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Enterocytozoon bieneusi is a zoonotic pathogen that infects a variety of hosts including humans, livestock, wildlife, companion animals, and birds, as well as being abundant in the environment. Humans and nonhuman animals could be infected with E. bieneusi via consumption of food or water that contains zoonotic and host-adapted genotypes. In this study, 288 fecal specimens were collected from farmed minks, blue foxes, and raccoon dogs, in Xinjiang, China. Enterocytozoon bieneusi was examined by PCR amplification based on sequence analysis of the internal transcribed spacer (ITS) region. The overall infection rate of E. bieneusi was 4.9% (14/288), with mink samples showing the highest infection rate (5.6%, 12/214), followed by blue foxes (2.9%, 1/35), and then raccoon dogs (2.6%, 1/39). Six E. bieneusi genotypes were identified, including D (n = 5), PigEBITS7 (n = 4), EbpA (n = 2), CAM5 (n = 1), WildBoar3 (n = 1), and a novel genotype XJMI-1 (n = 1). Phylogenetic analysis showed that all E. bieneusi genotypes belonged to group 1, which composed of over 300 genotypes and most of them have been identified in human and variety of animals, suggesting a risk of zoonotic transmission from farmed wildlife to humans., Graphical abstract Image 1, Highlights • Six genotypes were identified, including three zoonotic genotypes (D, EbpA ad PigEBIST7). • Farmed wildlife maybe a potential source of E. bieneusi infection for other animals and humans.

Background Enterocytozoon bieneusi is a zoonotic gastrointestinal pathogen and can infect both humans and animals. The coypu (Myocastor coypus) is a semi-aquatic rodent, in which few E. bieneusi infections have been reported and the distribution of genotypes and zoonotic potential remains unknown. Methods A total of 308 fresh fecal samples were collected from seven coypu farms in China to determine the infection rate and the distribution of genotypes of E. bieneusi from coypus using nested-PCR amplification of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene. Results Enterocytozoon bieneusi was detected with an infection rate of 41.2% (n = 127). Four genotypes were identified, including three known genotypes (CHN4 (n = 111), EbpC (n = 8) and EbpA (n = 7)) and a novel genotype named CNCP1 (n = 1). Conclusions The rare genotype CHN4 was the most common genotype in the present study, and the transmission dynamics of E. bieneusi in coypus were different from other rodents. To the best of our knowledge, this is the first report of E. bieneusi infections in coypus in China. Our study reveals that E. bieneusi in coypus may be a potential infection source to humans.

The objective of this study was to determine the infection rate and genetic diversity of Cryptosporidium spp. in minks, foxes, and raccoon dogs, farmed in the Xinjiang Uygur Autonomous Region, Northwest China. Fresh fecal specimens were collected from individual cages of farmed minks (n = 214), blue foxes (n = 35), and raccoon dogs (n = 39) and examined using nested PCR based on the Cryptosporidium spp. small subunit rRNA gene. Cryptosporidium spp. was detected in 35 cages (12.2%, 35/288), with a higher infection rate detected in raccoon dogs (20.5%) compared with minks (12.1%) and blue foxes (2.9%). Sequence analysis showed that Cryptosporidium canis was the only species identified in blue foxes and raccoon dogs, while in the 26 Cryptosporidium-positive mink specimens, Cryptosporidium mink genotype (n = 17), C. canis (n = 7), and Cryptosporidium parvum (n = 2) were identified. Further analysis based on the 60-kDa glycoprotein (gp60) gene determined that both C. parvum isolates belonged to the subtype IIdA15G1, while eight of the 17 Cryptosporidium mink genotype isolates were a novel subtype that we have named XeA5G1. To the best of our knowledge, this is the first report of C. parvum subtype IIdA15G1 infection in minks. Since all the Cryptosporidium species/genotypes identified in minks, foxes, and raccoon dogs from Xinjiang have been previously found in humans, our results suggest that these fur animals may play a role in the transmission of zoonotic Cryptosporidium.

