豫西地区莱姆病血清学及病原学研究

目的　了解豫西地区有关人群血清中是否存在抗莱姆病螺旋体抗体及可能存在的生物媒介。方法　采用间接或直接免疫荧光技术 ,对人血清相关抗体及蜱中肠带菌情况进行研究。结果　豫西地区人群中存在莱姆病自然感染 ,人群感染率为 13.5 3% ;蜱类调查显示 ,长角血蜱和微小牛蜱的中肠带菌阳性率为 2 6 .0 0 % ,这两种蜱可能为传播媒介 ;并从褐家鼠和黄胸鼠的血清中也检测到莱姆病抗体。结论　初步显示豫西地区存在莱姆病疫源地。     

镰形扇头蜱半饱血雌蜱cDNA文库的构建

目的 为了筛选抗蜱及蜱传疾病基因工程疫苗的抗原候选分子,构建了镰形扇头蜱半饱血雌蜱cDNA表达文库。方法 用Frizol试剂提取镰形扇头蜱半饱血雌蜱总RNA,经反转录合成cDNA第一链,应用长链PCR(LD-PCR)扩增方法,合成双链cDNA。双链cDNA(ds cDNA)在Sfi I内切酶的作用下,形成两端分别带有Sfi I A和Sfi I B的粘性末端。经CHROMA SPIN-400柱纯化,收集500bp以上的双链cDNA片段,将其连接于带有Sfi工A和Sfl I B末端的λTriplEx2噬菌体载体,经体外包装后,以XL1-Blue为受体菌构建cDNA噬菌体表达文库。结果 经测定,库容量为4.2×106,重组率为96％。扩增后的文库滴度为2.04×1010pfu/ml。通过对文库中随机挑选的15个克隆进行序列分析,获得一个编码线粒体ATP酶第6亚基蛋白的全长cDNA序列。结论 结果显示,镰形扇头蜱半饱血雌蜱cDNA表达文库已构建成功。     

Transovarial Transmission of Babesia ovis by Rhipicephalus sanguineus and Hyalomma marginatum

Background

Rhipicephalus sanguineus and Hyalomma marginatum are the most common species in sheep herds in Northeast of Iran. There is preliminary evidence that these species may be the vectors of Babesia ovis in Iran. We carried out two experiments in Mashhad area, Khorasan Razavi Province to determine whether B. ovis could be transovarially transmitted by R. sanguineus and H. marginatum.

Methods

In experiment 1, adults of laboratory reared H. marginatum and R.sanguineus were infected with B. ovis isolated from naturally infected sheep in Mashhad area by feeding the ticks on the sheep inoculated intravenously by infected blood samples. The inoculated sheep showed clinical signs with parasitaemia while the adult ticks were engorging on them. The engorged females were collected and kept at 28°C and 85% relative humidity in incubator. Then, larval, nymphal and adult stages derived from engorged females were used to infest the clean sheep. In experiment 2, two splenectomized sheep were infested only with the same adult ticks of two species.

Results

Examination of smears and PCR of blood samples to detect of B. ovis in infested sheep in two experiments were negative.

Conclusion

It seems that R. sanguineus and H. marginatum can not transovarially transmit B. ovis in sheep.     

A comparison of the efficacy of two commercial acaricides (fipronil and amitraz) with Azadirachta indica (neem) on the brown dog tick (Rhipicephalus sanguineus) from canines in Trinidad

The brown dog tick (Rhipicephalus sanguineus) is prevalent on canids in Trinidad. It is directly (by causing anaemia) and indirectly (by acting as a vector of tick‐borne pathogens) responsible for morbidity and mortalities in the canine population. The most commonly used commercial acaricides available to pet owners in Trinidad are amitraz and fipronil. Often, these acaricides may be abused and misused in a desperate attempt to rid pets of ticks. The objective of this study was to compare the efficacy of amitraz and fipronil with the herbal alternative, neem (Azadirachta indica). Triplicate in vitro trials utilizing the Larval Packet Test (LPT) were conducted using three concentrations (low, recommended and high) of fipronil (0.025%, 0.05% and 0.1%), amitraz (0.01%, 0.02% and 1%), neem oil (10%, 20% and 40%) and neem leaf extract (0.25%, 0.5% and 2%) for each trial. Statistical analysis using the mixed‐effect Poisson regression analysis indicated that there was a significant difference (p < .05) in the survival of ticks pre‐treatment versus post‐treatment with amitraz, fipronil and all controls when compared to the neem oil. Fipronil and amitraz caused ≥99% mortality for all concentrations used in this study. Mortalities for neem oil and neem leaf extract ranged from 72.7% to 82% and 38% to 95.3%, respectively, with the greatest percentage of mortalities occurring at the lower concentrations. Neem oil and neem leaf extract can be used as alternative acaricides, and however, they are less efficacious against the brown dog tick than amitraz and fipronil.     

