我国部分地区蜱传斑点热分子流行病学调查研究

Molecular epidemiological investigation of tick - borne spotted fever in some parts of China

目的为进一步了解我国斑点热群立克次体存在的多样性,发现可能存在的斑点热群立克次体新成员、潜在媒介和动物宿主。方法建立了扩增斑点热群立克次体190kDa rOmpA基因片段的PCR检测、鉴定方法,并用此方法检测了采自福建省和内蒙古自治区的蜱、动物和人血液标本。对扩增于越原血蜱、森林革蜱和FNH97未鉴定菌株的PCR产物采用PHYLIP软件包进行了序列分析。同时,为了寻找特异的斑点热群立克次体分类检测方法,建立了针对四种斑点热群立克次体的半巢式PCR方法,并对斑点热立克次体阳性标本进行了分类检测。结果采用 190kDa rOmpA.701/70p引物可以从7株斑点热群立克次体中的6株扩增出外膜蛋白A基因片段(小蛛立克次体除外)。并从采自福建省和内蒙古自治区的多种蜱及野生动物、人血液标本中扩增出了斑点热立克次体DNA片段,其中越原血蜱、卵形硬蜱、中华硬蜱、豪猪血蜱、森林革蜱、野鼠血块和人群血块的阳性率分别为15.69％、56.94％、8.70％、7.70％、43.56％、82.51％和0.98％。对来自越原血蜱和森林革蜱以及FNH97菌株的斑点热立克次体190kDa外膜蛋白A630bp左右核苷酸片段序列分析和推测的氨基酸序列分析结果表明:福建越原血蜱立克次体(福建立克次体)核苷酸序列与日本立克次体的该序列同源性最高(94％)。推测氨基酸序列同源性也与该立克次体最高(89％);内蒙古森林革蜱立克次体(森林革蜱立克次体)核苷酸序列与扇头蜱立克次的该序列同源性最高(97％),推测氨基酸序列的同源性也最高(95％)。遗传发育分析,这两种立克次体分别与日本立克次体和扇头蜱立克次体均为同一个分支。序列中限制性核酸内切酶的位点也显示了对应的相似性。但是它们的核苷酸和推测的氨基酸序列与康氏立克次体和西伯利亚立克次体以及内蒙古立克次体(HA-91)差别较大。提示,这两种蜱携带的立克次体可能是我国尚未发现的斑点热群立克欢体新成员。用初步分类引物对蜱标本、血液标本检测结果显示:以“福建立克次体”序列设计的引物检测越原血蜱阳性率为8.49％,卵形硬蜱阳性率为20.83％,中华硬蜱阳性率为4.35％,豪猪血蜱为阴性;以西伯利亚立克次体序列设计的引物检测卵形硬蜱阳性率为 24.39％,越原血蜱阳性率为 5.56％,中华硬蜱和豪猪血蜱均为阴性。以内蒙革蜱立克次体序列设计的引物扩增森林革蜱阳性率为43.56％。结论通过本次研究,在以下方面获得了新的认识:越原血蜱、卵形硬蜱、豪猪血蜱和中华硬蜱是福建南方蜱传斑点热立克次体的媒介或潜在媒介,其中卵形硬蜱、豪猪血蜱和中华硬蜱为我国首次证实的携带斑点热立克次体;福建的越原血蜱和内蒙古的森林革蜱分别携带一种未知斑点热群立克次体,并分别与日本立克次体和肩头蜱立克次体近缘;取自斑点热立克次体rOmpA基因的引物用于PCR,作为一种快速简便手段可直接用于斑点热立克次体初步分类和分子流行病学调查;我国可能存在斑点热群立克次体的多个成员及其自然疫源地。

To leam the distribution and variation of spotted fever group rickettsiae in China and find new members of rickettsiae, potential tick vectors and animal reservoirs, and to establish a method of the detection and identification of SFGR, Investigations on SFGR were carried out in some parts of China. Methods We developed a PCR method with a pair of primer from 190kDa rOmpA gene, which is specific for SFGR. Using the method, we detected the ticks, rodents and human blood samples in Fujian province and Inner Mongolia of China. The sequences of two PCR amplicons from H. yeni, D. silvarum and an unidentification strain(FNH97) of spotted fever group rickettsiae were analysed. The phyloge-netic relationships among SGFR rickettsiae were inferred from the comparison of both the nucleotide and derived anoint sequences, using neighbor - joining method of PHYLIP. To discover an effective and specific method to identify spotted fever group rickettsiae strain, four semi - nest PCR methods were tested. With the methods we detected the positive PCR amplicons of ticks, animals and human blood samples, respectively. Results We found that six of seven spotted fever group rickettsiae strains could be amplified with a 629bp ?632bp PCR. However, no evidence of earring spotted fever group rickettsiae rOmpA DNA has been shown in the tests from the O. tsutsug-umushi, the C. burnetii. and the rickettsiae akari. The rate of rodents was 82.51% . The infection rate of human was 0. 98%. The ticks positive rate was 15. 69%, 56. 94%, 8. 70%. 7.70% and 43.56%, respectively. Sequence analyses showed that three sequences were different from each other. Autoploidy of nucleotide and amino acid between ' rickettsia Fujian' and rickettsia japonica was the most significant. However, Rickettsia D. silvarum was closed to rickettsia rhipi-cephali. The FHN97 strain was identical with rickettsia sibiri-ca . A part of nucleotide in l90kDa rOmpA gene (217 - 701 site) were detected by a pair of primer Pf1/701 from 'rickettsia fujian'. The positive rate of H. yeni, I. ovatus and I. sinensis was 8.49%, 20.83% and 4.35%. H. hystricis was negative. The positive rate of I. ovatus and Ha. yeni with a pair of prime Ps1/701 ( from rickettsia sibrica ) was 24. 39 % and 5.56%. I. sinensis, H. hystricis were negative. The positive rate of D . silvarum with a pair of primer Pn1/701 (from rickettsia D . silvarum) was 43. 56% . Conclusions Our findings suggest that H. yeni, I. sinensis, H. hystrcis are the vectors and potential vectors transmitting SFGR in Fujian province of south China, and D. silverum is the vector of rickettsia D. silvarum in Inner Mongolia of north China. 'Rickettsia fujian' and rickettsia D. silvarum are unknown members of spotted fever gruop rickettsiae in China. The nest - PCR developed in the tests can be used for detection and identification of SFGR. The results of serological test showed that there were rickettsia conorii, rickettsiae sibirica and rickettsia akari in Fujian province of south China.