长角血蜱和微小扇头蜱的形态与分子生物学鉴定

目的 建立鉴定长角血蜱和微小扇头蜱的形态学和分子生物学相结合的方法.方法 采集自湖北和河南省布尼亚新病毒疫区家养动物体表寄生的蜱及草丛、灌木中的蜱;先以形态学鉴定,后用PCR方法扩增得到蜱的12S rDNA,测序后进行同源性和系统进化分析.结果 形态学方法鉴定采集到两种蜱:长角血蜱(Haemaphysalis longicornis)和微小扇头蜱(Rhipicephalus microplus).蜱的12S rDNA经克隆、测序,用PAUP 4.0软件构建系统发生树,湖北、河南省两种蜱的12S rDNA序列分别与长角血蜱和微小扇头蜱聚类,与形态学鉴定结果一致.结论 在传统形态学分类的基础上结合分子生物学鉴定方法能更准确地鉴定蜱的种类.

Abstract：

Objective To establish a method combined morphology and molecular marker for identifying Haemaphysalis longicomis and Rhipicephalus microplus. Methods Ticks were collected from domestic animals and wild environment in epidemic area of Hubei and Henan provinces where cases of fever with thrombocytopenia syndrome were prevalent. We classified the ticks by morphology characteristics before 12S rDNA of ticks were amplified by PCR and subsequently sequenced. Phylogenetic tree was constructed by PAUP4.0. Results The ticks belonged to Haemaphysalis longicomis and Rhipicephalus microplus through observation and analysed by the morphological characteristics of the ticks. 12S rDNA was cloned and sequenced while data confirmed the morphological identification of the results. Conclusion The method based on morphology that combined with molecular marker seemed a good method for the identificaton of ticks.     

新疆北疆部分地区硬蜱伯氏疏螺旋体基因型研究

目的调查和鉴定新疆北疆石河子市、沙湾县、伊宁县和察布查尔县媒介蜱,并确定其携带的莱姆病螺旋体的基因型。方法选择4县市6个牧区为调查点,采集羊源寄生硬蜱,结合形态学与16S rRNA进行蜱种鉴定;采用BSK-H培养基对莱姆病螺旋体分离培养,并用硝酸银染色和巢式PCR法进行病原检测,阳性产物测序结果比对,并与11种GenBank参考序列比对,确定莱姆病螺旋体的基因型。结果6个调查点共采集寄生硬蜱900多只,经形态学与16S rRNA鉴定分别为图兰扇头蜱、刻点血蜱、亚洲璃眼蜱和边缘革蜱;从中分别选取样本硬蜱共102只,分24管,分离培养后结合巢式PCR和硝酸银染色共获得16管阳性培养物。5S～23S rRNA基因问隔区测序比对分析表明,所有菌株为伯氏疏螺旋体与国际报道的B.burgdoq''eri Sensu Stricto(B31)基因型具有高度同源性,同源性为98.6%～99.5%;OspC基因型分析与上述结果一致。结论4县市分离获得伯氏疏螺旋体,基因型为B.burgdooCeri Sensu Stricto,且存在生物多样性。从图兰扇头蜱中分离到伯氏疏螺旋体为我国首次报道。     

新疆边境地区伊宁县图兰扇头蜱形态学及分子生物学鉴定

目的 对新疆边境地区伊宁县图兰扇头蜱进行形态学和分子生物学鉴定.方法 在新疆边境地区的伊宁县随机采集畜牧体表寄生蜱324只,进行形态学初步筛选,从中选取6只形态差异较大的图兰扇头蜱,进行线粒体16S rRNA和线粒体细胞色素C氧化酶亚基Ⅰ (CO Ⅰ)基因序列扩增、测序,并与GenBank登录的图兰扇头蜱参考序列进行遗传进化分析.结果 形态学鉴定采集的伊宁县蜱类皆为图兰扇头蜱,两段序列的碱基组成均显示较高的A+T比例(16SrRNA基因为73.8％,CO Ⅰ基因为70.34％),在变异程度上16S rRNA基因较CO Ⅰ基因明显保守.结论 首次应用形态学、16S rRNA和CO Ⅰ基因测序分析表明新疆伊宁县采集蜱皆为图兰扇头蜱,并证明图兰扇头蜱具有生物多样性.     

