Cryptosporidium spp. in large-scale sheep farms in China: prevalence and genetic diversity.

Cryptosporidium spp. are significant zoonotic intestinal parasites that induce diarrhea and even death across most vertebrates, including humans. Previous studies showed that sheep are important hosts for Cryptosporidium and that its distribution in sheep is influenced by geography, feeding patterns, age, and season. Environmental factors also influence the transmission of Cryptosporidium. Molecular studies of Cryptosporidium in sheep have been conducted in only a few regions of China, and studies into the effect of sheep-housing environments on Cryptosporidium transmission are even rarer. To detect the prevalence of Cryptosporidium in large-scale sheep-housing farms, a total of 1241 fecal samples were collected from sheep, 727 environmental samples were taken from sheep housing, and 30 water samples were collected in six regions of China. To ascertain the existence of the parasite and identify the species of Cryptosporidium spp., we conducted nested PCR amplification of DNA extracted from all samples using the small-subunit (SSU) rRNA gene as a target. For a more in-depth analysis of Cryptosporidium spp. subtypes, C. xiaoi-and C. ubiquitum-positive samples underwent separate nested PCR amplification targeting the 60 kDa glycoprotein (gp60) gene. The amplification of the Cryptosporidium spp. SSU rRNA gene locus from the whole genomic DNA of all samples yielded a positive rate of 1.2% (20/1241) in fecal samples, 0.1% (1/727) in environmental samples, and no positive samples were found in water samples. The prevalence of Cryptosporidium spp. infection in large-scale housed sheep was 1.7%, which was higher than that in free-ranging sheep (0.0%). The highest prevalence of infection was found in weaning lambs (6.8%). Among the different seasons, the peaks were found in the fall and winter. The most prevalent species were C. xiaoi and C. ubiquitum, with the former accounting for the majority of infections. The distribution of C. xiaoi subtypes was diverse, with XXIIIc (n = 1), XXIIId (n = 2), XXIIIe (n = 2), and XXIIIl (n = 4) identified. In contrast, only one subtype, XIIa (n = 9), was found in C. ubiquitum. In this study, C. xiaoi and C. ubiquitum were found to be the predominant species, and Cryptosporidium was found to be present in the environment. These findings provide an important foundation for the comprehensive prevention and management of Cryptosporidium in intensively reared sheep. Furthermore, by elucidating the prevalence of Cryptosporidium in sheep and its potential role in environmental transmission, this study deepens our understanding of the intricate interactions between animal health, environmental contamination, and public health dynamics.

Giardia duodenalis in Hu sheep: occurrence and environmental contamination on large-scale housing farms.

Giardia duodenalis is a common zoonotic intestinal parasitic protozoan and sheep are among its hosts; however, limited information is available on sheep kept in large-scale housing. The Hu sheep is a first-class protected local livestock breed in China. In this study, we investigated the seasonal dynamics of G. duodenalis infection in Hu sheep and the environmental contamination of large-scale sheep farms. We collected 474 fecal samples and 312 environmental samples from Hu sheep on a large-scale sheep farm in Henan, China. The prevalence of G. duodenalis was determined by nested PCR targeting the ß­giardin (bg) gene. The assemblages and multilocus genotypes (MLGs) were investigated based on analyses of three genetic loci, i.e. bg, glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi). To detect mixed infections of different assemblages, assemblage A/E-specific PCRs were performed to amplify the tpi gene. The prevalence of G. duodenalis infection in sheep was 17.9% (81/474) and the positivity rate in environmental samples was 0.96% (3/312). Genetic analysis revealed the presence of two assemblages (assemblages A and E), with assemblage E being detected in both fecal and environmental samples, and assemblage A detected only in fecal samples. A total of 23 MLGs were obtained in fecal and environmental samples, all of which belonged to assemblage E. These results indicate the seasonal dynamics of G. duodenalis infection in sheep and environmental contamination on large-scale housing sheep farms and provide an important reference for the prevention and control of G. duodenalis on large-scale housing sheep farms.