海南地区血红扇头蜱的生物学特性及对宿主的影响

目的 对野外采集的蜱进行形态学与分子生物学鉴定,确定种属并完成该蜱的实验室人工饲养,研究该蜱虫的生物学特性及其对宿主的影响。方法 在体视镜显微镜下观察蜱的形态学特征,并结合分子生物学的方法扩增蜱虫线粒体16srRNA目的基因,进一步确定蜱的种属;利用改进的实验室人工蜱喂养系统在新西兰白兔体表寄生蜱虫,分析其各时期的形态学变化和生活史特性,最后用全自动血细胞分析仪测定寄生与未寄生新西兰白兔血液的14项血常规指标。结果 根据蜱形态学分析,雌雄蜱的假头基呈六角扇形,腹部肛沟明显,雌蜱腹部可看到明显的生殖孔和肛门,具有明显的缘垛;PCR扩增16srRNA 1.5%琼脂糖凝胶在约460 bp处有目的条带,确定野外采集蜱为血红扇头蜱（Rhipicephalus sanguineus）。在相对温度28 ℃,相对湿度（90±5）%的实验室环境下,血红扇头蜱发育1个完整的世代需要61~83 d,并表现出宿主单一性,即在不更换宿主的情况下,幼蜱、若蜱和成蜱均在新西兰白兔上寄生; 新西兰白兔寄生蜱6次后,体质量和体温降低,白细胞数（WBC）、淋巴细胞百分比率（LYM）等升高。结论 在相对温度28 ℃,相对湿度（90±5）%的实验室环境下,血红扇头蜱完成一个世代的发育需要61~83 d,并且寄生后新西兰兔表现出体质量体温降低,有炎性感染、轻度脱水和贫血症状。     

Molecular detection and genetic diversity of Bartonella species in large ruminants and associated ectoparasites from the Brazilian Cerrado

Currently, five Bartonella species and an expanding number of Candidatus Bartonella species have globally been reported in ruminants. Likewise, different Bartonella genotypes were identified. However, studies relating to ruminant‐associated Bartonella in Brazil are scarce. The current study aimed to assess the prevalence and genetic diversity of Bartonella in cattle, buffaloes and associated ectoparasites in Brazil. For this purpose, EDTA‐blood samples from 75 cattle and 101 buffaloes were sampled. Additionally, 128 Rhipicephalus microplus and one Amblyomma sculptum ticks collected from cattle, and 197 R. microplus, one A. sculptum and 170 lice (Haematopinus tuberculatus) collected from buffaloes were included. Bartonella DNA was initially screened through an HRM real‐time PCR assay targeting the 16S–23S internal transcribed spacer (ITS), and the positive samples were submitted to an additional HRM assay targeting the ssrA gene. The HRM‐positive amplicons were sequenced, and the nucleotide identity was assessed by BLASTn. Bartonella spp.‐positive DNA samples were analysed by conventional PCR assays targeting the gltA and rpoB genes, and then, the samples were cloned. Finally, the phylogenetic positioning and the genetic diversity of clones were assessed. Overall, 21 of 75 (28%) cattle blood samples and 13 of 126 (10.3%) associated ticks were positive for Bartonella bovis. Out of 101 buffaloes, 95 lice and 188 tick DNA samples, one (1%) buffalo and four (4.2%) lice were positive for Bartonella spp. Conversely, none of the ticks obtained from buffaloes were positive for Bartonella. The Bartonella sequences from buffaloes showed identity ranging from 100% (ITS and gltA) to 94% (ssrA) with B. bovis. In contrast, the Bartonella DNA sequences from lice were identical (100%) to uncultured Bartonella sp. detected in cattle tail louse (Haematopinus quadripertusus) from Israel in all amplified genes. The present study demonstrates the prevalence of new B. bovis genotypes and a cattle lice‐associated Bartonella species in large ruminants and their ectoparasites from Brazil. These findings shed light on the distribution and genetic diversity of ruminant‐ and ectoparasite‐related Bartonella in Brazil.     

Experimental vaccination of sheep and cattle against tick infestation using recombinant 5′‐nucleotidase

Limited prior evidence suggests that 5′‐nucleotidase, an ectoenzyme principally located in the Malpighian tubules of the tick Rhipicephalus (Boophilus) microplus, could be an effective antigen in an anti‐tick vaccine. To assess this, recombinant 5′‐nucleotidase was expressed in Escherichia coli and used in vaccination trials with both sheep and cattle. Vaccinated sheep were challenged with freshly moulted adult ticks. Those with high titres of anti‐nucleotidase antibodies showed significant protection against tick infestation, although protection was less than that found with the previously characterized antigen, Bm86. Cattle were vaccinated, in separate groups, with 5′‐nucleotidase, Bm86 and both antigens combined. Cattle, as the natural host, were challenged with larval ticks. Although Bm86 showed typical efficacy, no significant protection was seen in cattle vaccinated with 5′‐nucleotidase. Cattle receiving a dual antigen formulation were no better protected than those receiving Bm86 alone. One possible reason for the difference between host species, namely antibody titre, was examined and shown to be an unlikely explanation. This demonstrates a limitation of using a model host like sheep in vaccine studies.     

Rickettsia sibirica isolation from a patient and detection in ticks, Portugal

We report the first isolation of Rickettsia sibirica (strain mongolotimonae) from the blood of a patient and detection by polymerase chain reaction (PCR) of the rickettsia in a Rhipicephalus pusillus tick collected from a dead mongoose (Herpestes ichneumon) in the Alentejo region, Portugal. We describe also the first PCR detection of a new Rickettsia strain that is related to R. sibirica.