Micro-RNA expression profile of BALB/c mouse glandular stomach in the early phase of Cryptosporidium muris infection.

Cryptosporidiosis is a zoonotic disease in humans and animals that is caused by infection with the oocysts of Cryptosporidium. MicroRNAs (miRNAs) are important players in regulating the innate immune response against parasitic infection. Public miRNAs data for studying pathogenic mechanisms of cryptosporidiosis, particularly in natural hosts, are scarce. Here, we compared miRNA profiles of the glandular stomach of C. muris-infected and uninfected BALB/c mice using microarray sequencing. A total of 10 miRNAs (including 3 upregulated and 7 downregulated miRNAs) with significant differential expression (|FC| ≥ 2 and P value < 0.05) were identified in the glandular stomach of BALB/c mice 8 h after infection with C. muris. MiRWalk and miRDB online bioinformatics tools were used to predict the target genes of differentially expressed miRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to annotate the target genes. GO analysis indicate that gene transcription-related and ion transport-related GO terms were significantly enriched. In addition, the KEGG analyses showed that the target genes were strongly related to diverse types of tumor disease progression and anti-pathogen immunity pathways. In the current study, we firstly report changes in miRNA expression profiles in the glandular stomach of BALB/c mice at the early phase of C. muris invasion. This dysregulation in miRNA expression may contribute to our understanding of cryptosporidiosis pathology. This study provides a new perspective on the miRNA regulatory mechanisms of cryptosporidiosis, which may help in the development of effective control strategies against this pathogen.

Acaricidal and repellent activities of ethanol extracts of nine chinese medicinal herbs against Rhipicephalus microplus (Acari: Ixodidae).

Rhipicephalus microplus is a major threat to the cattle industry worldwide. The intensive use of acaricides and repellents has resulted in drug resistance. Hence, effective and eco-friendly pest control alternatives are urgently needed, especially from natural plant resources. In this study, the acaricidal and repellent activities of nine herbs against the larvae and eggs of R. microplus were evaluated. The results showed that ethanol extracts of star anise (Illicium verum), chaulmoogra (Hydnocarpus anthelmintica), motherwart (Leonurus artemisia), mandarin orange peel (citri reticulatae pericarpium, i.e., peel of Citrus reticulata fruit), and stemona (Stemona sessilifolia) had good contact acaricidal activities of 100, 98, 94, 88 and 86%, respectively, whereas star anise and clove (Syzygium aromaticum) had good fumigant acaricidal activities of 98 and 96%, respectively. The hatching inhibition rate of star anise against R. microplus eggs was 100%. All nine herbs had good real-time repellent rates, but only castor bean and star anise had repellent effects after 48 h (81.3 and 79.6%, respectively). This is the first report of the acaricidal and repellent activities of these medicinal herbs against R. microplus. Ethanol extracts of these herbs might be considered as potential alternatives to chemical acaricides for control of R. microplus.

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

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

Aflatoxin B1 causes oxidative stress and apoptosis in sheep testes associated with disrupting rumen microbiota.

Aflatoxin B1 (AFB1) is an unavoidable environmental pollutant commonly found in feed and foodstuffs. It is the most toxic one of all the aflatoxins, which can cause severe impairment to testicular development and function. Yet, the underlying mechanisms of reproductive toxicity in rams sheep remain inconclusive. The study was designed to explore the effects of AFB1 on sheep testes through rumen-microbiota, oxidative stress and apoptosis. Six-month-old male Dorper rams (n = 6) were orally administrated with 1.0 mg/kg AFB1 (dissolved in 20 mL 4% ethanol) 24 h before the experiment. At the same time, rams in the control group (n = 6) were intragastrically administrated with 20 mL 4% ethanol. It was observed that acute AFB1 poisoning had significant (p < 0.05) toxin residue in the testis and could cause testicular histopathological damage. AFB1 stimulated the secretion of plasma testosterone level through regulating testosterone synthesis-related genes (StAR, 3ß-HSD, CYP11A1, and CYP17A1), which are accompanied by the increase of oxidative stress and testicular apoptosis that had a close relationship with the regulation of testosterone secretion. Interestingly, we observed rumen dysbacteriosis and decreased the abundances of Prevotella, Succiniclasticum, CF231, Ruminococcus, and Pseudobutyrivibrio in AFB1-exposed sheep, which were negatively correlated to the testosterone synthesis-related gene levels. Taken together, our findings indicated that AFB1 induced testicular damage and testicular dysfunction, which is related to testicular oxidative stress and apoptosis involved in rumen dysbacteriosis in sheep.

