Tandem mass tag (TMT)-based proteomic analysis of Cryptosporidium andersoni oocysts before and after excystation.

BACKGROUND: Cryptosporidium andersoni initiates infection by releasing sporozoites from oocysts through excystation. However, the proteins involved in excystation are unknown. Determining the proteins that participate in the excystation of C. andersoni oocysts will increase our understanding of the excystation process. METHODS: Cryptosporidium andersoni oocysts were collected and purified from the feces of naturally infected adult cows. Tandem mass tags (TMT), coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic analysis, were used to investigate the proteomic expression profiles of C. andersoni oocysts before and after excystation. RESULTS: Proteomic analysis identified a total of 1586 proteins, of which 17 were differentially expressed proteins (DEPs) upon excystation. These included 10 upregulated and seven downregulated proteins. The 17 proteins had multiple biological functions associated with control of gene expression at the level of transcription and biosynthetic and metabolic processes. Quantitative real-time RT-PCR of eight selected genes validated the proteomic data. CONCLUSIONS: This study provides information on the protein composition of C. andersoni oocysts as well as possible excystation factors. The data may be useful in identifying genes for diagnosis, vaccine development, and immunotherapy for Cryptosporidium.

Serum metabolomics in chickens infected with Cryptosporidium baileyi.

BACKGROUND: Cryptosporidium baileyi is an economically important zoonotic pathogen that causes serious respiratory symptoms in chickens for which no effective control measures are currently available. An accumulating body of evidence indicates the potential and usefulness of metabolomics to further our understanding of the interaction between pathogens and hosts, and to search for new diagnostic or pharmacological biomarkers of complex microorganisms. The aim of this study was to identify the impact of C. baileyi infection on the serum metabolism of chickens and to assess several metabolites as potential diagnostic biomarkers for C. baileyi infection. METHODS: Ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) and subsequent multivariate statistical analysis were applied to investigate metabolomics profiles in the serum samples of chickens infected with C. baileyi, and to identify potential metabolites that can be used to distinguish chickens infected with C. baileyi from non-infected birds. RESULTS: Multivariate statistical analysis identified 138 differential serum metabolites between mock- and C. baileyi-infected chickens at 5 days post-infection (dpi), including 115 upregulated and 23 downregulated compounds. These metabolites were significantly enriched into six pathways, of which two pathways associated with energy and lipid metabolism, namely glycerophospholipid metabolism and sphingolipid metabolism, respectively, were the most enriched. Interestingly, some important immune-related pathways were also significantly enriched, including the intestinal immune network for IgA production, autophagy and cellular senescence. Nine potential C. baileyi-responsive metabolites were identified, including choline, sirolimus, all-trans retinoic acid, PC(14:0/22:1(13Z)), PC(15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(16:1(9Z)/24:1(15Z)), phosphocholine, SM(d18:0/16:1(9Z)(OH)) and sphinganine. CONCLUSIONS: This is the first report on serum metabolic profiling of chickens with early-stage C. baileyi infection. The results provide novel insights into the pathophysiological mechanisms of C. baileyi in chickens.

Establishment and preliminary application of nanoparticle-assisted PCR assay for detection of Cryptosporidium spp.

Cryptosporidium is an important intestinal protozoan parasite that causes diarrhoea in humans and animals. To rapidly and specifically detect Cryptosporidium spp., we designed a pair of primers based on the small subunit ribosomal RNA (SSU rRNA) gene of Cryptosporidium spp. to be used in a new nanoparticle-assisted PCR (nano-PCR) assay. The minimum detectable concentration (1.02 pg) of this nano-PCR was 10 times more sensitive than conventional PCR using the same primer pair. The DNA samples of C. parvum, C. baileyi, C. xiaoi, C. ryanae, and C. andersoni were successfully detected by the nano-PCR. No amplifications were evident with DNA samples of some common intestinal pathogens, including Eimeria tenella, Blastocystis sp., Giardia lamblia, Enterocytozoon bieneusi, and Balantidium coli. To validate the clinical usefulness of the novel nano-PCR, a total of 40 faecal samples from goats, camels, calves, and chickens were examined. The positive rate of Cryptosporidium spp. was 27.5% (11/40), which was consistent with that of an established nested PCR. These results indicate that the novel nano-PCR assay enables the rapid, specific, and accurate detection of Cryptosporidium infection in animals. The findings provide a technical basis for the clinical diagnosis, prevention, and control of cryptosporidiosis.

