Molecular Prevalence and Genotype Detection of Babesia microti in Multiple Wild Rodent Species and Suncus murinus From Subtropical Mountainous Regions of South-Central Zhejiang, China.

Human babesiosis, an emerging zoonosis with ongoing geographic expansion, is primarily caused by Babesia microti, a tick-borne apicomplexan parasite that resides within host erythrocytes. While small mammals serve as its recognized reservoir hosts, the relative contribution of sympatric wild rodents and shrews in subtropical mountain endemic areas of Eastern China remains poorly characterized. In South-central Zhejiang, an endemic focus of human babesiosis, systematic data on local small-mammal hosts remain limited. In this study, 630 small mammals, comprising six rodent species and the shrew Suncus murinus, were captured across six counties in Zhejiang Province of China. Host identity was confirmed by morphological examination and mitochondrial cytochrome b (cytb) barcoding. Whole-blood DNA was screened for B. microti by TaqMan real-time quantitative PCR (qPCR); positive specimens were further analyzed by nested PCR (NPCR) targeting the 18S rRNA gene, followed by sequencing for genotype identification. The overall qPCR-positive rate was 3.0% (19/630). Geographically, B. microti infections were detected in four out of six surveyed counties, with Jinyun County showing the highest prevalence of 6.9%, followed by Qingyuan (4.0%), Wencheng (3.2%), and Qingtian (3.0%); no positive samples were identified in the Yunhe and Jingning counties. Among the sampled species, Niviventer lotipes showed the highest prevalence (11.7%, 12/103), followed by S. murinus (3.8%, 2/52), Alexandromys fortis (3.7%, 3/81), Apodemus agrarius (1.0%, 1/99), and Rattus norvegicus (0.5%, 1/222). No positive results were detected in R. tanezumi (n = 62) or Leopoldamys edwardsi (n = 11). Fifteen of the 19 samples yielded positive amplification by NPCR and were successfully sequenced. All 15 sequenced isolates were assigned to the Kobe genotype and exhibited close genetic relatedness to previously reported local human-derived and tick-derived strains. The dominance of a single haplotype (n = 14) across host species and counties indicates genetically homogeneous circulation of the Kobe genotype within local small-mammal communities, with host species rather than sampling county as the significant determinant of infection risk. These findings suggest that N. lotipes may be an important reservoir host in this region, and shrews may represent understudied secondary hosts, supporting the inclusion of both rodents and shrews in regional babesiosis surveillance.