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。     

泰勒焦虫人工感染蜱试验

<正> 在印度,牛热带焦虫的病原是泰勒焦虫,主要传播媒介是小亚璃眼蜱。Bhattacharyulu、chaudhri与Gill 1975年报道:将幼虫和若虫喂养在感染牛体表、继而幼虫和若虫感染了焦虫病,其若虫与成虫造成本病传播。本试验试图用泰勒焦虫人工感染小亚璃眼蜱。     

残缘璃眼蜱人工培育及环形泰勒虫的实验性传播

将采自内蒙古自治区的残缘璃眼蜱（Hyalomma detritum)饲喂于牛体，观察了成虫，幼虫，若虫在实验室饲喂条件下的生活周期及滞育现象。通过蜱传播试验证实，残缘璃眼蜱饥饿幼虫，若虫吸入病原，其蜕化之若虫，成虫能将环形泰勒虫（Theileria annulata) 传播给敏感动物，而幼虫阶段吸入病原，如若虫阶段在免体饱血，所蜕化产生的饥饿成虫不传播该病。     

牛血孢子虫单一种的分离技术及其保藏方法的研究——卵形巴贝斯虫的传播试验

将实验室培育的“洁净”长角血蜱的幼虫、若虫、成虫及微小牛蜱的幼虫,先后分别释放到人工感染卵形巴贝斯虫单一种牛体上不同部位事先粘贴好的布袋中,使其自行叮咬吸血。待饱血脱落后亦分别收集,置28℃、相对湿度约90％的条件下蜕皮或产卵孵化。而后用不同世代和各变态期的蜱,分别叮咬感染除脾和非除脾健康易感牛。结果表明,长角血蜱的当代若虫和成虫对卵形巴贝斯虫没有传播能力,幼虫和若虫吸入的病原亦不能经卵传递;饱血雌虫吸入的病原可经卵传递,次代幼虫、若虫和成虫三个阶段都具有传播能力。次代感染幼虫经兔体后的若虫和成虫也具有感染力。 微小牛蜱不能传播该种病原。     

甘肃省羊泰勒虫媒介蜱的传播试验

用采自羊泰勒虫病疫区甘肃省永靖且的麻点璃眼蜱饥饿成虫叮咬健康羊体，试验羊体内未发现羊泰勒虫；用其饱血雌虫孵化的幼虫叮咬羊泰勒虫感染羊，收集饱血若虫，再用其饥饿成虫叮咬健康羊体，被叮咬羊体内也未查出羊泰勒虫，证明麻点璃眼蜱不传播羊泰勒虫。用采自羊泰勒虫病疫区张家川和永靖县的青海血蜱经人工孵化的幼虫叮咬羊泰勒虫自然感染羊，收集饱血幼虫蜕皮后，用其若虫叮咬健康羊，40d后在被叮咬羊血片中发现羊泰勒虫，但未显示明显的临床症状。用采自羊泰勒虫病疫区羊体的青海血蜱成虫、若虫、幼虫同时叮咬健康羊，18d时在试验羊体内发现羊泰勒虫，试验羊表现贫血、黄疸、稽留热、肩前淋巴结肿大等特征性症状，出现心包积液，肝、脾、肾肿胀及出血的典型病理变化。由此确定，甘肃省的羊泰勒虫是由青海血蜱传播的。     

牛的巴贝斯虫18S rRNA基因序列比较研究

对中国已报道的8株牛的巴贝斯虫（包括1株牛巴贝斯虫、1株双芽巴贝斯虫、1株大巴贝斯虫、3株卵形巴贝斯虫和2株巴贝斯虫未定种）的18S rRNA基因序列进行了测定与比较。自感染动物的血液中纯化虫体，提取基因组DNA，PCR扩增靶基因，然后将其连接到pGEM—T Easy载体上，进行克隆测序。研究结果显示：牛的巴贝斯虫18S rRNA基因大小在1653～1699bp之间；用所测得的和自GenBank下载的各种动物的巴贝斯虫18S rRNA基因序列构建了系统发生树，发现由刻点血蜱传播的大巴贝斯虫伊犁株与由长角血蜱传播的3株卵形巴贝斯虫存在明显差别，应属于2个独立种；由小亚璃眼蜱传播的牛巴贝斯虫未定种不同于目前已报道的任何种类，在中国应为一个新种。因而，中国存在5种牛的巴贝斯虫，即：牛巴贝斯虫，双芽巴贝斯虫、大巴贝斯虫，卵形巴贝斯虫和巴贝斯虫未定种。     

