Hualin Fu

First Report of the Human-Pathogenic Enterocytozoon bieneusi from Red-Bellied Tree Squirrels (Callosciurus erythraeus) in Sichuan, China.

Enterocytozoon bieneusi is a common opportunistic pathogen causing diarrhea and enteric disease in a variety of animal hosts. Although it has been reported in many animals, there is no published information available on the occurrence of E. bieneusi in red-bellied tree squirrels. To understand the occurrence, genetic diversity, and zoonotic potential of E. bieneusi in red-bellied tree squirrels, 144 fecal specimens from Sichuan province, China, were examined by PCR amplification and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi. The overall infection rate of E. bieneusi 16.7% (24/144) was observed in red-bellied tree squirrels. Altogether five genotypes of E. bieneusi were identified: three known genotypes D (n = 18), EbpC (n = 3), SC02 (n = 1) and two novel genotypes CE01, CE02 (one each). Multilocus sequence typing (MLST) analysis employing three microsatellite (MS1, MS3, MS7) and one minisatellite (MS4) revealed 16, 14, 7 and 14 positive specimens were successfully sequenced, and identified eight, three, three and two genotypes at four loci, respectively. In phylogenetic analysis, the three known genotypes D, EbpC, and SC02 were clustered into group 1 with zoonotic potential, and the two novel genotypes CE01 and CE02 were clustered into group 6. The present study firstly reported the occurrence of E. bieneusi in red-bellied tree squirrels in China, and the E. bieneusi genotypes D and EbpC were found in humans previously. These results indicate that red-bellied tree squirrels may play a potential role in the transmission of E. bieneusi to humans.

Presence of zoonotic Cryptosporidium scrofarum, Giardia duodenalis assemblage A and Enterocytozoon bieneusi genotypes in captive Eurasian wild boars (Sus scrofa) in China: potential for zoonotic transmission

BackgroundCryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi are the main causal pathogens of gastrointestinal disease. However, there are limited reports about the prevalence of these organisms in captive Eurasian wild boars worldwide. Therefore, we examined the occurrence and identified the species/assemblages/genotypes of these pathogens in captive Eurasian wild boars, and estimated the zoonotic potential.FindingsOf 357 fecal samples collected from captive Eurasian wild boars in China, 155 (43.4%) were infected with Cryptosporidium, G. duodenalis and/or E. bieneusi. The infection rates significantly differed in different areas, but did not differ between wild boars kept indoors and outdoors. Three (0.8%), 11 (3.1%) and 147 (41.2%) fecal samples were positive for Cryptosporidium, G. duodenalis and E. bieneusi, respectively. Sequence analysis of SSU rRNA gene revealed that all of the Cryptosporidium strains belonged to C. scrofarum. Based on the sequence analysis of the β-giardia gene of G. duodenalis, assemblages E and A were characterized. Fourteen E. bieneusi genotypes comprising five novel (WildBoar 7–11) and eight known (EbpC, F, CHG19, CHC5, PigEBITS5, D, RWSH4, SC02) genotypes were identified by ITS sequencing. EbpC was the most frequent genotype, detected in 85 specimens. Phylogenetic analysis revealed that all 14 genotypes belonged to Group 1.ConclusionsThis first report on the occurrence of Cryptosporidium, G. duodenalis and E. bieneusi in captive wild boars in China indicates that the presence of zoonotic species/assemblages/genotypes poses a threat to public health. The findings suggest that wild boars could be a significant source of human infection and water pollution.

Multi-locus genotypes of Enterocytozoon bieneusi in captive Asiatic black bears in southwestern China: High genetic diversity, broad host range, and zoonotic potential

Enterocytozoon bieneusi is an obligate eukaryotic intracellular parasite that infects a wide variety of vertebrate and invertebrate hosts. Although considerable research has been conducted on this organism, relatively little information is available on the occurrence of E. bieneusi in captive Asiatic black bears. The present study was performed to determine the prevalence, genetic diversity, and zoonotic potential of E. bieneusi in captive Asiatic black bears in zoos in southwestern China. Fecal specimens from Asiatic black bears in four zoos, located in four different cities, were collected and analyzed for the prevalence of E. bieneusi. The average prevalence of E. bieneusi was 27.4% (29/106), with the highest prevalence in Guiyang Zoo (36.4%, 16/44). Altogether, five genotypes of E. bieneusi were identified among the 29 E. bieneusi-positive samples, including three known genotypes (CHB1, SC02, and horse2) and two novel genotypes named ABB1 and ABB2. Multi-locus sequence typing using three microsatellites (MS1, MS3, and MS7) and one minisatellite (MS4) revealed V, III, V, and IV genotypes at these four loci, respectively. Phylogenetic analysis showed that the genotypes SC02 and ABB2 were clustered into group 1 of zoonotic potential, the genotypes CHB1 and ABB1 were clustered into a new group, and the genotype horse2 was clustered into group 6 of unclear zoonotic potential. In conclusion, this study identified two novel E. bieneusi genotypes in captive Asiatic black bears, and used microsatellite and minisatellite markers to reveal E. bieneusi genetic diversity. Moreover, our findings show that genotypes SC02 (identified in humans) and ABB2 belong to group 1 with zoonotic potential, suggesting the risk of transmission of E. bieneusi from Asiatic black bears to humans and other animals.

