Characterization of CpCaM, a protein potentially involved in the growth of Cryptosporidium parvum.

Cryptosporidium parvum is an important apicomplexan parasite causing severe diarrhea in both humans and animals. Calmodulin (CaM), a multifunctional and universal calcium-binding protein, contributes to the growth and development of apicomplexan parasites, but the role of CaM in C. parvum remains unknown. In this study, the CaM of C. parvum encoded by the cgd2_810 gene was expressed in Escherichia coli, and the biological functions of CpCaM were preliminarily investigated. The transcriptional level of the cgd2_810 gene peaked at 36 h post infection (pi), and the CpCaM protein was mainly located around the nucleus of the whole oocysts, in the middle of sporozoites and around the nucleus of merozoites. Anti-CpCaM antibody reduced the invasion of C. parvum sporozoites by 30.69%. The present study indicates that CpCaM is potentially involved in the growth of C. parvum. Results of the study expand our knowledge on the interaction between host and Cryptosporidium.

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.

Molecular characterization of Balantioides coli in pigs from Shaanxi province, northwestern China.

Balantioides coli (syn. Balantidium coli) is an important zoonotic but usually neglected protozoa infecting human and a great number of animals, and the pig was considered to be the most important natural host and reservoir. However, no information about the infection of B. coli in pigs in northwestern China was available. In the present study, the prevalence and genetic diversity of B. coli in pigs in Shaanxi province were investigated. A total of 560 fecal samples were collected from pigs of four age groups in five different geographical regions and analyzed by using PCR targeting the ITS1-5.8S rRNA-ITS2 gene fragment. The infection of B. coli was detected in all age groups and regions, with the total prevalence of 16.8% (94/560). Significant differences (P < 0.01) in prevalence were found among four investigated age groups, with the highest in fatteners (38.8%) and the lowest in adults (5.7%). The prevalence was also significantly (P < 0.01) different among pigs from five sampling regions. Sequence analysis revealed two genetic variants, namely, A and B, in these investigated pigs, and both of them were detected in all age groups and regions, with the latter as the predominant one. Further, sixty-eight different haplotypes were found, with 19 and 49 belonged to genetic variants A and B, respectively. The findings in the present study indicated wide distribution and high diversity of B. coli in pigs in Shaanxi province and provided fundamental data for implementing control strategies on B. coli infection in pigs as well as other hosts in this province.

Genome-wide analysis of differentially expressed profiles of mRNAs, lncRNAs and circRNAs during Cryptosporidium baileyi infection.

BACKGROUND: Cryptosporidium baileyi is the most common Cryptosporidium species in birds. However, effective prevention measures and treatment for C. baileyi infection were still not available. Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) play important roles in regulating occurrence and progression of many diseases and are identified as effective biomarkers for diagnosis and prognosis of several diseases. In the present study, the expression profiles of host mRNAs, lncRNAs and circRNAs associated with C. baileyi infection were investigated for the first time. RESULTS: The tracheal tissues of experimental (C. baileyi infection) and control chickens were collected for deep RNA sequencing, and 545,479,934 clean reads were obtained. Of them, 1376 novel lncRNAs were identified, including 1161 long intergenic non-coding RNAs (lincRNAs) and 215 anti-sense lncRNAs. A total of 124 lncRNAs were found to be significantly differentially expressed between the experimental and control groups. Additionally, 14,698 mRNAs and 9085 circRNAs were identified, and significantly different expressions were observed for 1317 mRNAs and 104 circRNAs between two groups. Bioinformatic analyses of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway for their targets and source genes suggested that these dysregulated genes may be involved in the interaction between the host and C. baileyi. CONCLUSIONS: The present study revealed the expression profiles of mRNAs, lncRNAs and circRNAs during C. baileyi infection for the first time, and sheds lights on the roles of lncRNAs and circRNAs underlying the pathogenesis of Cryptosporidium infection.

Field synopsis and meta-analyses of genetic epidemiological evidence for Kashin-Beck disease, an endemic osteoarthropathy in China.