褐黄血蜱与铃头血蜱的分子生物学鉴定

目的建立2种形态学特征非常相近的褐黄血蜱及铃头血蜱的分子生物学鉴定方法,探讨它们的系统发生关系。方法在湖北省从动物体表采集寄生蜱,形态学鉴定完毕后,采用PCR方法从2种血蜱基因组中扩增12SrDNA、16SrDNA及线粒体细胞色素氧化酶亚基Ⅰ(COⅠ)基因,测序后进行同源性分析,用PAUP4.0软件分别构建系统发生树并进行系统进化分析。结果 2种血蜱12SrDNA、16SrDNA及COⅠ基因之间的同源性分别为90.8%、90.4%和86.8%,湖北省采集的褐黄血蜱3个基因片段与已知褐黄血蜱的同源性分别为100%、99.5%和99.7%。用这2种蜱的12SrDNA、16SrDNA及COⅠ基因的核苷酸序列分别构建系统发生树,湖北省采集的褐黄血蜱与已知的褐黄血蜱聚在一起,褐黄血蜱及铃头血蜱均形成独立的分支,但用3个基因构建的系统发生树中血蜱属不同种间的亲缘关系不同。结论对于形态学特征相近蜱种的鉴定,在传统形态学分类的基础上结合分子生物学鉴定方法能更准确地鉴定蜱的种类,也能更好地了解其进化关系,为蜱传播疾病的预防控制提供依据。     

河南省蜱种类和地理分布及季节消长调查

目的掌握河南省蜱的种类、地理分布和季节消长。方法 2011-2012年在全省范围内采用布旗法和体表查捉法分别调查野外游离蜱和动物体表寄生蜱。结果全省共采集到蜱2科6属8种,分别是长角血蜱、雉鸡血蜱、褐黄血蜱、微小扇头蜱、中华革蜱、中华硬蜱、残缘璃眼蜱和翘缘锐缘蜱,其中长角血蜱遍布全省。优势种长角血蜱3-4月开始出现,7-8月达到密度最高值,之后逐渐消退,10月以后进入蛰伏,越冬后至次年再次出现。结论蜱在河南省的分布广泛,种类较多,优势种长角血蜱的活动以夏、秋季为高峰期。     

西藏察隅县蜱的分子鉴定

目的 利用分子生物学方法分析西藏察隅县蜱类组成。方法 对察隅3个乡镇10个行政村饲养的家畜和丛林环境中的蜱进行调查采样,提取蜱DNA,对COI基因进行PCR扩增和测序,通过与GenBank数据库在线比对和系统发育进化树构建进行蜱虫种属鉴定。结果 经COI基因序列分析,共鉴定出蜱虫7种,其中牛身上的寄生蜱虫主要为微小扇头蜱,同时也发现了西藏血蜱、长角血蜱、卵形硬蜱,丛林环境中的游离蜱虫有微小扇头蜱、锐跗硬蜱、克拉斯比尔血蜱。结论 西藏察隅县蜱虫分布广泛,本研究可为察隅县蜱虫调查和疾病防控提供重要参考。     

新疆和硕地区荒漠景观中无形体感染的流行病学调查

目的明确无形体病在新疆南部和硕地区人群、山羊与媒介蜱种群中流行状况,探明该地区无形体种属多样性。方法采集当地健康人和山羊的血液样本,捕捉蜱样本;利用间接免疫荧光方法检测血清无形体特异性IgG抗体;通过巢式PCR方法检测无形体16S rDNA基因片段,通过序列比对分析确定无形体种属特征。结果和硕地区农牧民、山羊血清中无形体特异性IgG抗体阳性率分别为43．31%(55/127)和27．50%(55/200);采集获得蜱共367只,包括3属4种,其中主要为亚洲璃眼蜱(47．41%)和短小扇头蜱(37．60%);蜱、羊血样本无形体核酸阳性率分别为5．00%(18/360)和4．49%(7/156),人血中未检出无形体;序列测定与比对分析表明,阳性扩增序列与嗜吞噬细胞无形体(Gu046565、GU064897、ABl96721)同源性最高,分别为99．2%、99．5%和99．5%,与中央无形体16s rDNA(Gu064903)序列一致,同源性为99．2%。结论新疆和硕地区荒漠景观中存在人嗜吞噬细胞无形体与中央无形体的流行,并可能通过亚洲璃眼蜱与短小扇头蜱传播。     

海南省硬蜱种类调查及一新纪录蜱种

目的了解海南省硬蜱种类。方法宿主体捡蜱法,采集硬蜱标本并分类鉴定。结果捕获硬蜱5属12种,其中长角血蜱为海南省新纪录种。结论进一步加强硬蜱调查工作,为蜱传疾病防治提供科学依据。     