Title: Giardia duodenalis chez les moutons Hu : occurrence et contamination environnementale dans les fermes d'élevage à grande échelle. Abstract: Giardia duodenalis est un protozoaire parasitaire intestinal zoonotique commun et les moutons font partie de ses hôtes, mais peu d'informations sont disponibles sur les moutons élevés dans des stabulations à grande échelle. Le mouton Hu est une race de bétail locale protégée de première classe en Chine. Dans cette étude, nous avons étudié la dynamique saisonnière de l'infection à G. duodenalis chez les moutons Hu et la contamination environnementale des élevages ovins à grande échelle. Nous avons collecté 474 échantillons fécaux et 312 échantillons environnementaux de moutons Hu dans une ferme ovine à grande échelle dans le Henan, en Chine. La prévalence de G. duodenalis a été déterminée par PCR nichée ciblant le gène de la ß­giardine (bg). Les assemblages et les génotypes multilocus (MLG) ont été étudiés sur la base de l'analyse de trois locus génétiques, à savoir bg, glutamate déshydrogénase (gdh) et triosephosphate isomérase (tpi). Pour détecter des infections mixtes de différents assemblages, des PCR spécifiques aux assemblages A et E ont été réalisées pour amplifier le gène tpi. La prévalence de l'infection à G. duodenalis chez les ovins était de 17,9 % (81/474) et le taux de positivité dans les échantillons environnementaux était de 0,96 % (3/312). L'analyse génétique a révélé la présence de deux assemblages (assemblages A et E), l'assemblage E étant détecté à la fois dans les échantillons fécaux et environnementaux, et l'assemblage A détecté uniquement dans les échantillons fécaux. Au total, 23 MLG ont été détectés dans des échantillons fécaux et environnementaux, tous appartenant à l'assemblage E. Ces résultats montrent la dynamique saisonnière de l'infection à G. duodenalis chez les ovins et la contamination environnementale dans les élevages ovins à grande échelle et fournissent une référence importante pour la prévention et le contrôle de G. duodenalis dans les élevages ovins à grande échelle.

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

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

Whole transcriptome analysis of HCT-8 cells infected by Cryptosporidium parvum.

BACKGROUND: Cryptosporidium species are zoonotic protozoans that are important causes of diarrhoeal disease in both humans and animals. Non-coding RNAs (ncRNAs) play an important role in the innate immune defense against Cryptosporidium infection, but the underlying molecular mechanisms in the interaction between human ileocecal adenocarcinoma (HCT-8) cells and Cryptosporidium species have not been entirely revealed. METHODS: The expression profiles of messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) in the early phase of infection of HCT-8 cells with Cryptosporidium parvum and at 3 and 12 h post infection were analyzed using the RNA-sequencing technique. The biological functions of differentially expressed RNAs (dif-RNAs) were discovered through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The targeting relationships between three ncRNAs and mRNAs were analyzed using bioinformatics methods, followed by building a competing endogenous RNA (ceRNA) regulatory network centered on miRNAs. RESULTS: After strictly filtering the raw data, our analysis revealed 393 dif-lncRNAs, 69 dif-miRNAs and 115 dif-mRNAs at 3 hpi, and 450 dif-lncRNAs, 129 dif-miRNAs, 117 dif-mRNAs and one dif-circRNA at 12 hpi. Of these, 94 dif-lncRNAs, 24 dif-miRNAs and 22 dif-mRNAs were detected at both post-infection time points. Eleven dif-lncRNAs, seven dif-miRNAs, eight dif-mRNAs and one circRNA were randomly selected and confirmed using the quantitative real-time PCR. Bioinformatics analyses showed that the dif-mRNAs were significantly enriched in nutritional absorption, metabolic processes and metabolism-related pathways, while the dif-lncRNAs were mainly involved in the pathways related to the infection and pathogenicity of C. parvum (e.g. tight junction protein) and immune-related pathways (e.g. cell adhesion molecules). In contrast, dif-miRNAs and dif-circRNA were significantly enriched in apoptosis and apoptosis-related pathways. Among the downregulated RNAs, the miRNAs has-miR-324-3p and hsa-miR-3127-5p appear to be crucial miRNAs which could negatively regulate circRNA, lncRNA and mRNA. CONCLUSIONS: The whole transcriptome profiles of HCT-8 cells infected with C. parvum were obtained in this study. The results of the GO and KEGG pathway analyses suggest significant roles for these dif-RNAs during the course of C. parvum infection. A ceRNA regulation network containing miRNA at its center was constructed for the first time, with hsa-miR-324-3p and hsa-miR-3127-5p being the crucial miRNAs. These findings provide novel insights into the responses of human intestinal epithelial cells to C. parvum infection.

Evaluation of the in vitro acaricidal activity of Chinese herbal compounds on the poultry red mite (Dermanyssus gallinae).