Multilocus genotyping of Giardia duodenalis in Bactrian camels (Camelus bactrianus) in China.

The protozoan parasite Giardia duodenalis is known to infect humans and a wide range of animals globally. However, no studies on G. duodenalis infection in Bactrian camels have been reported. In the present study, in order to examine the prevalence and genetic diversity of G. duodenalis in Bactrian camels, 852 fecal samples were collected from 24 sampling sites in three geographical areas (Gansu province, Inner Mongolia, and Xinjiang Uygur autonomous regions) of northwestern China, and subjected to multilocus sequence typing (MLST) analysis targeting the 18S rRNA, ß-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes. About 84 fecal samples tested positive for Giardia infection, with an overall prevalence of 9.8%, including three samples from camel calves with diarrhea. Significant differences (χ2 = 80.7, df = 2, P < 0.01) in the prevalence were found in Bactrian camels belonging to three geographical areas, with the highest (33.3%) in Gansu province and the lowest (4.2%) in Xinjiang Uygur autonomous region. Furthermore, significantly different prevalences (χ2 = 34.2, df = 2, P < 0.01) were revealed among age groups, with the highest (35.7%) in camels aged 3 to 6 years old, and the lowest (7.5%) in camels aged > 6 years old. Sequence analysis identified two assemblages, including zoonotic assemblage A and ungulate-adapted assemblage E, with the latter as the dominant G. duodenalis assemblage in each age group and at all sampling sites having positive samples except Hotan. Genetic variations were detected among G. duodenalis isolates in these camels, and eight, three, and seven haplotypes were identified at loci bg, gdh, and tpi, respectively, forming two multilocus genotypes (MLGs) of zoonotic assemblage A and one MLG of assemblage E. To the best of our knowledge, this is the first report on G. duodenalis infection in Bactrian camels, and the data indicate that G. duodenalis have a broad host range.

Genotyping and Zoonotic Potential of Enterocytozoon bieneusi in Pigs in Xinjiang, China.

Enterocytozoon bieneusi is an obligate intracellular fungus, infecting various invertebrate and vertebrate hosts, it is common in humans and causes diarrhea in the immunocompromised. In the present study, 801 fecal specimens were collected from pigs on seven large-scale pig farms in Xinjiang, China. Nested polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) gene showed that the overall E. bieneusi infection rate was 48.6% (389/801). The E. bieneusi infection rates differed significantly among the collection sites (20.0-73.0%) (χ2 = 75.720, df = 6, p < 0.01). Post-weaned pigs had the highest infection rate (77.2%, 217/281), followed by fattening pigs (67.4%, 87/129) and pre-weaned suckling pigs (35.5%, 60/169). Adult pigs had the lowest infection rate (11.3%, 25/222). The E. bieneusi infection rates also differed significantly among age groups (χ2 = 246.015, df = 3, p < 0.01). Fifteen genotypes were identified, including 13 known genotypes (CHC, CS-1, CS-4, CS-7, CS-9, D, EbpA, EbpC, EbpD, H, PigEb4, PigEBITS5, and WildBoar8) and two novel genotypes (XJP-II and XJP-III). Among them, six genotypes (CS-4, D, EbpA, EbpC, H, and PigEBITS5) have been reported in humans. Phylogenetic analysis showed that all the genotypes belonged to Group 1 of E. bieneusi. These findings suggest that pigs may play an important role in transmitting E. bieneusi infections to humans.

Prevalence and Population Genetics Analysis of Enterocytozoon bieneusi in Dairy Cattle in China.

Enterocytozoon bieneusi, an obligate intracellular pathogen, can infect various hosts. In this study, 3527 dairy cattle fecal specimens were collected from different geographic locations in China (including 673 from Shandong province, 1,440 from Guangdong province and 1,414 from Gansu province) and examined for the presence of E. bieneusi using polymerase chain reactions targeting the ribosomal internal transcribed spacer (ITS). The dominant genotypes identified were further subtyped by multilocus sequence typing. The overall prevalence of E. bieneusi was 14.2% (501/3527), with a significant difference in prevalence among the different geographical locations (P < 0.001). Our logistic regression analysis showed that all four variables (farming model, location, age, and clinical manifestations) had strong effects on the risk of contracting E. bieneusi. Sequence analysis revealed 11 genotypes: eight known genotypes (J, I, BEB4, BEB10, D, EbpC, CM19, and CM21) and three novel genotypes (named here as CGC1, CGC2, and CGC3). Genotypes J and I, the commonest, were found on all farms across the three provinces. Our linkage disequilibrium analysis showed a clonal population structure in the E. bieneusi dairy cattle population but the ITS genotypes had different population structures. Phylogenetic and haplotype network analysis showed the absence of geographical segregation in the E. bieneusi dairy cattle populations. Instead, they revealed the presence of host adaptation to the E. bieneusi populations in various animals. Our findings augment the current understanding of E. bieneusi transmission dynamics.

