新疆南部图兰扇头蜱及卵携带R.raoultii的分子检测

【目的】世界上蜱类3科18属899种,中国有2科10属117种,新疆至少有2科10属45种,占全国蜱种类的1/3之多,分布也极其广泛。蜱直接危害和传播多种病原,且一些病原可经卵垂直传播,给畜牧业造成巨大经济损失,还严重威胁公共卫生安全。图兰扇头蜱是新疆南部荒漠及半荒漠地区常见种和优势种。确定新疆南部图兰扇头蜱及其卵是否携带立克次体,对该蜱及其传播立克次体病的防控意义重大。【方法】对新疆生产建设兵团塔里木畜牧科技重点实验室在新疆南部阿拉尔市某羊场收集的饱血雌性扇头蜱,置于一定湿度和一定温度的环境中产卵,随机取5个独立的卵样品及其对应的5只雌蜱为研究对象。通过对雌蜱及其卵分别处理后提取基因组DNA,进行PCR扩增蜱12S r RNA基因和立克次体16S r RNA基因,并对扩增产物测序,利用BLAST在线平台和多个分子生物学软件进行序列分析。【结果】5只雌蜱12S r RNA基因PCR扩增全部阳性,测序获得的4段蜱12S r RNA基因序列完全一致;Blast分析与Gen Bank数据库中图兰扇头蜱12S r RNA基因序列相似性高达99%以上,且高相似性前五基因序列均为图兰扇头蜱,其中包括来自新疆绵羊的图兰扇头蜱;本研究蜱12S r RNA基因序列提交Gen Bank数据库获得登录号为MG744514,与来自于Gen Bank数据库图兰扇头蜱、血红扇头蜱、微小牛蜱、边缘革蜱、草原革蜱、长角血蜱、小亚璃眼蜱、亚洲璃眼蜱、残缘璃眼蜱、全沟硬蜱及外围群尘螨的22个12S r RNA基因序列的进化树显示,研究所获得的蜱12S r RNA基因序列与图兰扇头蜱进化关系最近,聚在同一个小分支;确定了该扇头蜱为图兰扇头蜱。5只产卵后的雌蜱和相应蜱所产的全部卵立克次体16S r RNA基因PCR扩增,有1只蜱和其产的卵样品阳性,蜱携带率为20%;雌蜱及其卵立克次体16S r RNA基因测序结果完全一致;Blast分析与Gen Bank数据库中Rickettsia raoultii 16S r RNA基因序列相似性高达99%以上,且高相似性前五基因序列为4个Rickettsia raoultii和1个Rickettsia sp.,其中包括来自新疆2011年的亚洲璃眼蜱和草原革蜱的立克次体;立克次体16S r RNA基因序列提交Gen Bank数据库获得登录号为MG744513,与来自于Gen Bank数据库的37个24种立克次体16S r RNA基因序列的进化树显示,研究获得的立克次体16S r RNA基因序列与Rickettsia raoultii进化关系最近,聚在同一个小分支,与其他15种立克次体同属于斑点热群立克次体;确定了本研究图兰扇头蜱及其卵均携带斑点热群立克次体R.raoultii。【结论】首次发现图兰扇头蜱及其卵携带R.raoultii。     

镰形扇头蜱组胺结合蛋白RhHBP基因的克隆和重组表达

为了进行抗蜱及蜱传病疫苗的研究,本试验根据GenBank中的镰形扇头蜱组胺结合蛋白(Rhipicephalus haemaphysaloideshistamine bindingprotein,RhHBP)基因cDNA序列,设计一对特异性引物(内含EcoRI/XhoI酶切位点),经RT-PCR扩增了镰形扇头蜱组胺结合蛋白RhHBP的cD-NA,大小为603 bp,将其克隆到pGEM-Teasy中并测序。在去除编码RhHBP信号肽的核苷酸序列后,将其亚克隆到pGEX-4T-1中,构建重组质粒pGEX-4T-1-RhHBP,将该重组质粒转化大肠杆菌BL21,经终浓度为1 mmol/L IPTG诱导其表达,表达的融合蛋白大小为49 ku,以包涵体的形式存在。Western Blot分析表明,该蛋白具有良好的免疫原性。     

