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Background: Cryptosporidium parvum is an enteric protozoan parasite with zoonotic importance and can cause cryptosporidiosis in humans as well as domestic and wild animals worldwide. The IId subtype family (SF) is one of the most prevalent subtypes of C. parvum. Some clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been developed to detect nucleic acid with high flexibility, sensitivity and specificity., Methods: By integrating recombinase polymerase amplification and the Cas12a/crRNA trans-cleavage system (termed ReCTC), we established end-point diagnostics by observing fluorescence readouts with the naked eye under blue light and on-site diagnostics using a lateral flow strip (LFS) biosensor., Results: Our ReCTC-based diagnoses can detect as little as a single copy of a cloned C. parvum 60-kDa glycoprotein (GP60) gene, 10 oocysts per gram (OPG), clinical fecal sample without tedious extraction of genomic DNA and have no cross-reactivity with other SFs of C. parvum or other common enteric parasitic protozoa., Conclusions: This study provided a new strategy for direct identification of the IId SF of C. parvum free of highly trained operators and expensive special equipment.

Anaplasma phagocytophilum, the bacterial pathogen responsible for tick-borne fever and human granulocytic anaplasmosis, can seriously affect the health of humans and a wide range of other mammals. In this study, we developed a recombinase polymerase amplification (RPA) assay to detect A. phagocytophilum in clinical samples. Following alignment of the relevant DNA sequences, a pair of specific primers based on the 16S rRNA gene was designed to specifically detect A. phagocytophilum. The assay was performed at a constant temperature of 38 °C for 30 min, with a final primer concentration of 0.4 μM. The specificity of the primers was confirmed when DNA from A. phagocytophilum was used as the positive control, and DNA from other related pathogens were used as the negative controls, with ddH 2 O acting as the blank control. The results showed that the primers did not cross-react with DNA from the other related pathogens. The assay's detection limit was 1.77 × 10 -5  ng/μl, a 10 × higher sensitivity level than that determined for nested PCR. The RPA assay's performance was evaluated using 44 clinical samples, and the prevalence results for A. phagocytophilum were found to not differ significantly between the RPA assay and the nested PCR. Thus, we have developed a specific, sensitive, rapid and cost-effective RPA method, requiring only a water bath, for the detection of A. phagocytophilum. The assay should be especially useful in resource-limited areas where access to laboratory equipment is limited., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Blastocystis, a widely distributed zoonotic protozoan infecting both humans and numerous animals, remains poorly understood with its potential medical and veterinary significance. This study examined the molecular occurrence and genetic variation of Blastocystis in children and calves in Bangladesh to explore cross-species transmission and disease burden. Methods: In total, 998 DNA samples were investigated, comprising 299 stool DNA from children and 699 fecal DNA from calves, using polymerase chain reaction and sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. Results: This study detected Blastocystis in 5.35% of the children and 14.74% of the calves. While slight variations in occurrence rates were observed across different study variables, none were statistically significant. The occurrence was highest among children under 5 years and calves aged 1–3 months. Regarding breed, the Holstein Friesian cross and the Jersey cross exhibited higher rates of infection. Conversely, occurrences were lower among children and calves in Gazipur district. The remaining parameters displayed nearly equivalent percentages of Blastocystis. The subtypes identified in children included ST1, ST3, and ST4, with ST1 comprising 50% of them. ST3 and ST4 were also found in calves, alongside ST10 (55.34%) being the most prevalent. Other subtypes found in calves were ST14, ST21, and ST24–ST26. Conclusions: This study found that Blastocystis is more common in calves than in children in Bangladesh, with genetic diversity of nine subtypes. The common occurrence of identical variants of two subtypes in both populations suggests potential zoonotic transmission, highlighting the necessity for further molecular investigations and comprehensive measures within the One Health framework to mitigate public health risks. [ABSTRACT FROM AUTHOR]

Background: A close connection between a protozoan parasite and the balance of the other gut microbes of the host has been demonstrated. The calves may be naturally co-infected with many parasites, and the co-effects of parasites on other intestinal microbes of calves remain unclear. This study aims to preliminarily reveal the relationship between intestinal parasites and other intestinal microbes in calves. Methods: Fecal samples were collected from four calves with bloody diarrhea, four calves with watery diarrhea, and seven normal calves, and the microbial flora of the samples were analyzed by whole-genome sequencing. Protozoal parasites were detected in the metagenome sequences and identified using polymerase chain reaction (PCR). Results: Cryptosporidium, Eimeria, Giardia, Blastocystis, and Entamoeba were detected by metagenomic analysis, and the identified species were Giardia duodenalis assemblage E, Cryptosporidium bovis, Cryptosporidium ryanae, Eimeria bovis, Eimeria subspherica, Entamoeba bovis, and Blastocystis ST2 and ST10. Metagenomic analysis showed that the intestinal microbes of calves with diarrhea were disordered, especially in calves with bloody diarrhea. Furthermore, different parasites show distinct relationships with the intestinal microecology. Cryptosporidium, Eimeria, and Giardia were negatively correlated with various intestinal bacteria but positively correlated with some fungi. However, Blastocystis and Entamoeba were positively associated with other gut microbes. Twenty-seven biomarkers not only were significantly enriched in bloody diarrhea, watery diarrhea, and normal calves but were also associated with Eimeria, Cryptosporidium, and Giardia. Only Eimeria showed a distinct relationship with seven genera of bacteria, which were significantly enriched in the healthy calves. All 18 genera of fungi were positively correlated with Cryptosporidium, Eimeria, and Giardia, which were also significantly enriched in calves with bloody diarrhea. Functional genes related to parasites and diseases were found mainly in fungi. Conclusions: This study revealed the relationship between intestinal protozoan parasites and the other calf gut microbiome. Different intestinal protozoan parasites have diametrically opposite effects on other gut microecology, which not only affects bacteria in the gut, but also is significantly related to fungi and archaea. [ABSTRACT FROM AUTHOR]