Widespread occurrence of Cryptosporidium infections in patients with HIV/AIDS: Epidemiology, clinical feature, diagnosis, and therapy.

Cryptosporidium infection is a serious threat for HIV/AIDS patients, causing severe diarrhea and even death. The overall prevalence of Cryptosporidium in HIV/AIDS patients was calculated as approximately 8.69% (7,799/89,724), with higher prevalence observed in individuals with diarrhea, individuals with low CD4+ T-lymphocyte counts, and antiretroviral therapy-naïve individuals. Cryptosporidium infection was not significantly associated with patient age or gender, national development levels, or continent of residence. Over the period from 2007 to 2017, Cryptosporidium prevalence was 10.09% (3,282/32,517); this figure was higher than that observed in each of the previous observation periods (1985-1995 and 1996-2006), suggesting that the prevalence of cryptosporidiosis has been increasing over time in HIV/AIDS patients. Ten Cryptosporidium species and genotypes have been identified from 1,252 isolates, with C. hominis, C. parvum, and C. meleagridis accounting for 93.53% of infections. Five subtypes each of C. hominis (Ia, Ib, Id, Ie, and If), C. parvum (IIa to IIe), and C. meleagridis (IIIa to IIIe) have been described by sequence analyses of the 60-kDa glycoprotein (gp60) gene. Variation in the clinical manifestations observed in HIV/AIDS patients might be attributed to infection by different Cryptosporidium species, genotypes and subtypes, as well as different sites of infection. New molecular and immunological diagnostic techniques are in development or already commercially available. High-throughput screening methods for development of new or repurposed therapeutics as well as novel parasite genetic manipulation strategies offer hope for improving human cryptosporidiosis therapies. Painstaking efforts by researchers as well as support from governments and funding agencies will be required to make lasting achievements in this field.

Expression Profiles of mRNA and lncRNA in HCT-8 Cells Infected With Cryptosporidium parvum IId Subtype.

Cryptosporidium parvum is one of the most important enteric protozoan pathogens, responsible for severe diarrhea in immunocompromised human and livestock. However, few effective agents were available for controlling this parasite. Accumulating evidences suggest that long non-coding RNA (lncRNA) played key roles in many diseases through regulating the gene expression. Here, the expression profiles of lncRNAs and mRNAs were analyzed in HCT-8 cells infected with C. parvum IId subtype using microarray assay. A total of 821 lncRNAs and 1,349 mRNAs were differentially expressed in infected cells at 24 h post infection (pi). Of them, all five types of lncRNAs were identified, including 22 sense, 280 antisense, 312 intergenic, 44 divergent, 33 intronic lncRNAs, and 130 lncRNAs that were not found the relationship with mRNAs' location. Additionally, real-time polymerase chain reactions of 10 lncRNAs and 10 mRNAs randomly selected were successfully confirmed the microarray results. The co-expression and target prediction analysis indicated that 27 mRNAs were cis-regulated by 29 lncRNAs and 109 were trans-regulated by 114 lncRNAs. These predicted targets were enriched in several pathways involved in the interaction between host and C. parvum, e.g., hedgehog signaling pathway, Wnt signaling pathway, and tight junction, suggesting that these differentially expressed lncRNAs would play important regulating roles during the infection of C. parvum IId subtype.

Novel genotypes and multilocus genotypes of Enterocytozoon bieneusi in pigs in northwestern China: A public health concern.

