Molecular characterization of Blastocystis spp. in Hotan Black chickens in southern Xinjiang.

To determine the infection status and assess the zoonotic potential of Blastocystis spp. in Hotan Black chickens in southern Xinjiang, China, fecal samples were collected from 617 chickens on 18 large-scale farms. The presence of Blastocystis spp. was determined using polymerase chain reaction based on the small subunit rRNA (SSU rRNA) locus. The results revealed an overall infection rate of 26.3% (162/617). Samples from Farm 1 in Luopu County showed the highest infection rate (76.3%, 29/38). The highest and lowest infection rates were detected in the <30-day (34.4%, 43/125) and > 90-day age groups (12.4%, 11/89), respectively. The infection rate decreased with increasing age. Statistical analysis showed significant differences in the infection rates of Blastocystis spp. among the different sampling sites (p < 0.05) and age groups (p < 0.05). Four Blastocystis spp. subtypes (ST6, ST7, ST10, and ST23) were identified. The infection rates of the zoonotic subtypes, ST6 and ST7, were 3.2% (20/617) and 22.2% (137/617), respectively. The presence of Blastocystis spp. and zoonotic subtypes provided evidence for the potential transmission of this pathogen between Hotan Black chickens and humans, especially in animal handlers in this area.

Molecular detection of Cryptosporidium in Alpine musk deer (Moschus chrysogaster) in Gansu Province, Northwest China.

Cryptosporidium spp. are protozoa commonly found in domestic and wild animals. Limited information is available on Cryptosporidium in deer worldwide. In this study, 201 fecal samples were collected from Alpine musk deer on three farms in Gansu Province, China. Detection and subtyping of Cryptosporidium were performed by PCR and sequence analysis of the SSU rRNA and gp60 genes. The prevalence of Cryptosporidium infection in Alpine musk deer was 3.9% (8/201), with infection rates of 1.0% (1/100), 2.8% (1/36), and 9.2% (6/65) in three different farms. All positive samples for Cryptosporidium were from adult deer. Two Cryptosporidium species were identified, including C. parvum (n = 2) and C. xiaoi (n = 6). The C. parvum isolates were subtyped as IIdA15G1, while the C. xiaoi isolates were subtyped as XXIIIa (n = 2) and XXIIIg (n = 4). The IIdA15G1 subtype of C. parvum was found for the first time in deer. These results provide important insights into the identity and human infectious potential of Cryptosporidium in farmed Alpine musk deer.

Glyphosate induces autophagy in hepatic L8824 cell line through NO-mediated activation of RAS/RAF/MEK/ERK signaling pathway and energy metabolism disorders.

Glyphosate is an herbicide commonly used worldwide, and its substantial use causes widespread pollution with runoff. However, research on glyphosate toxicity has mostly remained at the embryonic level and existing studies are limited. In the present study, we investigated whether glyphosate can induce autophagy in hepatic L8824 cells by regulating energy metabolism and rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular regulated protein kinases (ERK) signaling by activating nitric oxide (NO). First, we selected 0, 50, 200, and 500 µg/mL as the challenge doses, according to the inhibitory concentration of 50% (IC50) of glyphosate. The results showed that glyphosate exposure increased the enzyme activity of inducible nitric oxide synthase (iNOS), which in turn increased the NO content. The activity and expression of enzymes related to energy metabolism, such as hexokinase (HK)1, HK2, phosphofructokinase (PFK), phosphokinase (PK), succinate dehydrogenase (SDH), and nicotinamide adenine dinucleotide with hydrogen (NADH), were inhibited, and the RAS/RAF/MEK/ERK signaling pathway was activated. This led to the negative expression of mammalian target of rapamycin (mTOR) and P62 in hepatic L8824 cells and the activation of the autophagy marker genes microtubule-associated proteins light chain 3 (LC3) and Beclin1 to induce autophagy. The above results were dependent on glyphosate concentration. To verify whether autophagy can be excited by the RAS/RAF/MEK/ERK signaling pathway, we treated L8824 cells with the ERK inhibitor U0126 and found that the autophagy gene LC3 was reduced due to the inhibition of ERK, thus demonstrating the reliability of the results. In conclusion, our results demonstrate that glyphosate can induce autophagy in hepatic L8824 cells by activating NO, thus regulating energy metabolism and the RAS/RAF/MEK/ERK signaling pathway.