Kashin-Beck disease (KBD) is a chronic degenerative osteoarthropathy with unclear etiology. To provide current evidence supporting a genetic predisposition for KBD, we conducted a systematic review and meta-analysis of published literature on the genetic epidemiology of KBD. The PubMed, China National Knowledge Infrastructure and Wan Fang Data were searched up to August 2015 for articles published in English and Chinese. Genome-wide and exome sequencing, linkage, and case-control association studies for any genetic variants associated with KBD were included. Meta-analysis was performed for all single nucleotide polymorphisms (SNPs) that were evaluated in two or more studies. The effect size was summarized as odds ratios (ORs) with 95 % confidence intervals (CIs) by fixed and random effects models. A total of 24 articles were systematically reviewed. Eleven short tandem repeats on chromosomes 2, 11 and 12, 34 SNPs in 12 genes, as well as copy number variant 452 were identified as KBD susceptibility factors in individual studies. The meta-analysis of the GPX1 rs1050450, DIO2 rs225014, TrxR2 rs5748469 and HLA-DRB1 rs7745040 failed to reveal any associations with KBD. However, the meta-analysis of HLA-DRB1 rs9275295 allele A was associated with KBD (OR = 1.737, 95 % CI: 1.002-3.012). In addition, seven haplotypes in GPX1, GPX4, HLA-DRB1 and GDF5 genes also showed significant associations with KBD. In conclusions, our study could identify a number of genetic markers associated with KBD. However, the evidence does not currently support a strong association between the specific variants and KBD because of the limited number of studies, and in the future, more rigorous studies are needed to confirm KBD's links with these variants.

Prevalence, Risk Factors and Multilocus Genotyping of Giardia intestinalis in Dairy Cattle, Northwest China.

Giardia intestinalis is a cosmopolitan protozoan parasite that can infect a range of animals, including dairy cattle. As information regarding the prevalence and genotyping of G. intestinalis infection in dairy cattle in northwestern China is limited, 2,945 feces samples from 1,224 dairy cattle in Gansu Province and from 1,614 in Ningxia Hui Autonomous Region (NXHAR) were examined between December 2012 and March 2014. The overall prevalence of G. intestinalis was 3.63% (107/2,945), with 2.63% and 4.38% in Gansu and NXHAR, respectively. Logistic regression analysis showed region, age and season to be significant risk factors for G. intestinalis infection. Assemblage analysis identified 106 assemblage E and one assemblage A at the triose phosphate isomerase (tpi) locus in this study. Intravariations were also detected at tpi, glutamate dehydrogenase (gdh) and beta giardin (bg) loci within assemblage E, showing seven, three, and five new subtypes, respectively. Moreover, 13 new multilocus genotypes (E20-E32) were observed in assemblage E. Effective strategies and measures should be taken to prevent and control giardiasis in Gansu and NXHAR.

Rapid detection and identification of four major Schistosoma species by high-resolution melt (HRM) analysis.

Schistosomiasis, caused by blood flukes belonging to several species of the genus Schistosoma, is a serious and widespread parasitic disease. Accurate and rapid differentiation of these etiological agents of animal and human schistosomiasis to species level can be difficult. We report a real-time PCR assay coupled with a high-resolution melt (HRM) assay targeting a portion of the nuclear 18S rDNA to detect, identify, and distinguish between four major blood fluke species (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma mekongi). Using this system, the Schistosoma spp. was accurately identified and could also be distinguished from all other trematode species with which they were compared. As little as 10(-5) ng genomic DNA from a Schistosoma sp. could be detected. This process is inexpensive, easy, and can be completed within 3 h. Examination of 21 representative Schistosoma samples from 15 geographical localities in seven endemic countries validated the value of the HRM detection assay and proved its reliability. The melting curves were characterized by peaks of 83.65 °C for S. japonicum and S. mekongi, 85.65 °C for S. mansoni, and 85.85 °C for S. haematobium. The present study developed a real-time PCR coupled with HRM analysis assay for detection and differential identification of S. mansoni, S. haematobium, S. japonicum, and S. mekongi. This method is rapid, sensitive, and inexpensive. It has important implications for epidemiological studies of Schistosoma.