江西省蜱虫种群分布特征

目的调查江西省蜱虫种群分布。方法采用形态学鉴定,对于形态鉴定存在争议的,采用分子生物学鉴定。结果在植被中采集到6种蜱虫,即长角血蜱、血红扇头蜱、越原血蜱、北岗血蜱、中华硬蜱和金泽革蜱。长角血蜱为优势种,占总蜱数的90.6%,越原血蜱和北岗血蜱是江西省新纪录种。蜱虫在草地中多见,占总采集数的68.2%,林地占9.4%,灌木丛占1.2%。从发育阶段来看,幼若蜱居多,分别占19.1%和68.2%,成虫较少,雌性占6.6%,雄性占6.0%。在宿主体表采集到1 513只蜱虫,归为4属13种,为长角血蜱、铃头血蜱、褐黄血蜱、钝刺血蜱、越原血蜱、北岗血蜱、豪猪血蜱、血红扇头蜱、镰形扇头蜱、微小扇头蜱、粒形硬蜱、中华硬蜱和龟形花蜱,龟形花蜱是江西省新纪录种。长角血蜱是江西省常见种(占总采集数的30.5%)和广布种。长角血蜱寄主广泛,其中野兔体表常见,侵害率为33.3%,犬体表较少见,侵害率为2.3%(χ~2=23.68,P=0.001 3)。此外,侵害野兔的长角血蜱占总长角血蜱总数的64%,远高于其他寄主。侵害宿主的蜱虫中,幼虫347只,占22.9%;若虫249只,占16.5%;雄虫404只,占26.7%;雌虫513只,占33.9%,性别均匀。结论本文首次报道了江西省蜱虫种群分布特征,将为该地区蜱虫防控提供重要参考。     

DNA条形码技术在鉴定绥芬河口岸日本血蜱和嗜群血蜱中的应用

目的建立用DNA条码技术对绥芬河口岸两种形态学特征非常相近的嗜群血蜱和日本血蜱的鉴定方法,探讨DNA条形码在蜱类鉴定中的实用性。方法在黑龙江绥芬河口岸采集游离血蜱103只,形态学鉴定完毕后,提取蜱样本基因组DNA,采用PCR方法从2种血蜱基因组中扩增线粒体细胞色素氧化酶亚基Ⅰ(COⅠ)基因,测序后进行同源性分析,用Mega6.0软件构建系统发生树并进行系统进化分析。结果经形态学鉴定嗜群血蜱89只,日本血蜱14只;2种血蜱COⅠ基因之间的同源性为87.5%,嗜群血蜱与GenBank中编号KR108867.1公布的嗜群血蜱CO Ⅰ序列相似性为99.8%;日本血蜱与GenBank中编号KR108879.1公布的日本血蜱CO Ⅰ序列相似性为99.8%,与形态学鉴定结果一致。结论对于形态学特征相近蜱种的鉴定,在传统形态学分类的基础上结合DNA条码技术鉴定方法能更准确地鉴定蜱的种类,也能更好地了解其进化关系,为蜱传播疾病的预防控制提供依据。     

GroEL gene typing and genetic diversity of Anaplasma bovis in ticks in Shaanxi,China

Anaplasma bovis, causative agent of bovine anaplasmosis, is usually identified by nested-PCR amplifying the rrs gene. However, it is difficult to determine the genetic relationship among different variants within A. bovis using this gene because of high conservation. In this study, two tick species, identified as Rhipicephalus microplus and Haemaphysalis longicornis based on morphological and molecular methods by analyzing COI gene, were collected from cattle, goat or sheep. Subsequently, A. bovis was initially detected by PCR amplifying the rrs gene in ticks in Shaanxi Province, China. The sequencing and Blast results showed that some false positive samples were found when only based on the amplification of partial rrs gene, presenting these sequences resembled those of other Alphaproteobacteria rather than A. bovis. Although major surface proteins genes were proposed and used successfully to identify members within Anaplasmataceae, these genes were unavailable for A. bovis. Hence, primers targeting the groEL gene were designed and a PCR assay was developed. The PCR products were sequenced and similarity and phylogenetic analysis suggested all these sequences are the groEL gene of A. bovis. In addition, phylogenetic analysis based on the groEL gene also revealed the genetic diversity of A. bovis worldwide, as well as in Shaanxi Province of China, which wasn't reflected by analyzing the rrs gene. In sum, groEL gene is important for molecular detection and phylogenetic analysis of A. bovis.     