Enterocytozoon bieneusi, the most important and common microsporidian species, inhabits in most animals and humans causing diarrhea and systemic diseases. The objectives of the present study were to examine the prevalence and genetic variability of E. bieneusi in pigs in Shaanxi province, northwestern China. A total of 560 pig faecal samples were collected from five different farms in Shaanxi province and molecularly characterized using multilocus genotyping (MLST) technology. High E. bieneusi infection rate (78.9%) was observed in these samples. 12 known and 22 possible novel ITS genotypes were identified, with the novel SZZD1 as the predominant genotype distributed in all age groups and pig farms. 32 (including 11 known and 21 novel ones) of them belong to the zoonotic group 1. MLST analysis showed that 109 ITS positive samples formed 87 distinct multilocus genotypes (MLGs). An incomplete linkage disequilibrium (LD) and clonal genetic structure of E. bieneusi were found in pigs in Shaanxi province. These findings indicated the complex population structures of E. bieneusi in pigs in Shaanxi province and provided baseline data for better understanding of the epidemiological status of E. bieneusi in this province.

Prevalence and genotypes of Enterocytozoon bieneusi in China.

Enterocytozoon bieneusi has been considered as the most frequently diagnosed microsporidian species in humans and various animal species, accounting for more than 90% of the cases of human microsporidiosis. Spores of this pathogen excreted from both symptomatic and asymptomatic hosts into environment also would be an important source of waterborne outbreak of microsporidiosis. Due to limited effective drugs available but with too much side effects to mammals (eg. toxic), accurate characterization of E. bieneusi in both humans and animals is essential to implement effective control strategies to this pathogen. In China, E. bieneusi infection was presented in humans and some animals with high prevalence. Analysis of genetic variations of the internal transcribed spacer (ITS) sequences found 361 genotypes in China, and some novel genotypes were identified in some specific hosts. Additionally, associations between infections and some risk factors were also observed. In the present article, we reviewed the current status of prevalence, genotypes, multilocus genotypes (MLGs) in humans, various animals and waters in China. These findings will provide basic information for developing effective control strategies against E. bieneusi infection in China as well as other countries.

Avirulence and lysozyme secretion in Paneth cells after infection of BALB/c mice with oocysts of Toxoplasma gondii strains TgCatCHn2 (ToxoDB#17) and TgCatCHn4 (ToxoDB#9).

Toxoplasma gondii has a complex life cycle and pathogenic mechanisms. Acute T. gondii infections in mice often result in death, whereas in chronic infections, the parasites may persist in the host tissues as intraneuronal or intramuscular cysts. However, the virulence of T. gondii strains in mice varies with its genetic background. The present study investigated the pathogenicity and pathological lesions of two T. gondii isolates from China: namely, TgCatCHn2 (ToxoDB#17) and TgCatCHn4 (ToxoDB#9). The virulent (ToxoDB#216) and avirulent (VEG) strains were employed as controls. Toxoplasmosis was induced by inoculating BALB/c mice with oocysts of the strains of T. gondii VEG, ToxoDB#216, TgCatCHn2 (ToxoDB#17), and TgCatCHn4 (ToxoDB#9), respectively. As a result, one oocyst of ToxoDB#216 could kill a mouse within 10 days post inoculation (DPI). The survival time of the mice for T. gondii TgCatCHn2 and TgCatCHn4 was >60 DPI for 106/mL oocysts, but this concentration (106/mL oocysts) of VEG strain could kill mice within 11 DPI. Compared with the strains of T. gondii ToxoDB#216 and VEG, the lesions in the small intestines of the strains of TgCatCHn2- and TgCatCHn4-infected mice were significantly smaller (P < 0.01). The positive area of T. gondii antigen in the ileum of mice infected with the strains of T. gondii VEG, TgCatCHn2 and TgCatCHn4 were significantly lower than that T. gondii ToxoDB#216 at 8 DPI (P < 0.01). Paneth cells (PCs) in the small intestines was eliminated by ToxoDB#216 (5-6 DPI) (P < 0.05). The strains of T. gondii TgCatCHn2-, TgCatCHn4- and VEG-infected mice, the number of PCs and granules decreased in the intestines, compared to T. gondii free mice, but the difference was not significant (8 DPI, P > 0.05). However, the granules in the PCs showed negative lysozyme expression in the intestines of mice infected with T. gondii TgCatCHn2 and TgCatCHn4. Thus, T. gondii strains of TgCatCHn2 (ToxoDB#17) and TgCatCHn4 (ToxoDB#9) were avirulent strains, they triggered an inhibition of lysozyme expression in the granules of PCs of the mouse intestine. These effects may in turn lead to intestinal dysbiosis, which may be related to further parasitic invasion of the intestines. The findings of the present study further expand the spectrum of the pathogenic features of various Chinese isolates of T. gondii.