Cryptosporidium suis infection in post-weaned and adult pigs in Shaanxi province, northwestern China.

Cryptosporidium spp., ubiquitous enteric parasitic protozoa of vertebrates, recently emerged as an important cause of economic loss and zoonosis. The present study aimed to determine the distribution and species of Cryptosporidium in post-weaned and adult pigs in Shaanxi province, northwestern China. A total of 1,337 fresh fecal samples of post-weaned and adult pigs were collected by sterile disposable gloves from 8 areas of Shaanxi province. The samples were examined by Sheather's sugar flotation technique and microscopy at × 400 magnification for Cryptosporidium infection, and the species in positive samples was further identified by PCR amplification of the small subunit (SSU) rRNA gene. A total of 44 fecal samples were successfully amplified by the nested PCR of the partial SSU rRNA, with overall prevalence of 3.3%. The average prevalence of Cryptosporidium infection in each pig farms ranged from 0 to 14.4%. Species identification by sequencing of SSU rRNA gene revealed that 42 (3.1%) samples were Cryptosporidium suis and 2 (0.15%) were Cryptosporidium scrofarum. C. suis had the highest prevalence (7.5%) in growers and the lowest in breeding pigs (0.97%). C. suis was the predominant species in pre-weaned and adult pigs, while C. scrofarum infected pigs older than 3 months only. A season-related difference of C. suis was observed in this study, with the highest prevalence in autumn (5.5%) and the lowest (1.7%) in winter. The present study provided basic information for control of Cryptosporidium infection in pigs and assessment of zoonotic transmission of pigs in Shaanxi province, China.

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.

Dicrocoelium chinensis and Dicrocoelium dendriticum (Trematoda: Digenea) are distinct lancet fluke species based on mitochondrial and nuclear ribosomal DNA sequences.

Lancet flukes parasitize the bile ducts and gall bladder of a range of mammals, including humans, causing dicrocoeliosis. In the present study, we sequenced and characterized the complete mitochondrial (mt) genomes as well as the first and second internal transcribed spacers (ITS-1 and ITS-2=ITS) of nuclear ribosomal DNA (rDNA) of two lancet flukes, Dicrocoelium chinensis and D. dendriticum. Sequence comparison of a conserved mt gene and nuclear rDNA sequences among multiple individual lancet flukes revealed substantial nucleotide differences between the species but limited sequence variation within each of them. Phylogenetic analysis of the concatenated amino acid and multiple mt rrnS sequences using Bayesian inference supported the separation of D. chinensis and D. dendriticum into two distinct species-specific clades. Results of the present study support the proposal that D. dendriticum and D. chinensis represent two distinct lancet flukes. While providing the first mt genomes from members of the superfamily Plagiorchioidea, the novel mt markers described herein will be useful for further studies of the diagnosis, epidemiology and systematics of the lancet flukes and other trematodes of human and animal health significance.

Characterization of the intergenic spacer rDNAs of two pig nodule worms, Oesophagostomum dentatum and O. quadrispinulatum.

The characteristics of the intergenic spacer rDNAs (IGS rDNAs) of Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical locations in Mainland China were determined, and the phylogenetic relationships of the two species were reconstructed using the IGS rDNA sequences. The organization of the IGS rDNA sequences was similar to their organization in other eukaryotes. The 28S-18S IGS rDNA sequences of both O. dentatum and O. quadrispinulatum were found to have variable lengths, that is, 759-762 bp and 937-1128 bp, respectively. All of the sequences contained direct repeats and inverted repeats. The length polymorphisms were related to the different numbers and organization of repetitive elements. Different types and numbers of repeats were found between the two pig nodule species, and two IGS structures were found within O. quadrispinulatum. Phylogenetic analysis showed that all O. dentatum isolates were clustered into one clade, but O. quadrispinulatum isolates from different origins were grouped into two distinct clusters. These results suggested independent species and the existence of genotypes or subspecies within pig nodule worms. Different types and numbers of repeats and IGS rDNA structures could serve as potential markers for differentiating these two species of pig nodule worms.

