阿拉山口口岸常见媒介生物本底调查及携带病原谱检测研究

摘要

目的：对阿拉山口口岸地区常见的媒介生物鼠、蚤、蠓、蚊和蜱进行本底调查和虫媒病监测，建立口岸地区媒介生物“DNA条形码”物种鉴定平台，解析阿拉山口口岸地区鼠、蚤、蠓、蚊和蜱携带传染病病原谱，为阐述重要疾病在人-宿主-媒介中的传播机制和重要媒介源传染病跨种/跨境传播规律奠定基础，同时为口岸地区虫媒传播动物疫病的风险评估提供基础资料。

方法：（1）采用鼠笼法、鼠夹法、网捕法、灯诱法、白布旗法和动物体表采集法等方法采集鼠、蚤、蠓、蚊和蜱。（2）采用形态学、分子生物学方法对采集的媒介生物进行种类鉴定，并进行基因序列系统发育分析。（3）采用血清学和分子生物学技术对非洲猪瘟、蓝舌病、水泡性口炎、莱姆病、无形体和埃立克体等虫媒病进行病原检测，对PCR阳性产物进行测序并登录Genbank数据库Blast 分析，使用Mega 6.0构建分子遗传进化树，进行系统发育分析。

结果：（1）采集鼠类样本4属9种384只，蚤类7种431只，蠓1属1种24017只，蚊4属10种27128只，蜱2科5属8种1336只。（2）分析并获取灰仓鼠和子午沙鼠COI基因序列，大沙鼠、灰仓鼠和西伯利亚五趾跳鼠16S rRNA基因序列，臀突客蚤线粒体COI和COII基因序列，凶小库蚊16S rRNA和ITS基因序列，里海伊蚊16S rRNA、28S rRNA和Cytb基因序列；短垫血蜱的16S rDNA，COI，12SrRNA，ITS基因序列，且分子生物学鉴定结果与形态学鉴定不符。（3）大沙鼠嗜吞噬细胞无形体核酸阳性率为3.39%；42.9%的臀突客蚤中检测到未定型的立克次体核酸；凶小库蚊沃尔巴克氏体核酸检出率为3.33%；在蜱中检测到伯氏疏螺旋体、埃立克体、无形体、斑点热群立克次体、Q热和巴贝斯虫核酸，阳性率分别为45.44%、14.95%、11.26%、12.04%、17.48%和12.82%。

结论：（1）在阿拉山口口岸地区首次采集到迷走按蚊、阿拉斯加脉毛蚊、带棒按蚊、图兰扇头蜱和囊形扇头蜱。大沙鼠、臀突客蚤、原野库蠓、凶小库蚊、亚洲璃眼蜱和边缘革蜱为优势种。（2）鼠、蚤、蚊和蜱进行分子信息研究，其中灰仓鼠和子午沙鼠COI基因序列，大沙鼠、灰仓鼠和西伯利亚五趾跳鼠16S rRNA 基因序列，臀突客蚤线粒体COI和COII基因部分序列，凶小库蚊16S rRNA和ITS基因序列，里海伊蚊16S rRNA、28S rRNA和Cytb基因序列和短垫血蜱的16S rDNA、COI、12SrRNA、ITS基因序列信息在国际上首次进行报道。（3）在阿拉山口口岸检测到沃尔巴克氏体、嗜吞噬粒细胞无形体、伯氏疏螺旋体、绵羊无形体、未定型埃立克体、Q热、饶氏立克次体、隐藏巴贝斯虫、弩巴贝斯虫和未定型巴贝斯虫核酸。在短垫血蜱中检测到嗜吞噬细胞无形体、饶氏立克次体和未定型的巴贝斯虫，提示虎鼬可能是无形体、立克次体和巴贝斯虫的潜在宿主。（4）阿拉山口口岸地区存在多种虫媒病，为多种虫媒病的自然疫源地，对野生动物、候鸟和旅客造成潜在危害。

关键词：媒介生物；本底调查；虫媒病；疾病监测；PCR

论文类型：B (应用研究)

Abstract

Objective: We surveied the population of the rodents, fleas, ticks, mosquitoes, ceratopogouidaes and detected the insect-borne disease (IBP) at Alashankou port in order to establishment the “DNA barcode” to identification the vector and resolve the pathogen spectrum in the rodents, fleas, ticks, mosquitoes and ceratopogouidaes and expound the propagation mechanism of IBP in human-host-vetor and the law of the IBP across species / cross-border propagation. Finally, we provide the basic information to assess the risk of IBP.

