Calculated phylogenetic distances for Rickettsia.

BackgroundCtenocephalides felis is a common ectoparasite of dogs and cats and can transmit a variety of pathogens including Bartonella and Rickettsia species. These bacteria, along with the known endosymbiont Wolbachia, are well-documented members of the C. felis microbiome, but species-level information is limited. Additionally, little is known about the variation in the C. felis microbiome in fleas from different sources and when different sequencing methods are applied to the same samples.ObjectiveThis study aimed to characterize the flea microbiome using both short-read (V3/V4) and long-read (full-length) 16S rRNA gene sequencing, determine whether long-read sequencing improves species-level identification especially in known pathogenic genera, and evaluate differences in microbial composition between fleas collected from cats, dogs, and environmental traps.MethodsFleas were collected from cats, dogs, and traps in flea-infested homes in Florida, pooled by source, and sequenced using short- (V3/V4) and long-read (full-length) 16S rRNA gene sequencing. Microbial prevalence and abundance were compared across sequencing approaches. Community composition was evaluated for differences between sources and houses. Candidate members of the flea microbiome were identified based on a combination of prevalence, abundance, and statistical signatures of potential contaminant origin. For Rickettsia and Bartonella, species-level taxonomic assignments were refined using a phylogenetic approach.ResultsWolbachia, Rickettsia, and Bartonella were the most prevalent and abundant taxa. Spiroplasma was identified as a fourth core member of the flea microbiome. Long-read sequencing enabled better, but not perfect, species-level classification of Bartonella and Rickettsia compared to short-read sequencing. Important relationships between specific ASVs and flea sources were identified, for example fleas from cats harbored higher abundances of B. clarridgeiae and B. henselae than fleas from traps.【背景】猫栉首蚤（Ctenocephalides felis）是犬与猫常见的体外寄生虫，可传播巴尔通体属（Bartonella）、立克次体属（Rickettsia）等多种病原体。上述细菌与已知内共生体沃尔巴克氏体（Wolbachia）均为猫栉首蚤微生物组中被充分记录的成员，但物种级别的相关信息仍较为匮乏。此外，针对不同来源跳蚤的微生物组变异情况，以及对同一样本采用不同测序方法时的微生物组差异，目前的研究认知仍十分有限。 【研究目的】本研究旨在通过短读长（V3/V4区）16S rRNA基因测序与长读长（全长）16S rRNA基因测序两种技术表征跳蚤微生物组，探究长读长测序是否可提升物种水平的鉴定效能，尤其是针对已知致病属，并评估从猫、犬及环境诱捕器采集的跳蚤之间的微生物组成差异。 【研究方法】研究人员从美国佛罗里达州受跳蚤侵扰的住宅中，分别从猫、犬及诱捕器采集跳蚤样本，按来源进行混样后，采用上述两种16S rRNA基因测序技术完成测序。对比不同测序方法下的微生物检出率与相对丰度差异；评估不同采集来源及不同住宅间的微生物群落组成差异；结合微生物检出率、相对丰度以及潜在污染物来源的统计学特征，筛选跳蚤微生物组的候选成员；针对立克次体属与巴尔通体属，采用系统发育分析方法优化其物种水平的分类学注释。 【研究结果】沃尔巴克氏体、立克次体属及巴尔通体属为检出率与相对丰度最高的类群。螺旋体属（Spiroplasma）被鉴定为猫栉首蚤微生物组的第四个核心成员。相较于短读长测序，长读长测序可实现更优但并非完美的巴尔通体属与立克次体属物种水平分类。研究明确了特定扩增子序列变异体（Amplicon Sequence Variant，ASV）与跳蚤采集来源间的重要关联：例如，相较于诱捕器采集的跳蚤，从猫身上采集的跳蚤中克拉里奇巴尔通体（B. clarridgeiae）与亨氏巴尔通体（B. henselae）的丰度显著更高。