MiR-4521 affects the propagation of Cryptosporidium parvum in HCT-8 cells through targeting foxm1 by regulating cell apoptosis.

Cryptosporidium parvum could regulate the expression of microRNAs of epithelial cells to facilitate its intracellular propagation. MiR-4521 has been reported to play an important role during the development and progression of tumors and infectious diseases by regulating cell proliferation, apoptosis, and autophagy. However, the implication of miR-4521 during C. parvum infection was still unknown. In this study, the expression of miR-4521 was found to be upregulated in HCT-8 cells infected with C. parvum from 8 h post-infection (pi) to 48 hpi, and its upregulation would be related with the TLR/NF-κB signal pathway during C. parvum infection. One potential target of miR-4521, foxm1, was down-regulated in HCT-8 cells from 24 hpi to 48 hpi, and the expression of foxm1 was negatively regulated by miR-4521. The target relationship between miR-4521 and foxm1 was further validated by using dual luciferase reporter assay. Further studies showed that miR-4521 promoted the propagation of C. parvum in HCT-8 cells through targeting foxm1 by regulating BCL2-mediating cell apoptosis. These results contribute to further understanding of the regulatory mechanisms of host miRNAs during Cryptosporidium infection.

Circular RNA ciRS-7 affects the propagation of Cryptosporidium parvum in HCT-8 cells via regulating miR-135a-5p/stat1 axis.

Cryptosporidium spp. are protozoan parasites that mainly inhabit intestinal epithelial cells, causing diarrheal diseases in humans and a great number of animals. Cryptosporidium parvum is the most common zoonotic species, responsible for nearly 45% of human cryptosporidiosis worldwide. Understanding the interaction mechanisms between C. parvum and host gastrointestinal epithelial cells has significant implications to control cryptosporidiosis. One up-regulated circRNA ciRS-7 was found previously by our group to promote in vitro propagation of C. parvum in HCT-8 cells. In the present study, miR-135a-5p, was found to be a miRNA target of ciRS-7. Cryptosporidium parvum infection induced significantly down-regulation of miR-135a-5p and dramatic up-regulation of its potential target stat1 gene at mRNA and protein levels. Dual luciferase reporter assays validated the physical interactions between miR-135a-5p and stat1, and between ciRS-7 and miR-135a-5p. Further study revealed that ciRS-7 could sponge miR-135a-5p to positively regulate the protein levels of STAT1 and phosphorylated STAT1 (p-STAT1) and thus promote C. parvum propagation in HCT-8 cells. Our findings further reveal the mystery of regulatory roles of host circRNAs during Cryptosporidium infection, and provide a novel insight to develop strategies to control cryptosporidiosis.

Temporal transcriptomic changes in microRNAs involved in the host immune response and metabolism during Neospora caninum infection.