Prevalence and sequence diversity of Balantioides coli in pigs in Xinjiang, China.

Balantioides coli is a common intestinal parasitic protozoan in pigs. In the present study, 801 fecal samples of pigs from seven farms in Xinjiang were analyzed based on the ITS1-5.8S rRNA-ITS2 gene. The prevalence of B. coli was 4.2% (34/801), with the highest prevalence of 18.9% (18/95) occurring in Alaer, Xinjiang. B. coli was detected in all age groups (pre-weaned pigs, post-weaned pigs, fattening pigs and sows), with the highest rate in fatteners (6.9%, 9/129) and the lowest (1.2%, 2/169) in pre-weaned pigs. Significant differences (P = 0.000) were found among sampling sites but not among age groups (P = 0.084). Sequence analysis indicated that 34 sequence variants, including sequence type A (n = 11) and sequence type B (n = 23), occurred in all age groups. In this study, the existence of sequence type A suggested that B. coli poses a potential threat to human health. More studies are needed to better understand the distributions and public health significance of B. coli in China.

Prevalence and genetic characterization of Theileria equi and Babesia caballi in grazing horses in Xinjiang, northwestern China.

Piroplasmosis is a disease that negatively affects equine health worldwide. Hence, 324 blood samples were collected from grazing horses in ten sites in Xinjiang and testing them for the presence of Theileria equi and Babesia caballi by PCR of the EMA-1 gene and BC48 gene, respectively. Of the 324 blood samples, 161 (49.7%) were positive for equine piroplasms. The prevalence of T. equi was 38.9% (126/324), while that of B. caballi was 30.2% (98/324). The T. equi and B. caballi co-infection rate was 19.4% (63/324). From the 126 EMA-1 gene sequences and 98 BC48 gene sequences we obtained, 21 and 27 genotypes were identified, respectively. The EMA-1 sequences together with the GenBank reference sequences grouped into four clusters, with those from the present study forming two distinct clusters. In contrast, the BC48 sequences formed eight clusters with the GenBank reference sequences, while those obtained in the present study formed five distinct clusters. Our results highlight the widespread distribution and abundant gene polymorphism of T. equi and B. caballi in grazing horses from Xinjiang.

Molecular characterization of Enterocytozoon bieneusi genotypes in wild Altai marmot (Marmota baibacina) in Xinjiang, China: host specificity and adaptation.

Enterocytozoon bieneusi is responsible for opportunistic infections leading to gastrointestinal diseases in humans and animals worldwide. A total of 334 fresh fecal samples were collected from wild Altai marmots (Marmota baibacina) in Xinjiang, China, and E. bieneusi was screened via PCR amplification of the internal transcribed spacer (ITS) region of the small submit ribosomal RNA (SSU rRNA). The results indicated that 22.8% (76/334) of the wild Altai marmot fecal samples were positive for E. bieneusi, and the highest positive rate was detected in Akqi (51.9%, 27/52), with a significant difference from other sampling sites (p < 0.01). Four known genotypes (BEB6, CHG3, GX2, and YAK1) and three novel genotypes (XJHT2 to XJHT4) were identified in the present study. Genotype XJHT3 was dominant and detected in 48 fecal samples. In the phylogenetic analysis, the novel genotypes XJHT2 and XJHT3 were clustered in Group 1 together with the known genotype YAK1, while genotypes CHG3 and BEB6 were clustered in Group 2. The novel genotype XJHT4 was clustered together with other rodent-derived genotypes and generated a novel Group 14. These data confirmed the host specificity and adaptation of E. bieneusi in rodents. These findings enrich our understanding of the prevalence and genetic diversity of E. bieneusi in wild Altai marmots in Xinjiang, China.