The poultry red mite Dermanyssus gallinae is an economically important pest in poultry farms worldwide, but an effective treatment option is lacking. The current study determined the effectiveness of six Chinese herbal medicines [Syzygium aromaticum (clove), Hibiscus syriacus (Hibiscus), Illicium verum (star anise), Leonurus artemisia (motherwort), Cinnamomum cassia (cinnamon), and Taraxacum sp. (dandelion)] against D. gallinae. Alcohol extracts were prepared via the solvent extraction method and the phenol, flavonoid, and tannin contents were determined. These active components were highest in S. aromaticum and lowest in H. syriacus, I. verum. No tannin content was detected in L. artemisia. All extracts showed contact toxicity against D. gallinae at a test concentration of 1 g/mL, with S. aromaticum and L. artemisia resulting in 100% mortality. S. aromaticum, L. artemisia, and I. verum showed the best efficacy (LC50 0.159, 0.200, and 0.292 g/mL, respectively). Different combinations of extracts showed an additive effect of I. verum LC90 + L. artemisia LC90. The acaricidal efficacy of this combination was tested against different developmental stages of D. gallinae, being most efficacious against nymphal and larval D. gallinae, with a corrected mortality rate of 100%. However, inhibition of egg hatching was only 53.69%. Taken together, these results highlight I. verum LC90 + L. artemisia LC90 as a promising compound with severe contact toxicity against D. gallinae. Given the wide cultivation of these species and their extensive use in foodstuffs and cosmetics as flavors and fragrances, they could be a cheap, readily available ecofriendly alternative to pesticides currently used in poultry farms.

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

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

Molecular detection and phylogeny of Anaplasma spp. closely related to Anaplasma phagocytophilum in small ruminants from China.

The genus Anaplasma comprises eight bacterial species that are obligate intracellular pathogens that affect human and animal health. The zoonotic species A. phagocytophilum is the causative agent of tick-borne fever in ruminants, and of granulocytic anaplasmosis in horses, dogs, and humans. Recently, novel strains related to A. phagocytophilum (A. phagocytophilum-like 1/Japanese variant and A. phagocytophilum-like 2/Chinese variant) have been identified. The aim of this study was to reveal the prevalence and phylogeny of A. phagocytophilum and related stains in small ruminants and ticks in China based on sequences of the 16S rRNA combined restriction fragment length polymorphism (RFLP) and groEL genes. PCR-RFLP and phylogenetic analyses based on the 16S rRNA gene showed the presence of A. phagocytophilum-like 1 and 2 variants in sampled animals from China, with prevalence rates of 22.6% (303/1338) and 0.7% (10/1338), respectively. Only A. phagocytophilum-like 1 DNA was found in Haemaphysalis longicornis. The phylogeny based on the groEL gene showed inclusion of A. phagocytophilum-like 1 and some A. phagocytophilum-like 2 strains in two unique clades distinct from, but related to, Japanese and Chinese strains of related A. phagocytophilum, respectively. One noteworthy result was that the SSAP2f/SSAP2r primers detected Ehrlichia spp. strains. Moreover, the A. phagocytophilum-like 1 and 2 strains should be considered in the differential diagnosis of caprine and ovine anaplasmosis. Further investigations should be conducted to provide additional epidemiological information about A. phagocytophilum and A. phagocytophilum-like variants in animals and ticks.

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.

Cryptosporidium parvum downregulates miR-181d in HCT-8 cells via the p50-dependent TLRs/NF-κB pathway.

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

Prevalence of Blastocystis infection in free-range Tibetan sheep and Tibetan goats in the Qinghai-Tibetan Plateau in China.

Blastocystis is one of the most common enteric parasites in humans and domestic animals. For Tibetan sheep and Tibetan goats, the traditional grazing methods still occupy a dominant position, and the close contact between humans and domestic animals increases the risk of infection by Blastocystis between herdsmen and livestock. However, less pertinent information is available for Tibetan sheep or Tibetan goats. In this study, 880 fecal specimens from Tibetan sheep and Tibetan goats were collected from 6 sampling sites in Tibet to test for Blastocystis using the polymerase chain reaction and sequencing analysis of the partial SSU rRNA gene. The infection rate of Blastocystis was 8.55% for Tibetan sheep (53/620) and 8.46% for Tibetan goats (22/260). The genetic analysis of 53 positive samples from Tibetan sheep identified 4 known subtypes (ST4, ST5, ST10, and ST14). Four known subtypes (ST1, ST5, ST6, and ST10) were identified in Tibetan goats. ST10 was the dominant subtype in Tibetan sheep and Tibetan goats, accounting for 65.33% (49/75) of total subtypes. ST1, ST4, ST5, and ST6 were recognized as belonging to zoonotic subtypes. This report provides a detailed data on the prevalence and subtype distribution of Blastocystis in Tibetan sheep and Tibetan goats in Tibet, which enriches the epidemiological data of Blastocystis infection in Tibetan sheep and Tibetan goats in China. Our results indicated that Tibetan sheep and Tibetan goats can be infected with multiple Blastocystis subtypes, including zoonotic subtypes. More research is needed among humans, livestock and wild animals in Tibet to better understand their role in the spread of Blastocystis. And, One Health measures need to be taken to control and prevent its zoonotic transmission.

