Prevalence and Molecular Characterization of the Zoonotic Enteric Protozoans Cryptosporidium spp., Enterocytozoon bieneusi, and Blastocystis from Pallas’s Squirrels (Callosciurus erythraeus) in Kanagawa Prefecture, Japan

ABSTRACT Pallas’s squirrel (Callosciurus erythraeus) was introduced in Japan in the 1930s and has since established itself in several areas across the country. Although wild Sciuridae populations have been demonstrated to be potential reservoirs for zoonotic enteric protozoa, epidemiological studies of such pathogens in Japan are scarce. Here, we examined 423 fecal samples from Pallas’s squirrels captured in Kanagawa Prefecture, Japan, using PCR and DNA sequencing to determine the occurrence of Cryptosporidium spp., Enterocytozoon bieneusi, and Blastocystis. The overall prevalence of Cryptosporidium spp., E. bieneusi, and Blastocystis was 4.3% (18/423 samples), 13.0% (55/423 samples), and 44.0% (186/423 samples), respectively. The prevalence of Blastocystis and E. bieneusi was significantly higher in spring (60.1% and 17.4%, respectively) than in winter (27.6% and 8.6%, respectively [P < 0.01]). Sequence analysis of Cryptosporidium spp., targeting the partial small subunit ribosomal RNA gene (SSU rDNA), showed 100% identity (541/541 bp) to Cryptosporidium ubiquitum, and analysis of the gp60 gene showed 99.76% (833/835 bp) identity to C. ubiquitum subtype XIIh. The sequences of the ribosomal internal transcribed spacer region of E. bieneusi and the partial SSU rDNA of Blastocystis were identified as E. bieneusi genotype SCC-2 and Blastocystis subtype 4, respectively. This study confirmed the presence of C. ubiquitum, E. bieneusi, and Blastocystis in Pallas’s squirrels in Kanagawa Prefecture. Because Pallas’s squirrels inhabit urban areas, living close to humans, the species may serve as a potential source of infection in human populations. IMPORTANCE Pallas’s squirrel is designated a “regulated organism” under the Invasive Alien Species Act in Japan, and municipal authorities are introducing control measures to reduce its populations. It has been suggested that wild mammals may play a role in contaminating the environment with zoonotic pathogens. The present study detected the enteric pathogens Cryptosporidium ubiquitum, Enterocytozoon bieneusi, and Blastocystis in the feces of Pallas’s squirrels inhabiting Kanagawa Prefecture, Japan. These pathogens persist in the environment and contaminate soils and water, which may potentially infect humans. Because Pallas’s squirrels in Kanagawa Prefecture are found in urban areas, where they are in close contact with human populations, continued monitoring of zoonotic diseases among squirrel populations will be important for evaluating the significance of wildlife in pathogen transmission.