Background: Cryptosporidium is second only to rotavirus as a cause of moderate-to-severe diarrhea in young children. There are currently no fully effective drug treatments or vaccines for cryptosporidiosis. MicroRNAs (miRNAs) are involved in regulating the innate immune response to Cryptosporidium parvum infection. In this study, we investigated the role and mechanism of miR-3976 in regulating HCT-8 cell apoptosis induced by C. parvum infection. Methods: Expression levels of miR-3976 and C. parvum burden were estimated using real-time quantitative polymerase chain reaction (RT-qPCR) and cell apoptosis was detected by flow cytometry. The interaction between miR-3976 and B-cell lymphoma 2-related protein A1 (BCL2A1) was studied by luciferase reporter assay, RT-qPCR, and western blotting. Results: Expression levels of miR-3976 were decreased at 8 and 12 h post-infection (hpi) but increased at 24 and 48 hpi. Upregulation of miR-3976 promoted cell apoptosis and inhibited the parasite burden in HCT-8 cells after C. parvum infection. Luciferase reporter assay indicated that BCL2A1 was a target gene of miR-3976. Co-transfection with miR-3976 and a BCL2A1 overexpression vector revealed that miR-3976 targeted BCL2A1 and suppressed cell apoptosis and promoted the parasite burden in HCT-8 cells. Conclusions: The present data indicated that miR-3976 regulated cell apoptosis and parasite burden in HCT-8 cells by targeting BCL2A1 following C. parvum infection. Future study should determine the role of miR-3976 in hosts' anti-C. parvum immunity in vivo. [ABSTRACT FROM AUTHOR]

Cryptosporidium spp. are zoonotic intestinal parasites that infect fish, birds, reptiles and mammals. Cryptosporidium spp. are common cause of diarrhea. In this study, a total of 1032 fecal samples were collected from the rectums of sheep and goats. The samples were analyzed using nested polymerase chain reaction (nested PCR) based on the small subunit ribosomal RNA (SSU rRNA) gene of Cryptosporidium spp. The average infection rate of Cryptosporidium spp. was 2.23% (n = 23), and three Cryptosporidium species were identified, namely Cryptosporidium ubiquitum (8/23), Cryptosporidium andersoni (5/23) and Cryptosporidium xiaoi (10/23). Subtyping of C. ubiquitum and C. xiaoi was carried out by DNA sequence analysis of the 60-kDa glycoprotein (gp60) gene. Eight C. ubiquitum isolates were identified as zoonotic subtype XIIa. Nine C. xiaoi isolates were identified as subtypes XXIIIc (n = 1), XXIIIf (n = 3) and XXIIIg (n = 5). Subtype XXIIIg was first found in Chinese sheep. C. ubiquitum subtype XIIa was found in both sheep and goats, suggesting that sheep and goats are important sources of C. ubiquitum infections. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) are involved in the regulation of both the innate and adaptive immune response to Cryptosporidium parvum infection. We previously reported that C. parvum upregulated miR‑942‑5p expression in HCT‑8 cells via TLR2/TLR4‑NF‑κB signaling. In the present study, the role of miRNA-942-5p in the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated HCT-8 cell apoptosis induced by C. parvum was investigated. Methods: Quantitative real-time polymerase chain reaction, western blotting, flow cytometry, and immunofluorescence were used for analysis. Results: Forced expression of miRNA-942-5p resulted in decreased apoptosis and an increased C. parvum burden in HCT-8 cells. The opposite results were observed using the suppressed expression of miRNA-942-5p. The miRNA-942-5p led to the translational suppression of IFI27 gene through targeting the 3'-untranslated region of the IFI27 gene. Moreover, overexpression of the IFI27 gene produced a high apoptotic ratio and low C. parvum burden. In contrast, a low apoptotic ratio and a high C. parvum burden were observed following downregulation of the IFI27 gene. Both miR-942-5p and the IFI27 gene influenced TRAIL and caspase-8 expression induced by C. parvum in HCT-8 cells. Moreover, TRAIL promoted HCT-8 cell apoptosis in a concentration-dependent manner. Conclusions: These data suggested that C. parvum induced the downregulation of IFI27 via relief of miR-942-5p-mediated translational suppression. IFI27 downregulation was affected the burden of C. parvum by regulating HCT-8 cell apoptosis through TRAIL-dependent pathways. Future studies should determine the mechanisms by which C. parvum infection increases miR-942-5p expression and the role of miR-942-5p in hosts' anti-C. parvum immunity in vivo. [ABSTRACT FROM AUTHOR]