Coccidia-Microbiota Interactions and Their Effects on the Host.

As a common parasitic disease in animals, coccidiosis substantially affects the health of the host, even in the absence of clinical symptoms and intestinal tract colonization. Gut microbiota is an important part of organisms and is closely related to the parasite and host. Parasitic infections often have adverse effects on the host, and their pathogenic effects are related to the parasite species, parasitic site and host-parasite interactions. Coccidia-microbiota-host interactions represent a complex network in which changes in one link may affect the other two factors. Furthermore, coccidia-microbiota interactions are not well understood and require further research. Here, we discuss the mechanisms by which coccidia interact directly or indirectly with the gut microbiota and the effects on the host. Understanding the mechanisms underlying coccidia-microbiota-host interactions is important to identify new probiotic strategies for the prevention and control of coccidiosis.

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 detection and phylogenetic analyses of Anaplasma spp. in Haemaphysalis longicornis from goats in four provinces of China.

Anaplasma species, which are distributed worldwide, are gram-negative obligate intracellular tick-borne bacteria that pose a threat to human and animal health. Haemaphysalis longicornis ticks play a vital role as vectors in the transmission of Anaplasma pathogens. However, the Anaplasma species carried by H. longicornis in China are yet to be characterized. In this study, 1074 H. longicornis specimens were collected from goats in four provinces of China from 2018 to 2019 and divided into 371 sample pools. All tick sample pools were examined for the presence of Anaplasma species via nested PCR amplification of 16S ribosomal RNA, major surface protein 4 (msp4), or citric acid synthase (gltA) genes, which were sequenced to determine the molecular and phylogenetic characteristics of the isolates. The overall Anaplasma spp-positive rate of H. longicornis was determined to be 26.68% (99/371). The percentage prevalence of A. phagocytophilum-like1, A. bovis, A. ovis, A. marginale, and A. capra were 1.08% (4/371), 13.21% (49/371), 13.21% (49/371), 1.35% (5/371), and 10.24% (38/371), respectively, and the co-infection rate of two or more types of Anaplasma was 6.47% (24/371). Phylogenetic analyses led to the classification of A. phagocytophilum into an A. phagocytophilum-like1 (Anaplasma sp. Japan) group. Anaplasma bovis sequences obtained in this study were 99.8-100% identical to those of an earlier strain isolated from a Chinese tick (GenBank accession no. KP314251). Anaplasma ovis sequences showed 99.3-99.6% identity to an A. ovis human strain identified from a Cypriot patient (GenBank accession no. FJ460443). Only one msp4 sequence of A. marginale was detected and was grouped with those of other A. marginale isolates, and these A. capra isolates obtained in this present study may be zoonotic. The detection and characterization of four Anaplasma species in H. longicornis in this study have added to the current knowledge of the parasite and provided data on multiple Anaplasma species with veterinary and medical significance from four provinces of China.

The Novel Zoonotic Pathogen, Anaplasma capra, Infects Human Erythrocytes, HL-60, and TF-1 Cells In Vitro.

Anaplasma capra, a species of the family Anaplasmataceae, is zoonotic tick-borne obligate intracellular bacteria. There have been no reports of human infection with this pathogen since 2015. Therefore, the zoonotic characteristics of A. capra need to be further studied. To verify the ability of A. capra to infect human cells, A. capra were inoculated in human erythrocytes, HL-60, and TF-1 cell lines in vitro. Cell smears were taken after inoculation, using Giemsa staining, transmission electron microscope (TEM), chromogenic in situ hybridization and immunocytochemistry for detection. In the Giemsa staining, many dark colored corpuscles or purple granules were seen in the inoculated erythrocytes, HL-60, and TF-1 cells. The results of chromogenic in situ hybridization show that there were brown precipitates on the surface of most erythrocytes. Immunocytochemistry results show many dark brown vacuolar structures or corpuscles in the cytoplasm of erythrocytes, HL-60, and TF-1 cell lines. The A. capra morulae were seen in the cytoplasm of both HL-60 and TF-1 in TEM, and their diameter was about 295-518 nm. Both dense-cored (DC) and reticulate cell (RC) form morulae could be seen. This study confirmed the ability of A. capra to infect human erythrocytes, HL-60, and TF-1. This study is of profound significance in further verifying the zoonotic characteristics of the pathogen and for establishing an in vitro cultivation model.