Veterinary parasitology teaching in China in the 21st century - Challenges, opportunities and perspectives.

China has made significant achievements in social-economic development in the last three decades, and the numbers of livestock and companion animals are rapidly increasing. Some advances have been made in the control and prevention of animal parasitic diseases, but there are still some significant challenges, particularly in relation to foodborne parasitic zoonoses and vector-borne diseases. In addition, new molecular (e.g., genomic and transcriptomic) technologies have been developed and are gradually being introduced into the veterinary parasitology field. Therefore, teaching of veterinary parasitology in Chinese universities has undergone significant changes over the years, in terms of topics, depth and breadth, and also in the ways in which courses are delivered. In this article, we describe the current status of veterinary parasitology teaching at the undergraduate and postgraduate levels in Chinese universities, summarise changes and improvement in veterinary parasitology teaching, and discuss the challenges and opportunities for veterinary parasitology teaching in the 21st century, including the use of new teaching technologies and the integration of the "One Health" concept into veterinary parasitology courses.

Antibody Detection, Isolation, Genotyping, and Virulence of Toxoplasma gondii in Captive Felids from China.

The felids are the only definitive hosts of Toxoplasma gondii, which could excrete oocysts into the environment and provide an infection source for toxoplasmosis in various warm-blooded animal species, particularly the captive felids that live close to human communities. The infection rate of the captive felids is a perfect standard in detecting the presence of Toxoplasma gondii oocysts in the environment. In this study, sera or tissue samples from zoo (1 young tiger, 2 adult tigers, 6 young lions), farm (10 masked palm civets), and pet hospital (28 cats) from Henan Province (China) were collected. The sera (n = 47) were tested for immunoglobulin G (IgG) antibodies against T. gondii by using modified agglutination test (MAT), whereas the hearts tissue (n = 40) were bioassayed in mice to isolate T. gondii strains. The genotype was distinguished by using PCR-RFLP of 10 loci (SAG1, SAG2, SAG3, GRA6, BTUB, L358, c22-8, PK1, c29-2, and Apico). The detection rate for the T. gondii antibody in captive felids was 21.3% (10/47). One viable T. gondii strain (TgCatCHn4) was obtained from a cat heart tissue, and its genotype was ToxoDB#9. The oocysts of ToxoDB#9 were collected from a T. gondii-free cat. The virulence of TgCatCHn4 was low and no cysts were detected in the brain of mice at 60 days post-inoculation. The finding of the present study suggested a widespread exposure of T. gondii for felids in Henan Province of central China, particularly those from the zoological gardens and homes. ToxoDB#9 was the predominant strain in China. Preventive measures against T. gondii oocyst contamination of various components of the environment should thus be implemented, including providing pre-frozen meat, well-cooked cat food, cleaned fruits and vegetables, monitoring birds and rodents, inactive T. gondii oocysts in felids feces, and proper hygiene.

Recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip for equipment-free detection of Cryptosporidium spp. oocysts in dairy cattle feces.

Cryptosporidium is a widespread protozoan parasite that infects a large number of vertebrate animals, resulting in varying degrees of diarrhea or even death. As dairy cattle feces is an important source of Cryptosporidium spp. infection, development of a handy and accurate detection method via its oocysts in dairy cattle feces would be interesting and necessary. We herein developed a quick detecting method using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip to detect DNA of Cryptosporidium oocysts in dairy cattle feces. The DNA was released by boiled water with 0.1 % N-lauroylsarcosine sodium salt (LSS). The established method was proven to be of higher sensitivity than normal polymerase chain reaction (PCR) amplification with the lowest detection of 0.5 oocyst per reaction, and specificity with no cross reactivity to other common protozoan species in the intestine of dairy cattle. The diagnostic method established herein is simple, rapid, and cost-effective, and has potential for further development as a diagnostic kit for the diagnosis of cryptosporidiosis of dairy cattle.

Infection rate of Giardia duodenalis, Cryptosporidium spp. and Enterocytozoon bieneusi in cashmere, dairy and meat goats in China.

