from Japan showed higher nucleotide homology,all reaching up to 93.9%. It＇s confirmed by molecular identification that the epidemic Ehrlichia infected Boophilus microplus from body surface of farm cattle in Lincang area of Yunnan Province were similar to Japan Yonaguni 206 strains,in addition,a new local Haemaphysalis may exist in this region.