Ben Said Mourad, Belkahia Hanène, El Mabrouk Narjesse, Saidani Mariem, Alberti Alberto, Zobba Rosanna, Cherif Amal, Mahjoub Tarek, Bouattour Ali, Messadi Lilia
Laboratoire de Microbiologie, Ecole Nationale de Médecine Vétérinaire, Institution de la Recherche et de l'Enseignement Supérieur Agricoles, Université de La Manouba, Sidi Thabet, Tunisia.
Dipartimento di Medicina Veterinaria, Università degli Studi di Sassari, Sassari, Italy.
Infect Genet Evol. 2017 Apr;49:226-233. doi: 10.1016/j.meegid.2017.01.023. Epub 2017 Jan 24.
Molecular diagnosis of Anaplasma platys and related strains (A. platys-like) in carnivores and ruminants is challenging due to co-infections with cross-reacting strains, and require post-amplification sequencing of the hemi-nested PCR products traditionally generated by targeting the groEL gene. In this study, a Restriction Enzyme Fragment Length Polymorphism (RFLP) assay coupled to hemi-nested groEL PCR was developed to discriminate among A. platys and genetically related strains. This novel approach was used for investigating A. platys-like infection in 963 domesticated ruminants (241 goats, 355 sheep, and 367 cattle) from 22 delegations located in North Tunisia. Overall prevalence rates of A. platys-like were 22.8, 11, and 3.5% in goats, sheep, and cattle, respectively. Alignment, identity comparison, and phylogenetic analysis of the groEL sequence variants obtained in this study confirmed RFLP data suggesting that Tunisian ruminants are infected by novel unclassified Anaplasma strains genetically related to A. platys. Compared to sequencing, RFLP assay allows fast detection of A. platys and A. platys-like pathogens in the same sample and has a potential value especially when screening ticks, cats and ruminants, which can be a common host for these two bacteria. This newly developed molecular technique would provide valuable molecular tool for epidemiological studies related to A. platys as well as remove concern over specificity of serological and molecular methods routinely used to identify diverse Anaplasma strains and species in wild and domestic ruminants.
由于与交叉反应菌株的共感染，对食肉动物和反刍动物中的扁平无形体及相关菌株（类扁平无形体）进行分子诊断具有挑战性，并且需要对传统上通过靶向groEL基因生成的半巢式PCR产物进行扩增后测序。在本研究中，开发了一种与半巢式groEL PCR相结合的限制性酶切片段长度多态性（RFLP）分析方法，以区分扁平无形体和遗传相关菌株。这种新方法用于调查来自突尼斯北部22个代表团的963头家养反刍动物（241只山羊、355只绵羊和367头牛）中的类扁平无形体感染情况。类扁平无形体在山羊、绵羊和牛中的总体患病率分别为22.8%、11%和3.5%。对本研究中获得的groEL序列变异进行比对、同一性比较和系统发育分析，证实了RFLP数据，表明突尼斯反刍动物感染了与扁平无形体遗传相关的新型未分类无形体菌株。与测序相比，RFLP分析能够在同一样本中快速检测扁平无形体和类扁平无形体病原体，尤其在筛查蜱、猫和反刍动物时具有潜在价值，因为这些动物可能是这两种细菌的共同宿主。这种新开发的分子技术将为与扁平无形体相关的流行病学研究提供有价值的分子工具，同时消除对常规用于鉴定野生和家养反刍动物中各种无形体菌株和物种的血清学和分子方法特异性的担忧。