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Background: Blastocystis is one of the most common intestinal parasites in humans and various animals worldwide. Few studies are available regarding the genetic characterization of Blastocystis infections in humans in China. Methods: In the present study, 609 fecal samples were collected from two- to six-year-old kindergarten children in southern Xinjiang and were examined by polymerase chain reaction (PCR). Results: The infection rate of Blastocystis was 14.3% (87/609); no significant difference was observed among counties and between sexes. Blastocystis subtypes ST1 (n = 38), ST2 (n = 8), and ST3 (n = 41) were identified by sequence analysis of the small subunit ribosomal RNA gene. Genetic polymorphisms were observed at the intra-subtype level, including seven variations for ST1 (ST1A to ST1G), four for ST2 (ST2A to ST2D), and two for ST3 (ST3A and ST3B); with ST1F and ST2B being new variations. Conclusions: ST1 and ST3 are the two common Blastocystis subtypes in the study area. More extensive studies in both humans and animals in different regions are needed to better characterize the transmission of Blastocystis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Anaplasma capra is an emerging pathogen, which can infect ruminants and humans. This study was conducted to determine the occurrence of A. capra in the blood samples of sheep and goats in China. Using nested polymerase chain reaction (nested-PCR) targeting the gltA gene and conventional PCR targeting the heat shock protein (groEL) gene and the major surface protein4 gene (msp4), A. capra was detected in 129 (8.9%) of 1453 sheep and goat blood samples. The positive rate was higher in goats (9.4%, 89/943) than in sheep (7.8%, 40/510) (χ2 = 1.04, p > 0.05, df = 1). For sheep, A. capra was found in 17 sites from 2 provinces. The prevalence was 28.6% in sheep from Liaoning province, which was higher than in Henan Province (7.3%). For goats, A. capra was detected in 35 sites from 7 provinces. The prevalence varied from 0 to 19.4% in the goat sites examined. The prevalence rates were 19.4, 19.3, 10, 8.8, 6.8, 1.8, and 0% in goats from Guizhou province, Henan Province, Inner Mongolia Autonomous Region, Shanxi Province, Xinjiang Uygur Autonomous Region, Yunnan province, and Gansu province, respectively. Based on the analysis of the A. capra citrate synthase gene (gltA), two variants were identified. Variant I showed a high sequence similarity to the A. capra , which were previously reported in sheep, goats, Ixodes persulcatus, Haemaphysalis longicornis, Haemaphysalis qinghaiensis , and humans. Variant II was only found in Luoyang, Anyang, and Sanmengxia, of Henan province. To our knowledge, this is the first detection of this variant of A. capra in sheep and goat blood in China. Phylogenetic analysis based on groEL and msp4 genes showed that the Anaplasma sp. sequences clustered independently from A. capra and other Anaplasma species with high bootstrap values. We found A. capra DNA in sheep and goats in China, providing evidence that sheep and goats can be infected by A. capra. We also found that this zoonotic pathogen is widely distributed in China. This study provides information for assessing the public health risks for human anaplasmosis. [ABSTRACT FROM AUTHOR]

Anaplasma phagocytophilum is a zoonotic pathogen and the causative agent of human granulocytic anaplasmosis in humans and tick-borne fever in various kinds of animals. In the present study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of A. phagocytophilum was developed using primers specific to 16S rRNA gene of this organism. The LAMP assay was performed at 65 C for 60 min and terminated at 80 C for 10 min. The optimal reaction conditions under which no cross-reaction was observed with other closely related tick-borne parasites ( Anaplasma bovis, Anaplasma ovis, Theileria luwenshuni, Babesia motasi, and Schistosoma japonicum) were established. The assay exhibited much higher sensitivity compared with conventional polymerase chain reaction (PCR) (1 copy vs. 1,000 copies). To evaluate the applicability of the LAMP assay, 94 field samples of sheep blood were analyzed for A. phagocytophilum infection by using LAMP, nested PCR, and conventional PCR assays at the same time. A positive LAMP result was obtained from 53 (56.4%) of the 94 samples, whereas only 12 (12.8%) and 3 (3.2%) tested positive by nested and conventional PCR, respectively. In conclusion, this LAMP assay is a specific, sensitive, and rapid method for the detection of A. phagocytophilum in sheep/goats. [ABSTRACT FROM AUTHOR]

Background. Chickens serve as an intermediate host for Toxoplasma gondii and Neospora caninum; infection of free-range (FR) chickens with these organisms is a useful indicator of soil and environmental contamination with oocysts. A total of 700 FR chicken serum samples and 300 heart samples were collected from Henan province from March to July 2015. Antibodies to T. gondii were found in 18.86% (132/700) of the chickens by modified agglutination test (cut-off 1 : 25), while 23.14% (162/700) were positive for N. caninum by indirect fluorescent antibody test (cut-off 1 : 25). T. gondii DNA was detected in the myocardium digestion liquids of 4/25 (16%) FR chickens. The PCR results of N. caninum DNA from FR chicken myocardium digestion liquids (n=25) were all negative. Attempts to isolate viable T. gondii were unsuccessful. The results showed that there were antibodies to T. gondii and N. caninum in FR chickens from Henan province. Accordingly, effective control of feces from cats and dogs and improved pets hygiene habits were needed. To the author’s knowledge, this is the first report of the presence of N. caninum antibody in chickens from China. [ABSTRACT FROM AUTHOR]