BACKGROUND: Neospora caninum infection is a major cause of abortion in cattle, which results in serious economic losses to the cattle industry. However, there are no effective drugs or vaccines for the control of N. caninum infections. There is increasing evidence that microRNAs (miRNAs) are involved in many physiological and pathological processes, and dysregulated expression of host miRNAs and the biological implications of this have been reported for infections by various protozoan parasites. However, to our knowledge, there is presently no published information on host miRNA expression during N. caninum infection. METHODS: The expression profiles of miRNAs were investigated by RNA sequencing (RNA-seq) in caprine endometrial epithelial cells (EECs) infected with N. caninum at 24 h post infection (pi) and 48 hpi, and the functions of differentially expressed (DE) miRNAs were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The transcriptome data were validated by using quantitative real-time polymerase chain reaction. One of the upregulated DEmiRNAs, namely chi-miR-146a, was selected to study the effect of DEmiRNAs on the propagation of N. caninum tachyzoites in caprine EECs. RESULTS: RNA-seq showed 18 (17 up- and one downregulated) and 79 (54 up- and 25 downregulated) DEmiRNAs at 24 hpi and 48 hpi, respectively. Quantitative real-time polymerase chain reaction analysis of 13 randomly selected DEmiRNAs (10 up- and three downregulated miRNAs) confirmed the validity of the RNA-seq data. A total of 7835 messenger RNAs were predicted to be potential targets for 66 DEmiRNAs, and GO and KEGG enrichment analysis of these predicted targets revealed that DEmiRNAs altered by N. caninum infection may be involved in host immune responses (e.g. Fc gamma R-mediated phagocytosis, Toll-like receptor signaling pathway, tumor necrosis factor signaling pathway, transforming growth factor-ß signaling pathway, mitogen-activated protein kinase signaling pathway) and metabolic pathways (e.g. lysine degradation, insulin signaling pathway, AMP-activated protein kinase signaling pathway, Rap1 signaling pathway, calcium signaling pathway). Upregulated chi-miR-146a was found to promote N. caninum propagation in caprine EECs. CONCLUSIONS: This is, to our knowledge, the first report on the expression profiles of host miRNAs during infection with N. caninum, and shows that chi-miR-146a may promote N. caninum propagation in host cells. The novel findings of the present study should help to elucidate the interactions between host cells and N. caninum.

High genotype diversity and zoonotic potential of Enterocytozoon bieneusi in yaks (Bos grunniens) from Ganzi Tibetan Autonomous Prefecture, Sichuan Province.

Enterocytozoon bieneusi is a common pathogen in humans and various animals, threatening the breeding industry and public health. However, there is limited information on the molecular characteristics of E. bieneusi in yaks, an economically important animal mainly domesticated in the Qinghai Tibet Plateau in China. In the present study, nested PCR targeting the ITS gene region was applied to investigate the positive rates and genetic diversity of E. bieneusi in 223 faecal samples of yaks from three locations in Ganzi Tibetan Autonomous Prefecture, Sichuan Province. The total positive rate of E. bieneusi was 23.8% (53/223). Significant differences in positive rates were identified among yaks from three locations (χ2 = 8.535, p = 0.014) and four age groups (χ2 = 17.259, p = 0.001), with the highest positive rates in yaks from Yajiang and aged < 6 months, respectively. Sequence analysis identified seven known (EbpC, LW1, LQ10, PigEBITS5, ESH-01, J and BEB4) and five novel (Ganzi1-5) ITS genotypes. Phylogenetic analysis showed eight genotypes (EbpC, LW1, LQ10, PigEBITS5, ESH-01, Ganzi1, Ganzi2 and Ganzi4) in group 1 and three genotypes (J, BEB4 and Ganzi3) in group 2, indicating high genotype diversity and zoonotic potential of E. bieneusi in yaks from Ganzi. Considering the increasing zoonotic genotypes in yaks in the present study compared with previous findings, interventions should be developed to reduce the potential transmission of E. bieneusi between humans and animals.

