Molecular Detection, Epidemiology and Genetic Diversity of Babesia microti Infecting Wild Rodents

Abstract

BACKGROUND: Rodents are considered an important natural reservoir of many zoonotic infectious agents, including parasitic Apicomplexa. To assess the potential risk of rodent-borne parasitic protozoa in Pakistan, the present study was designed to determine the molecular prevalence and phylogenetic characteristics of Babesia microti in blood samples collected from Rattus rattus (N = 137), Mus musculus (N = 67) and Rattus norvegicus (N = 80). METHODS: Rodents were trapped from seven districts of Pakistan between October 2023 and September 2024. PCR amplified a 238 bp fragment from the 18S rRNA gene of B. microti in 35 out of 284 (12%) of the screened rats and mice. DNA sequencing, BLAST and phylogenetic analysis were performed to confirm the presence and the phylogenetic similarity of the isolates. Complete blood counts were also analysed to evaluate haematological changes. RESULTS: All three rodent species were found infected with this parasite but no significant association was observed between infection status and rodent sex or species. Statistical analysis showed that the prevalence of B. microti in R. norvegicus differed significantly between the sampling sites (p = 0.01), ranging from 0% in Buner, Dera Ghazi Khan and Rajanpur to 42% in Sargodha. DNA sequencing and BLAST analysis confirmed the presence of B. microti in rodent blood samples, with 100% sequence similarity to GenBank reference sequences. Phylogenetic analysis showed that Pakistani isolates were genetically similar and clustered with the 18S rRNA nucleotide sequences that were isolated from rodents in France, Russia and Japan. B. microti-infected rodents had disturbed total white and red blood cell counts and associated parameters. CONCLUSIONS: In conclusion, we are reporting a moderate prevalence of B. microti in three wild Pakistani rodent species for the very first time. Further epidemiological studies are required to evaluate the potential zoonotic risk posed by B. microti-infected rodents in Pakistan.