Introduction

Rickettsia felis is the causative agent of flea-borne spotted fever (FBSF), an emerging zoonosis. Although there is evidence of FBSF in Greece, fleas, the classic vectors of R. felis, have not been adequately studied. Thus, the aim of this study was to detect and characterize bacteria of genus Rickettsia and especially R. felis from common fleas parasitizing domestic cats and dogs in Greece and evaluate the efficiency of established molecular techniques.

Materials and methods

DNA of flea-pools (samples) by animal-host was investigated by quantitative real-time PCRs (qPCR), and 16S metagenomics (16S). Determination of Rickettsia spp., Rickettsia felis-like organisms (RFLOs), and R. felis was based on a combination of qPCRs targeting gltA and ompB genes, 16S automated metagenomics and manual comparison of 16S sequences for >99% similarity with the publicly available 16S R. felis GenBank sequences using the Basic Local Alignment Search Tool (BLAST_>99). Information for the animal-hosts was available and statistically analyzed.

Results

Among 100 flea-pools, R. felis was detected in 14 samples with a combination of six, five and three assays in 10, two and two samples, respectively. The sensitivity of the assays for Rickettsia genus (16S, and genus specific qPCRs) ranged from 62.5% to 93.8% and the specificity from 65.0% to 100%. R. felis-targeting qPCRs for gltA and ompB demonstrated sensitivity and specificity of 92.9% and 100%, and 100.0% and 87.5%, respectively. 16S metagenomics using the assay software was not able to identify R. felis positive specimens, although manual BLAST_>99 did identify the species, but demonstrated sensitivity of 92.9% and specificity of 65.0%. No association of the detection rate of Rickettsia genus or R. felis, with the epidemiological data collected, was identified.

Conclusions

These observations suggest the occurrence of R. felis in fleas from pets in Attica, Greece, but PCR and sequencing assays varied considerably in sensitivity and specificity and a consensus methodology for assigning the positivity status is required to be established.

中文翻译：

---

希腊普通跳蚤中猫立克次体的分子检测及基因型方法的比较评价

介绍

猫立克次体是跳蚤传播斑疹热 (FBSF) 的病原体，这是一种新出现的人畜共患病。虽然在希腊，跳蚤FBSF的证据，经典矢量R.猫，都没有得到充分的研究。因此，本研究的目的是检测和表征立克次体属的细菌，尤其是来自寄生于希腊家猫和狗的普通跳蚤的R. felis，并评估已建立的分子技术的效率。

材料和方法

通过定量实时 PCR (qPCR) 和 16S 宏基因组学 (16S) 研究了动物宿主的跳蚤池（样品）的 DNA。的测定立克次氏体属，立克次氏体蚤样生物（RFLOs），和R.蚤基于qPCRs靶向的组合的gltA和OMPB基因，16S自动宏基因组学和16S序列的手工对比为> 99％的相似性与公开可用的16S R. felis GenBank 序列使用基本局部比对搜索工具 (BLAST _>99 )。可以获得动物宿主的信息并进行统计分析。

结果

在 100 个跳蚤池中，分别在 10 个、两个和两个样本中进行了六、五和三种检测的组合，在 14 个样本中检测到了R. felis。立克次体属（16S 和属特异性 qPCR）的检测灵敏度为62.5% 至 93.8%，特异性为 65.0% 至 100%。gltA和ompB 的R. felis靶向 qPCR 的灵敏度和特异性分别为 92.9% 和 100%，以及 100.0% 和 87.5%。使用检测软件的 16S 宏基因组学无法识别R. felis阳性标本，尽管手动 BLAST _>99确实识别了物种，但表现出 92.9% 的敏感性和 65.0% 的特异性。没有发现立克次体属或猫立克次氏体的检出率与收集到的流行病学数据之间存在关联。

结论

这些观察结果表明，在希腊阿提卡的宠物跳蚤中存在R. felis，但 PCR 和测序分析在灵敏度和特异性方面差异很大，需要建立一种确定阳性状态的共识方法。