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。     

气候变化对我国微小扇头蜱分布区的影响

目的 分析影响我国微小扇头蜱地理分布的环境因子及微小扇头蜱在我国的潜在适生区,分析气候变化对我国微小扇头蜱分布的影响.方法 检索我国微小扇头蜱地理分布相关的国内外公开发表文献,提取地理位置信息.结合环境因子,应用ArcGIS 10.7软件和最大熵模型,对微小扇头蜱在我国的适生区分布和影响其分布的主导环境因子进行预测.结...     

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.     

镰形扇头蜱IgG结合蛋白基因的克隆与表达分析

本实验以镰形扇头蜱吸血前后雄蜱唾液腺的抑制消减杂交cDNA文库中的EST数据为基础,应用RACE方法从镰形扇头蜱体内克隆出一个IgG(IgG binding protein,IGBP)基因的全长序列,该基因全长683bp,编码178个氨基酸,预测分子量为19.5kDa,经同源性比较该基因与具尾扇头蜱的IGBP-MB基因序列的同源性高达92%。用RT-PCR方法分析了该基因表达的性别特异性、各个器官、不同发育阶段的表达情况,结果表明,该基因表达没有性别特异性;IGBP基因在唾液腺、壳这2个器官有所表达,但在肠中却没有表达;在蜱的各个发育阶段均有表达。     

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.