新疆小亚璃眼蜱的鉴定及其携带牛环形泰勒虫病原DNA检测

通过对璃眼蜱形态学及璃眼蜱属特异性基因(ITS-2)和其携带病原基因(Tams1)PCR扩增及其PCR产物的克隆、测序、同源性比较等,鉴定和检测采自新疆吐鲁番地区疑似牛体表璃眼蜱(n=50)及其携带病原状况。结果显示,采集的蜱虫经形态学鉴定均为小亚璃眼蜱,PCR扩增的璃眼蜱种属特异性ITS-2基因和环形泰勒虫Tams1基因序列大小分别为491 bp和534 bp。本试验获得的ITS-2序列与Gen Bank中已记载的小亚璃眼蜱ITS-2序列一致,与形态学鉴定结果相符,故这些璃眼蜱属于小亚璃眼蜱种(Hyalomma anatolicum)。在检测的50只璃眼蜱中,携带牛环形泰勒虫的有3只,阳性率为6%(3/50),并发现所扩增的携带病原目的基因序列与已登录记载的牛环形泰勒虫基因序列相似性为99%。结果表明,在传统形态学分类的基础上,借助分子生物学方法对璃眼蜱种属鉴定及无典型硬蜱种的鉴定具有简易、准确度高等特点,而且在璃眼蜱种属确定的基础上分离其携带的病原更有实际意义。     

小亚璃眼蜱形态学鉴定及其携带牛环形泰勒虫病原的检测

为对吐鲁番地区璃眼蜱的精确鉴定提供依据,同时为该蜱分类以及携带病原溯源奠定基础。本研究对采集的疑似患牛体表璃眼蜱(n=120)采用形态学和ITS2基因特异性PCR方法鉴定出其种,再通过Tams1基因特异性PCR方法鉴定出其携带病原情况。结果显示,经形态学鉴定均为小亚璃眼蜱,PCR结果显示扩增的璃眼蜱ITS2基因和环形泰勒虫Tams1基因序列分别与Gen Bank中已记载的小亚璃眼蜱ITS-2、环形泰勒虫Tams1基因序列一致。在检测的120枚璃眼蜱中,携带牛环形泰勒虫病原的有11只,阳性率为9.17%(11/120)。结果表明,在传统形态学分类的基础上,借助分子生物学方法对璃眼蜱种属鉴定及无典型硬蜱种的鉴定不但具有简易、准确度高等特点,而且在璃眼蜱种属确定的基础上分离其携带的病原更有实际意义。     

托克逊县牛环形泰勒虫媒介蜱形态鉴定及病原检测

为了解托克逊县璃眼蜱及其携带病原情况,采用形态学方法和分子生物学方法对璃眼蜱形态学及其携带病原牛环形泰勒虫的基因(Tams1)进行PCR扩增,鉴定和检测采自托克逊地区疑似牛体表璃眼蜱(473枚)及其携带牛环形泰勒虫的感染情况。结果表明:采集的硬蜱经形态学鉴定为小亚璃眼蜱(248枚)和亚洲璃眼蜱(225枚)。小亚璃眼蜱携带牛环形泰勒虫有20枚,阳性率为8.06%(20/248);亚洲璃眼蜱携带牛环形泰勒虫有16枚,阳性率为7.11%(16/225)。4个乡采集的璃眼蜱均有不同程度携带牛环形泰勒虫的情况,其中伊拉湖乡牛环形泰勒虫阳性率最高,为8.40%(11/131)。说明托克逊县的气候环境适合媒介蜱的孳生,是当地牛环形泰勒虫病广泛流行的重要因素。     

小亚璃眼蜱和亚洲璃眼蜱对环形泰勒虫传播能力的研究

将实验室培养的小亚璃眼蜱（Hyalomma anatolicum anatolicum)和亚洲璃眼蜱（Hyalomma asiaticum asiaticum)清洁幼虫和若虫饲喂于感染环形泰勒虫的牛体，收集饱血幼虫和若虫，用所孵化的若虫和成虫感染试验牛。结果显示，小亚璃眼蜱幼虫、若虫阶级感染，所发育之若虫、成虫均可传播环形泰勒虫；而亚洲璃眼蜱各阶级的传播试验结果均为阴性。     