Dominant infection of Cryptosporidium baileyi in broiler chickens in Zhejiang Province, China.

Cryptosporidium is a common enteric parasite in chickens. A total of 812 fecal specimens were collected from 11 broiler farms in Zhejiang Province, China, and analyzed by nested PCR amplification based on the small subunit ribosomal RNA (SSU rRNA) gene. The overall infection rate of Cryptosporidium was 6.3% (51/812), and five of 11 farms were Cryptosporidium positive. Broilers aged > 90 days accounted for the highest infection rate of 16.1% (6/56), followed by those aged 30-60 days (10.6%, 38/358) and 60-90 days (4/378, 1.1%). Two Cryptosporidium species were identified by sequence analysis, with the predominant species being C. baileyi (96.1%, 49/51) and the minor infection being C. meleagridis (3.9%, 2/51). Based on the 60-kDa glycoprotein (gp60) gene, two C. meleagridis-positive isolates were identified as one known subtype, IIIbA24G1R1. This study indicated the common occurrence of C. baileyi in broiler chickens in this region and low zoonotic transmission potential of Cryptosporidium to humans.

Molecular evaluation of Cryptosporidium spp. in sheep in southern Xinjiang, China.

Cryptosporidium spp. are diarrheagenic intestinal parasites with multiple hosts worldwide. A total of 1252 fresh fecal samples of sheep were collected from 10 large-scale farms in southern Xinjiang. Based on the small subunit ribosomal (SSU rRNA) gene of Cryptosporidium, 100 Cryptosporidium-positive samples (8.0%, 100/1252) were detected by PCR. Nine out of 10 farms were positive for Cryptosporidium, with the highest infection rate being 18.4% (23/125) on farm 9 in Qira. The infection rates of Cryptosporidium in pre-weaned lambs, weaned lambs, fattening sheep, and adult sheep were 20.3% (61/301), 10.3% (34/329), 0.9% (3/327), and 0.7% (2/295), respectively. Three Cryptosporidium species were identified, namely, C. xiaoi (n = 61), C. parvum (n = 22), and C. ubiquitum (n = 17). Of them, C. xiaoi was detected on all positive farms and in different age groups of sheep. The subtypes of C. parvum and C. ubiquitum were identified by PCR at the 60 kDa glycoprotein (gp60) gene. Two C. parvum subtypes were identified: IIdA19G1 (n = 21) and IIdA15G1 (n = 1). One C. ubiquitum subtype was identified with XIIa (n = 17). These results indicated the common transmission and genetic diversity of Cryptosporidium in sheep in southern Xinjiang, and further investigations are needed on the zoonotic potential of C. parvum and C. ubiquitum in this region.

Di(2-ethylhexyl) phthalate and microplastics cause necroptosis and apoptosis in hepatocytes of mice by inducing oxidative stress.

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer and an endocrine disruptor. Microplastics (MPs) are pathogenic small plastic particles and abundant in the aqueous environment. The problem of residual hazards of plastic products is worthy of study, especially the joint exposure of a variety of plastic-related products to the toxic effect. We used 200 mg/kg DEHP and 10 mg/L MPs to establish exposure model in vivo and 2 mM DEHP and 200 µg/L MPs to establish AML12 cell exposure model in vitro. In vivo study results showed that compared with the control group (NC) group, DEHP and MPs significantly increased the contents of malondialdehyde and hydrogen peroxide, and significantly decreased the contents of glutathione and the activity of superoxide dismutase, total antioxidant capacity, catalase and glutathione peroxidase. The level of oxidative stress was further aggravated after combined exposure. The reactive oxygen species level of AML12 exposed to DEHP and MPs in vitro was significantly higher than NC group, and the combined exposure was significantly higher than the single exposure. The in vivo and in vitro also confirmed that DEHP and MPs could significantly increase the mRNA and protein levels of apoptosis markers and necroptosis markers and there was an additive effect. After N-acetylcysteine treatment in vitro, the above-mentioned oxidative stress level and cell damage decreased significantly. This study provided a reference for advocating the reduction of the mixed use of plastic products, and provided a basis for preventing the harm of plastic products residues.