Comparative analyses of mitochondrial and nuclear genetic markers for the molecular identification of Rhipicephalus spp.

The genus Rhipicephalus (Acari: Ixodidae) comprises a large number of vectors of pathogens of substantial medical and veterinary concern; however, species identification based solely on morphological features is often challenging. In the present study, genetic distance within selected Rhipicephalus species (i.e., Rhipicephalus bursa, Rhipicephalus guilhoni, Rhipicephalus muhsamae, Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus), were investigated based on molecular and phylogenetic analyses of fragments of the mitochondrial 16S, 12S and cytochrome c oxidase subunit 1 (cox1) genes, as well as of the whole sequences of the ribosomal internal transcribed spacer-2 (ITS-2) region. Mean values of inter-specific genetic distance (e.g., up to 12.6%, 11.1% and 15.2%), as well as of intra-specific genetic distance (e.g., 0.9%, 0.9% and 1%), calculated using the Kimura-2 parameter substitution model with uniform rates among sites for 16S, 12S and cox1 genes, respectively, confirmed the differentiation of the rhipicephaline species herein examined. The molecular identification was also supported by the distinct separation of species-specific clades inferred from the phylogenetic analyses of all mitochondrial sequences. Conversely, little interspecific divergence was detected amongst ribosomal ITS-2 sequences (i.e., up to 2.8%) for species belonging to the R. sanguineus complex, which resulted in the ambiguous placement of selected R. sanguineus s.l. and R. turanicus sequences in the corresponding phylogenetic tree. Results from this study confirm the suitability of mtDNA markers for the reliable identification of ticks within the Rhipicephalus genus and provide a framework for future studies of taxonomy, speciation history and evolution of this group of ticks.     

Rhipicephalus ticks infected with Rickettsia and Coxiella in Southern Switzerland (Canton Ticino).

Ticks of the Rhipicephalus sanguineus species complex may be vector of various pathogens including Rickettsia conorii (the etiological agent of the Mediterranean spotted fever) and Coxiella burnetii (cause of the Query (Q) fever). R. sanguineus ticks have been imported in several parts of central and northern Europe, especially in environments such as kennels and houses providing the appropriate microclimatic conditions and the blood source necessary for their survival. Since 1940 these ticks have occasionally been recorded in Switzerland. In Ticino (the southern part of Switzerland), they have been reported since 1980 and their probable establishment in this area has been suggested in the '90s. By means of PCR and direct sequencing, we tested the identity of these ticks (using 12S rDNA gene) and the occurrence of Rickettsia spp. (using 16S rDNA, gltA and OmpA genes) as well as Coxiella sp. (using 16S rDNA). The results indicated that in Ticino, two different tick species coexist, i.e. R. sanguineus sensu stricto and Rhipicephalus turanicus. A few individuals of R. sanguineus sensu stricto are infected with Rickettsia massiliae/Bar29, which are strains of unknown pathogenicity. Coxiella sp., an endosymbiont of Rhipicephalus ticks, has also been identified in both tick species. Due to climatic changes towards global warming, imported tick species may therefore adapt to new area and might be considered as epidemiological markers for a number of infectious agents transmitted by them.     

Distribution of ticks in Bosnia and Herzegovina

Ticks are one of the important groups of parasites, whose populations have increased in Europe in the last 20 years and, as vectors of many emerging diseases, are a major threat to human and animal health. Bosnia and Herzegovina has both climate and environmental conditions favorable for ticks. Given this risk, the aim of this study was to identify tick species currently present in this country and to compare the findings with previous studies, in order to update information on their spatial distribution and other parameters of occurrence.From March 2017 to April 2020, ticks were collected in 42 sampling areas (municipalities) throughout the country. Prespecified identification keys were used for identifying the ticks to the species level.A total of 6193 tick specimens from the Ixodidae family were collected from animal hosts or from vegetation. Ixodes ricinus was the most dominant tick species found. However, our results indicate a decrease in the relative contribution of this tick species to the overall abundance of tick populations. Dermacentor marginatus, which almost doubled in abundance compared to earlier studies, is the second most common tick species. We further confirmed the presence of Dermacentor reticulatus, Rhipicephalus bursa, Rhipicephalus sanguineus sensu lato, Hyalomma marginatum and Haemaphysalis punctata. Ixodes hexagonus, Ixodes canisuga and Haemaphysalis concinna, which were previously reported at low abundance were not found amongst our specimens.     