Multilocus genotyping of Enterocytozoon bieneusi derived from nonhuman primates in southwest China

Enterocytozoon bieneusi has been increasingly reported in non-human primates (NHPs) in recent years, and this has garnered attention. However, reports of E. bieneusi infections in NHPs are limited worldwide. To appreciate the genetic diversity and assess the zoonotic potential during the transmission of human microsporidiosis, we examined a total of 369 fecal samples from NHPs and performed PCR amplification of the ITS gene of E. bieneusi. An infection rate of 12.5% (46/369) was detected in NHPs, with three known genotypes (D, PigEBITS7, and SC02) and a novel genotype (SCM01) characterized. Phylogenetic analysis indicated that all four genotypes in our study were classified as zoonotic group 1. Multilocus genotyping of positive E. bieneusi strains revealed that 36, 37, 30, and 29 specimens were successfully amplified and sequenced to generate 16, six, four, and five types of MS1, MS3, MS4, and MS7 loci, respectively. Twenty-four specimens were successfully amplified and sequenced at all four loci, forming 13 multilocus genotypes (MLGs). The occurrence of zoonotic genotypes suggests that zoonotic transmission of E. bieneusi between humans and NHPs has probably occurred and NHPs could be a source of human microspordiosis.

Occurrence and Genetic Characteristics of Cryptosporidium hominis and Cryptosporidium andersoni in Horses from Southwestern China

A total of 333 fecal specimens from horses in southwestern China were genotyped based on analysis of the small subunit rRNA (SSU rRNA) gene. Cryptosporidium hominis and Cryptosporidium andersoni were identified in 2 and 4 stool specimens, respectively. The identification of C. hominis was confirmed by sequence analysis of the 70‐kDa heat shock protein (HSP70) and oocyst wall protein (COWP) genes. Subtyping analysis of the 60‐kDa glycoprotein (GP60) gene sequence of C. hominis revealed a new rare subtype Id, named IdA15; only three Id isolates have been reported in humans to date. Multilocus sequence typing (MLST) analysis indicated that the C. andersoni subtype was A6, A5, A2, and A1 at the four minisatellite loci (MS1, MS2, MS3, and MS16, respectively). This is the first report to identify the presence of C. andersoni and C. hominis in horses in southwestern China and the first to identify a rare zoonotic subtype Id of C. hominis in horses. These findings suggest that infected horses may act as potential reservoirs of Cryptosporidium to transmit infections to humans.

Transcriptional Regulation and Adaptation to a High-Fiber Environment in Bacillus subtilis HH2 Isolated from Feces of the Giant Panda

In the giant panda, adaptation to a high-fiber environment is a first step for the adequate functioning of intestinal bacteria, as the high cellulose content of the gut due to the pandas vegetarian appetite results in a harsh environment. As an excellent producer of several enzymes and vitamins, Bacillus subtilis imparts various advantages to animals. In our previous study, we determined that several strains of B. subtilis isolated from pandas exhibited good cellulose decomposition ability, and we hypothesized that this bacterial species can survive in and adapt well to a high-fiber environment. To evaluate this hypothesis, we employed RNA-Seq technology to analyze the differentially expressed genes of the selected strain B. subtilis HH2, which demonstrates significant cellulose hydrolysis of different carbon sources (cellulose and glucose). In addition, we used bioinformatics software and resources to analyze the functions and pathways of differentially expressed genes. Interestingly, comparison of the cellulose and glucose groups revealed that the up-regulated genes were involved in amino acid and lipid metabolism or transmembrane transport, both of which are involved in cellulose utilization. Conversely, the down-regulated genes were involved in non-essential functions for bacterial life, such as toxin and bacteriocin secretion, possibly to conserve energy for environmental adaptation. The results indicate that B. subtilis HH2 triggered a series of adaptive mechanisms at the transcriptional level, which suggests that this bacterium could act as a probiotic for pandas fed a high-fiber diet, despite the fact that cellulose is not a very suitable carbon source for this bacterial species. In this study, we present a model to understand the dynamic organization of and interactions between various functional and regulatory networks for unicellular organisms in a high-fiber environment.