小亚璃眼蜱对牛巴贝斯虫未定种和环形泰勒虫传播的试验研究

利用蜱传播试验确定小亚璃眼蜱对中国新分离的牛的巴贝斯虫未定种和环形泰勒虫的传播能力与传播方式，进而明确其在中国牛梨形虫病传播中的流行病学意义。试验结果表明：小亚璃眼蜱可在雌虫阶段受到巴贝斯虫未定种的感染，并可在次代若虫（2／2）和成虫（3／3）阶段将病原传播给试验牛；小亚璃眼蜱幼虫和若虫吸入环形泰勒虫，饱血脱落的幼虫和若虫所蜕化发育的饥饿若虫（2／2）和成虫（2／2）均可将病原传递给敏感动物；感染巴贝斯虫未定种的小亚璃眼蜱饱血雌虫所孵育出的次代幼虫和若虫仍可被环形泰勒虫感染，所发育出的若虫（2／2）和成虫（2／2）可在一次传播试验中将环形泰勒虫和巴贝斯虫未定种同时传播给试验动物。     

新疆部分地区牛环形泰勒虫优势种媒介蜱的分子生物学鉴定及种群结构分析

璃眼蜱可传播牛环形泰勒虫病。为了解2018～2019年吐鲁番和阿勒泰部分地区牛体表璃眼蜱区系分布特点、种类组成及相对优势度,明确璃眼蜱种群分布和活动范围的变化,试验采用统计学方法和分子生物学方法对牛体表媒介璃眼蜱种类及群落结构进行详细的分析。结果显示,鉴定出璃眼蜱4种,其中小亚璃眼蜱的相对优势度最高,物种均匀度指数为0.925 0,群落多样性指数为1.282 3,优势度指数为0.299 4。调查结果对媒介璃眼蜱及蜱传牛环形泰勒虫病的流行和流行区的变化提供参考。     

阿合奇县色乡牦牛环形泰勒虫病流行病学调查报告

牛环形泰勒焦虫病在新疆多发,但有关牦牛环形泰勒虫病的报道甚少,尤其是南疆克州几乎未见报道。笔者于2006年3-5月期间,为了摸清牦牛环形泰勒焦虫病的传播媒介及感染率进行本次调查研究。经常规寄生虫病原学检查,发现在阿合奇县色乡采样的30头样品牦牛中20头为阴性,10头为阳性,其感染率为33.33%;其主要传播媒介是小亚璃眼蜱与亚洲璃眼蜱;确诊为牦牛环形泰勒虫病(Yak the ileriosis annulata)。基本查清了牦牛环形泰勒虫病在克州阿合奇县色乡流行情况,为制定综合防治措施提供科学依据。     

环形泰勒虫TRAP-A蛋白酵母双杂交诱饵质粒的构建与鉴定

为了筛选出环形泰勒虫TRAP蛋白在虫体入侵媒介蜱唾液腺过程中相互作用蜱源蛋白,构建了能够用于酵母双杂交筛选系统的重组诱饵质粒pGBKT7-TRAP-A。本研究以环形泰勒虫裂殖体为材料,根据环形泰勒虫TRAP-A结构域的基因序列设计引物,经过PCR扩增获得576 bp的基因片段,将其连接到线性化载体pGBKT7上。经双酶切鉴定和序列分析以及重组诱饵质粒在酵母双杂交系统中的自激活、细胞毒性及其表达情况检测,结果显示,本研究成功构建了酵母双杂交重组诱饵质粒pGBKT7-TRAP-A,构建的诱饵质粒对Y2HGold酵母菌无自激活活性和细胞毒性,且构建的诱饵质粒pGBKT7-TRAP-A可以在Y2HGold酵母菌内正确表达。表明所构建的诱饵质粒pGBKT7-TRAP-A可以用于筛选小亚璃眼蜱唾液腺酵母双杂交c DNA文库,获得可能与环形泰勒虫TRAP-A蛋白相互作用的蜱源蛋白。     