The burden of cryptosporidiosis due to Cryptosporidium parvum is well documented in HIV-positive patients in Ethiopia. However, the role of animals in zoonotic transmission of the disease is poorly understood. The aim of this study was to determine the prevalence and genotypes of Cryptosporidium species in dairy calves; to assess the role of cattle in zoonotic transmission in central Ethiopia. A total of 449 fecal samples were collected and screened using modified Ziehl-Neelson staining method and PCR targeting the small-subunit (SSU) rRNA gene. The prevalence of Cryptosporidium was 9.4% (42/449) and 15.8% (71/449) as detected by microscopy and nested PCR, respectively. The prevalence of infection varied significantly across the study areas with the higher prevalence being observed in Chancho 25.4% (30/118). Crossbred calves had significantly higher prevalence of Cryptosporidium than indigenous zebu. Genotyping results revealed the presence of C. andersoni (76.1%), C. bovis (19.7%) and C. ryanae (4.2%). The occurrence of these Cryptosporidium species appeared to be age-related. C. andersoni constituted 92.1% of the Cryptosporidium infection in calves older than 3 months. Sequence analysis also showed the existence of intra-species variation at SSU rRNA gene. Findings of the current study indicate that cattle may not be an important source of zoonotic cryptosporidiosis in central Ethiopia. Further molecular studies are needed to support this observation from other part of the country. [ABSTRACT FROM AUTHOR]

To appreciate the genetic diversity and zoonotic implications of Enterocytozoon bieneusi in nonhuman primates (NHPs) in zoos, we genotyped E. bieneusi in captive NHPs in seven zoos located at six major cities in China, using ribosomal internal transcribed spacer (ITS)-based PCR and sequence analyses. A total of 496 fecal specimens from 36 NHP species under nine families were analyzed and E. bieneusi was detected in 148 (29.8%) specimens of 25 NHP species from six families, including Cercopithecidae (28.7%), Cebidae (38.0%), Aotidae (75.0%), Lemuridae (26.0%), Hylobatidae (50.0%) and Hominidae (16.2%) (P = 0.0605). The infection rates were 29.0%, 15.2%, 18.2%, 37.3%, 29.2%, 37.7% and 44.8% in Shijiazhuang Zoo, Wuhan Zoo, Taiyuan Zoo, Changsha Wild Animal Zoo, Beijing Zoo, Shanghai Zoo and Shanghai Wild Animal Park, respectively (P = 0.0146). A total of 25 ITS genotypes were found: 14 known (D, O, EbpC, EbpA, Type IV, Henan-IV, BEB6, BEB4, Peru8, PigEBITS5, EbpD, CM1, CM4 and CS-1) and 11 new (CM8 to CM18). Genotype D was the most prevalent one (40/148), followed by CM4 (20/148), CM1 (15/148), O (13/148), CM16 (13/148), EbpC (11/148). Of them, genotypes D, EbpC, CM4 and O were widely distributed in NHPs (seen in 9 to 12 species) whereas genotypes CM1 and CM16 were restricted to one to three NHP species. In phylogenetic analysis, 20 genotypes (121/148, 81.8%), excluding genotypes BEB4, BEB6, CM9, CM4 and CM18, belonged to group 1 with zoonotic potential. New genotype CM9 clustered in group 2 with BEB4 and BEB6. The remaining two genotypes CM4 and CM18 formed new cluster (group 9) in between two other genotypic clusters found in primates. The findings of high diversity in E. bieneusi genotypes and their zoonotic potentiality concluded the importance of captive NHPs as reservoir hosts for human microsporidiosis. [ABSTRACT FROM AUTHOR]

Enteric protozoa are frequently found in snakes. Nevertheless, few studies regarding genetic characterization of these parasites have been carried out. We describe here the first molecular survey of protozoan pathogens from snakes in China and the first report on Enterocytozoon bieneusi genotyping in snakes in the world. Here, 240 fecal specimens were collected from two species of captive snakes, Naja naja (Indian cobra) and Ptyas mucosus (Oriental rat snake), in Guangxi Province, China, and examined by PCR amplification of the small subunit-ribosomal RNA of enteric protozoa and the internal transcribed spacer of ribosomal RNA of E. bieneusi. Cryptosporidium serpentis was identified in three specimens (2.1 %) of Oriental rat snakes. Caryospora was found in 5.4 % specimens, including eight from cobras (8.1 %) and five from rat snakes (3.6 %), and represented six new species- Caryospora sp. SKC-2014a to Caryospora sp. SKC-2014 f. Three new Eimeria species, Eimeria sp. SKE-2014a to Eimeria sp. SKE-2014c, were detected in three specimens (2.1 %) from rat snakes. Additionally, Sarcocystis sp. SKS-2014 was detected in one specimen from a cobra. The infection rates of E. bieneusi were 3.0 % in cobras and 5.7 % in rat snakes. Sequence analysis of 11 PCR products revealed the presence of six E. bieneusi genotypes-two known genotypes (type IV and Henan V) and four new genotypes (CRep-1 to CRep-4). All six E. bieneusi genotypes belonged to the zoonotic group (group 1). This result raised the possibility that E. bieneusi could be present in animals consumed by snakes. This should be taken into consideration to better understand the diversity of the parasite, its transmission through the predator-prey relationship, and public health implications. [ABSTRACT FROM AUTHOR]