Enterocytozoon bieneusi, an obligate intracellular pathogen of the intestinal epithelium, is commonly identified in humans and many other animals and is ubiquitous in water sources and the environment generally. To determine the molecular prevalence of E. bieneusi in edible bullfrogs (Lithobates catesbeiana) and evaluate the possibility of its potential zoonotic transmission to humans via food or water, the intestinal contents of 295 bullfrogs were intermittently collected from two open markets in Aksu, China. The samples were screened for the internal transcribed spacer by polymerase chain reaction amplifications, revealing that 20.7% (61/295) of them were infected with E. bieneusi, with no significant differences found between the two sampling locations (p > 0.05). Twenty-two different E. bieneusi genotypes were identified, including one known genotype (EbpC) and 19 novel ones (named BLC1 to BLC19). The zoonotic genotype EbpC was identified in most of the E. bieneusi-positive samples (65.6%, 40/61). The remaining genotypes were identified in either one or three samples each. Our phylogenetic analysis showed that 20 of the E. bieneusi genotypes belonged to Group 1. As far as we are aware, this is the first report of E. bieneusi infections in edible bullfrogs. Our findings suggest that E. bieneusi can be maintained in edible bullfrogs and potentially transmitted via food or water. It is possible that these amphibians are unsuspected zoonotic reservoirs of E. bieneusi. Keywords: Enterocytozoon bieneusi, Bullfrogs, Genotype, Zoonotic

Cryptosporidium and Enterocytozoon bieneusi are important intestinal pathogens that infect humans and various animals. Few reports are available regarding the infections of the two pathogens in Père David's deer. In this study, polymerase chain reaction (PCR) confirmed Cryptosporidium infection in two (1.6%) and E. bieneusi in 45 (35.2%) of 128 fecal samples collected from Père David's deer in the National Nature Reserve of Shishou, Hubei Province, China. C. parvum (n = 1) and Cryptosporidium deer genotype (n = 1) were identified using the small subunit rRNA (SSU rRNA) gene. The C. parvum was further subtyped as IIdA20G1 by sequencing analysis of the 60-kDa glycoprotein (gp60) gene. The identity of E. bieneusi was confirmed by an internal transcribed spacer (ITS) gene; the HLJD-V (n = 42) and MWC_d1 (n = 3) genotypes were identified, with the former clustering in group 2 and the latter in group 1. These data suggest that the Père David's deer were infected with host-specific and/or zoonotic genotypes of these pathogens, implicating Père David's deer could be a potential source of human Cryptosporidium infection., Graphical abstract Image 1, Highlights • C. parvum subtype IIdA20G1 was firstly identified in Pere David's deer. • Genotypes HLJD-V and MWC_d1 were detected from 45 E. bieneusi-positive samples. • The present study implicated Père David's deer could be a potential source of human infection.

Enterocytozoon bieneusi is an emerging zoonotic intestinal pathogen that infects humans and various animal species. Here, we aimed to determine the infection rate and genetic characteristics of E. bieneusi from bamboo rats from different regions of China using nested polymerase chain reaction-based amplification of the internal transcribed spacer region of the rRNA gene. A total of 435 bamboo rats fecal samples were collected from individual tank from Guangdong, Hunan, Jiangxi, Chongqing, and Guangxi, southeastern China. E. bieneusi was detected on 22 tanks (5.1%, 22/435), with a higher infection rate being observed among samples from Guangdong Province (10.9%, 5/46) compared with those from Hunan (9.3%, 10/107), Jiangxi (6.7%, 6/90), Chongqing (2.0%, 1/50), and Guangxi (0%, 0/142) (P

To assess the prevalence and molecular characteristics of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in grazing adult sheep from Xinjiang Uygur Autonomous Region, China, 318 fecal samples were collected and screened for the presence of these parasites by polymerase chain reaction. The overall infection rate for the three pathogens was 13.5% (43/318), with observed individual infection rates of 0.9% (3/318), 7.5% (24/318), and 6.3% (20/318) for Cryptosporidium spp., G. duodenalis, and E. bieneusi, respectively. Three Cryptosporidium species were identified amongst the samples, including C. xiaoi (n = 1), C. ubiquitum (n = 1), and C. parvum (n = 1), with gp60-based subtyping analysis identifying C. parvum as subtype IIdA15G1 and C. ubiquitum as subtype XIIa. Eight E. bieneusi genotypes were identified based on internal transcribed spacer region sequencing, including six known (BEB6, CHG1, CHG3, CHS3, CHS8, and COS-I) and two novel (designated XJS1 and XJS2) genotypes. All G. duodenalis-positive samples were identified as assemblage E based on small subunit rRNA (n = 24) and gdh (n = 10) gene sequence analysis. These data support the occurrence of host adaptation by Cryptosporidium spp., G. duodenalis, and E. bieneusi in sheep, and the zoonotic risk may posed by these parasites in Xinjiang, China.