小亚璃眼蜱、亚洲璃眼蜱和残缘璃眼蜱的分子生物学鉴定

本研究旨在建立小亚璃眼蜱、亚洲璃眼蜱和残缘璃眼蜱的分子生物学鉴定方法,并探讨它们的系统发生关系。在新疆、内蒙古从动物体表采集寄生蜱,形态学鉴定后,PCR扩增测序获得3种璃眼蜱的16S rRNA及线粒体色素氧化酶亚基Ⅰ基因(cytochrome oxidaseⅠ,COⅠ)序列后进行同源性分析。用Mega 5.0和Mrbayes 3.2软件分别构建系统进化树,亚洲璃眼蜱、小亚璃眼蜱样本16S rRNA序列与GenBank中已知相应蜱虫16S rRNA序列聚类,而残缘璃眼蜱COⅠ序列与GenBank中已知残缘璃眼蜱COⅠ序列聚类,与形态学鉴定结果一致。在传统形态学分类的基础上,结合分子生物学鉴定方法能简易、准确鉴定小亚璃眼蜱、亚洲璃眼蜱和残缘璃眼蜱。     

中哈边境阿拉山口口岸输入性亚洲璃眼蜱的种类鉴定与分析

对1批从哈萨克斯坦进口羊毛中检获的蜱样品进行种类鉴定。应用16S rDNA基因和COI基因两条DNA条形码技术进行鉴定分析。结果 16S rDNA序列分析显示与新疆亚洲璃眼蜱(KC203351)同源性最接近(99%);COI基因序列分析显示与新疆伊犁亚洲璃眼蜱(KF527440)同源性最接近(99%)。结论:经DNA条形码比对,确定为亚洲璃眼蜱,应加强对外来医学媒介生物防控,严防"外来病"入侵。     

Natural infection rates of Hyalomma anatolicum anatolicum with theileria in Sudan

Hyalomma anatolicum anatolicum ticks were collected around cattle pens at two locations close to Khartoum. They were assessed for theileria infection by four methods. Salivary glands were stained whole with methyl green pyronin and examined for parasite masses. Adult ticks were partially fed on rabbits, ground up in medium, and the suspensions were examined in Giemsa stained smears and by inoculation into bovine lymphocyte cultures. Ticks were fully fed on calves which were monitored for developing theileriosis. H a anatolicum were found infected with parasite masses similar to those seen in experimental infections with T annulata. At one site 38 per cent of 102 ticks were infected and the mean number of parasite masses per tick for the whole sample was 37. At the other site 86 per cent of 156 ticks were similarly infected and the mean parasite masses per tick was 19.5. Suspensions of sporozoites contained sporozoites typical of those found in experimental preparations of T annulata. Sporozoites harvested from ticks from both locations infected and transformed normal bovine lymphocyte cultures. H a anatolicum ticks from both locations produced fatal theileria infection in susceptible calves.     

The production of nymphs ofHyalomma anatolicum anatolicum for experimental infection withTheileria annulata

Summary Methods are described for the production of nymphs ofHyalomma anatolicum anatolicum for later use in transmission experiments withTheileria annulata where the timing of the application of nymphs to infected cattle needs to be accurately controlled. Larvae are fed on the torsos of rabbits where approximately 98% undergo a two-host feeding cycle. This cycle is interrupted 2 days after the first larvae moult into nymphs by killing the rabbits and the nymphs are then collected with a suction pump. Nymphs produced by the interrupted larval feeding method feed well on cattle, in regard to timing of detachment and weight, compared with nymphs produced by interrupted feeding on rabbits

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La Produccion De Ninfas DeHyalomma Anatolicum Anatolicum Para La Infeccion Experimental ConTheileria Annulata