Cercopithifilaria spp. in ticks of companion animals from Asia: new putative hosts and vectors

Cercopithifilaria bainae, Cercopithifilaria grassi, and Cercopithifilaria sp. II sensu Otranto et al., 2013 tick borne filarioids are typically found in dogs. Among them, Cercopithifilaria bainae has a worldwide distribution according to the occurrence of its tick vector, Rhipicephalus sanguineus sensu lato (s.l.). Nevertheless, in Asian countries, despite the wide presence of this tick species, data on Cercopithifilaria spp. are scant. Therefore, this study aimed to assess the occurrence of these dermal filarioids in ixodid ticks collected on dogs and cats from Asian countries, providing a better epidemiological picture on their distribution in this continent. Ticks (n = 687) of the species Rhipicephalus sanguineus s. l. (n = 667), Rhipicephalus haemaphysaloides (n = 8), Haemaphysalis longicornis (n = 7), Haemaphysalis campanulata (n = 1), Haemaphysalis wellingtoni (n = 2), Haemaphysalis hystricis (n = 1), and Ixodes sp. (n = 1) were collected on dogs and cats under the frame of previous studies in China, India, Indonesia, Malaysia, Singapore, Taiwan, Thailand, the Philippines and Vietnam. Tick samples were molecularly screened for Cercopithifilaria spp. by conventional PCR and real-time PCR using two pair of primers targeting partial sequences of cytochrome c oxidase 1 gene. Overall, Cercophitifilaria spp. DNA was detected in 9.5% (n = 65/687) of the tick specimens tested, with C. bainae being the most prevalent species (8.9%), followed by C. grassii (0.6%). Most Cercophitifilaria spp. positive ticks were collected on dogs (92.3%; 60/65); whereas ticks collected on cats represented 7.7% of the positive specimens. In addition, Cercopithifilaria spp. were mostly detected in R. sanguineus s.l. ticks (96.9%; 63/65), followed by Rhipicephalus haemaphysaloides (3.1%; 2/65). Data herein presented demonstrate the occurrence of dermal tick borne filarioids of the genus Cercopithifilaria in several Asian countries, with C. bainae being the most prevalent species. We also report for the first time the molecular detection of C. bainae in R. sanguineus s.l. ticks collected on cats, as well as in R. haemaphysaloides ticks, suggesting that the biological cycle of this filarioid species may involve other intermediate and definitive hosts than R. sanguineus s.l. and dogs. However, confirmatory studies on the role of other tick species and domestic cats on the biology of C. bainae are advocated.     

Geodemographic analysis of Borrelia burgdorferi sensu lato using the 5S–23S rDNA spacer region

BackgroundLyme borreliosis is the predominant tick-borne disease in the Northern hemisphere, with considerable heterogeneity in clinical manifestations. Here, we evaluated one genetic marker for its use in population genetic based analysis. For that we collected molecular and epidemiological records of Borrelia burgdorferi sensu lato isolates from ticks, animals and humans at various sites in The Netherlands and worldwide.MethodsThe 5S–23S rDNA (rrfA–rrlB) intergenic spacer region (IGS) from 291 Dutch Borrelia positive ticks was sequenced and compared to Borrelia sequences from GenBank. We estimated several population genetic measures to test the neutrality of the marker. We also assessed the ability of this marker to discriminate between Eurasian Borrelieae at a finer geographical resolution, and to detect population expansion per genospecies.ResultsThe most prevalent genospecies in The Netherlands was Borrelia afzelii, whereas Borrelia garinii, B. burgdorferi sensu stricto, Borrelia spielmanii and Borrelia valaisiana were found less frequently. The result of the Ewens–Watterson–Slatkin test was consistent with neutral selection of IGS region. Estimated pairwise fixation indices (Fst) were significantly different from zero between The Netherlands, the rest of Europe, Russia and Asia for B. afzelii and Borrelia garinii. Estimated Fu’s Fs were significantly negative for B. afzelii and B. garinii.ConclusionsAt least seven B. burgdorferi s.l. genospecies circulate in Ixodes ricinus population in The Netherlands. The population genetic analyses of IGS region can resolve subpopulations within a genospecies and detect a large excess of rare genetic variants at the genospecies level. A genetic trace of population expansion for B. afzelii and B. garinii is consistent with the reported increase in Lyme borreliosis incidence in European countries.