Few data are available on the molecular characterization of Cryptosporidium spp. in chickens and ducks in China. In this study, 2579 faecal samples from 46 chicken farms and eight Pekin duck farms in 21 prefectures in Henan Province were examined. The overall infection rate of Cryptosporidium was 10.6% (163/1542) in layer chickens (10 out of 17 farms), 3.4% (16/473) in broilers (five out of 29 farms), and 16.3% (92/564) in Pekin ducks (four out of eight farms), respectively. The highest infection rates were observed in 31-day-old to 60-day-old layer chickens (24.6%) and 11-day-old to 30-day-old Pekin ducks (40.3%). The season of highest prevalence in chickens was spring (15.6%) and the lowest was winter (P<0.01). One hundred and eighty-seven Cryptosporidium-positive samples were analysed by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of the small subunit rRNA gene, and 55 were further analysed by DNA sequencing of the PCR products. Two Cryptosporidium species were identified: Cryptosporidium baileyi (184/187) on 15 chicken farms and four duck farms, and Cryptosporidium meleagridis (3/187) on three layer chicken farms. C. baileyi was the predominant Cryptosporidium species, found in all age groups of chickens and all Cryptosporidium-positive ducks examined, whereas C. meleagridis was only identified in 31-day-old to 120-day-old layer chickens. Considering the large size of the chicken industry and the close contact between chickens and humans, and that C. meleagridis is the third most common Cryptosporidium parasite in humans, then C. meleagridis could potentially become an emerging zoonosis in some areas in China. [ABSTRACT FROM AUTHOR]

The distribution and public health significance of Cryptosporidium species/genotypes in pigs differ among geographic areas and studies. To characterize the prevalence of cryptosporidiosis in pigs in Henan, China, a total of 1,350 fecal samples from 14 farms in ten prefectures in Henan Province were examined. The overall prevalence of Cryptosporidium was 8.2% (111/1,350), with the highest infection rate (79/383 or 20.6%) in 1-2-month-old piglets and the lowest infection rates in 3-6-month-old pigs. Cryptosporidium-positive samples from 108 animals were analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of the small subunit rRNA gene, and 35 were further analyzed by DNA sequencing of the PCR products. Two Cryptosporidium species/genotype were identified, including Cryptosporidium suis (94/108) and the Cryptosporidium pig genotype II (14/108). C. suis infection was more common in younger piglets whereas the pig genotype II was relatively common in older pigs. These findings suggest that pigs are not a major source of zoonotic Cryptosporidium in the study area. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • Sheep have played a role in the spread of C. parvum and C. ubiquitum in Xinjiang, China • Identified Eight E. bieneusi genotypes that belonged to cattle host-specific Group 2. • All the G. duodenalis -positive specimens belonged to assemblage E. • Two novel E. bieneusi genotypes were named as XJS1 and XJS2. [ABSTRACT FROM AUTHOR]

Abstract Giardia duodenalis is a gastrointestinal parasitic protozoan with important veterinary and public health implications worldwide. In this study, a total of 1440 fecal specimens were collected from dairy calves in Guangdong Province, China. The overall prevalence of G. duodenalis was 2.2% (31/1440) by polymerase chain reaction (PCR). The prevalence was 8.4% and 1.1% in pre- and post-weaned calves, respectively. Both assemblages A (6.5%, 2/31) and E (93.5%, 29/31) were found based on sequence analysis of the SSU rRNA gene. All G. duodenalis -positive samples were assayed with PCR followed by sequencing the beta giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes, and 20, 23, and 18 sequences were obtained, respectively. Multilocus genotyping yielded 10 assemblage E multilocus genotypes (MLGs). All assemblage E MLGs were genetically distinct from those obtained in cattle from Henan, Xinjiang, Shanghai, and Shaanxi in China. This finding was also supported by F ST , N m , and median-joining network analysis. Results of this study indicated that G. duodenalis was found in low prevalence in dairy calves in Guangdong Province, southern China; nevertheless, the infected calves might represent a public health risk in this region. Additionally, the differences in assemblage E MLGs from the five analyzed regions revealed significant geographic segregation of G. duodenalis in China. Graphical abstract Unlabelled Image Highlights • Both assemblages A and E of Giardia duodenalis were found in dairy calves. • The infected calves might represent a public health risk in this region. • Geographic segregation has shown in assemblage E of G. duodenalis in China. [ABSTRACT FROM AUTHOR]

Abstract Cryptosporidium spp., Giardia duodenalis , and Enterocytozoon bieneusi are globally ubiquitous pathogens. However, little is known about the occurrence and distribution of Cryptosporidium spp., G. duodenalis , and E. bieneusi in Tibetan sheep. In the present study, fecal specimens of 177 Tibetan sheep were examined by nested PCR. 4.5% (n = 8), 1.7% (n = 3) and 34.5% (n = 61) of the Tibetan sheep were positive for Cryptosporidium spp., G. duodenalis , and E. bieneusi , respectively. Cryptosporidium ubiquitum was the only species found by small subunit (SSU) rRNA-based PCR, and subtyping of C. ubiquitum (n = 6) by 60-kDa glycoprotein (gp60) revealed that all positive isolates belonged to zoonotic XIIa subtype 2. Multilocus genotyping at the SSU rRNA and β-giardin ( bg ) genes suggested that three G. duodenalis positive specimens belonged to assemblage E. Sequences analysis of internal transcribed spacer (ITS) gene characterized four E. bieneusi genotypes which all belonged to Group 2, one novel CGS1 (n = 3), and three known: CM7 (n = 34), BEB6 (n = 22), and CHS3 (n = 2). We employed multilocus sequence typing (MLST) using three microsatellites (MS1, MS3 and MS7), one minisatellite (MS4), and sequence analysis of MLST yielded 3, 2, 2 and 2 genotypes at the MS1, MS3, MS4, and MS7 loci, respectively, forming 4 MLGs. Our findings elucidate the occurrence and distribution of three zoonotic pathogens in Tibetan sheep in China. More subsequent and detailed data are required to better understand the transmission of Cryptosporidium spp., G. duodenalis , and E. bieneusi in sheep. Graphical abstract Unlabelled Image Highlights • Only Cryptosporidium ubiquitum was found in Tibetan sheep, all positive isolates belonged to zoonotic XIIa subtype 2. • Giardia positive specimens belonged to assemblage E. • Four E. bieneusi genotypes were identified, one novel and three known. • Four E. bieneusi MLGs were identifiied based on multilocus sequence typing tool. [ABSTRACT FROM AUTHOR]