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。     

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。     

克拉玛依地区牛焦虫病蜱媒鉴定

克拉玛依地区有牛5000多头,近年来连续暴发牛焦虫病,发病率在20％左右,尽管每年给牛打牛焦虫疫苗,对发病牛进行治疗,死亡率仍高达1％。牛焦病病原有3种,分别为牛环形泰勒焦虫、牛双芽巴贝斯焦虫和牛巴贝斯焦虫;其传播媒介分别为璃眼蜱、牛蜱和硬蜱,3个属的虫体发育史不同,在防治方面采取的措施也有所不同,为了查明克拉玛依地区牛焦虫的传播媒介,     

小亚璃眼蜱源性牛环形泰勒虫裂殖子表面抗原(Tams1)基因检测及同源性分析

针对牛环形泰勒虫裂殖子表面抗原(Tams1)基因设计特异性引物,对提取的奶牛全血DNA和小亚璃眼蜱组织DNA进行环形泰勒虫病原的PCR扩增,并对PCR产物进行克隆、测序和同源性分析。结果显示,血源性和小亚璃眼蜱蜱源性环形泰勒虫Tams1基因序列大小分别为534bp和546 bp,经NCBI BLAST后发现,血液源性环形泰勒虫与环形泰勒虫印度株同源性为99%,而蜱源性环形泰勒虫与环形泰勒虫意大利株有99%的同源性,表明新疆地区存在环形泰勒虫不同株型的流行。     

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.     

Experimental transmission of an unnamed bovine Babesia by Hyalomma spp., Haemaphysalis longicornis and Boophilus microplus

Experiments were undertaken to determine the mode of transmission to cattle of an unnamed Babesia sp. by Hyalomma anatolicum anatolicum, Hyalomma detritum, Hy. rufipes koch, Haemaphysalis longicornis and Boophilus microplus. The unnamed Babesia species designated Babesia U sp. was isolated by infesting cattle with nymphs from female Hy. a. anatolicum ticks collected from Xinjiang province. Adults of laboratory reared Hy. a. anatolicum, Hy. detritum and Hy. rufipes koch were infected with Babesia U sp. by feeding on infected cattle, isolated with nymphal ticks of Hy. a. anatolicum derived from females collected from field. The experiments revealed that Hy. a. anatolicum was capable of transmitting Babesia U sp. transovarially in larval (2 of 4 calves), nymphal (6 of 6 calves) and adult (3 of 8 calves) stages, with prepatent periods of 16, 12, and 8 days, respectively, and that this Babesia was also transovarially transmitted by both the nymphal and adult stages of Hy. detritum and Hy. rufipes. Attempts to transmit this Babesia U sp. transovarially with Hae. longicornis and B. microplus, and transstadially with Hyalomma spp., were carried out, and the results proved to be negative.     

Experiments on transmission of an unidentified Theileria sp. to small ruminants with Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum

Experiments on the transmission of an unidentified Theileria sp. infective for small ruminants by Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum were carried out. Three Theileria-free batches of adult, larvae, and nymphs of laboratory reared H. qinghaiensis and Hy. a. anatolicum ticks were infected by feeding them on sheep infected with Theileria sp. The Theileria sp. was originally isolated from adult ticks of H. qinghaiensis, by inoculation of blood stabilates or tick transmission. H. qinghaiensis has been shown to be capable of transmitting the Theileria sp. infective for small ruminants transstadially to sheep and goats. The nymphs developed from the larvae engorged on the sheep infected with the parasite transmitted the pathogen to splenectomized sheep with prepatent periods of 30, 31 days, respectively; but the subsequent adult ticks of H. qinghaiensis derived from the nymphs did not transmit the pathogen to sheep. However, adults developed from the nymphs engorged on the sheep infected with the parasite transmitted the pathogen to sheep with prepatent periods of 24-27 days. The larvae, nymphs and adult ticks derived from female H. qinghaiensis ticks engorged on infected sheep were not able to transmit the parasite transovarially. The same experiments were done with Hy. a. anatolicum, but examination for presence of piroplasma of Theileria sp. from all animals were negative, demonstrating that Hy. a. anatolicum could not transmit the organism to sheep or goats.     

The effects of bovine tick resistance on the susceptibility of Hyalomma anatolicum anatolicum to infection with Theileria annulata (Ankara)

Two Bos taurus calves were made resistant to tick infestation by exposing them to approximately 500 rabbit-reared nymphs of Hyalomma anatolicum anatolicum twice at a 2-week interval. These two calves, together with a tick-susceptible control calf, were inoculated with a stabilate of Theileria annulata (Ankara). Patent infection resulted in all three calves. Seven-hundred and fifty gerbil-reared nymphs were then applied on each of these calves as well as another tick-susceptible calf that was Theileria free. This infestation was carried out on Day 8 post-inoculation. Ticks that dropped on Day 13 post-inoculation were examined to note the development of T. annulata in them and the histological changes that occurred in the gut and salivary glands. During the second phase of feeding, the gut epithelia of the ticks from the tick-resistant calves were less active. There were no notable differences in the characteristics of the developmental stages of T. annulata between the ticks from the tick-resistant calves and those from the susceptible calf. However, ticks from one calf that acquired a higher level of tick resistance were significantly less susceptible to infection by T. annulata. Bovine tick resistance therefore compromises the vector capacity of H. a. anatolicum and this may be of epidemiological significance in the endemic areas of tropical theileriosis.     