Genetic diversity of Blastocystis subtypes in the Alpine musk deer (Moschus chrysogaster) in Gansu province, northwestern China.

Blastocystis sp. is a gastrointestinal pathogen that is frequently found in humans and animals worldwide. In this study, 201 fecal samples were collected from captive Alpine musk deer (Moschus chrysogaster) at three farms in Gansu province. Blastocystis was detected and subtyped by amplifying and sequencing the small subunit ribosomal DNA gene. The overall prevalence of Blastocystis was 39.8% (80/201). Five known Blastocystis subtypes (STs), including ST1 (n = 1), ST4 (n = 12), ST10 (n = 50), ST14 (n = 6), and ST24 (n = 11) were identified using subtyping and evolutionary analysis. ST10 was the most common ST observed in each farm. This study showed the infection status and genetic characteristics of Blastocystis in M. chrysogaster. Based on the surveyed data, because various potentially zoonotic STs, such as ST1, ST4, ST10, ST14, and ST24, were detected, it is believed that the zoonotic risk of Blastocystis from the Alpine musk deer in this area cannot be ignored.

Seasonal monitoring of Cryptosporidium species and their genetic diversity in neonatal calves on two large-scale farms in Xinjiang, China.

To find out whether and how the prevalence and genetic diversity of Cryptosporidium in neonatal calves vary with the season, 380 fecal samples from neonatal calves on two large-scale farms in Xinjiang (Alar and Wensu) were studied using molecular biology techniques. Cryptosporidium was detected in 48.7% (185/380) of the samples and was most frequent in summer (56.8%), followed by spring (50.0%), winter (46.8%), and autumn (41.7%; p > 0.05). Calves with diarrhea seem to be more likely infected by Cryptosporidium than those without diarrhea (p < 0.01). We also found that C. parvum (n = 173), C. bovis (n = 7), and C. ryanae (n = 3) were the Cryptosporidium species detected in this study, and co-infections of these three species (n = 2) were also identified. Two subtypes (IIdA14G1 and IIdA15G1) of C. parvum were identified, and both can infect human. These results also show that neonatal calves commonly suffer diarrhea caused by C. parvum throughout the year.

Cineole regulates Wnt/ß-catenin pathway through Nrf2/keap1/ROS to inhibit bisphenol A-induced apoptosis, autophagy inhibition and immunosuppression of grass carp hepatocytes.

Bisphenol A (BPA), an environmental pollutant, can cause multiple organ tissue damage by inducing oxidative stress. Cineole (CIN) is a terpene oxide existing in a variety of plant essential oils, which has anti-inflammatory, analgesic, and antioxidant effects. This study examined the effects of 200 nM BPA and 20 µM CIN on apoptosis, autophagy, and immunology in grass carp hepatocytes (L8824). The treatments were categorized as NC, CIN, BPA + CIN, and BPA. The findings demonstrated that BPA exposure could increase ROS levels and oxidative stress-related indicators, decrease the expression of the Nrf2/keap1 pathway and the Wnt/ß-catenin pathway, increase the expression of genes involved in the apoptotic pathway (Bax and Caspase3), and decrease the expression of the anti-apoptotic gene Bcl-2 by lowering mitochondrial membrane potential. BPA also reduced the expression of genes linked to autophagy (ATG5, Beclin1, LC3). Changes in immunological function after BPA exposure were also shown by changes in the amounts of antimicrobial peptides (HEPC, ß-defensin, LEAP2) and cytokines (INF-γ, IL-1ß, IL-2, and TNF-α). After the co-treatment of CIN and BPA, CIN can inhibit BPA-induced apoptosis and recover from autophagy and immune function to a certain extent by binding to keap1 to exert an anti-oxidative regulatory effect of Nrf2 incorporation into the nucleus. Molecular docking provides strong evidence for the interaction of CIN ligands with keap1 receptors. Therefore, these results indicated that CIN could inhibit BPA-induced apoptosis, autophagy inhibition and immunosuppression in grass carp hepatocytes by regulating the Wnt/ß-catenin pathway with Nrf2/keap1/ROS. This study provided further information to the risk assessment of the neuroendocrine disruptor BPA on aquatic organisms and offered suggestions and resources for further research into the function of natural extracts in the body's detoxification process.