湖北省水牛巴贝斯虫病调查研究(Ⅵ)——镰形扇头蜱体内牛巴贝斯虫虫样体的观察

人工感染牛巴贝斯虫的去脾脏水牛犊2头,当血液中出现巴贝斯虫体,染虫率达0.2%时,于一耳内放入饥饿成蜱,使之吸血。雌蜱吸饱血离休后,收集并分别放在28℃相27℃的恒温箱中,每隔24小时取蜱剪肢,涂片检查。发现置28℃的蜱在72小时,其血淋巴中出现虫样体,96—120小时,虫样体出现最多,144小时仍可见到。成熟的虫样体大小为12.0—15.0×1.8—3.5μm;置37℃的蜱其血淋巴中始终未能查到虫样体。     

The interaction between the immune response of rabbits to heterologous antigens and a primary infestation with Rhipicephalus evertsi evertsi

Rhipicephalus evertsi evertsi feeding on hosts inoculated with sheep red blood cells (SRBC) and bovine serum albumin (BSA) suppressed the primary antibody response to the two antigens. In addition, while the ticks paralysed most hosts in the studies, fatality associated with this toxicosis occurred only in rabbits which had received SRBC, either alone or with BSA. Only those hosts inoculated with BSA developed any resistance against the ticks, manifested by a slight reduction of engorged weights and development of anti-tick antibodies. These results suggest that R. e. evertsi infestation induces a degree of reduced host immune responsiveness to heterologous antigens.     

Effects of a Novel Pesticide Resistance Management Strategy on Tick Control in a Smallholding Exotic-breed Dairy Herd in Kenya

Effects of a novel pesticide resistance management strategy on tick control are evaluated in this study. The study is based on a temporal analysis of tick management practices on a smallholding in western Kenya. Results are reported of an innovation to tackle individual resistance in a pair of alternative pesticides using relay application. Incidence of tick-borne diseases at the farm were reduced from 79.6% per annum to 4.5% and no cases were observed in the last two years of the study. Negative cross-resistance is believed to be the mechanism in play for this effective tick control practice. Tick-borne disease control and management costs were halved in comparison to application of a single ineffective pesticide at the same treatment frequency. The acaricide relay strategy is suitable for smallholdings and is expected to significantly extend the useful lifespan of the pesticide pair.     

Will Culling White‐Tailed Deer Prevent Lyme Disease?

White‐tailed deer play an important role in the ecology of Lyme disease. In the United States, where the incidence and geographic range of Lyme disease continue to increase, reduction of white‐tailed deer populations has been proposed as a means of preventing human illness. The effectiveness of this politically sensitive prevention method is poorly understood. We summarize and evaluate available evidence regarding the effect of deer reduction on vector tick abundance and human disease incidence. Elimination of deer from islands and other isolated settings can have a substantial impact on the reproduction of blacklegged ticks, while reduction short of complete elimination has yielded mixed results. To date, most studies have been conducted in ecologic situations that are not representative to the vast majority of areas with high human Lyme disease risk. Robust evidence linking deer control to reduced human Lyme disease risk is lacking. Currently, there is insufficient evidence to recommend deer population reduction as a Lyme disease prevention measure, except in specific ecologic circumstances.     

镰形扇头蜱-新基因的分离、克隆、表达和抗蜱免疫保护作用

目的分离、克隆镰形扇头蜱一新基因。方法本实验从镰形扇头蜱半饱血雌蜱唾液腺cDNA文库的100个EST（Expressed Sequence Tag）序列中选取一个带有polyA尾的序列，经5’RACE（Rapid Amplification of cDNA Ends）得到此序列的全长基因，命名为RhHp2，基因全长1002bp，开放阅读框（ORF）从31bp至879bp，共848bp，编码282个氨基酸。经序列比较，RhHp2基因与其它序列在核苷酸水平上基本无同源性，但在氨基酸水平上与其它物种同源性最高可达50．81％。RhHp2基因连接于PET32a（＋）载体后克隆于表达菌B121，1mM IPTG诱导表达，产生分子量约为55kD的融合重组蛋白，并以不溶性包涵体形式存在。经纯化和体外复性的重组蛋白三次免疫新西兰兔后，将镰形扇头蜱未吸血成蜱和若蜱接种于此免疫兔耳，并对接种48h后的蜱上体数、蜱的饱血数量、饱血时间、饱血重量等进行记录。结果发现蜱的吸血行为受到一定影响，若蜱在48h的减虫率（上体数减少）为58％，在整个吸血过程中的死亡数增加，成蜱的饱血体重显著降低。结论此结果表明RhHp2基因的重组表达产物具有一定的免疫保护作用。     