Resumen Se describen métodos para la producción de ninfas deHyalomma anatolicum anatolicum, para usarlas posteriormente en experimentos de transmisión deTheileria annulata, en donde el tiempo de aplicación de las ninfas en el ganado infectado necesita un control riguroso. Las larvas se alimentan en conejos en donde aproximadamente el 98% desarrolla un ciclo de alimentación de dos huéspedes. Este ciclo se interrumpe 2 días despues de la muda de las primeras larvas a ninfas, sacrificando los conejos y recogiendo las ninfas con una bomba de succión. Las ninfas producidas interrumpiendo el ciclo de alimentación de las larvas, se alimentan muy bien en ganado, en relación con el tiempo en que se desprenden y el peso, en comparación con las ninfas producidas interrumpiendo el ciclo de alimentación en conejos.

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Production De Nymphes DeHyalomma Anatolicum Anatolicum Pour L'Infection Experimentale AvecTheileria Annulata

Résumé Des méthodes sont décrites pour produire des nymphes deHyalomma anatolicum anatolicum destinées à des expériences ultérieures de transmission deTheileria annulata; expériences dans lesquelles le moment de l'application des nymphes sur du bétail infecté doit être contrôlé avec précision.Les larves sont nourries sur thorax de lapins et près de 98 p. 100 y poursuivent un cycle à deux hôtes. Deux jours après la transformation des premières larves en nymphes, le cycle est interrompu par sacrifice des lapins et les nymphes sont récoltées à l'aide d'une pompe aspirante.Les nymphes obtenues par interruption du repas larvaire se nourrissent bien sur le bétail, en comparaison de celles obtenues par repas interrompus sur lapins, compte tenu du moment du détachement et de leur poids.

     

Immunoprotective Efficacy of a Purified 39 kDa Nymphal Antigen of Hyalomma anatolicum anatolicum

Soluble nymphal antigens (HNAg) were purified by immunoaffinity chromatography using CNBr-activated Sepharose 4B coupled with immunoglobulin ligands from animals immunized with HNAg and 69–71% protected against challenge infestations, and 8% recovery of the purified protein (Aff-HNAg) was obtained. Following immunization of crossbred calves (Bos indicus×Bos taurus) with 1600 g of Aff-HNAg in three divided doses, significant rejections of larvae (p<0.001, 84.2%), nymphs (p<0.05, 61.4%) and adults (p<0.05, 58.7%) were recorded. No significant changes were recorded in the engorgement weights of the larvae and nymphs, but there was a significant (p<0.05) reduction in the weight of the engorged adults. Immunization conferred a significant decrease in the numbers of resultant nymphs (p<0.001) and adults (p<0.001) that had fed on the immunized animals. SDS-PAGE analysis identified a 39 kDa protein, previously isolated from larvae of Hyalomma anatolicum anatolicum, as the antigen responsible for the induction of resistance against all the stages of the tick.     

Evaluation of purified Theileria annulata sporozoite antigen from the tick Hyalomma anatolicum anatolicum

Rabbits immunized with purified sporozoites from Hyalomma anatolicum anatolicum produced antibody to Theileria annulata specific antigens as measured by the indirect fluorescent antibody test. Nonspecific fluorescence which compounded the interpretation of the test was observed. Incorporation of Eriochrome black and Evans blue as a counterstain in the diluent buffer for the conjugate significantly reduced the background fluorescence.     

Relative role of male and female Hyalomma anatolicum anatolicum ticks in Theileria transmission

The relative role of male and female Hyalomma anatolicum anatolicum ticks in the epidemiology of Theileria transmission was studied by detecting Theileria sporozoites in the dissected salivary glands of 568 ticks by the methyl green pyronin staining method. Detailed frequency distribution of Theileria-positive acini in the salivary glands of the 264 (46.48%) positive ticks from a field collection in Haryana indicated that the number of infected salivary acini per positive tick was greater in females than in males. This suggests that female ticks have a more important role in Theileria transmission than male ticks. This finding assumes greater significance in the light of the observation that the natural male:female ratio is also in favour of female ticks.     