Evaluation of the in vitro acaricidal activity of ethanol extracts of seven Chinese medicinal herbs on Ornithonyssus sylviarum (Acari: Macronyssidae).

Ornithonyssus sylviarum (Acari: Macronyssidae) is a common ectoparasite that feeds on the blood of poultry. Following infestation, this mite will cause symptoms such as weight loss, anemia, and decreased egg production. To explore green and safe drugs for the prevention and treatment of O. sylviarum, this study evaluated the effects of ethanol extracts of seven Chinese medicinal herbs-Leonurus artemisia (motherwort), Illicium verum (star anise), Cinnamomum cassia (cinnamon), Hibiscus syriacus, Artemisia argyi (Chinese mugwort), Taraxacum sp. (dandelion), and Syzygium aromaticum (clove)-on O. sylviarum at different life stages. The results showed that different methods of administration affected the acaricidal efficacy of these plant extracts on O. sylviarum. After 6 h of administration with the fumigation method, the acaricidal efficacy of S. aromaticum on adults, nymphs and larvae of O. sylviarum reached 100%. 30 min after administration with the infiltration method, S. aromaticum, H. syriacus and L. artemisia showed acaricidal effects on adults and nymphs of O. sylviarum reaching 100%. In another experiment evaluating the inhibition of egg hatching of O. sylviarum with alcohol extracts of these seven herbs, at 48 h after treatment, A. argyi and C. cassia showed inhibition rates of 19.4%. The results of this study indicate that S. aromaticum induced mortality at all stages of O. sylviarum, whereas A. argyi was found to be the most effective at inhibiting the mite's egg hatching among the seven herbs. These herbs can therefore be used as potential substitutes for chemical pesticides to prevent and control O. sylviarum. These results provide practical knowledge for the control of O. sylviarum.

Aflatoxin B1 alters meat quality associated with oxidative stress, inflammation, and gut-microbiota in sheep.

Aflatoxin B1 (AFB1) is an unavoidable contaminant in animal feed and agricultural products. AFB1 has been found to impair the liver and kidney function of sheep. However, few data are available, which explain the toxic damage of AFB1 exposure on meat quality. In the study, male Dorper RAMS sheep (6-month-old) were orally administrated with AFB1 at the dose of 1 mg/kg body weight once. The body temperature, serum biochemistry, meat quality-related parameters, oxidation indicators in meat and serum, the mRNA expression of pro-inflammatory cytokines and anti-inflammatory, and microbiota composition of feces were measured 24 h after AFB1 exposure. The results showed that the body temperature was slightly increased, the mental state of mutton sheep was suppressed, and biochemical indicators were significantly changed after AFB1 exposure. AFB1 impaired mutton quality reflected by the structure of muscle fibers was changed, and increased muscle drip loss and lightness (L*), and decreased muscle redness (a*). Moreover, we found that AFB1 caused changes in the oxidative stress indicators T-SOD, T-AOC, MDA, GSH level, and GSH/GSSG ratio, and inflammation damage of mutton reflected by increasing pro-inflammatory TNF-α and reducing anti-inflammatory IL-10 mRNA levels, disrupts the secretion of inflammatory factors, and changed the composition of gut microbiota reflected by significantly increased Firmicutes/Bacteroidetes ratio and decreased the abundances of Butyrivibrio, which are related to the quality of the mutton. In summary, gut microbiota participates in AFB1 to damage mutton quality, which may be co-mediated by oxidative stress, inflammation, and gut microbiota.