Cryptosporidium is one of the most prevalent zoonotic parasites and is responsible for the high burden of diarrheal disease across the globe. Rodents are globally overpopulated and are reservoirs for a variety of zoonotic pathogens. Bamboo rats are a common species of rodent that are bred for meat and wool in China. However, the genetic characterization of Cryptosporidium in bamboo rats in China is limited. The aim of this study was to determine the occurrence and genetic characterization of Cryptosporidium in bamboo rats from South Central China. From February2017to February 2018, 435 fecal samples were collected from bamboo rats in 13 farms located in 12 cities in South Central China. All fecal specimens were examined for Cryptosporidium by PCR, and through sequencing the partial small subunit of ribosomal DNA (SSU rRNA). C. parvum-positive samples were further subtyped through analysis of the 60-kDa glycoprotein (gp60) gene sequence. Meanwhile, all the new Cryptosporidium genotypes samples were selected for further sequence characterization at the 70-kDa heat shock protein (HSP70) gene and oocyst wall protein (COWP) gene as well as gp60 gene. Infection rates of 2.1% (9/435) were recorded for Cryptosporidium. Sequence analysis confirmed the presence of two Cryptosporidium species including C. parvum (n = 2), C. occultus (n = 1) and two new Cryptosporidium genotypes termed Cryptosporidium bamboo rat genotype I (n = 5) and Cryptosporidium bamboo rat genotype II (n = 1). Two subtypes of C. parvum were identified including IIdA15G1 and IIpA19 (one each).The discovery of zoonotic Cryptosporidium species/genotypes in bamboo rats suggests they have significant zoonotic potential and pose a threat to human health. The novel sequences discovered provide new insight into genotypic variations in Cryptosporidium in bamboo rats. Keywords: Cryptosporidium, Bamboo rat, Molecular detection, Zoonotic, Public health

Background Enterocytozoon bieneusi, a zoonotic pathogen, has the potential to infect both immunocompromised and immunocompetent humans. It is found in large number of animals; however, not much is known regarding its prevalence in equine animals, particularly donkeys. This is the first molecular epidemiological evaluation of E. bieneusi in 178 free-ranging donkeys from five countrysides; and 502 farmed donkeys from 18 farms in 12 cities of Xinjiang, China by Nested PCR. Results E. bieneusi was detected in 2.5% (17/680) donkeys, with 2.6% (13/502) in farmed and 2.2% (4/178) in free-ranging ones. Sequence analysis identified eight ITS genotypes, all belonging to zoonotic Groups 1 or 2, including six known genotypes: horse1 (n = 5), D (n = 3), NCD-2 (n = 3), BEB6 (n = 2), BEB4 (n = 1), and NIAI (n = 1); and two new genotypes: XJD1 (n = 1) and XJD2 (n = 1). Conclusions This is the first report confirming the presence of E. bieneusi in donkeys in Xinjiang, China, and indicates the possibility of zoonotic transmission of this pathogenic parasite.

Bovine anaplasmosis is caused by a group of obligate intracellular bacteria belonging to the genus Anaplasma, which are transmitted by ticks. This study was conducted to determine the prevalences and molecular characterization of Anaplasma spp. in dairy cattle in the upper reaches of the Tarim River in Xinjiang, China. Using polymerase chain reaction (PCR) and sequencing approaches, DNA of Anaplasma spp. was detected in 16 of 493 (3.2%) blood samples from dairy cattle. Positive rates were 0.2% (1/493), 0.4% (2/493), 0.2% (1/493), 2.4% (12/493) and 2.4% (12/493) for A. bovis, A. ovis, A. phagocytophilum like strain, A. phagocytophilum and A. platys like strain, respectively. Anaplasma phagocytophilum and A. platys like strain co-infection was detected in 12 samples. To our knowledge, this is the first report of A. ovis infection in dairy cattle in Xinjiang. This study provides new data on the prevalences of Anaplasma spp. in cattle in Xinjiang, which will help to formulate appropriate control strategies for these pathogens in this area.

Enterocytozoon bieneusi is a widely distributed human and animal pathogen. However, few data are available on the distribution of E. bieneusi genotypes in racehorses. In this study, 621 fecal specimens were collected from racehorses at 17 equestrian clubs in 15 Chinese cities. E. bieneusi was detected via nested polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) gene. The overall infection rate of E. bieneusi was 4.8% (30/621). Statistically significant differences were found in the prevalence of this parasite among the equestrian clubs (χ2 = 78.464, df = 16, p < 0.01) and age groups (χ2 = 23.686, df = 1, p < 0.01), but no sex bias was found among the racehorses for the E. bieneusi infections (χ2 = 1.407, df = 2, p > 0.05). Ten E. bieneusi genotypes were identified, including seven known genotypes (EbpC, EbpA, Peru6, horse1, horse2, CAF1, and TypeIV) and three novel genotypes (HBH-1, SXH-1, and BJH-1). Phylogenetic analysis showed that EbpC, EbpA, Peru6, horse2, CAF1, TypeIV, BJH-1, and SXH-1 belonged to Group 1 of E. bieneusi, HBH-1 belonged to Group 2, and horse2 belonged to Group 6. Our findings advance the current knowledge of E. bieneusi prevalence and genotypes in racehorses in China.

