Service de Microbiologie et Immunologie, Ecole Nationale de Médecine Vétérinaire, Université de Manouba, 2020 Sidi Thabet, Tunisia; Institut National Agronomique de Tunis, Université de Carthage, Tunisia; Ministère de la Défense Nationale, Direction Générale de la Santé Militaire, Service Vétérinaire, Tunis, Tunisia.
Service de Microbiologie et Immunologie, Ecole Nationale de Médecine Vétérinaire, Université de Manouba, 2020 Sidi Thabet, Tunisia.
Ticks Tick Borne Dis. 2019 Oct;10(6):101260. doi: 10.1016/j.ttbdis.2019.07.004. Epub 2019 Jul 12.
In camels and their infesting ectoparasites, specific detection of pathogenic Anaplasma platys and genetically related strains (A. platys-like strains) remains problematic. This requires sequencing of the hemi-nested PCR products specific to A. platys and related strains. In this study, a PCR/RFLP method, earlier developed for specific detection of A. platys-like strains in animal species other than camels, was adapted in order to subtype A. platys-like strains isolated from camels and their ticks and to differentiate them from pathogenic A. platys without going through a sequencing step. This approach was used for investigating the infections with A. platys and related strains in 412 Camelus dromedarius camels and 334 feeding ticks from five Tunisian governorates. Microscopic examination using Giemsa-stained blood smears was performed in order to specify which types of cells were infected. Ticks were identified as Hyalomma dromedarii (n = 164, 49%), H. impeltatum (n = 161, 48.3%) and H. excavatum (n = 9, 2.7%). A. platys was not detected in any of the tested camels or ticks. The overall prevalence of A. platys-like strains was 5.6% (23/412) in camels and microscopic examination of infected cells showed a tropism for neutrophil granulocytes. One tick identified as H. dromedarii out of 327 analyzed ticks was found to be infected with A. platys-like strains (0.3%). Alignment, identity comparison and phylogenetic analysis of the 16S rRNA partial sequences obtained in this study suggest that Tunisian dromedaries and feeding ticks are infected with different Anaplasma strains genetically related to A. platys. Sequence analysis and phylogenetic study based on the groEL gene confirm the RFLP results and show that camel strains formed a separate sub-cluster relatively close to A. platys-like strains infecting Tunisian cattle. This adapted RFLP assay allows fast and specific detection of pathogenic A. platys and A. platys-like strains in camels and infesting ticks and has the intrinsic potential of revealing co-infections with these two types of bacteria in the same sample, reducing the time and costs associated with cloning and sequencing during molecular diagnosis.
在骆驼及其寄生的外寄生虫中，对致病性安氏巴贝斯虫和遗传相关菌株（安氏巴贝斯虫样菌株）的特异性检测仍然存在问题。这需要对特定于安氏巴贝斯虫和相关菌株的半巢式 PCR 产物进行测序。在这项研究中，我们对先前开发的用于检测除骆驼以外的动物物种中安氏巴贝斯虫样菌株的 PCR/RFLP 方法进行了调整，以便对从骆驼及其蜱中分离出的安氏巴贝斯虫样菌株进行亚型分类，并在不进行测序步骤的情况下将其与致病性安氏巴贝斯虫区分开来。该方法用于调查来自突尼斯五个省的 412 只单峰驼和 334 只采食蜱中的安氏巴贝斯虫和相关菌株感染情况。使用吉姆萨染色血涂片进行显微镜检查，以确定感染的细胞类型。鉴定出的蜱为：单峰驼璃眼蜱（n=164，49%）、钝缘璃眼蜱（n=161，48.3%）和荒地璃眼蜱（n=9，2.7%）。在测试的骆驼或蜱中均未检测到安氏巴贝斯虫。安氏巴贝斯虫样菌株的总流行率为 412 只骆驼中的 5.6%（23/412），感染细胞的显微镜检查显示嗜中性粒细胞趋向性。在分析的 327 只蜱中，有一只鉴定为单峰驼璃眼蜱的蜱被发现感染了安氏巴贝斯虫样菌株（0.3%）。本研究获得的 16S rRNA 部分序列的比对、同源性比较和系统发育分析表明，突尼斯单峰驼和采食蜱感染了与安氏巴贝斯虫相关的不同安氏巴贝斯虫菌株。基于 groEL 基因的序列分析和系统发育研究证实了 RFLP 结果，并表明骆驼株形成了一个相对靠近感染突尼斯牛的安氏巴贝斯虫样菌株的单独亚群。这种适应性 RFLP 检测方法可快速、特异性地检测骆驼和寄生蜱中的致病性安氏巴贝斯虫和安氏巴贝斯虫样菌株，具有在同一样本中揭示这两种类型细菌共感染的内在潜力，从而减少分子诊断中克隆和测序相关的时间和成本。