First identification and multilocus genotyping of Giardia duodenalis in pet chipmunks ( Eutamias asiaticus ) in Sichuan Province, southwestern China

BackgroundGiardia duodenalis is a flagellated parasite that causes diarrhea in humans and other animals. Although G. duodenalis is found in companion animals worldwide, information regarding the prevalence and genetic characteristics of G. duodenalis in pet chipmunks in China is limited. The present study therefore aimed to investigate the prevalence and genotypes of G. duodenalis in pet chipmunks in Sichuan province, southwestern China, as well as to assess zoonotic potential of revealed assemblages.ResultsA total of 279 fecal samples were collected from pet chipmunks in seven pet shops and one breeding facility in Sichuan province, southwestern China. The prevalence of G. duodenalis was 8.6% (24/279), as determined by nested PCR detection of the beta giardin (bg) gene. Giardia duodenalis assemblages and subtypes were determined using multilocus genotyping of the bg, triosephosphate isomerase (tpi), and glutamate dehydrogenase (gdh) loci. Two assemblages were identified: potentially zoonotic assemblage A (54.2%, 13/24) and rodent-specific assemblage G (45.8%, 11/24). A total of 24, 17 and 17 sequences of the bg, gdh and tpi loci, respectively, were successfully obtained, which formed four, four and three subtypes, respectively. Moreover, four assemblage A (MLGs A1-A4) and three assemblage G (MLGs G1-G3) multilocus genotypes were identified.ConclusionsTo our knowledge, this is the first study that investigated G. duodenalis in pet chipmunks in China. Detection of assemblage A in pet chipmunks and in previous studies in humans suggests a possible role of chipmunks as a reservoir for human giardiasis in Sichuan Province, China.

Multilocus genotyping of Giardia duodenalis in captive non-human primates in Sichuan and Guizhou provinces, Southwestern China

[This corrects the article DOI: 10.1371/journal.pone.0184913.].

First report and multilocus genotyping of Enterocytozoon bieneusi from Tibetan pigs in southwestern China

Enterocytozoon bieneusi is a common intestinal pathogen and a major cause of diarrhea and enteric diseases in a variety of animals. While the E. bieneusi genotype has 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. E. 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. Age group demonstrated the prevalence of E. bieneusi range from 24.4%(aged 0 to 1 years) to 44.4%(aged 1 to 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). Phylogenetic analysis showed these four genotypes clustered to group 1 with zoonotic potential. Multilocus sequence typing (MLST) analysis three microsatellites (MS1, MS3, MS7) and one minisatellite (MS4) revealed 47, 48, 23 and 47 positive specimens were successfully sequenced, and identified ten, ten, five and five genotypes at four loci, respectively. This study indicates the potential danger of E. bieneusi to Tibetan pigs in southwestern China, and offers basic data for preventing and controlling infections.

Human-Pathogenic Enterocytozoon bieneusi in Captive Giant Pandas ( Ailuropoda melanoleuca ) in China

Human and animal infections of Enterocytozoon bieneusi (E. bieneusi) have consistently been reported worldwide, garnering public attention; however, the molecular epidemiology of E. bieneusi in the giant panda remains limited. We surveyed captive giant pandas in China for the presence of E. bieneusi by using PCR and sequence analysis of the ribosomal internal transcribed spacer (ITS) revealing a 34.5% positive rate, with seven known genotypes (SC02, EpbC, CHB1, SC01, D, F, and Peru 6) and five novel genotypes (SC04, SC05, SC06, SC07, and SC08) identified. We similarly analyzed water samples, and E. bieneusi was detected in two samples, with genotype SC02 identified. Phylogenetic analysis revealed that CHB1 did not cluster with any recognized group, while the remaining genotypes belonged to group 1. The predominance of zoonotic group 1 genotypes indicates a public health threat that giant pandas could spread E. bieneusi to humans. The identification of E. bieneusi in water samples suggests giant pandas could contribute to water contamination. Effective control measures are therefore needed to minimize the contamination of the water and prevent a human microsporidiosis outbreak.