Identification of Anaplasma spp. in Tian Shan wapiti deer (Cervus elaphus songaricus) in Xinjiang, China.

Anaplasma spp. are important zoonotic tick-borne pathogens that impact on human health. There are few reports on the prevalence and molecular genetic characteristics of Cervidae species in China. The purpose of this study, therefore, was to investigate the presence of Anaplasma spp. in blood samples of Tian Shan wapiti (Cervus elaphus songaricus) in the Xinjiang Uygur Autonomous Region of China, and conduct phylogenetic analyses. A total of 50 blood samples (wild deer n = 26, and captive deer n = 24) were collected from the deer. PCR was used to detect Anaplasma spp. in the blood samples. Forty percent (20) of the samples were found to contain Anaplasma spp. Three Anaplasma species DNA were detected in deer blood samples: A. bovis (n = 13), A. ovis (n = 18), and A. phagocytophilum (n = 11). Among the 20 Anaplasma spp. positive samples, 14 were mixed infection of two or three pathogens. The prevalence of Anaplasma species in wild deer was significantly higher than that of captive deer, 73.1% (19) vs 4.2% (1) respectively, (p < 0.01). Two A. ovis sequence types (AB1, and AB2), three A. ovis sequence types (AO1-AO3), and one A. phagocytophilum sequence type (AP1) were obtained in this study. The sequences of AO1 shared 100% identity with a human isolate from Cyprus. Our results suggest that wild deer are more likely to become infected with Anaplasma spp. than captive individuals, and thus, could potentially transmit pathogens to humans.

Seasonal dynamics of Anaplasma spp. in goats in warm-temperate zone of China.

Anaplasma are tick-borne obligate intracellular bacteria that can endanger human and animal health, and until now, there have been few reports on the seasonal dynamics of Anaplasma species in China. In this study, a total of 491 goat blood samples were collected in spring (n = 124), summer (n = 135), autumn (n = 110), and winter (n = 122) from Shaanxi provinces. Single and mixed infections of Anaplasma spp. from warm-temperate regions of China were analyzed according to seasons using a nested PCR method. Positive samples were sequenced to observe the molecular and phylogenetic characteristics of the Anaplasma species, and we determined the co-infection rates of Anaplasma spp. for each season. A molecular survey of Anaplasma phagocytophilum, A. bovis, A. ovis, and A. capra in goats showed average prevalences of 71.6 % (maximum 86.7 % in summer and minimum 48.4 % in winter), 62.2 % (minimum 38.7 % in spring and maximum 94.1 % in summer), 25.5 % (minimum 0% in summer and maximum 51.6 % in spring), and 26.6 % (minimum 8.2 % in winter and maximum 55.6 % in summer), respectively. In the phylogenetic analysis, A. phagocytophilum and A. capra occupied two separate groups, Chinese A. bovis and foreign isolates appeared to be geographically isolated, and all A. ovis isolates were in the same branch as the previously described sequences. The survey indicated that goats in warm-temperate regions of China are frequently exposed to Anaplasma spp. all year round, and thus prevention and treatment efforts for anaplasmosis in the region should be strengthened.

The first detection of Anaplasma capra, an emerging zoonotic Anaplasma sp., in erythrocytes.

ABSTRACT An emerging infectious disease caused by "Anaplasma capra" was reported in a 2015 survey of 477 hospital patients with a tick-bite history in China. However, the morphological characteristics and parasitic location of this pathogen are still unclear, and the pathogen has not been officially classified as a member of the genus Anaplasma. Anaplasma capra-positive blood samples were collected, blood cells separated, and DNA of whole blood cells, erythrocytes, and leukocytes extracted. Multiplex PCR detection assay was used to detect whole blood cell, erythrocytes and leukocytes, DNA samples, and PCR identification, nucleic acid sequencing, and phylogenetic analyses based on A. capra groEL, 16S rRNA, gltA, and msp4 genes. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), Wright-Giemsa staining, chromogenic in situ hybridization (CISH), immunocytochemistry, and indirect immunofluorescence assay (IFA) were used to identify the location and morphological characteristics of A. capra. Multiple gene loci results demonstrated that erythrocyte DNA samples were A. capra-positive, while leukocyte DNA samples were A. capra-negative. Phylogenetic analysis showed that A. capra is in the same clade with the A. capra sequence reported previously. SEM and TEM showed one or more pathogens internally or on the outer surface of erythrocytes. Giemsa staining, CISH, immunocytochemistry, and IFA indicated that erythrocytes were A. capra-positive. This study is the first to identify the novel zoonotic tick-borne Anaplasma sp., "Anaplasma capra," in host erythrocytes. Based on our results, we suggest revision of Genus Anaplasma and formally name "A. capra" as Anaplasma capra sp. nov.