Amebiasis is a major public-health concern. It has a global distribution, and is listed as the third leading parasitic cause of human mortality. To survey the prevalence and assess the potential zoonotic transmission of Entamoeba spp. in nonhuman primates (NHPs) in China, 2688 fresh fecal specimens were collected from NHPs reared in farms or zoos/parks or free ranging in 13 districts. The overall prevalence of Entamoeba spp. infection determined with microscopy analysis was 39.4% (1059/2688). Higher infection rates were detected in the free ranging group (41.1%, 169/411) and in animals < 1 year old (58.7%, 556/947). Gene fragments were successfully amplified 463 (87.2%) out of the 531 selected specimens (approximately half of the total microscopy-positive specimens). Polymerase chain reaction (PCR) amplification identified 386 (83.4%) Entamoeba dispar and 287 (62.0%) E. coli infections, and among these, 210 (45.4%) were mixed infections. And that the E. dispar and E. coli had also been detected in humans. In phylogenetic analysis, the E. dispar and E. coli sequences clustered with reference E. dispar and E. coli , respectively. In conclusion, nonhuman primates infected with Entamoeba species, with high prevalence and zoonotic potential, should be considered when evaluating the maintenance of Entamoeba spp. and its transmission between animal reservoirs and humans. [ABSTRACT FROM AUTHOR]

Enterocytozoon bieneusi is the most common microsporidia species in humans and has a variety of animal hosts. To assess the prevalence and molecular characteristics of E. bieneusi in dairy calves in the Xinjiang Uyghur Autonomous Region of China, 514 fecal samples were collected from 15 farms and examined by polymerase chain reaction based on the internal transcribed spacer of the ribosomal RNA gene of E. bieneusi. The overall prevalence of E. bieneusi in calves was 16.5% (85/514). No significant difference in prevalence was observed between pre- and post-weaned calves. Sequence analysis of ITS nucleotide sequences identified six known genotypes (BEB4, CC4, D, I, J, and EbpC), five of these previously detected in humans. Genotype J was the most prevalent genotype (57/85) and was identified on 11 farms. The high prevalence of zoonotic E. bieneusi genotypes in dairy calves suggests they are a potential source of zoonotic infection in humans. [ABSTRACT FROM AUTHOR]

• Average infection of E. bieneusi in ruminants was 16.0% in northwest China. • Diverse E. bieneusi ITS genotypes were identified, with total of 13 genotypes observed (10 known and 3 novel genotypes). • Genotype BEB6 was predominant, followed by subtype COS-I, J, and CHC8. This study was conducted to examine the molecular characteristics and assess the zoonotic potential of Enterocytozoon bieneusi in ruminants in northwest China. A total of 1581 fresh fecal samples were collected from eight categories of ruminants. The E. bieneusi was screened and genotyped via nested polymerase chain reaction (PCR) amplification targeting the internal transcribed spacer (ITS) of the small subunit rRNA (ssu rRNA) gene. The result indicated that the average infection rate of E. bieneusi in ruminants was 16.0% (253/1581), with infection rates of E. bieneusi in Mongolian sheep, Mongolian goats, Chifeng cattle, red deer, alpine musk deer, and blue sheep at 21.8% (169/777), 8.2% (46/561), 25.9% (28/108), 13.2% (5/38), 20.0% (4/20), and 6.3% (1/16), respectively. The infections of E. bieneusi varied by different categories. For the different age groups, the infection rates in lambs (29.3%, 108/369) and calves (57.1%, 8/14) were significantly higher than that in ewes (21.1%, 215/1020) and cows (21.3%, 20/94). For the molecular characterization, diverse E. bieneusi ITS genotypes were identified, with a total of 13 genotypes were observed, including 10 known genotypes (BEB6, COS-I, J, CHC8, I, CHG1, BEB4, CHG3, CHS7, and NCF2) and 3 novel genotypes (CNR1 to CNR3). Genotype BEB6 was predominant (59.7%, 151/253). Phylogenetic analysis revealed that most E. bieneusi ITS genotypes clustered into group 2 and only one (NCF2) genotype belonged to group 1. The zoonotic genotypes identified in ruminants in the present study indicated the zoonotic potential of E. bieneusi. In addition, simultaneous identification of genotypes, such as BEB6, COS-I, and BEB4, in the same eco-geographical system indicated some host multiplicity transmission potential of E. bieneusi. [ABSTRACT FROM AUTHOR]