Genotyping of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi from sheep and goats in China.

BACKGROUND: Few studies have molecularly characterized the potential zoonotic protozoa, Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in sheep and goats in China, therefore total 472 fecal samples were collected from eight provinces and infection rates of three protozoa were determined by PCR analysis of corresponding loci. All PCR positive samples were sequenced to identify the genotype. RESULTS: The overall infection rates for Cryptosporidium, G. duodenalis, and E. bieneusi were 1.9% (9/472), 20.6% (97/472), and 44.5% (210/472), respectively. C. xiaoi (n = 5), C. ubiquitum (n = 3), and C. anderson (n = 1) were identified in goats. 97 G. duodenalis strains were successfully detected, and assembly E (n = 96) and assembly A (n = 1) were identified. Two novel G. duodenalis multilocus genotype (MLGs) were identified, with one belonging to subgroup AI and the other to subgroup E5. Nine known genotype (BEB6, CD6, CHC8, CHG3, CHG5, Peru6, CHG1, CHG2, and COS-I) and four new genotype (CHG26, CHG27, CHG28, and CHS18) were identified in E. bieneusi, with CHG3 dominant in this group. CONCLUSIONS: The present results highlight the role of sheep and goats as reservoir hosts for this three gastrointestinal pathogens. In summary, we provided a platform for more detailed research on genotyping or subtyping intestinal pathogens to better understand their risks and modes of transmission.

Molecular characterization of Cryptosporidium spp. and Giardia duodenalis in pet dogs in Xinjiang, China.

Cryptosporidium spp. and Giardia duodenalis are opportunistic zoonotic protozoa transmitted through several routes. In this study, a total of 604 fecal samples were collected from pet dogs in Xinjiang, China to detect the presence of Cryptosporidium spp. and G. duodenalis by PCR amplification of the small subunit (SSU) rRNA gene. The incidence rate of Cryptosporidium spp. or G. duodenalis was 5.3% (32/604). Among the collection sites, a higher number of Cryptosporidium spp. or G. duodenalis positive dogs were detected in Hotan (9.9%, 21/213) and Shihezi (9.8%, 4/41) were higher than those in Aksu (4.5%, 1/22), Urumqi (2.1%, 4/191), and Korla (1.5%, 2/137). Among the sources, dogs in pet shops (7.4%, 19/256) showed a significantly higher incidence rate than those in pet hospitals (3.0%, 4/134) and pet kennels (2.3%, 5/214). When the data were examined by age, dogs < 1 year of age (6.1%, 28/459) were more likely to be infected by Cryptosporidium spp. or G. duodenalis than older (≥ 1 year) dogs (2.8%, 4/145). No significant differences were observed when animals were grouped by sex (5.0%, 14/278 for males; 5.5%, 18/326 for females). Sequence analysis revealed that the Cryptosporidium spp. (n = 10) in dogs were identified as C. canis. The G. duodenalis detected belonged to assemblages A (n = 1), C (n = 14), and D (n = 7). Among the identified 22 G. duodenalis isolates, eight samples were subtyped according to ß-giardin (bg) and the results were consistent with the identified assemblages. To our knowledge, this is the first report of Cryptosporidium spp. and G. duodenalis infections in domesticated canines in Xinjiang, China. The C. canis and G. duodenalis assemblage A identified in pet dogs in the present study were previously associated with infections in humans, indicating the potential for zoonotic transmission.

