Cryptosporidium spp. and Enterocytozoon bieneusi are two well-known protist pathogens which can result in diarrhea in humans and animals. To examine the occurrence and genetic characteristics of Cryptosporidium spp. and E. bieneusi in pet red squirrels (Sciurus vulgaris), 314 fecal specimens were collected from red squirrels from four pet shops and owners in Sichuan province, China. Cryptosporidium spp. and E. bieneusi were examined by nested PCR targeting the partial small subunit rRNA (SSU rRNA) gene and the ribosomal internal transcribed spacer (ITS) gene respectively. The infection rates were 8.6% (27/314) for Cryptosporidium spp. and 19.4% (61/314) for E. bieneusi. Five Cryptosporidium species/genotypes were identified by DNA sequence analysis: Cryptosporidium rat genotype II (n = 8), Cryptosporidium ferret genotype (n = 8), Cryptosporidium chipmunk genotype III (n = 5), Cryptosporidium rat genotype I (n = 4), and Cryptosporidium parvum (n = 2). Additionally, a total of five E. bieneusi genotypes were revealed, including three known genotypes (D, SCC-2, and SCC-3) and two novel genotypes (RS01 and RS02). Phylogenetic analysis revealed that genotype D fell into group 1, whereas the remaining genotypes clustered into group 10. To our knowledge, this is the first study to report Cryptosporidium spp. and E. bieneusi in pet red squirrels in China. Moreover, C. parvum and genotype D of E. bieneusi, previously identified in humans, were also found in red squirrels, suggesting that red squirrels may give rise to cryptosporidiosis and microsporidiosis in humans through zoonotic transmissions. These results provide preliminary reference data for monitoring Cryptosporidium spp. and E. bieneusi infections in pet red squirrels and humans.Cryptosporidium spp. and Enterocytozoon bieneusi, causative agents of cryptosporidiosis and microsporidiosis, are two important opportunistic intestinal pathogens that can infect vertebrate and invertebrate, posing a significant threat to public health 1 . Humans are infected with these pathogens mainly via the fecal-oral route with anthroponotic and zoonotic transmission or via food-borne and water-borne transmission 2,3 . Clinical manifestations of infection with these pathogens are often inconsistent due to the variabilities in the health condition of infected hosts 4,5 . In healthy individuals, these pathogens usually cause asymptomatic infection or self-limiting diarrhea 6 . However, infection may also result in chronic or life-threatening diarrhea in immunocompromised individuals, such as patients with acquired immunodeficiency syndrome and patients who had undergone organ transplantation 7 . In addition to humans, there are a variety of animals that can act as hosts for these two pathogens, including various mammals, reptiles, birds, amphibians and insects 8 . Therefore, Cryptosporidium spp. and

Background Blastocystis sp. is an anaerobic protozoan that parasitizes many animal hosts and the human gastrointestinal tract, and its pathogenicity is controversial. Captive wildlife may be potential reservoirs for human infection with Blastocystis sp. The present study was performed to investigate the prevalence and subtype distribution of Blastocystis sp. in zoo animals in Sichuan Province, southwestern China. Methods A total of 420 fresh fecal samples were collected from 54 captive wildlife species in four zoos in southwestern China between June 2017 and September 2019. The prevalence and subtype (ST) genetic characteristics of Blastocystis sp. were determined by PCR amplification of the barcode region of the SSU rRNA gene and phylogenetic analysis. Results Overall, 15.7% (66/420) of the animal samples and 20.7% (14/54) of the species tested were shown to be infected with Blastocystis sp. The highest prevalence of Blastocystis sp. was found in Panzhihua Zoo (24.3%), which was significantly higher than that in Chengdu Zoo (6.9%), and Xichang Zoo (2.9%) (P < 0.05). There are also significant differences in the prevalence of Blastocystis sp. among different species (P < 0.05), and the highest of Blastocystis sp. prevalence was observed in white-cheeked gibbon, black great squirrel, and red giant flying squirrel (100%). Subtype analysis of Blastocystis sp. revealed nine subtypes, including six zoonotic STs (ST1-5, and ST8) and three animal-specific STs (ST10, ST14, and ST17), with ST17 as the predominant subtype (26/66) in Blastocystis sp.-positive isolates. Conclusions To our knowledge, this is the first report on the prevalence and subtype distribution of Blastocystis sp. among captive wildlife in zoos in southwestern China. This study highlights that these animals may serve as reservoirs for human Blastocystis sp. infections.

Blastocystis is a common enteric protist that colonizes humans and a wide range of animals. Although some studies have reported incidences of Blastocystis in humans and animals in China, there is no information available on the prevalence of Blastocystis in giant pandas, red pandas, or bird species. The aims of the present study were to determine the prevalence, subtype distribution, and genetic characterizations of Blastocystis in these animals in a captive situation in southwestern China, as well as assess the zoonotic potential of Blastocystis isolates. A total of 168 fecal specimens, including 81 from giant pandas, 23 from red pandas, 38 from black swans, 11 from ruddy shelducks, and 15 from green peafowl were collected at the Chengdu Research Base of Giant Panda Breeding in Sichuan province. The overall minimum prevalence of Blastocystis was 11.3% (19/168) based on PCR amplification of the barcode region of the SSU rRNA gene. The highest prevalence of Blastocystis was observed in ruddy shelduck (18.2%) and the lowest was found in green peafowl (6.7%). The prevalence of Blastocystis in giant pandas >5.5 years of age was higher than that in younger giant pandas. Two potentially zoonotic subtypes (ST1 and ST8) were identified, and ST1 (n = 12) was found to be more prevalent than ST8 (n = 7). To the best of our knowledge, this is the first report of the prevalence and subtypes of Blastocystis in giant pandas, red pandas, and bird species in China. The findings of this study will improve our understanding of the genetic diversity and public health potential of Blastocystis.

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