Effects of permethrin and amitraz on gas exchange and water loss in unfed adult females of Amblyomma americanum (Acari: Ixodidae)

Effects of two acaricides, permethrin and amitraz, with different modes of action, on gas exchange and water balance of the lone star tick, Amblyomma americanum, were examined using a flow-through carbon dioxide (CO₂) and water vapor analyzer. Untreated adult female ticks exhibited a distinct discontinuous gas exchange pattern (DGEP) with no measurable water loss. Similarly, ticks with acetone (a solvent) showed little change in gas exchange and water loss. Topical treatment of ticks with a lethal dose of permethrin caused immediate major water loss and long-lasting high frequency DGEP, with increased amplitude of CO₂ release. A small water loss spike was observed for each CO₂ release. Treatment of ticks with amitraz abolished the DGEP and elicited a long-lasting continuous gas exchange pattern, indicating significantly increased tick metabolism. No detectable water loss was observed during amitraz-elicited continuous gas exchange. Treatment of ticks with a mixture of sub-lethal doses of permethrin and amitraz caused a similar initial major water loss, as in permethrin-treated ticks, followed by a second period of water loss activity at 10–14 h after treatment. Mixture of the two acaricides caused immediate onset of major chaotic CO₂ release activities with a significantly elevated baseline CO₂ emission level. The standard metabolism rate (SMR, VCO₂) of acetone-treated ticks (0.452 μl h⁻¹) was similar to that of the untreated ticks (0.461 μl h⁻¹). Compared to untreated control ticks, permethrin-, amitraz-, and the acaricide mixture-treated ticks exhibited significantly increased SMR (1.054, 1.392 and 1.520 μl h⁻¹ respectively (P < 0.05). Results obtained from this study clearly demonstrated the detrimental effects of permethrin and amitraz on CO₂ gas exchange and water balance in A. americanum ticks. The data also revealed subtle differences between permethrin and amitraz in their effects on tick metabolism, specifically gas exchange and water loss. In addition, the study may provide insight into the modes of action of these two acaricides in tick physiology. The results may also help understand the mechanism of synergism between permethrin and amitraz that was reported previously in other ticks and insects.     

Reviewing molecular adaptations of Lyme borreliosis spirochetes in the context of reproductive fitness in natural transmission cycles

Lyme borreliosis (LB) is caused by a group of pathogenic spirochetes – most often Borrelia burgdorferi, B. afzelii, and B. garinii – that are vectored by hard ticks in the Ixodes ricinus-persulcatus complex, which feed on a variety of mammals, birds, and lizards. Although LB is one of the best-studied vector-borne zoonoses, the annual incidence in North America and Europe leads other vector-borne diseases and continues to increase. What factors make the LB system so successful, and how can researchers hope to reduce disease risk – either through vaccinating humans or reducing the risk of contacting infected ticks in nature? Discoveries of molecular interactions involved in the transmission of LB spirochetes have accelerated recently, revealing complex interactions among the spirochete-tick-vertebrate triad. These interactions involve multiple, and often redundant, pathways that reflect the evolution of general and specific mechanisms by which the spirochetes survive and reproduce. Previous reviews have focused on the molecular interactions or population biology of the system. Here molecular interactions among the LB spirochete, its vector, and vertebrate hosts are reviewed in the context of natural maintenance cycles, which represent the ecological and evolutionary contexts that shape these interactions. This holistic system approach may help researchers develop additional testable hypotheses about transmission processes, interpret laboratory results, and guide development of future LB control measures and management.