Longitudinal surveillance of Cryptosporidium spp. in broiler chickens in Xinjiang, northwest China: genetic diversity of Cryptosporidium meleagridis subtypes.

Cryptosporidium spp. are common enteric parasites in humans and animals. Herein, 175 faecal specimens were collected from a broiler farm in Xinjiang, China, including seven repeated samplings at 10-day intervals of broilers aged 10 to 70 days. Cryptosporidium was detected and identified by PCR-RFLP analysis. The overall infection rate of Cryptosporidium in broilers was 23.4% (41/175), with the highest infection rate of 48.0% (12/25) at 40 days of age, and no infection was detected at 10 days of age. Two Cryptosporidium species were confirmed, namely, C. baileyi (3.4%, 6/175) and C. meleagridis (20%, 35/175). In total, 21 of 35 C. meleagridis isolates were successfully subtyped based on the gp60 gene, and one known subtype, IIIgA22G3R1 (n = 1), and three novel subtypes, IIIbA25G1R1 (n = 10), IIIgA24G3R1 (n = 9) and IIIgA25G2R1 (n = 1), were identified. Our findings highlight the genetic diversity of C. meleagridis in Xinjiang and the potential endemic characteristics of the subtypes.

Occurrence and molecular characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. in captive wild animals in zoos in Henan, China.

BACKGROUND: Captive wild animals in zoos infected with Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. can be sources of zoonotic infections and diseases. Therefore, to investigate the distribution of these pathogens in captive wild animals of zoos in Henan, China, a total of 429 fresh fecal samples were collected from six zoos in Henan, China. The infection rates of Cryptosporidium spp., G. duodenalis, E. bieneusi, and Blastocystis sp. were determined by PCR analysis of corresponding loci. Positive results for Cryptosporidium (C. parvum and C. hominis) were subtyped based on the (gp60) gene. RESULTS: The overall prevalence was 43.1% (185/429), and the prevalence of Cryptosporidium, Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. were 2.8% (12/429), 0.5% (2/429), 20.8% (89/429), and 19.1% (82/429), respectively. Five Cryptosporidium species, namely, C. hominis, C. parvum, C. muris, C. andersoni, and C. macropodum, were identified in this study. Cryptosporidium parvum was further subtyped as IIdA19G1. Two Giardia duodenalis assemblages (A and E) were also identified. A total of 20 Enterocytozoon bieneusi genotypes were detected, including 18 known (BEB6, D, HND-1, CD7, SDD1, Henan-IV, KIN-1, CHK1, Peru8, Henan-V, CHG11, CHG-1, CHS9, CHG21, Type-IV, CHC9, CM5, and CHB1) and 2 novel genotypes (CHWD1 and CHPM1). A total of nine subtypes of Blastocystis sp. (ST1, ST2, ST3, ST5, ST6, ST7, ST10, ST13, and ST14) were identified in captive wild animals in zoos in the present study. Cryptosporidium andersoni, nine Enterocytozoon bieneusi genotypes, and five Blastocystis subtypes were here first identified in new hosts. CONCLUSIONS: Our study has expanded the host ranges of these four pathogens. The data indicate that animals in zoos can commonly be infected with these four zoonotic pathogens, and animals in zoos are potential sources of zoonotic infections in humans.

Morphological and molecular characterization of Cystoisospora yuensis n. sp. and Cystoisospora rastegaievae (Protozoa: Eimeriidae) in amur hedgehogs, Erinaceus amurensis (Schrenk, 1859).

