微小牛蜱中肠内容物菌群结构分析

Analysis of microflora in midgut contents from Boophilus microplus ticks

目的分析不同阶段的雌、雄微小牛蜱中肠内容物的细菌种群结构,为蜱及蜱传病防治提供科学依据。方法无菌收集微小牛蜱中肠内容物,以试剂盒提取细菌总DNA,通过通用引物PCR扩增16SrDNA V3区并进行DGGE电泳,选取9条优势条带进行回收克隆测序。结果微小牛蜱中肠优势菌9种,包括拉乌尔特立克次氏体,克雷伯菌,假单胞菌,生癌肠杆菌,阪崎肠杆菌,斯洛伐克立克次氏体,欧文氏菌,泛生菌,柯克斯氏体,其中,克雷伯菌、斯洛伐克立克次氏体、柯克斯氏体见于不同阶段的雌、雄微小牛蜱肠道;欧文氏菌、生癌肠杆菌仅见于雄成蜱,而泛生菌仅见于雌成蜱;阪崎肠杆菌只见于饱成蜱;雌蜱产卵后,肠道泛生菌、拉乌尔特立克次氏体优势消失;产卵5d后,蜱肠道菌群结构基本稳定。结论不同阶段的雌、雄微小牛蜱中肠内容物的细菌种群结构存在一定程度的差别。

Objective To analyze microflora in midgut contents from Boophilus microplus ticks of different sexes and stages in order to provide a scientific basis for prevention of ticks and tick borne diseases. Method Midgut contents were collected and a kit was used to extract total DNA under sterile conditions. The sequence of the 16S rRNA V3 region was amplified with universal primers and analyzed using DGGE. Nine predominant bands were obtained, and the 16S rRNA gene was cloned and sequenced. Results Nine strains of bacteria were identified, including Rickettsia raoultii, Klebsiella spp, Pseudornonas spp, Enterobacter cancerogenus, Cronobacter spp, Rickettsia slovaca, Erwinia chrysanthemi, Pantoea spp, and Coxiella spp, Klebsiella spp, Rickettsia slovaca, and Coxiella spp. were the predominant bacteria in B. rnicroplus of both sexes and different stages. Erwinia spp and E. cancerogenus were only present in male ticks and Pantoea spp was only present in females. Cronobacter spp were isolated from fed ticks. Pantoea spp. and Rickettsia raoultii were no longer the predominant bacteria once females had laid eggs. The composition of intestinal flora was essentially consistent 5 days after eggs were laid. Conclusion There were some differences in microflora in midgut contents from B. microplus ticks of different sexes and stages.