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With over 1,400 species worldwide, bats represent the second largest order of mammals after rodents, and are known to host major zoonotic pathogens. Here, we estimate the presence of pathogens in autochthonous bat populations. First, we set out to check our samples for PCR amplification efficiency by assessing the occurrence of inhibited PCR reactions from different types of bat samples with amplifying the housekeeping gene β-actin. Second, we investigated the presence of five targeted pathogens in a French bat population using PCR. We targeted viral RNA of Canine distemper virus, Alphacoronavirus, Lyssavirus, Rotavirus and bacterial Leptospira DNA. To do so, we screened for these viruses in bat faecal samples as well as in oropharyngeal swab samples. The presence of Leptospira was assessed in urine, kidney, lung and faecal samples. Results showed a frequency of inhibited reactions ranging from 5 to 60% of samples, varying according to the sample itself and also suspected to vary according to sampling method and the storage buffer solution used, demonstrating the importance of the sampling and storage on the probability of obtaining negative PCR results. For pathogen assessment, rotavirus and alphacoronavirus RNA were detected in Myotis myotis, Myotis daubentonii, Myotis emarginatus and Rhinolophus ferrumequinum bats. Rotaviruses were also detected in Barbastella barbastellus. The presence of alphacoronavirus also varied seasonally, with higher frequencies in late summer and October, suggesting that juveniles potentially play an important role in the dynamics of these viruses. Leptospira DNA was detected in M. myotis and M. daubentonii colonies. The 16S rRNA sequences obtained from Leptospira positive samples showed 100% genetic identity with L. borgpetersenii. Neither canine distemper virus nor lyssavirus RNA were detected in any of the tested samples. This study is the first to show the presence of Leptospira in autochthonous French bats in addition to coronavirus and rotavirus RNA previously reported in European autochthonous bats.全球范围内现有超过1400个蝙蝠物种，是仅次于啮齿类的第二大哺乳动物类群，且已知其可携带重大人兽共患病原体。 本研究旨在评估本土蝙蝠种群中的病原体存在情况：首先，我们通过靶向扩增持家基因β-肌动蛋白（β-actin），检测不同类型蝙蝠样本中的PCR抑制反应发生率，以此校验样本的PCR扩增效率；其次，我们采用PCR技术，对法国本土蝙蝠种群中的5种目标病原体开展检测。本次靶向检测的病原体包括：犬瘟热病毒（Canine distemper virus）的病毒RNA、甲型冠状病毒（Alphacoronavirus）、狂犬病病毒属（Lyssavirus）、轮状病毒（Rotavirus），以及钩端螺旋体（Leptospira）的细菌DNA。为完成该检测，我们分别对蝙蝠粪便样本与口咽拭子样本进行了上述病毒的筛查；针对钩端螺旋体，则同步检测了尿液、肾脏、肺脏及粪便样本。 检测结果显示，PCR抑制反应的发生率介于5%至60%之间，其比例因样本类型而异，且推测同时受采样方法及所用保存缓冲液的影响，这证实了采样与保存流程对获得阴性PCR结果概率的重要性。在病原体检测层面，研究在鼠耳蝠（Myotis myotis）、道氏鼠耳蝠（Myotis daubentonii）、凹缘鼠耳蝠（Myotis emarginatus）以及大菊头蝠（Rhinolophus ferrumequinum）体内检出了轮状病毒与甲型冠状病毒RNA；轮状病毒还在欧洲山蝠（Barbastella barbastellus）体内被检出。甲型冠状病毒的检出率存在季节性差异，夏末及10月的检出率更高，这提示幼体可能在这类病毒的传播动力学中发挥关键作用。本研究还在M. myotis与M. daubentonii的蝙蝠群落中检出了钩端螺旋体DNA，从该类阳性样本中获得的16S rRNA序列，与博格霍夫钩端螺旋体（L. borgpetersenii）的遗传同源性达100%。所有受试样本中均未检出犬瘟热病毒与狂犬病病毒属的RNA。本研究为首次在法国本土蝙蝠体内检出钩端螺旋体，同时也在欧洲本土蝙蝠此前已报道的冠状病毒与轮状病毒RNA基础上，补充了相关检测结果。