Title: Grande diversité génotypique et potentiel zoonotique d'Enterocytozoon bieneusi chez les yaks (Bos grunniens) de la préfecture autonome tibétaine de Ganzi, province du Sichuan. Abstract: Enterocytozoon bieneusi est un agent pathogène courant chez l'homme et chez divers animaux, menaçant l'industrie de l'élevage et la santé publique. Cependant, il existe peu d'informations sur les caractéristiques moléculaires d'E. bieneusi chez les yaks, un animal important pour l'économie, principalement domestiqué sur le plateau du Qinghai au Tibet en Chine. Dans la présente étude, une PCR imbriquée ciblant la région du gène ITS a été appliquée pour étudier la positivité et la diversité génétique d'E. bieneusi dans 223 échantillons fécaux de yaks provenant de trois sites de la préfecture autonome tibétaine de Ganzi, province du Sichuan. Le taux total de positivité pour E. bieneusi était de 23,8 % (53/223). Des différences significatives dans les taux positifs ont été identifiées parmi les yaks de trois emplacements (χ2 = 8,535, P = 0,014) et de quatre groupes d'âge (χ2 = 17,259, P = 0,001), avec les taux positifs les plus élevés respectivement chez les yaks de Yajiang et ceux âgés de moins de 6 mois. L'analyse de séquence a identifié sept génotypes ITS connus (EbpC, LW1, LQ10, PigEBITS5, ESH-01, J et BEB4) et cinq nouveaux (Ganzi1­5). L'analyse phylogénétique a montré huit génotypes (EbpC, LW1, LQ10, PigEBITS5, ESH-01, Ganzi1, Ganzi2 et Ganzi4) dans le groupe 1 et trois génotypes (J, BEB4 et Ganzi3) dans le groupe 2, indiquant une diversité génotypique élevée et un potentiel zoonotique d'E. bieneusi chez les yaks de Ganzi. Compte tenu de l'augmentation des génotypes zoonotiques chez les yaks dans la présente étude par rapport aux résultats précédents, des interventions devraient être développées pour réduire la transmission potentielle d'E. bieneusi entre les humains et les animaux.

Retrotransposon-microsatellite amplified polymorphism, an electrophoretic approach for studying genetic variability among Schistosoma japonicum geographical isolates.

In the present study, retrotransposon-microsatellite amplified polymorphism (REMAP) was used to examine genetic variability among Schistosoma japonicum isolates from different endemic provinces in mainland China, using S. japonicum from Japan and the Philippines for comparison. Of the 50 primer combinations screened, eight produced highly reproducible REMAP fragments. Using these primers, 190 distinct DNA fragments were generated in total, of which 147 (77.37%) were polymorphic, indicating considerable genetic variation among the 43 S. japonicum isolates examined. The percentage of polymorphic bands (PPB) among S. japonicum isolates from mainland China, Japan, and the Philippines was 77.37%; PPB values of 18.42% and 53.68% were found among isolates from southwestern (SW) China and the lower Yangtze/Zhejiang province in eastern (E) China, respectively. Based on REMAP profiles, unweighted pair-group method with arithmetic averages (UPGMA) dendrogram analysis revealed that all of the S. japonicum samples grouped into three distinct clusters: parasites from mainland China, Japan, and the Philippines were clustered in each individual clade. Within the mainland China cluster, SW China isolates (from Sichuan and Yunnan provinces) grouped together, whereas worms from E China (Zhejiang, Anhui, Jiangxi, Jiangsu, Hunan, and Hubei provinces) grouped together. These results demonstrated that the REMAP marker system provides a reliable electrophoretic technique for studying genetic diversity and population structures of S. japonicum isolates from mainland China, and could be applied to other pathogens of human and animal health significance.

High prevalence of Eimeria infection in dairy goats in Shaanxi province, northwestern China.

A survey of dairy goats for infection with Eimeria species of coccidia was conducted in the Shaanxi province, northwestern China between December and November 2010, including Saanen and Guanzhong breeds. A total of 584 fecal samples (250 and 334 from Saanen and Guanzhong dairy goats, respectively) in six farms were collected. Eimeria oocysts were seen in 568 (97.3%) fecal samples, with six species, namely Eimeria jolchijevi, Eimeria arloingi, Eimeria alijevi, Eimeria caprina, Eimeria hirci, and Eimeria christenseni. The most prevalent were E. arloingi in Saanen and Guanzhong dairy goats, with an overall prevalence of 83.3% and 84.4%, and the lowest prevalence were E. christenseni (26.9%) and E. hirci (20.7%) for Saanen and Guanzhong Dairy goats, respectively. Two or more Eimeria species were commonly presented in all the age groups; 80.0% and 81.4% of positive Saanen and Guanzhong dairy goats carried more than two species, and 1.6% and 6.5% of two breeds had six species. The results of the present survey suggested that Eimeria infection is wide and severe in the Saanen and Guanzhong dairy goats, which suggested that integrated strategies should be implemented to prevent and control coccidial infection in dairy goats in this province.