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.     

Effect of temperature on transtadial transmission of Theileria annulata in Hyalomma anatolicum anatolicum ticks.

The effect of temperature on the transtadial transmission of Theileria annulata in Hyalomma anatolicum anatolicum was studied. Variation in temperature (4-40 degrees C) had a significant effect on moulting rate of the ticks and transmission of theilerial parasites from nymphs to resultant adults. The temperatures above 40 degrees C and below 12 degrees C prevented moulting. Maximum infection levels were obtained in salivary glands of adult ticks when the infected engorged nymphs were incubated at 24-28 degrees C. The infection rate in salivary glands was assessed using a methyl green pyronin technique.     

Vaccine potential of recombinant antigens of Theileria annulata and Hyalomma anatolicum anatolicum against vector and parasite

In an attempt to develop vaccine against Hyalomma anatolicum anatolicum and Theileria annulata, three antigens were expressed in prokaryotic expression system and protective potentiality of the antigens was evaluated in cross bred calves. Two groups (grs. 1 and 4) of male cross-bred (Bos indicus × Bos taurus) calves were immunized with rHaa86, a Bm86 ortholog of H. a. anatolicum, while one group of calves (gr. 2) were immunized with cocktails of two antigens viz., surface antigens of T. annulata (rSPAG1, rTaSP). One group each was kept as negative controls (grs. 3 and 5). The animals of groups 1, 2 and 3 were challenged with T. annulata infected H. a. anatolicum adults while the animals of groups 1, 3, 4 and 5 were challenged with uninfected adult ticks. A significantly high (p<0.05) antibody responses to all the three antigens were detected in immunized calves, but the immune response was comparatively higher with rHaa86 followed by rTaSP and rSPAG1. Upon challenge with T. annulata infected ticks, animals of all groups showed symptoms of the disease but there was 50% survival of calves of group 1 while all non immunized control calves (group 3) and rSPAG1+rTaSP immunized calves died. The rHaa86 antigen was found efficacious to protect calves against more than 71.4-75.5% of the challenge infestation. The experiment has given a significant clue towards the development of rHaa86 based vaccine against both H. a. anatolicum and T. annulata.     

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.     

Detection of Theileria and Babesia species in ticks collected from cattle

The present study was carried out to detect tick species that infest cattle, and Theileria and Babesia species transmitted by these ticks in Kayseri province (Turkey). A total of 300 cattle were examined for tick infestations. Of the 300 cattle, 117 (39%) were infested with ticks. A total of 1160 ticks belonging to 11 Ixodid genera were collected from the infested animals and their shelters. The most prevalent tick species was Boophilus annulatus 26.37% (306/1160) followed by Hyalomma marginatum marginatum 21.12% (245/1160) and Rhipicephalus turanicus 18.7% (217/1160). The collected ticks were separated into 43 tick pools, according to their species. These pools were examined for bovine Theileria and Babesia species (Theileria sp., Babesia sp., Theileria annulata, T. buffeli/orientalis, Babesia bigemina, B. bovis and B. divergens) by using the reverse line blotting method (RLB). Of the 43 tick pools examined, 6 (14%) were infected with B. bigemina, 4 (9.3%) with T. annulata, and 1 (2.3%) with Babesia sp., whereas 1 (2.3%) displayed mixed infection with T. annulata + B. bigemina. The sequence and phylogenetic analyses of Babesia sp., which could not be identified to the species level by RLB, were performed. In the phylogenetic tree, Babesia sp. (Kayseri 1) grouped with Babesia sp. (Kashi 2), Babesia sp. (Kashi 1), Babesia sp. (Xinjiang) and B. orientalis with 96.8-100% identity.     

A comparison of susceptibilities to infection of four species of Hyalomma ticks with Theileria annulata

In this comparative study unfed nymphs of four Hyalomma tick species (Hyalomma anatolicum anatolicum, Hyalomma anatolicum excavatum, Hyalomma detritum and Hyalomma marginatum marginatum) were allowed to engorge on calves experimentally infected with Theileria annulata. The infection prevalence in the salivary glands of the adult female and male ticks of each Hyalomma species used in the study were assessed. The infection prevalence with T. annulata was high and did not vary markedly in the four Hyalomma tick species. The mean number of infected acini per tick in female and male ticks was different with female ticks having higher numbers of infected acini than the male ticks. The sex difference was more significant between H.a. anatolicum and H.a. excavatum than between H. detritum and H.m. marginatum. This study clarifies the roles of four Hyalomma tick species, and their sex, in the development of T. annulata.