Method: (1) During from 2014 to 2015, we used the rodent trapping and mousetrap capture to investigate rodent, the net trap associating with lamp to investigate the mosquito and ceratopogouidaes, the free-ticks were collected by drag-flag method, and blood feeding ticks and fleas were collected from wild. (2) The morphological and COI, 16S rDNA, Cytb and ITS identification were carried out based on representative vetors. (3) The African Swine Fever, Bluetongue, Vesicular Stomatitis, Lyme Disease, Anaplasmosis and Ehrlichiosis and other insect-borne disease were detected by the Elisa, PCR or RT-PCR. The PCR products were sequenced with BLAST and phylogenetically analyzed by Mage 6.0.

Result: （1）The dominant vetor species were Rhombomys opimus, Xenopsylla minax, Hyalomma asiaticum asiaticum, Dermacentor marginatus, Culex modestus and Cerodentopogonidae homotomus, respectively. (2) We found that the identificatation of the 16S rRNA sequence of Rhombomys opimus, Cricetulus migrodentorius and Allactaga sibirica, the COI and COII sequence of Xenopsylla minax, the 16S rRNA and ITS sequence of Culex modestus, the 16S rRNA, 28S rRNA and Cytb sequence of Ochlerotatus Culex and the 16S rDNA, COI, 12SrRNA and ITS sequence of Haemaphysalis erinacei weren’t match with the morphological. (3) Anaplasma DNA was detected in 3.39% rodents , and the Rickettsia DNA was detected in 42.9% fleas, and the Wolbachia DNA was detected in 3.33% mosquitoes. The anavage positive rate of Borrelia spp.，Anaplasma, Erhlichia，Rickettsia，Coxiella burnetii and Babesia were 45.44%, 14.95%, 12.82%, 12.04%,17.48% and 11.26% in ticks , respectively.

Conclusions:（1）The Anopheles vagus，Culiseta alaskaensis，Anopheles claviger，Rhipicephalus turanicus，Rhipicephalus bursa for the first time were found at Alashankou port.（2）We firstly reported the 16S rRNA sequence of Rhombomys opimus, Cricetulus migrodentorius and Allactaga sibirica, the COI and COII sequence of Xenopsylla minax, the 16S rRNA and ITS sequence of Culex modestus, the 16S rRNA, 28S rRNA and Cytb sequence of Ochlerotatus Culex and the 16S rDNA, COI, 12SrRNA and ITS sequence of Haemaphysalis erinacei.（3）The Borrelia burgdorferi sensu stricto, Anaplasma ovis, Anaplasma phagocytophilum, Coxiella burnetii, Babesia caballi, Babesia occultans, Babesia spp., Wolbachia , Erhlichia spp., and Rickettsia raoultii nucleic acid were detected in Alashankou. Our findings suggest that H. erinacei parasitizing wild marbled polecat may serve as reservoir and carrier for Anaplasma phagocytophilum，Rickettsia raoultii and Babesia spp.in areas around the China-Kazakhstan border.（4）Notably, IBP isolates from Alashankou showed genetic diversity related to the routes followed by migrodentory birds in the area. The results of this study suggest that Alashankou acts as a natural reservoir for multiple IBPs originating from various lineages. IBPs in Alashankou may pose risks to the health of terrestrial mammals, migrant birds, and human tourists. International cooperodention is needed to survey IBPs and diseases at the level of the tick–wildlife interface.

Key word: vectors; background investigation; insect-borne disease; disease surveillance; PCR Type of thesis: B ( Basic research )