Novel Anaplasma Variants in Small Ruminants From Central China.

Anaplasma capra is an emerging zoonotic pathogen that pose a risk to the health of human and veterinary animal. Numerous variants in a variety of domestic and wild animals had been reported since its discovery and confirmation in humans in 2015 and its first detection from goat blood during 2012-2013. In order to find out more A. capra variants data of A. capra in central China, 16S rRNA, gltA, groEL, and msp4 genes of this pathogen were amplified from sheep and goat samples collected during 2011-2015 and phylogenetic analysis of these sequences were conducted. The results of 16S rRNA and gltA manifested that partial sequences obtained in this study were 100% identical with A. capra isolates, while phylogenetic analysis results of groEL and msp4 showed that the obtained sequences were independent with all other Anaplasmas, formed separate branches on the evolutionary trees. What needed to be emphasized was that the 16S rRNA and gltA gene sequences of X51 (KX505302 and KX450269), a sample from Shandong in 2011, were found to be 100% identical with A. capra. Therefore, we could speculate that the occurrence of A. capra may be earlier than its first discovery and report. And the A. capra isolates in central China were novel variants which were different from known genotypes.

Cryptosporidium parvum upregulates miR-942-5p expression in HCT-8 cells via TLR2/TLR4-NF-κB signaling.

BACKGROUND: Micro (mi)RNAs are small noncoding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression. This study investigated host miRNA activity in the innate immune response to Cryptosporidium parvum infection. METHODS: In vitro infection model adopts HCT-8 human ileocecal adenocarcinoma cells infected with C. parvum. The expression of miR-942-5p was estimated using quantitative real-time polymerase chain reaction (qPCR). The TLRs-NF-κB signaling was confirmed by qPCR, western blotting, TLR4- and TLR2-specific short-interfering (si)RNA, and NF-κB inhibition. RESULTS: HCT-8 cells express all known toll-like receptors (TLRs). Cryptosporidium parvum infection of cultured HCT-8 cells upregulated TLR2 and TLR4, and downstream TLR effectors, including NF-κB and suppressed IκBα (nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha). The expression of miR-942-5p was significantly upregulated at 4, 8, 12 and 24 h post-infection, and especially at 8 hpi. The results of TLR4- and TLR2-specific siRNA and NF-κB inhibition showed that upregulation of miR-942-5p was promoted by p65 subunit-dependent TLR2/TLR4-NF-κB pathway signaling. CONCLUSIONS: miR-942-5p of HCT-8 cells was significantly upregulated after C. parvum infection, especially at 8 hpi, in response to a p65-dependent TLR2/TLR4-NF-κB signaling. TLR4 appeared to play a dominant role.

Molecular detection of Anaplasma spp. in dairy cattle in southern Xinjiang, China.

Bovine anaplasmosis is caused by a group of obligate intracellular bacteria belonging to the genus Anaplasma, which are transmitted by ticks. This study was conducted to determine the prevalences and molecular characterization of Anaplasma spp. in dairy cattle in the upper reaches of the Tarim River in Xinjiang, China. Using polymerase chain reaction (PCR) and sequencing approaches, DNA of Anaplasma spp. was detected in 16 of 493 (3.2%) blood samples from dairy cattle. Positive rates were 0.2% (1/493), 0.4% (2/493), 0.2% (1/493), 2.4% (12/493) and 2.4% (12/493) for A. bovis, A. ovis, A. phagocytophilum like strain, A. phagocytophilum and A. platys like strain, respectively. Anaplasma phagocytophilum and A. platys like strain co-infection was detected in 12 samples. To our knowledge, this is the first report of A. ovis infection in dairy cattle in Xinjiang. This study provides new data on the prevalences of Anaplasma spp. in cattle in Xinjiang, which will help to formulate appropriate control strategies for these pathogens in this area.