Development of a duplex PCR assay for detecting Theileria luwenshuni and Anaplasma phagocytophilum in sheep and goats.

Coinfections with the tick-borne pathogens Theileria luwenshuni and Anaplasma phagocytophilum can cause significant economic losses in sheep and goat farming. The difficulty in detecting these two pathogens by microscopic examination warrants the development of a rapid detection test to discriminate them. In this study, a duplex polymerase chain reaction (PCR) assay was developed to simultaneously detect T. luwenshuni and A. phagocytophilum. Alignment of the sequences from related pathogens allowed us to design a primer pair targeting the 18S ribosomal RNA gene in T. luwenshuni and generate a target product of 962 bp, whereas a previously reported species-specific primer (SSAP2f/SSAP2r) for A. phagocytophilum was used in the same reaction to generate a product of 641 bp. Genomic DNA from T. luwenshuni and A. phagocytophilum was 10-fold serially diluted for testing PCR sensitivity. Under the optimal PCR conditions we established, the lower limit of detection of the assay was 29.13 fg/µL for T. luwenshuni and 1.53 fg/µL for A. phagocytophilum, and PCR primers used in this study were confirmed to be 100% species-specific using other hemoparasites previously identified by other methods. No significant difference was found between conventional and duplex PCR protocols used to detect the two species. Our study provides an effective, sensitive, specific, and accurate tool for the diagnosis and epidemiological surveillance of mixed infections of the two pathogens in sheep and goats.

Molecular Characterization of Giardia duodenalis and Enterocytozoon bieneusi Isolated from Tibetan Sheep and Tibetan Goats Under Natural Grazing Conditions in Tibet.

In the present study, fecal samples from a total of 620 Tibetan sheep and 260 Tibetan goats from six counties in Tibet were examined by nested PCR. The results showed that the overall infection rates of Giardia duodenalis and Enterocytozoon bieneusi were 0.8% (5/620) and 15% (93/620), respectively, in Tibetan sheep, and 0% (0/260) and 9.6% (25/260), respectively, in Tibetan goats. Based on sequence analysis of the SSU rRNA, tpi, bg, and gdh genes of G. duodenalis, only assemblage E was identified. Based on sequence analysis of the ribosomal internal transcriptional spacer (ITS) region of E. bieneusi, a total of 12 genotypes (three novel and nine known) were detected, and these clustered into two separate phylogenetic groups. Genotypes CHG19, EbpA, EbpC, H, PigEBITS5, and CTS3 clustered into Group 1 with high zoonotic potential, while genotypes BEB6, CHC8, CHG1, I, CTS1, and CTS2 fell within the host-specific Group 2. Ten genotypes were detected in Tibetan sheep, and two genotypes were found in Tibetan goats. The current study indicated that E. bieneusi infections are widespread among these livestock, and Tibetan goats may play an important role as a reservoir of zoonotic E. bieneusi genotypes.

MicroRNA expression profile of HCT-8 cells in the early phase of Cryptosporidium parvum infection.

BACKGROUND: Cryptosporidium parvum is an important zoonotic parasitic disease worldwide, but the molecular mechanisms of the host-parasite interaction are not fully understood. Noncoding microRNAs (miRNAs) are considered key regulators of parasitic diseases. Therefore, we used microarray, qPCR, and bioinformatic analyses to investigate the intestinal epithelial miRNA expression profile after Cryptosporidium parvum infection. RESULTS: Twenty miRNAs were differentially expressed after infection (four upregulated and 16 downregulated). Further analysis of the differentially expressed miRNAs revealed that many important cellular responses were triggered by Cryptosporidium parvum infection, including cell apoptosis and the inflammatory and immune responses. CONCLUSIONS: This study demonstrates for the first time that the miRNA expression profile of human intestinal epithelium cells is altered by C. parvum infection. This dysregulation of miRNA expression may contribute to the regulation of host biological processes in response to C. parvum infection, including cell apoptosis and the immune responses. These results provide new insight into the regulatory mechanisms of host miRNAs during cryptosporidiosis, which may offer potential targets for future C. parvum control strategies.