Cryptosporidiosis, microsporidiosis, and giardiasis contribute significantly to the high burden of zoonotic diarrhea worldwide. Goats constitute an important species in animal agriculture by providing cashmere wool, meat, and dairy products for human consumption. However, zoonotic pathogens with the potential to cause morbidity and to degrade production have been reported frequently in goats recently. The present study examined 629 fecal specimens from goats, including 315 cashmere goats, 170 dairy goats and 144 meat goats, in multiple cities of Shaanxi and Henan provinces, northwestern and central China, to investigate the infection rate and species/assemblages/genotypes of Giardia duodenalis, Cryptosporidium spp. and Enterocytozoon bieneusi. Of these samples, 274 (43.6%) were positive for three zoonotic pathogens, including 80 (12.7%), 104 (16.5%) and 179 (28.5%) for G. duodenalis, Cryptosporidium spp. and E. bieneusi, respectively. Infections with G. duodenalis, Cryptosporidium spp. and E. bieneusi existed in meat, dairy and cashmere goats, with the highest infection rate of each pathogen being observed in meat goats. DNA sequencing of the SSU rRNA gene from 104 Cryptosporidium-positive specimens revealed existence of Cryptosporidium xiaoi, and the zoonotic parasites Cryptosporidium parvum and Cryptosporidium ubiquitum. Genotyping of G. duodenalis based on the triosephosphate isomerase (TPI) gene identified parasites from zoonotic assemblage A in four cashmere goats and the animal-adapted assemblage E in a group of 76 goats that included cashmere, dairy and meat animals. Polymorphisms in the ribosomal internal transcribed spacer characterized E. bieneusi genotype CHG1 and a novel genotype named as SX1 in both dairy and cashmere goats, genotypes CHS7 and COSI in meat goats, the genotype CHG2 in dairy goats, and the human-pathogenic genotype BEB6 in dairy and meat goats. This is the first detailed study to compare infection rate of the zoonotic protozoan pathogens in cashmere, dairy and meat goats in China. Our research discovered Cryptosporidium spp. and E. bieneusi infections, each with zoonotic potential in meat goats, and G. duodenalis and Cryptosporidium spp. in cashmere goats raising a significant public health concern.

Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi in Captive Non-Human Primates in Qinling Mountains.

Non-human primates (NHPs) are confirmed as reservoirs of Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi. In this study, 197 fresh fecal samples from 8 NHP species in Qinling Mountains, northwestern China, were collected and examined using multilocus sequence typing (MLST) method. The results showed that 35 (17.8%) samples were positive for tested parasites, including Cryptosporidium spp. (3.0%), G. intestinalis (2.0%), and E. bieneusi (12.7%). Cryptosporidium spp. were detected in 6 fecal samples of Macaca mulatta, and were identified as C. parvum (n=1) and C. andersoni (n=5). Subtyping analysis showed Cryptosporidium spp. belonged to the C. andersoni MLST subtype (A4, A4, A4, and A1) and C. parvum 60 kDa glycoprotein (gp60) subtype IId A15G2R1. G. intestinalis assemblage E was detected in 3 M. mulatta and 1 Saimiri sciureus. Intra-variations were observed at the triose phosphate isomerase (tpi), beta giardin (bg), and glutamate dehydrogenase (gdh) loci, with 3, 1, and 2 new subtypes found in respective locus. E. bieneusi was found in Cercopithecus neglectus (25.0%), Papio hamadrayas (16.7%), M. mulatta (16.3%), S. sciureus (10%), and Rhinopithecus roxellana (9.5%), with 5 ribosomal internal transcribed spacer (ITS) genotypes: 2 known genotypes (D and BEB6) and 3 novel genotypes (MH, XH, and BSH). These findings indicated the presence of zoonotic potential of Cryptosporidium spp. and E. bieneusi in NHPs in Qinling Mountains. This is the first report of C. andersoni in NHPs. The present study provided basic information for control of cryptosporidiosis, giardiasis, and microsporidiosis in human and animals in this area.

First report of zoonotic Cryptosporidium spp., Giardia intestinalis and Enterocytozoon bieneusi in golden takins (Budorcas taxicolor bedfordi).