Organisms of the genera Anaplasma and Theileria are important intracellular bacteria and parasites that cause various tick-borne diseases, threatening the health of numerous animals as well as human beings. In the present study, a 12-month-old male wild South African giraffe ( Giraffa camelopardalis giraffa ) originating from South Africa, and living in Zhengzhou Zoo (located in the urban district of Zhengzhou in the provincial capital of Henan), suddenly developed an unknown fatal disease and died 1 day after the onset of the clinical signs. By microscopic examination of Giemsa-stained blood smears combined with nested PCR and DNA sequence analysis, Anaplasma phagocytophilum , Anaplasma bovis and a novel Theileria spp. were found in the blood of this giraffe. The six other Cervidae animals in the zoo and three ruminants living in the same colony house with them were found to be negative for both Anaplasma and Theileria in their blood specimens. We report on the first case of an A. phagocytophilum infection and the occurrence of a novel Theileria spp. in the blood of a giraffe. This is the first reported case of a multi-infection of A. bovis , A. phagocytophilum and Theileria spp. in a giraffe, as revealed by microscopic examination of blood smears and the results of nested PCR and DNA sequencing. [ABSTRACT FROM AUTHOR]

822 fecal samples from cattle in six areas of Beijing were examined with microscopy for Cryptosporidium oocysts and Giardia cysts. The overall infection rates for Cryptosporidium spp. and Giardia duodenalis were 2.55% and 1.09%, respectively. Cryptosporidium was only detected in calves and heifers, whereas G. duodenalis was found in all age groups. Cryptosporidium spp. were characterized with a PCR-restriction fragment length polymorphism analysis and DNA sequence analysis of the small subunit (SSU) rRNA gene. Two Cryptosporidium species were identified: Cryptosporidium parvum ( n = 12) and Cryptosporidium andersoni ( n = 9). Six C. parvum isolates were successfully subtyped with the gp60 gene and three subtypes were detected: IIdA19G1 ( n = 1), IIdA17G1 ( n = 1), and IIdA15G1 ( n = 4). Subtype IIdA17G1 is reported for the first time in cattle worldwide. Nine G. duodenalis isolates were analyzed by sequencing the triosephosphate isomerase ( tpi ) gene, and only G. duodenalis assemblage E was identified. Therefore, the predominance of C. parvum detected in calves was identical to that found in the Xinjiang Uyghur and Ningxia Hui Autonomous Regions, but differed considerably from that in Henan, Heilongjiang, and Shannxi Provinces. In contrast, the predominance of G. duodenalis assemblage E was more or less similar to its predominance in other areas of China or countries. Our findings confirm the unique character of the C. parvum IId subtypes in China. More systematic studies are required to better understand the transmission of Cryptosporidium and G. duodenalis in cattle in China. [ABSTRACT FROM AUTHOR]

A total of 514 fecal samples were collected from dairy calves on 15 farms in the Xinjiang Uyghur Autonomous Region in Northwestern China and were screened for Cryptosporidium spp. by PCR. The overall prevalence of Cryptosporidium was 16.0% (82/514), and the infection rate was 15.6% (37/237) and 16.2% (45/277) in pre- and post-weaned calves, respectively. PCR-restriction fragment length polymorphism (RFLP) and DNA sequence analyses of 82 positive fecal samples revealed the presence of four Cryptosporidium species; of these, 22 were Cryptosporidium parvum , 20 were Corynebacterium bovis , 9 were Cryptosporidium ryanae , 25 were Cryptosporidium andersoni , 2 were mixed C. bovis / C. parvum , and 4 were mixed C. bovis / C. ryanae infections. In pre-weaned calves, C. parvum was the most common species (22/37, 59.5%). In contrast, C. andersoni was the dominant species (23/45, 51.1%) in post-weaned calves. Subtyping analysis based on the 60-kDa glycoprotein ( gp60 ) gene successfully identified 15 C. parvum isolates as being in the IId family; 11 were IIdA15G1 and 4 were IIdA14G1. Recent findings describing the C. parvum IId subtypes as the dominant group in humans and animals in China indicate that dairy calves may be an important source of zoonotic Cryptosporidium in China. [ABSTRACT FROM AUTHOR]

Non-human primates (NHPs) are commonly infected with Cryptosporidium spp. and Giardia duodenalis . However, molecular characterisation of these pathogens from NHPs remains scarce. In this study, 2,660 specimens from 26 NHP species in China were examined and characterised by PCR amplification of 18S rRNA, 70 kDa heat shock protein ( hsp70 ) and 60 kDa glycoprotein ( gp60 ) gene loci for Cryptosporidium ; and 1,386 of the specimens by ssrRNA, triosephosphate isomerase ( tpi ) and glutamate dehydrogenase ( gdh ) gene loci for Giardia . Cryptosporidium was detected in 0.7% (19/2660) specimens of four NHP species including rhesus macaques (0.7%), cynomolgus monkeys (1.0%), slow lorises (10.0%) and Francois’ leaf monkeys (6.7%), belonging to Cryptosporidium hominis (14/19) and Cryptosporidium muris (5/19). Two C. hominis gp60 subtypes, IbA12G3 and IiA17 were observed. Based on the tpi locus, G. duodenalis was identified in 2.2% (30/1,386) of specimens including 2.1% in rhesus macaques, 33.3% in Japanese macaques, 16.7% in Assam macaques, 0.7% in white-headed langurs, 1.6% in cynomolgus monkeys and 16.7% in olive baboons. Sequence analysis of the three targets indicated that all of the Giardia -positive specimens belonged to the zoonotic assemblage B. Highest sequence polymorphism was observed at the tpi locus, including 11 subtypes: three known and eight new ones. Phylogenetic analysis of the subtypes showed that most of them were close to the so-called subtype BIV. Intragenotypic variations at the gdh locus revealed six types of sequences (three known and three new), all of which belonged to so-called subtype BIV. Three specimens had co-infection with C. hominis (IbA12G3) and G. duodenalis (BIV). The presence of zoonotic genotypes and subtypes of Cryptosporidium spp. and G. duodenalis in NHPs suggests that these animals can potentially contribute to the transmission of human cryptosporidiosis and giardiasis. [ABSTRACT FROM AUTHOR]