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

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

Molecular characterization and distribution of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi from yaks in Tibet, China.

BACKGROUND: With worldwide distribution and importance for veterinary medicine, Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi have been found in a wide variety of vertebrate hosts. At present, few available molecular data can be used to understand the features of genetic diversity of these pathogens in areas without or less intensive farming. Dominated by grazing, Tibet is a separate geographic unit in China and yaks are in frequent contact with local herdsmen and necessary for their daily life. Therefore, to investigate the distribution of these pathogens in yaks of Tibet, 577 fecal specimens were screened using nested PCR for the presence and genotypes of the three intestinal pathogens. RESULTS: The overall prevalence of Cryptosporidium spp., G. duodenalis, and E. bieneusi were 1.4% (8/577), 1.7% (10/577), and 5.0% (29/577), respectively. Cryptosporidium andersoni (n = 7) and Cryptosporidium bovis (n = 1) were detected by sequence analysis of the SSU rRNA gene. Genotyping at the SSU rRNA and triosephosphate isomerase genes suggested that all G. duodenalis positive specimens belonged to assemblage E. Sequence analysis of the internal transcribed spacer gene identified six known E. bieneusi genotypes: BEB4 (n = 11), I (n = 6), D (n = 5), J (n = 2), CHC8 (n = 1), and BEB6 (n = 1). One subtype (A5,A4,A2,A1) for C. andersoni and three multilocus genotypes for E. bieneusi were identified by multilocus sequence typing. CONCLUSIONS: We report for the first time the status of three enteric pathogens infection simultaneously for grazing yaks in Tibet. Yaks in our study are likely to impose a low zoonotic risk for humans. The molecular epidemiology data add to our knowledge of the characteristics of distribution and transmission for these pathogens in Tibet and their zoonotic potential and public health significance.

Rapid and sensitive detection of Anaplasma phagocytophilum using a newly developed recombinase polymerase amplification assay.

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 ddH2O 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.

Occurrence, Molecular Characterization, and Assessment of Zoonotic Risk of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Pigs in Henan, Central China.

Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common gastrointestinal pathogens in humans and animals. Little is known about them and the range of species/assemblages/genotypes occurring in domestic pigs in China. Here, we present data on the occurrence and molecular diversity of these pathogens detected in the feces from farms in Henan, central China. Of 897 fecal samples tested, 28 (3.1%), 15 (1.7%), and 408 (45.5%) samples were positive for Cryptosporidium, G. duodenalis, and E. bieneusi, respectively. Cryptosporidium and G. duodenalis were most frequently detected in piglets, while E. bieneusi was markedly more prevalent in fattening pigs. Sequence analysis of SSU rRNA gene revealed that positive Cryptosporidium strains belonged to C. suis (n = 18) and C. scrofarum (n = 10). Giardia duodenalis assemblages E (n = 9), assemblages A (n = 3), and assemblages C (n = 3) were characterized based on the sequence analysis of tpi gene. Thirteen E. bieneusi genotypes comprising four novel (pigHN-I to pigHN-IV) and nine known (EbpC, EbpA, pigEbITS5, LW1, H, CM8, G, CHG19, and CHS5) genotypes were identified by ITS sequence analysis of a large proportion (n = 200) of E. bieneusi-positive samples. EbpC was the most frequent genotype, detected in 60 specimens. All 13 genotypes identified in this study clustered in zoonotic Group 1. The findings indicate that the presence of zoonotic species/assemblages/genotypes of these pathogens poses a threat to public health, suggesting that pigs in Henan province could be a significant source of human infection and water pollution.

A canine model of experimental infection with Cryptosporidium canis.

