褐黄血蜱中肠内容物菌群结构分析

Identification of the microflora in the midgut content from Haemaphysalis flava

为了解褐黄血蜱的带菌情况及其公共卫生意义,本研究在无菌条件下采集褐黄血蜱中肠内容物,提取细菌总DNA为模板,采用通用引物PCR扩增细菌16S r DNA V3区,经DGGE电泳回收DGGE 16个优势条带,并选取其中13个条带进行测序分析,结果显示,选取条带分别与立克次氏体属、柯克斯氏体属、假单胞菌属、葡萄球菌属、大肠杆菌属等高度相似。采用传统细菌学方法,从中肠内容物培养分离到2株细菌,经鉴定其中1株为蜡样芽胞杆菌。对各地区褐黄血蜱半饱血雌成蜱和吸血雄成蜱中肠菌群结构分析结果表明其带菌情况基本相同,但饱血雌成蜱3个地区间具有较大的差异;而饱血雌蜱和其它两种状态下的蜱肠道菌群也有差异。贝纳柯克斯氏体、菊欧文氏菌、肺炎克雷伯菌及软腐果胶杆菌是褐黄血蜱中肠内的优势菌。本研究利用PCR-DGGE技术分析褐黄血蜱中肠内容物,为蜱传播疾病的研究奠定基础。

In order to investigate bacterial community profile, the microflora in the midgut contents were analyzed from Haemaphysalis flaga by using PCR-DGGE （denaturing gradient gel electrophoresis） method. The microbial DNA was directly extracted from the contents of the mid-gut （collected under aseptic conditions） from H.flava. Afterwards, 16S rDNA V_3 region of bacteria from contents of the midgut was amplified by PCR with universal primers using the total microbial DNA as template, and the result of DGGE showed that there were 16 bands, of which 13 randomly selected bands were sequenced and they were highly similar to those from Rickettsia, Coxiella, Pseudomonas, Staphylococcus, Enterobacte, etc. Moreover, two kinds of bacteria were isolated from midgut contents by traditional bacteriological method and one of the bacteria was Bacillus cereus. These results demonstrated that constitution of the microflora was basically similar between partially fed female and feding male H.flava. However, there was obvious difference （constitution of the microflora） between partially fed females and engorgement females. Moreover, C.burnetii, E.chrysanthemi, K.pneumoniae and P.carotovorum and P.carotovorum are the dominant bacteria in the midgut of H.Flava.