Immunization of cattle with nymphal Hyalomma anatolicum anatolicum extracts: effects on tick biology

Antigens derived from partially engorged nymphs of Hyalomma anatolicum anatolicum were used in immunizing crossbred (Bos indicus×Bos taurus) cattle against larval, nymphal and adult H. a. anatolicum and H. dromedarii. The cattle were either infected with Theileria annulata at low parasitaemia or were uninfected. Whole nymphal extract (WNE), nymphal membrane antigens (NMA) and nymphal soluble antigens (NSA) were used for immunization. The group immunized with WNE showed significant and better rejection of H. a. anatolicum ticks as compared to calves immunized with either NMA or NSA. The moulting rates of both engorged larvae and nymphs remained unaffected. Nymphs which engorged on the immunized calves were fully susceptible to infection by T. annulata as indicated by the intensity and abundance of Theileria infections in the resulting adult ticks from immunized and unimmunized Theileria infected cattle. These ticks also transmitted fatal theileriosis to susceptible calves.     

High infection rates of the tick Hyalomma anatolicum anatolicum with Trypanosoma theileri

A crossbred calf (3 months old) obtained from a farm where regular control of ticks was practised and found to be free of blood parasites was inoculated with 20 ml pooled blood collected from four field cattle which had very low Trypanosoma theileri parasitaemias (one parasite per 70 microl blood as determined by the haematocrit centrifugation technique). Trypanosoma theileri was present in the blood 6 days after injection and a peak parasitaemia of 42 parasites per 70 microl blood was recorded by day 12. Hyalomma anatolicum anatolicum nymphs were applied on the ears of the calf on day 8 and they dropped engorged by days 13 and 14. The resulting adult ticks were examined for the presence of T. theileri by severing a leg and making a smear of the clear haemolymph which exuded from the wound. The smear was fixed in methanol and stained with Giemsa stain. The infection rate with T. theileri in the ticks was 43.3% (26 out of 60). The intensity of infection was very high and various developmental stages of the flagellates were observed (epimastigotes, sphaeromastigotes, trypomastigotes and other intermediate stages). The haemolymph from 12 ticks was also collected in tissue culture medium and the trypanosomes survived for 25 weeks before eventually dying. The results demonstrated unequivocally the high vectorial capacity of the tick H. a. anatolicum for T. theileri.     

Transmission of a Trypanosoma sp. to cattle by the tick Hyalomma anatolicum anatolicum

Hyalomma anatolicum anatolicum engorged nymphs and flat adults were collected from two areas in northern Sudan. Various developing stages of Trypanosoma theileri-like flagellates were observed in the engorged nymphs, freshly moulted adults and mature adults partially engorged on rabbits. When these ticks were applied to two calves, one calf became infected with the trypanosome. The parasites were observed for one day in the enlarged lymph node nearest to the tick-feeding site 5 days after the tick application. Subsequently the trypanosomes were re-isolated in vitro from the infected calf. Inoculation of a ground-up tick supernatant suspension from the infected batch of ticks containing 10(4) trypanosomes into a calf did not produce a patent infection.     

Infectivity of ground-up tick supernates prepared from Theilerai annulata infected Hyalomma anatolicum anatolicum.

Groups of Theileria annulata infected Hyalomma anatolicum anatolicum adults prefed on a calf for 1 to 6 days were separately ground in tissue culture medium-199, supplemented with bovine albumin powder, Fraction-V. Supernatant fluid was collected, made up with additional medium, so that each millilitre represented material from 25 ticks, and was injected subcutaneously into groups of cross-bred male calves. The results indicated that ground-up tick supernate (GUTS) prepared from unfed ticks was not infective, whereas that prepared from 1 to 6 days prefed ticks was infective. GUTS prepared from 3 days prefed ticks appeared to contain highest infectivity and 1 ml of it induced fatal theileriosis.