Cryptosporidium is a genus of protozoal parasites that affects the gastrointestinal epithelium of a variety of hosts. Several models of experimental infection have been described to study the susceptibility, infectivity and pathogenicity among different Cryptosporidium species and isolates. This study aimed to establish an experimental infection of Cryptodporidium canis in canids. Infectivity and pathogenicity have been measured by evaluating the clinical status, pattern of oocyst excretion and histological examination. Results showed that C. canis was not infective for immunocompetent dogs or mice with severe combined immunodeficiency syndrome (SCID). Oocysts were first detected in the feces of immunosuppressed dogs on day 3 post-infection (p.i.), with levels peaking twice on days 10 and 17 p.i. during the patent period. cryptosporidial developmental stages were found in the duodenum and jejunum of dogs in histological sections stained with hematoxylin and eosin (H & E) and using scanning electron microscopy (SEM). Histopathological changes in the intestinal tract of infected dogs were characterized by epithelial metaplasia and dilatation; the integrity of intestinal mucosal epithelial cells was distinctly damaged with whole sheets of cilia sloughed away. Ultrastructural observation data were consistent with histological observations. Based on these findings, the canine model described in this work will be useful to evaluate clinical, parasitological and histological aspects of C. canis infection and will be useful for the further understanding of cryptosporidiosis, drug development, and vaccine development.

Morphological and molecular characterisation of Isospora butcherae n. sp. in a silvereye (Zosterops lateralis) (Latham, 1801).

A new Isospora (Apicomplexa:Eimeriidae) species is described from a silvereye (Zosterops lateralis) in Western Australia. Sporulated oocysts of this species are spherical, 24.2 (23.1-25.2) × 23.3 (22.8-23.9) µm, with a shape index (length/width) of 1.02, and with a smooth bi-layered oocyst wall, 1.2 µm thick (outer layer 0.9 µm, inner 0.3 µm). A polar granule is present, but the oocyst residuum and a micropyle are absent. The ovoid-shaped sporocysts are 16.1 (15.7-17.3) × 10.5 (15.7-17.3) µm and have a shape index of 1.53. A hemidome-shaped Stieda and a rectangular-shaped substieda body are present. A sporocyst residuum is present and composed of numerous granules of different sizes scattered among the sporozoites. The oocysts from this isolate are morphologically different from those of all known Isospora spp. This coccidian parasite was molecularly characterised at the 18S, 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) gene. At the 18S locus, based on 1210 bp of sequence, this new isolate exhibited 99.9, 99.8, 99.7 and 99.5% similarity to I. sp. MAH-2013a (KF648870) from a superb starling (Lamprotornis superbus) in Canada, I. sp. MS-2003 (AY33157) from a Southern cape sparrow (Plocepasser mahali) in America, I. sp. Tokyo (AB75786) from Japan and I. sp. respectively. Further analysis of a subgroup of 300 bp long 18S sequences (n = 11), including I. anthochaerae and the other three Isospora characterised from birds in Western Australia, revealed that I. butcherae n. sp. exhibited 98.3% similarity to both I. sp. MAH-2013a (KF648870) and I. MS-2003 (AY33171). At the 28S locus, this new isolate exhibited 97.3% similarity with I. sp. MS-2003 from a California towhee (Melozone crissalis). At the COI locus, this new isolate exhibited 99.8% similarity to I. neochmiae from a red-browed finch. Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora butcherae n. sp. after Mrs. June Butcher for her lifelong dedication as a wildlife rehabilitator. Graphical abstract ᅟ.

Molecular characterization of hemotropic mycoplasmas (Mycoplasma ovis and 'Candidatus Mycoplasma haemovis') in sheep and goats in China.