Enterocytozoon bieneusi is the mainly pathologies or intestinal disorders that causes approximately 90% of reported cases of human microsporidiosis. To understand the prevalence and genotype distribution of E. bieneusi in the Xinjiang Uygur Autonomous Region, China, 609 fecal samples were collected from children in kindergarten in Southern Xinjiang and screened for this pathogen by PCR and sequencing of the internal transcribed spacer (ITS). Thirty-six fecal samples (5.9%, 36/609) were positive for E. bieneusi, with the highest prevalence observed in children from Yopurga (17.5%, 11/63). Nine genotypes were identified, of which six were known (A, CHN6, D, EbpA, KB-1, and NIA1) and three were novel (CXJH1, CXJH2 and CXJH3). Genotype NIA1 was most prevalent (52.8%, 19/36), followed by genotypes D (16.7%, 6/36), A (8.3%, 3/36), and EbpA (8.3%, 3/36). The remaining five genotypes were detected in one sample each. Phylogenetic analysis revealed that the E. bieneusi isolates clustered into two groups, one consisting of six genotypes (Group 1: A, CXJH1, D, EbpA, KB-1, and NIA1) and another consisting of three genotypes (Group 2: CHN6, CXJH2, and CXJH3). Our results confirmed that infection of E. bieneusi unusual dominant genotype NIA1 occurs in children in Xinjiang, China. Further epidemiological studies must be conducted to clarify potential sources of E. bieneusi infection in this area., Author summary This study reports the infection rates and genetic characteristics of Enterocytozoon bieneusi in 609 children in kindergarten in Southern Xinjiang, China. All samples were screened for this pathogen with PCR, based on the internal transcribed spacer (ITS) of E. bieneusi. Thirty-six fecal samples (5.9%, 36/609) were positive, with the highest prevalence observed in children from Yopurga (17.5%, 11/63). Three novel genotypes were identified (CXJH1, CXJH2 and CXJH3). Phylogenetic analysis revealed that the E. bieneusi isolates clustered into two groups: Group 1 (A, CXJH1, D, EbpA, KB-1, and NIA1) and Group 2 (CHN6, CXJH2, and CXJH3). Genotype NIA1 used to be detected in HIV-positive patients, however, it was most prevalent (52.8%, 19/36) among the nine genotypes identified in this study. Additionally, we confirmed the zoonotic potential of E. bieneusi genotype D and this is the first report of human infection by E. bieneusi genotypes KB-1 and CHN6.

A total of 321 rabbit fecal samples were collected from 10 farms in the Xinjiang Uyghur Autonomous Region, China. The prevalence of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in the samples was 3.4% (11/321), 1.9% (6/321), and 2.8% (9/321), respectively. Small subunit ribosomal RNA (SSU rRNA) gene sequence analysis identified all 11 Cryptosporidium-positive samples as C. cuniculus. Further subtyping based on the 60-kDaglycoprotein locus (gp60) identified five of the C. cuniculus isolates as subtype VbA24. G. duodenalis genotypes were determined by multilocus sequence typing of the SSU rRNA, triosephosphate isomerase, β-giardin and glutamate dehydrogenase loci, which confirmed that six G. duodenalis isolates belonged to subtype BIV of assemblage B. Analysis of the internal transcribed spacer region, showed that five, three, and one E. bieneusi isolates belonged to genotypes J, BEB8, and Type IV, respectively. These results suggest that Cryptosporidium spp., G. duodenalis, and E. bieneusi isolates from rabbits in China have zoonotic potential.