Genetic study of Cryptosporidium spp., Giardia intestinalis and Enterocytozoon bieneusi at species/assemblage/genotype/subtype level facilitates understanding their mechanical transmissions and underpins their control. A total of 191 fresh faecal samples were collected from golden takins in China and examined using multilocus sequence typing (MLST). Cryptosporidium spp. was detected in 15 faecal samples (7.9%), including Cryptosporidium parvum (2/15) and Cryptosporidium andersoni (13/15). MLST tool identified C. andersoni subtypes (A1, A4, A4, A1) and (A4, A4, A4, A1), and C. parvum gp60 gene subtype IId A19G1. The prevalence of G. intestinalis infection was 8.9% (17/191) and assemblage analysis identified 14 assemblage E and three assemblage B. Intra-variations were observed at triose phosphate isomerase (tpi), beta giardin (bg) and glutamate dehydrogenase (gdh) loci within the assemblage E, showing seven, three and three new subtypes in respective locus. Ten and one multilocus genotypes (MLGs) were present in assemblages E and B, respectively. E. bieneusi infection was positive in 14.7% (28/191) of the examined specimens, with three genotypes known (BEB6, D and I) and four novel internal transcribed spacer (ITS) genotypes (TEB1-TEB4). The present study revealed, for the first time, the presence of zoonotic C. parvum IId A19G1, G. intestinalis assemblage B and E. bieneusi genotype D and four novel genotypes in golden takins in China. These findings expand the host range of three zoonotic pathogens and have important implications for controlling cryptosporidiosis, giardiasis and microsporidiosis in humans and animals.

First report of Enterocytozoon bieneusi from giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens) in China.

Enterocytozoon bieneusi is an emerging and opportunistic enteric pathogen triggering diarrhea and enteric disease in humans and animals. Despite extensive research on this pathogen, the prevalence and genotypes of E. bieneusi infection in precious wild animals of giant and red pandas have not been reported. In the present study, 82 faecal specimens were collected from 46 giant pandas (Ailuropoda melanoleuca) and 36 red pandas (Ailurus fulgens) in the northwest of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an overall infection rate of 10.98% (9/82) was observed in pandas, with 8.70% (4/46) for giant pandas, and 13.89% (5/36) for red pandas. Two ITS genotypes were identified: the novel genotype I-like (n=4) and genotype EbpC (n=5). Multilocus sequence typing (MLST) employing three microsatellites (MS1, MS3 and MS7) and one minisatellite (MS4) showed that nine, six, six and nine positive products were amplified and sequenced successfully at four respective loci. A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that the genotype EbpC fell into 1d of group 1 of zoonotic potential, and the novel genotype I-like was clustered into group 2. The present study firstly indicated the presence of E. bieneusi in giant and red pandas, and these results suggested that integrated strategies should be implemented to effectively protect pandas and humans from infecting E. bieneusi in China.

Genotyping Cryptosporidium andersoni in cattle in Shaanxi Province, Northwestern China.

The present study examined the prevalence and genotypes of Cryptosporidium andersoni in cattle in Shaanxi province, China. A total of 2071 fecal samples (847 from Qinchuan cattle and 1224 from dairy cattle) were examined for the presence of Cryptosporidium oocysts, and 70 samples (3.4%) were C. andersoni-positive and those positive samples were identified by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) and the Cryptosporidium oocyst wall protein (COWP) genes. C. andersoni was the only species found in the examined cattle in this province. Fifty-seven C. andersoni isolates were characterized into 5 MLST subtypes using multilocus sequence typing analysis, including a new subtype in the native beef breed Qinchuan cattle. All of these C. andersoni isolates presented a clonal genetic structure. These findings provide new insights into the genetic structure of C. andersoni isolates in Shaanxi province and basic data of Cryptosporidium prevalence status, which in turn have implications for controlling cryptosporidiosis in this province.

Molecular characterization of Cyclospora-like organisms from golden snub-nosed monkeys in Qinling Mountain in Shaanxi province, northwestern China.

Cyclospora spp. have been identified as one of the most important intestinal pathogens causing protracted diarrhea in animals and human beings. To determine the Cyclospora species in the non-human primate Rhinopithecus roxellanae, a total of 71 fecal samples from 19 endangered snub-nosed monkeys in Shaanxi province were collected and examined using Sheater's sugar flotation technique and by sequencing the fragments of 18S rDNA. Only two Cyclospora isolates from 2 golden snub-nosed monkeys (R. roxellanae) were obtained and identified between July 2011 and August of 2012. The sequences of the 18S rDNA for the two Cyclospora isolates were 477 bp, with no nucleotide variation between them. Phylogenetic analysis based on the 18S rDNA sequences revealed that the two Cyclospora isolates were posited into the clade Cyclospora spp. and sistered to C. colobi. These results first showed that Cyclospora infection occurred in R. roxellanae in hot and rainy weather, which would provide useful information for further understanding the molecular epidemiology of Cyclospora spp. and the control of Cyclospora infection in non-human primates as well as in human beings.