BACKGROUND: Hemotropic mycoplasmas (hemoplasmas) are emerging zoonotic pathogens with a worldwide distribution that can cause mild to severe hemolytic anemia, icterus, ill-thrift, infertility, and poor weight gain. However, understanding of the molecular epidemiology of hemoplasmas (Mycoplasma ovis and 'Candidatus Mycoplasma haemovis') is limited in sheep and goats, and the hemoplasma strain/species/variant 'Candidatus M. haemovis' was poorly studied throughout the world and had never been detected in China until now. Thus, the aim of the present study was to determine the molecular prevalence of hemoplasmas, including M. ovis and 'Candidatus M. haemovis' in sheep and goats from seven provinces and one autonomous region of China. METHODS: A total of 1364 blood samples were collected from sheep and goats in seven provinces and one autonomous region of China. All blood samples were tested for hemoplasmas (M. ovis and 'Candidatus M. haemovis') by nested PCR amplification based on 16S rRNA gene. Positive specimens underwent nucleotide sequencing and phylogenetic analysis. RESULTS: Overall, 610 specimens (44.7%, 610/1364) were shown to be hemoplasmas (M. ovis and 'Candidatus M. haemovis') -positive by nested PCR amplification based on 16S rRNA gene. The prevalence in goats was 44.1% (379/860), and 45.8% (231/504) in sheep, while that in grazing small ruminants was 54.4% (396/728) and 33.6% (214/636) in house feeding small ruminants. Sequencing of the nearly complete 16S rRNA gene was successful for the 103 randomly selected positive specimens from different farms in different sampling sites of China. Among them, analysis of the 16S rRNA gene sequences identified M. ovis (n = 56) and 'Candidatus M. haemovis' (n = 47). Two (KU983740 and KU983746) of the four novel genotypes obtained in this study were closely related to M. ovis, while the other two genotypes (KU983748 and KU983749) had high identity with 'Candidatus M. haemovis'. Remarkably, the genotype (KU983740) of M. ovis in sheep and goats in this study fell in a clade with two human hemoplasmas from USA (KF313922 and GU230144) and shared 99.8%-99.9% with them. CONCLUSIONS: In this study, 'Candidatus M. haemovis' was first detected in Chinese sheep and goats and hemoplasmas (M. ovis and 'Candidatus M. haemovis') are highly prevalent, and widely distributed in China.

Development of duplex PCR for simultaneous detection of Theileria spp. and Anaplasma spp. in sheep and goats.

Theileria spp. and Anaplasma spp., which are important tick-borne pathogens (TBPs), impact the health of humans and animals in tropical and subtropical areas. Theileria and Anaplasma co-infections are common in sheep and goats. Following alignment of the relevant DNA sequences, two primer sets were designed to specifically target the Theileria spp. 18S rRNA and Anaplasma spp. 16S rRNA gene sequences. Genomic DNA from the two genera was serially diluted tenfold for testing the sensitivities of detection of the primer sets. The specificities of the primer sets were confirmed when DNA from Anaplasma and Theileria (positive controls), other related hematoparasites (negative controls) and ddH2O were used as templates. Fifty field samples were also used to evaluate the utility of single PCR and duplex PCR assays, and the detection results were compared with those of the PCR methods previously published. An optimized duplex PCR assay was established from the two primer sets based on the relevant genes from the two TBPs, and this assay generated products of 298-bp (Theileria spp.) and 139-bp (Anaplasma spp.). The detection limit of the assay was 29.4 × 10-3 ng per µl, and there was no cross-reaction with the DNA from other hematoparasites. The results showed that the newly developed duplex PCR assay had an efficiency of detection (P > 0.05) similar to other published PCR methods. In this study, a duplex PCR assay was developed that can simultaneously identify Theileria spp. and Anaplasma spp. in sheep and goats. This duplex PCR is a potentially valuable assay for epidemiological studies of TBPs in that it can detect cases of mixed infections of the pathogens.

Enterocytozoon bieneusi Genotypes in Grazing Horses in China and their Zoonotic Transmission Potential.

In present study, 262 fecal specimens were collected from 12 groups of grazing horses in the Xinjiang Uyghur Autonomous Region of China. The specimens were subjected to PCR and sequencing analyses of the ribosomal internal transcribed spacer (ITS). The overall prevalence of E. bieneusi in horses was 30.9% (81/262). No significant differences in prevalence were observed between horses of different ages or sexes. Nineteen genotypes were identified: 15 known genotypes (BEB6, CHG19, CM6, CM7, CM8, CS-1, CS-4, D, EpbA, EbpC, G, horse1, horse2, O, and Peru8) and four new genotypes (XJH1-XJH4). Six of these genotypes were previously detected in humans: BEB6, D, EbpA, EbpC, O, and Peru8. Genotype EbpC was the most prevalent (21/81), followed by EpbA (20/81), BEB6 (9/81), CM6 (4/81), horse1 (4/81), O (4/81), G (3/81), CHG19 (2/81), CM7 (2/81), horse2 (2/81), and XJH1 (2/81), whereas the remaining eight genotypes were seen in one specimen each. In a phylogenetic analysis, 14 genotypes (65/81, 80.2%), excluding genotypes BEB6, CM7, horse2, XJH1, and XJH4, belonged to group 1, which have zoonotic potential. The high diversity in the E. bieneusi genotypes and their zoonotic potential suggest that grazing horses are a potential source of zoonotic infection in humans.