Twenty-four fecal samples were collected from captive amur hedgehogs (Erinaceus amurensis) in Zhengzhou, China. Based on morphological and molecular analysis, the overall prevalence of Cystoisospora was 62.5% (15/24). These samples contained two types of coccidian oocysts, including C. rastegaievae (50.0%, 12/24) and a new species named C. yuensis n. sp. (12.5%, 3/24). Sporulated oocysts (n = 30) of C. yuensis n. sp. are ovoid, (20.6 ± 1.4) µm × (20.9 ± 0.9) µm, with a shape index (length/width) of 1.0 and a smooth and bi-layered oocyst wall, 1.3 µm thick (outer layer 0.8 µm, inner 0.5 µm). A polar granule is present, but micropyle cap, micropyle, and oocyst residuum are absent. The sporocysts are ovoid-shaped, (9.3 ± 0.6) µm × (8.5 ± 1.1) µm, with a shape index (length/width) of 1.1. Stieda, substieda bodies, and refractile bodies are absent. Residuum is scattered and distributed around the entire sporocysts. At the 18S rRNA locus, C. yuensis n. sp. exhibited the highest identity to C. timoni (99.3%) from a slender-tailed meerkat. It has 98.0% identity at the 28S rRNA locus and 99.3% at the ITS locus. Based on morphological and molecular data, this isolate is a new species of Cystoisospora. Additionally, we have provided data on the prevalence of C. rastegaievae in China and sequences of the 18S rRNS, 28S rRNA, and ITS loci.

The Potential Role of Synanthropic Rodents and Flies in the Transmission of Enterocytozoon bieneusi on a Dairy Cattle farm in China.

Enterocytozoon bieneusi causes microsporidiosis, a condition with complex epidemiology involving both direct and indirect transmission routes. To assess the potential role of synanthropic rodents and flies in the transmission of this pathogen, a total of 277 cattle fecal samples, 199 synanthropic rodents, and 50 batches of 20 flies were collected from a cattle farm. These samples were screened for the presence of E. bieneusi by PCR and sequencing of the internal transcribed spacer (ITS) region of the rRNA gene. The positive rates of cattle, synanthropic rodents, and flies were 11.9% (33/277), 4.0% (8/199) and 12.0% (6/50), respectively. Nineteen genotypes were identified, including 11 known genotypes (BEB6, I, COS-I, EbpC, D, J, CHS5, CHG1 to CHG3 and CHG14) and eight novel genotypes (named CHC9 to CHC16). The dominant genotype detected in the present study, BEB6, was found in all three categories of hosts. Moreover, human pathogenic genotypes D and EbpC were also observed in both synanthropic rodents and flies. These results demonstrate that synanthropic rodents and flies may act as biological disseminator or mechanical vector in the transmission of microsporidiosis to humans. Efforts should be made to minimize threats from these commensal animals to public health.

Multilocus Typing of Enterocytozoon bieneusi in Pig Reveals the High Prevalence, Zoonotic Potential, Host Adaptation and Geographical Segregation in China.

Enterocytozoon bieneusi is one of the most frequently diagnosed Microsporidia of humans and most animals. However, there is no information on E. bieneusi infection of pigs in Tibet and Henan, China. In this study, 1,190 fecal samples were collected from pigs in Tibet and Henan and screened for the presence of E. bieneusi. The overall prevalence of E. bieneusi infection was 54.2% (645/1,190), with differences in prevalence observed among geographical areas, ages, and pig breeds. Moreover, 10 E. bieneusi genotypes were identified based on internal transcribed spacer region genotyping, including eight known genotypes (EbpC, EbpA, CHG19, CHC5, Henan-III, I, D, and H) and two novel genotypes (XZP-I and XZP-II). Multilocus sequence typing revealed 18, 7, 17, and 13 genotypes at minisatellite/microsatellite loci MS1, MS3, MS4, and MS7, respectively. Strong linkage disequilibrium (LD) and few numbers of recombination events, suggest a clonal structure of the E. bieneusi population examined in this study. The low pairwise genetic distance (FST ) and gene flow (Nm) values indicated limited gene flow in the E. bieneusi population from different hosts, with phylogenetic, structure, and median-joining network analyses all indicating the existence of host and geographical isolation. The identification of isolates belonging to nine human-pathogenic genotypes indicates that pigs play an important role in the dissemination of E. bieneusi, improving our present understanding of E. bieneusi epidemiology in the studied region.

Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Rabbits in Xinjiang, China.

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, ß-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.