Cryptosporidiosis is prevalent in domesticated, caged, and wild birds. Cryptosporidium baileyi , an ascendant species of avian Cryptosporidium , is an important pathogen. It causes respiratory disease in chickens, especially chickens younger than 50 days. In this study, SEM, histological, semi-quantitative PCR, and nested PCR techniques were used to explore the impact of different inoculation routes on sites of C. baileyi infection in chickens. Results showed that inoculation with sporozoites or oocysts via the rectum was an effective means of causing infection. This may provide an important reference for the development of the transfection system of C. baileyi in chickens. Numerous endogenous stages of C. baileyi were observed in the bursas of Fabricius (BF) and cloacas of chickens inoculated with sporozoites or oocysts via the rectum, but no parasite was seen in the tracheas of any of these chickens. In chickens infected with oocysts via the crop, the number of parasites in the BF was approximately 23-fold more than in the trachea. All blood samples collected after inoculation were negative for C. baileyi . These data show that C. baileyi was not transferred by blood circulation between the BF and respiratory tract. Different routes of inoculation were here found to distinctly affect sites of parasitism in chickens. These findings may facilitate further understanding of the biology of C. baileyi and efforts to control avian cryptosporidiosis. [ABSTRACT FROM AUTHOR]

Cryptosporidium spp. can cause diarrhea and even death in humans and animals. Host microRNAs (miRNAs) play an important role in the post-transcriptional regulation of the innate immune response to Cryptosporidium infection. To study host miRNA activity in the innate immune response to C. parvum infection, we examined the expression of miR-181d in HCT-8 cells infected with C. parvum and found that it was significantly downregulated, while TLR2, TLR4, NF-κB, and myD88 involved in the TLR/NF-κB signaling pathway were significantly upregulated at the early stages of C. parvum infection. We transfected cells with short-interfering RNAs (siRNA) as TLR2 , TLR4 , and NF-κB inhibitors. Analysis by quantitative real-time polymerase chain reaction (qPCR) and western blot confirmed that C. parvum downregulates miR-181d expression via the p50 subunit-dependent TLR2/TLR4-NF-κB signaling pathway in HCT-8 cells. This study provides a new theoretical foundation to elucidate the regulatory mechanism of host miRNAs against Cryptosporidium infection. • The miR-181d was inhibited via TLRs/NF-κB signaling induced by C. parvum. • Both TLR2 and TLR4 are involved in regulating the expression of miR-181d. • NF-κB p50 subunit negatively regulates miR-181d induced by C. parvum. [ABSTRACT FROM AUTHOR]

Abstract: The prevalence of Cryptosporidium spp. was investigated in scale quail (Coturnix coturnix japonica) farms in Henan Province, China between September 2006 and August 2007. One thousand eight hundred and eighteen fecal samples from 47 quail farms in five areas were collected for the examination of Cryptosporidium oocysts. The overall prevalence of Cryptosporidium was 13.1% (95% CI 13.1±1.6%) (29 of 47 farms), with 72–100-day-old quails having the highest prevalence (23.6%, 95% CI 23.6±2.6%) (χ 2 =64.91; ρ <0.01). The highest prevalence was observed in autumn (21.8%, 95% CI 21.8±3.1%) and the lowest in winter (χ 2 =74.83; ρ <0.01). Two hundred and thirty-nine Cryptosporidium-positive samples were analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene, and 42 were further analyzed by DNA sequencing of the PCR products. Two Cryptosporidium species were identified, Cryptosporidium baileyi in 237 birds on 29 farms, and potentially zoonotic Cryptosporidium meleagridis in only two birds on two farms. These findings may suggest that quails are not a major source of zoonotic Cryptosporidium in the study area. [Copyright &y& Elsevier]

Abstract: Few data are available on the molecular characterization of Cryptosporidium spp. in ostriches. The objective of this study was to determine the prevalence of Cryptosporidium species or genotypes in ostriches. A total of 452 fecal samples from five farms, a zoo, and an animal rescue center in Zhengzhou, Henan Province, China were examined for Cryptosporidium oocysts by microscopy of wet mount of fecal materials concentrated by the Sheather''s sugar flotation technique. Fifty-three samples were Cryptosporidium-positive from four farms, with an overall prevalence of 11.7%. The percentage of animals shedding oocysts was 0, 16.2%, 7.2%, and 0 in 1–3 weeks, 4–8 weeks, 3–12 months, and more than 12 months ostriches, respectively (χ 2 =17.74; ρ <0.01). PCR-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene of the 53 Cryptosporidium-positive samples showed the presence of only Cryptosporidium baileyi, which was confirmed by DNA sequencing of the SSU rRNA PCR products from 16 positive samples. Cross-transmission studies demonstrated that the C. baileyi isolate could infect chickens and quails. Thus, ostriches are commonly infected with C. baileyi that is genetically and biologically similar to C. baileyi found in other birds. [Copyright &y& Elsevier]