Morphological and molecular characterization of Isospora manorinae n. sp. in a yellow-throated miner (Manorina flavigula wayensis) (Gould, 1840).

A new Isospora (Apicomplexa:Eimeriidae) species is described from a single yellow-throated miner bird (Manorina flavigula) (subspecies M. f. wayensis) in Western Australia. Sporulated oocysts (n = 32) of this isolate are spherical to subspherical, 22.8 (20.3-23.8) × 18.3 (17.7-18.7) µm, with a shape index (length/width) of 1.25 (1.2-1.3); and a smooth and bilayered oocyst wall, 1.3 µm thick (outer layer 0.9 µm, inner 0.4 µm). A polar granule is present, but the micropyle and oocyst residuum are absent. The sporocysts are lemon-shaped, 15.5 (14.6-15.8) × 9.5 (9.5-10.2) µm, with a shape index of 1.6. Stieda and substieda bodies are present, the Stieda body being knob-like and the substieda body being subspherical-shaped. A sporocyst residuum is present and composed of numerous granules of different size scattered among the sporozoites, a spheroid or subspheroid refractile body is present in the sporozoite. Morphologically, the oocysts from this isolate are different from those of all known valid Isospora spp. Molecular analysis was conducted at 3 loci; the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) gene. At the 18S locus, this new isolate exhibited 99.2% similarity to Isospora gryphoni and three other Isospora spp. Further analysis of a subgroup of 300 bp long 18S sequences (8), including Isospora anthochaerae was conducted. This new isolate grouped in a clade with I. anthochaerae and exhibited 99.3% similarity. At the 28S locus, this new isolate grouped with I. anthochaerae with which it shared 99.1% similarity. At the COI locus, this new isolate exhibited 96.8% similarity to Isospora sp. JCI-2015 from a spectacled warbler (Sylvia conspicillata) in Spain. Further analysis from a subgroup of shorter COI sequences (n = 13) was performed and this new isolate exhibited 99.1% similarity to I. anthochaerae. Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora manorinae n. sp. after its host, the yellow-throated miner (Manorina flavigula wayensis).

First molecular evidence for the presence of Anaplasma DNA in milk from sheep and goats in China.

Anaplasmosis, a disease caused by bacteria in the genus of Anaplasma, imposes economic constraints on animal breeders and also threatens human health. In the present study, we investigated the presence of Anaplasma spp. DNA in milk collected from sheep and goats in China. A total of 120 milk samples and 414 field-sampled blood specimens from sheep and goats were analyzed by PCR and DNA sequencing. Anaplasma ovis was detected in 12 milk samples (three from sheep and nine from goats). The blood specimens corresponding to the A. ovis DNA-positive milk were analyzed for Anaplasma DNA presence, and A. ovis DNA was identified in 10 out of the 12 blood specimens. One goat's milk sample was Anaplasma bovis DNA-positive, as was the corresponding blood sample. Anaplasma phagocytophilum was found in a milk sample and blood sample from one goat. One milk sample from Xinmi in Henan province was simultaneously positive for A. bovis and A. phagocytophilum; however, the corresponding blood was negative for both species. DNA sequencing of the PCR products and phylogenetic analysis confirmed that the sequences from the milk samples matched those of the corresponding blood samples. This is the first report to detect the Anaplasma DNA in milk samples under natural condition, and represents the first evidence of the presence of A. ovis, A. bovis and A. phagocytophilum DNA in milk from sheep and goats.