[Construction and expression of an eukaryote vector of 14-3-3 protein in Toxoplasma gondii].

OBJECTIVE: To construct and express the eukaryotic expression vector of 14-3-3 protein of Toxoplasma gondii RH stain. METHODS: The structure and physicochemical property of 14-3-3 protein were predicted by bioinformatics analysis tools. The desired gene fragment was amplified from total RNA in T. gondii RH strain by RT-PCR, and sub-cloned into pcDNA3.0 to construct recombinant plasmid pcDNA3.0/14-3-3. After PCR confirming, double restriction enzyme digestion and DNA sequencing, the eukaryotic expression vector pcDNA3.0/14-3-3 was transfected into HeLa cells and the target protein was detected by Western blotting. RESULTS: The prediction of its gene sequence and amino acid sequence suggested that the 14-3-3 protein was acid soluble protein with five conserved regions, existing as homo- or hetero-dimers. The amplified gene fragment was about 800 bp, and the inserted fragment in pcDNA3.0/14-3-3 was 801 bp by sequencing, which had 99% homology to the 14-3-3 gene sequence of T. gondii in GenBank (Accession No. AB012775.1). Western blotting showed that there was more 14-3-3 protein expressed in the pcDNA3.0/14-3-3-transfected HeLa cells than not-transfected and mock transfected cells. Its relative molecular mass (Air) was about 30 000. CONCLUSION: The eukaryotic expression vector pcDNA3.0/14-3-3 is constructed and expressed in eukaryotic cells.

Neospora caninum infection induced mitochondrial dysfunction in caprine endometrial epithelial cells via downregulating SIRT1.

BACKGROUND: Infection of Neospora caninum, an important obligate intracellular protozoan parasite, causes reproductive dysfunctions (e.g. abortions) in ruminants (e.g. cattle, sheep and goats), leading to serious economic losses of livestock worldwide, but the pathogenic mechanisms of N. caninum are poorly understood. Mitochondrial dysfunction has been reported to be closely associated with pathogenesis of many infectious diseases. However, the effect of N. caninum infection on the mitochondrial function of hosts remains unclear. METHODS: The effects of N. caninum infection on mitochondrial dysfunction in caprine endometrial epithelial cells (EECs), including intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) contents, mitochondrial DNA (mtDNA) copy numbers and ultrastructure of mitochondria, were studied by using JC-1, DCFH-DA, ATP assay kits, quantitative real-time polymerase chain reaction (RT-qPCR) and transmission electron microscopy, respectively, and the regulatory roles of sirtuin 1 (SIRT1) on mitochondrial dysfunction, autophagy and N. caninum propagation in caprine EECs were investigated by using two drugs, namely resveratrol (an activator of SIRT1) and Ex 527 (an inhibitor of SIRT1). RESULTS: The current study found that N. caninum infection induced mitochondrial dysfunction of caprine EECs, including accumulation of intracellular ROS, significant reductions of MMP, ATP contents, mtDNA copy numbers and damaged ultrastructure of mitochondria. Downregulated expression of SIRT1 was also detected in caprine EECs infected with N. caninum. Treatments using resveratrol and Ex 527 to caprine EECs showed that dysregulation of SIRT1 significantly reversed mitochondrial dysfunction of cells caused by N. caninum infection. Furthermore, using resveratrol and Ex 527, SIRT1 expression was found to be negatively associated with autophagy induced by N. caninum infection in caprine EECs, and the intracellular propagation of N. caninum tachyzoites in caprine EECs was negatively affected by SIRT1 expression. CONCLUSIONS: These results indicated that N. caninum infection induced mitochondrial dysfunction by downregulating SIRT1, and downregulation of SIRT1 promoted cell autophagy and intracellular proliferation of N. caninum tachyzoites in caprine EECs. The findings suggested a potential role of SIRT1 as a target to develop control strategies against N. caninum infection.