Pucciarelli Alessia, Cardillo Lorena, Viscardi Maurizio, Picazio Gerardo, D'Alessio Nicola, Sgroi Giovanni, Rinaldi Antonio, Veneziano Vincenzo, Fusco Giovanna, de Martinis Claudio
Unit of exotic and vector-borne diseases, Department of Animal Health, Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute, 2, Portici, Naples, 80055, Italy.
Unit of Wildlife and Regional Wildlife Observatory, Department of Animal Health, Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute, 2, Portici, Naples, 80055, Italy.
Vet Res Commun. 2025 Jul 1;49(4):240. doi: 10.1007/s11259-025-10808-7.
Candidatus Midichloria mitochondrii (Ca. M. mitochondrii), an endosymbiont intracellular bacterium living in the mitochondria of several tick species, especially ixodid species, has been proposed as a potential marker for monitoring tick-bite exposure. Therefore, the present study aimed to develop two different diagnostic methods, TaqMan-based real-time PCR (rt-PCR) and Droplet Digital PCR (dd-PCR), targeting the 16 S rRNA gene and gyrB gene for the detection of Ca. M. mitochondrii in different wildlife species from several areas of southern Italy. Both techniques were validated using 10-fold serial dilutions of a sequenced positive control up to reach 10 final dilution. Among wildlife field samples, both the techniques identified Ca. M. mitochondrii DNA, although dd-PCR showed higher sensitivity, being able to detect the target DNA in a higher dilution and in several spleen samples scored negative by rt-PCR. Noteworthy, these molecular methods revealed for the first time the presence of Ca. M. mitochondrii DNA in red foxes (Vulpes vulpes), Eurasian badgers (Meles meles), otters (Lutra lutra), porcupines (Hystrix cristata), European hares (Lepus europaeus), and alpacas (Vicugna pacos), suggesting a tick-bite exposure of these animals in the study area.
“拟线粒体中绿菌”（Ca. M. mitochondrii）是一种胞内共生细菌，存在于几种蜱虫的线粒体中，尤其是硬蜱科物种，它已被提议作为监测蜱虫叮咬暴露的潜在标志物。因此，本研究旨在开发两种不同的诊断方法，即基于TaqMan的实时荧光定量PCR（rt-PCR）和数字PCR（dd-PCR），针对16S rRNA基因和gyrB基因来检测意大利南部几个地区不同野生动物物种中的“拟线粒体中绿菌”。两种技术均使用测序阳性对照进行10倍系列稀释直至达到10个最终稀释度来进行验证。在野生动物野外样本中，两种技术均检测到了“拟线粒体中绿菌”的DNA，尽管dd-PCR显示出更高的灵敏度，能够在更高的稀释度下以及在rt-PCR判定为阴性的几个脾脏样本中检测到目标DNA。值得注意的是，这些分子方法首次揭示了在赤狐（Vulpes vulpes）、欧亚獾（Meles meles）、水獭（Lutra lutra）、豪猪（Hystrix cristata）、欧洲野兔（Lepus europaeus）和羊驼（Vicugna pacos）中存在“拟线粒体中绿菌”的DNA，这表明这些动物在研究区域内曾被蜱虫叮咬。
