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: Microsporidia are common opportunistic parasites in humans and animals, including rabbits. However, only limited epidemiology data concern about the prevalence and molecular characterization of Enterocytozoon bieneusi and Encephalitozoon spp. in rabbits. This study is the first detection and genotyping of Microsporidia in pet rabbits in China. Results: A total of 584 faecal specimens were collected from rabbits in pet shops from four cities in Sichuan province, China. The overall prevalence of microsporidia infection was 24.8% by nested PCR targeting the internal transcribed spacer (ITS) region of E. bieneusi and Encephalitozoon spp. respectively. E. bieneusi was the most common species (n = 90, 15.4%), followed by Encephalitozoon cuniculi (n = 34, 5.8%) and Encephalitozoon intestinalis (n = 16, 2.7%). Mixed infections (E. bieneusi and E. cuniculi) were detected in five another rabbits (0.9%). Statistically significant differences in the prevalence of microsporidia were observed among different cities (χ 2 = 38.376, df = 3, P < 0.01) and the rabbits older than 1 year were more likely to harbour microsporidia infections (χ 2 = 9.018, df = 2, P < 0.05). Eleven distinct genotypes of E. bieneusi were obtained, including five known (SC02, I, N, J, CHY1) and six novel genotypes (SCR01, SCR02, SCR04 to SCR07). SC02 was the most prevalent genotype in all tested cities (43.3%, 39/90). Phylogenetic analysis showed that these genotypes were clustered into group 1-3 and group 10. Meanwhile, two genotypes (I and II) were identified by sequence analysis of the ITS region of E. cuniculi. Conclusion: To the best of our knowledge, this is the first report of microsporidia infection in pet rabbits in China. Genotype SC02 and four novel genotypes were classified into potential zoonotic group 1, suggesting that pet rabbits may cause microsporidiosis in humans through zoonotic transmissions. These findings provide preliminary reference data for monitoring microsporidia infections in pet rabbits and humans.

Cryptosporidium spp. are opportunistic pathogens that cause diarrhea in a variety of animal hosts. Although they have been reported in many animals, no information has been published on the occurrence of Cryptosporidium spp. in red-bellied tree squirrels (Callosciurus erythraeus). A total of 287 fecal specimens were collected from Sichuan province in China; the prevalence of Cryptosporidium spp., measured by nested-PCR amplification of the partial small-subunit (SSU) rRNA gene, was 1.4% (4/287). Three different Cryptosporidium species or genotypes were identified: Cryptosporidium parvum (n = 1), Cryptosporidium wrairi (n = 1), and Cryptosporidium rat genotype II (n = 2). The present study is the first report of Cryptosporidium infection in red-bellied tree squirrels in China. Although there is a relatively low occurrence of Cryptosporidium, the presence of C. parvum and C. wrairi, which were previously reported in humans, indicates that red-bellied tree squirrels may be a source of zoonotic cryptosporidiosis in China.

Enterocytozoon bieneusi is a common intestinal pathogen in a variety of animals. While E. bieneusi genotypes have become better-known, there are few reports on its prevalence in the Tibetan pig. This study investigated the prevalence, genetic diversity, and zoonotic potential of E. bieneusi in the Tibetan pig in southwestern China. Tibetan pig feces (266 samples) were collected from three sites in the southwest of China. Feces were subjected to PCR amplification of the internal transcribed spacer (ITS) region. Enterocytozoon bieneusi was detected in 83 (31.2%) of Tibetan pigs from the three different sites, with 25.4% in Kangding, 56% in Yaan, and 26.7% in Qionglai. Prevalence varies according to age group, from 24.4% (age 0–1 years) to 44.4% (age 1–2 years). Four genotypes of E. bieneusi were identified: two known genotypes EbpC (n = 58), Henan-IV (n = 24) and two novel genotypes, SCT01 and SCT02 (one of each). We compare our results with a compilation of published results on the host range and geographical distribution of E. bieneusi genotypes in China. Phylogenetic analysis showed these four genotypes clustered to group 1 with zoonotic potential. Multilocus sequence typing (MLST) analysis of three microsatellites (MS1, MS3, MS7) and one minisatellite (MS4) was successful in 47, 48, 23 and 47 positive specimens and identified 10, 10, 5 and 5 genotypes at four loci, respectively. This study indicates the potential danger of E. bieneusi to Tibetan pigs in southwestern China, and offers basic advice for preventing and controlling infections.

24Enterocytozoon bieneusi is a common intestinal pathogen and a major cause of 25 diarrhea and enteric diseases in a variety of animals. While the E. bieneusi genotype 26 has become better-known, there are few reports on its prevalence in the Tibetan pig. 27 This study investigated the prevalence, genetic diversity, and zoonotic potential of E. 28 bieneusi in the Tibetan pig in southwestern China. Tibetan pig feces (266 samples) 29 were collected from three sites in the southwest of China. Feces were subjected to 30 PCR amplification of the internal transcribed spacer (ITS) region. E. bieneusi was 31 detected in 83 (31.2%) of Tibetan pigs from the three different sites, with 25.4% in 32 Kangding, 56% in Yaan and 26.7% in Qionglai. Age group demonstrated the 33 prevalence of E. bieneusi range from 24.4%( aged 0 to 1 years) to 44.4%( aged 1 to 2 34 years). Four genotypes of E. bieneusi were identified: two known genotypes EbpC 35 (n=58), Henan-IV (n=24) and two novel genotypes, SCT01 and SCT02 (one of each). 36Phylogenetic analysis showed these four genotypes clustered to group 1 with zoonotic 37 potential. Multilocus sequence typing (MLST) analysis three microsatellites (MS1, 38 MS3, MS7) and one minisatellite (MS4) revealed 47, 48, 23 and 47 positive 39 specimens were successfully sequenced, and identified ten, ten, five and five 40 genotypes at four loci, respectively. This study indicates the potential danger of E. 41 bieneusi to Tibetan pigs in southwestern China, and offers basic data for preventing 42 and controlling infections.43 44 45 Microsporidia are obligate intracellular eukaryotic pathogens, classified as fungi, 46 which are composed of approximately 1300 species in 160 genera[1]. To date, 17 47 microsporidia species are known to infect humans, and of these, E. bieneusi is the 48 most prevalent, accounting for over 90% of cases of human microsporidiosis[2]. 49 Since its first detection in an HIV/AIDS patient in 1985, a growing literature attests to 50 E. bieneusi expanding range of hosts [3-5]. In humans, infection by microsporidia 51 results in self-limiting diarrhea and malabsorption, most seriously, 52 immunocompromised and immunocompetent patients more susceptible to E. bieneusi 53 infection[6]. Normally, fecal-oral routes serve as the main infection pathways in 54 humans and animals, while human inhalation of E. bieneusi spores has also been 55 documented[7, 8]. 56 PCR-based molecular techniques may be used to analyze the E. bieneusi genome, 57 and for diagnosis. Based on the nested PCR amplification of internal transcribed 58 spacers (ITS) of small subunits of ribosomal rRNA (SSU rRNA), over 240 E. 59bieneusi genotypes have been identified globally [9][10][11]. Phylogenetic analysis reveals 60 that these genotypes clustered into nine groups. Group 1 is considered zoonotic, and is 61 composed of genotypes from humans and a few animals, while groups 2-9 have 62 particular host associations or are found in wastewater [5, 11]. To better comprehend 63 E. bieneusi genetic diversity and molecu...