Three novel octonucleated Entamoeba lineages in pet fancy rats (Rattus norvegicus) in China: evidence from...

Entamoeba spp. are anaerobic protists primarily inhabiting the digestive tracts of vertebrates, some of which are pathogenic or zoonotic. However, genetic information on Entamoeba spp. in rodents, including rats, remains limited. This study investigated the prevalence and molecular characterization of Entamoeba in pet fancy rats in China. Fecal samples were collected from a total of 152 fancy rats from four pet shops in two provinces of China. Microscopy and PCR targeting the small subunit ribosomal RNA (SSU rRNA) gene were used for detection. Lineage differentiation was evaluated using compensatory base change (CBC) analysis of the SSU rRNA secondary structure, with base-pair assignments anchored to the published Entamoeba muris AB445018 secondary-structure model. Phylogenetic analysis was applied to infer the evolutionary relationships of the identified lineages. The overall prevalence of Entamoeba was 8.6% (13/152). Three distinct conditional lineages (CLs) were identified, designated Entamoeba CL-Rat1 (n = 4), CL-Rat2 (n = 5), and CL-Rat3 (n = 4). At least one manually verified CBC was detected in each pairwise comparison among the three lineages and between each lineage and the included reference taxa, supporting their recognition as independent evolutionary lineages. Notably, six CBCs differentiated CL-Rat1 from its closest relative RL11, two CBCs separated CL-Rat3 from E. muris AB445018, and CL-Rat2 was separated from all other octonucleated reference taxa by one or more CBCs. Phylogenetic analyses further resolved their evolutionary positions: CL-Rat1 clustered closely with Entamoeba RL11 from a field vole, with 18.4-18.7% sequence divergence; CL-Rat2 represented a deeply divergent octonucleated lineage, showing >27.3% sequence divergence from all other octonucleated taxa; CL-Rat3 was placed as sister to the E. muris reference sequence AB445018, with 11.9-12.3% sequence divergence. The three lineages showed similar cyst morphology, although CL-Rat1 cysts were slightly larger and possessed a near-central karyosome. No zoonotic Entamoeba histolytica/Entamoeba dispar DNA was detected. This is the first report on molecular characterization of octonucleated Entamoeba in pet fancy rats in China. These results reveal considerable cryptic genetic diversity among rodent Entamoeba and support the evolutionary distinctness of the three identified lineages.