Background Enterocytozoon bieneusi is an obligate, intracellular fungus and is commonly reported in humans and animals. To date, there have been no reports of E. bieneusi infections in Bactrian camels (Camelus bactrianus). The present study was conducted to understand the occurrence and molecular characteristics of E. bieneusi in Bactrian camels in China. Results Of 407 individual Bactrian camel fecal specimens, 30.0% (122) were E. bieneusi-positive by nested polymerase chain reaction (PCR) based on internal transcriber spacer (ITS) sequence analysis. A total of 14 distinct E. bieneusi ITS genotypes were obtained: eight known genotypes (genotype EbpC, EbpA, Henan-IV, BEB6, CM8, CHG16, O and WL17), and six novel genotypes (named CAM1 to CAM6). Genotype CAM1 (59.0%, 72/122) was the most predominant genotype in Bactrian camels in Xinjiang, and genotype EbpC (18.9%, 23/122) was the second-most predominant genotype. Phylogenetic analysis revealed that six known genotypes (EbpC, EbpA, WL17, Henan-IV, CM8 and O) and three novel genotypes (CAM3, CAM5 and CAM6) fell into the human-pathogenic group 1. Two known genotypes (CHG16 and BEB6) fell into the cattle host-specific group 2. The novel genotypes CAM1, CAM 2 and CAM4 cluster into group 8. Conclusions To our knowledge, this is the first report of E. bieneusi in Bactrian camels. The host-specific genotype CAM1 was the predominant genotype, which plays a negligible role in the zoonotic transmission of E. bieneusi. However, the second-most predominant genotype, EbpC, has greater zoonotic potential.

The prevalence and zoonotic potential of Cryptosporidium in donkeys is poorly understood. Here, 680 fecal specimens were collected from 178 free-ranging and 502 farmed donkeys in Xinjiang, China. Cryptosporidium was identified using PCR amplification of the small subunit of ribosomal DNA. Cryptosporidium-positive isolates were subtyped using PCR analysis of the 60 kDa glycoprotein gene (gp60). The overall prevalence of Cryptosporidium was 2.4% (16/680), with 3.2% (16/502) in farmed donkeys and 0% (0/178) in free-ranging donkeys. Cryptosporidium hominis (n = 13), C. parvum (n = 1) and Cryptosporidium horse genotype (n = 2) were identified. The C. hominis isolates belonged to two subtypes, IkA16 (n = 9) and IkA16G1 (n = 4). The subtype of C. parvum was IIdA15G1, whereas the two Cryptosporidium horse genotype isolates were of subtype VIaA15G4. The predominance of C. hominis in donkeys suggests that these animals are infected through human contact.Prévalence et identification génotypique des Cryptosporidium chez les ânes (Equus asinus asinus) en liberté et en élevage au Xinjiang, Chine.La prévalence et le potentiel zoonotique de Cryptosporidium chez les ânes sont mal connus. Dans cet article, 680 spécimens fécaux ont été prélevés de 178 ânes en liberté et 502 ânes en élevage dans le Xinjiang, en Chine. Les Cryptosporidium ont été identifiés en utilisant une amplification par PCR de la petite sous-unité de l’ADN ribosomique. Les isolats positifs pour Cryptosporidium ont été sous-typés en utilisant une analyse PCR du gène de la glycoprotéine 60 kDa (gp60). La prévalence globale de Cryptosporidium était de 2,4 % (16/680), avec 3,2 % (16/502) chez les ânes d’élevage et 0 % (0/178) chez les ânes en liberté. Cryptosporidium hominis (n = 13), C. parvum (n = 1) et Cryptosporidium génotype chevalin (n = 2) ont été identifiés. Les isolats de C. hominis appartenaient à deux sous-types, IkA16 (n = 9) et IkA16G1 (n = 4). Le sous-type de C. parvum était IIdA15G1, tandis que les deux isolats de Cryptosporidium génotype chevalin étaient du sous-type VIaA15G4. La prédominance de C. hominis chez les ânes suggère que ces animaux sont infectés par contact humain.

Enterocytozoon bieneusi, an obligate intracellular pathogen, can infect a wide variety of hosts. This study aimed to determine the prevalence and molecular characteristics of E. bieneusi in alpacas (Vicugna pacos) in China. A total of 185 alpaca fecal samples were collected from five herds in Tacheng, Wensu, Hejing, Qinghe, and Nilka counties in Xinjiang Uygur Autonomous Region. Enterocytozoon bieneusi was detected by nested PCR of the internal transcribed spacer (ITS) region. Twenty-eight fecal samples (15.1%, 28/185) were positive for E. bieneusi, with the highest prevalence in alpacas from Qinghe (42.9%, 15/35). Four E. bieneusi genotypes were identified, which included two known (P and ALP3) and two novel (ALP7 and ALP8) genotypes. Genotype ALP3 was the dominant genotype (57.1%, 16/28), followed by genotypes P (32.1%, 9/28), ALP7 (7.1%, 2/28), and ALP8 (2.6%, 1/28). Phylogenetic analysis revealed that three genotypes (P, ALP7, and ALP3) clustered into group 1, whereas genotype ALP8 clustered into group 8. This is the first report of E. bieneusi infection and genetic diversity in alpacas from Xinjiang, China.