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Abstract Background The parasitic protozoan Giardia duodenalis is an important cause of diarrheal disease in humans and animals that can be spread by fecal–oral transmission through water and the environment, posing a challenge to public health and animal husbandry. Little is known about its impact on large-scale sheep farms in China. In this study we investigated G. duodenalis infection of sheep and contamination of the environment in large-scale sheep farms in two regions of China, Henan and Ningxia. Methods A total of 528 fecal samples, 402 environmental samples and 30 water samples were collected from seven large-scale sheep farms, and 88 fecal samples and 13 environmental samples were collected from 12 backyard farms. The presence of G. duodenalis was detected by targeting the β-giardin (bg) gene, and the assemblage and multilocus genotype of G. duodenalis were investigated by analyzing three genes: bg, glutamate dehydrogenase (gdh) and triphosphate isomerase (tpi). Results The overall G. duodenalis detection rate was 7.8%, 1.4% and 23.3% in fecal, environmental and water samples, respectively. On the large-scale sheep farms tested, the infection rate of sheep in Henan (13.8%) was found to be significantly higher than that of sheep in Ningxia (4.2%) (P 0.05). Investigations of sheep at different physiological stages revealed that late pregnancy ewes showed the lowest infection rate (1.7%) and that young lambs exhibited the highest (18.8%). Genetic analysis identified G. duodenalis belonging to two assemblages, A and E, with assemblage E being dominant. A total of 27 multilocus genotypes were identified for members of assemblage E. Conclusions The results suggest that G. duodenalis is prevalent on large-scale sheep farms in Henan and Ningxia, China, and that there is a risk of environmental contamination. This study is the first comprehensive examination of the presence of G. duodenalis on large-scale sheep farms in China. Challenges posed by G. duodenalis to sheep farms need to be addressed proactively to ensure public health safety. Graphical Abstract

Abstract 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.

Abstract Coccidia of the genus Eimeria are important pathogens that cause coccidiosis in livestock and poultry. Due to the expansion of intensive farming, coccidiosis has become more difficult to control. In addition, the continued use of anti-coccidiosis drugs has led to drug resistance and residue. Some herbs used in traditional Chinese medicine (TCM) have been shown to alleviate the clinical symptoms of coccidiosis, while enhancing immunity and growth performance (GP) of livestock and poultry. Previous in vitro and in vivo studies have reported that the TCM herb Portulaca oleracea exhibited anti-parasitic activities. In total, 36 female Hu lambs were equally divided into six treatment groups: PL (low-dose P. oleracea), PH (high-dose P. oleracea), PW (P. oleracea water extract), PE (P. oleracea ethanol extract), DIC (diclazuril), and CON (control). The treatment period was 14 days. The McMaster counting method was used to evaluate the anti-coccidiosis effects of the different treatments. Untargeted metabolomics and 16S rRNA gene sequencing were used to investigate the effects of treatment on the gut microbiota (GM) and GP. The results showed that P. oleracea ameliorated coccidiosis, improved GP, increased the abundances of beneficial bacteria, and maintained the composition of the GM, but failed to completely clear coccidian oocysts. The Firmicutes to Bacteroides ratio was significantly increased in the PH group. P. oleracea increased metabolism of tryptophan as well as some vitamins and cofactors in the GM and decreased the relative content of arginine, tryptophan, niacin, and other nutrients, thereby promoting intestinal health and enhancing GP. As an alternative to the anti-coccidiosis drug DIC, P. oleracea effectively inhibited growth of coccidia, maintained the composition of the GM, promoted intestinal health, and increased nutrient digestibility.

Meat from sheep offers an abundance of essential amino acids and trace elements essential for optimal human health and a delectable culinary delight. Because it has fewer calories and a lower cholesterol content than other meats, this succulent meat is not only delicious but also a nutritious choice. Globally, discriminating consumers have expressed profound appreciation for its irresistible flavor and nutritious composition. High-quality sheep breeds and lamb quality are in the spotlight as the market for sheep meat grows. Nevertheless, the demand for rapid growth and the use of antibiotics and other drugs have led to a shortage of high-quality mutton on the market. In the face of this emergency phenomenon, people add organic matter to the growth of mutton to improve the quality of mutton. This paper discusses the comprehensive evaluation methods of meat quality; summarizes the relationship between the nutritional components of meat and diet; discusses the genetic factors affecting meat quality attributes; feed nutrition, feeding methods, mutton storage methods, and related measures to improve the quality of mutton; and provides information on the current status of mutton and the challenges of ensuring high-quality meat supply in the future.

Abstract 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. Graphical Abstract

Abstract 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.

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

Clostridium butyricum has been widely considered an antibiotic substitute in recent years. It can promote growth performance, improve the immune response and enhance the intestinal barrier function of the host. In the present study, 1-d-old Arbor Acres (AA) broilers were fed C. butyricum (1 × 109 cfu/kg) for 28 d. The transcriptomic characteristics of epithelial cells of the cecal mucosa were determined by RNA-sequence, and the cecal microbiota composition was explored by 16S ribosomal RNA gene sequencing. The changes in the intestinal mucosa of broilers were then analyzed by tissue staining. Gene Ontology (GO) annotations identified substance transport and processes and pathways that might participate in intestinal development and cell viability. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the differentially expressed genes are involved in numerous pathways related to amino acid and vitamin metabolism and antioxidant and defensive functions, among others. The relative expression of some genes associated with intestinal barrier function (claudins 2, 15, 19, and 23, tight junction proteins 1, 2, and 3 and mucin 1) was significantly increased in the treatment group (P

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.

Abstract 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.

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.

Abstract 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.

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.

Abstract 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.

Abstract 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.

The genus Anaplasma (Rickettsiales: Anaplasmataceae), which includes the species Anaplasma capra, Anaplasma bovis, Anaplasma ovis, and Anaplasma phagocytophilum, is responsible for a wide variety of infections in both human and veterinary health worldwide. Multiple infections with these four Anaplasma pathogens have been reported in many cases. We introduce a novel multiplex PCR for the simultaneous detection of A. capra, A. bovis, A. ovis, and A. phagocytophilum, based on species-specific primers against the groEL (A. capra and A. bovis), msp4 (A. ovis), and 16S rRNA (A. phagocytophilum) genes. To verify the specificity of the PCR reactions, we evaluated four sets of primers to analyze samples containing different blood pathogens. The sensitivity of the multiplex PCR was evaluated by amplifying 10-fold dilutions of total genomic DNA extracted from sheep blood infected with A. capra, A. bovis, A. ovis, or A. phagocytophilum. The reproducibility of the assay was evaluated by testing 10-fold dilutions of total genomic DNA extracted from sheep blood infected with these pathogens from 100 to 10–3 ng/μL per reaction in triplicate on three different days. A total of 175 field blood DNA samples were used to evaluate the reproducibility of multiplex PCR compared with the simplex PCRs. PCR primers used in this study were confirmed to be 100% species-specific using blood pathogens previously identified by other methods. The lower limit of detection of the multiplex PCR with good repeatability enabled the detection of A. capra, A. bovis, A. ovis and A. phagocytophilum at concentrations of 3 × 10–5, 5 × 10–7, 2 × 10–5, and 7 × 10–7 ng/μL, respectively. There was no significant difference between conventional and multiplex PCR protocols used to detect the four Anaplasma species (P > 0.05). The results of the multiplex PCR revealed that the A. capra groEL gene, the A. bovis groEL gene, the A. ovis msp4 gene, and the A. phagocytophilum 16S rRNA gene were reliable target genes for species identification in clinical isolates, being specific for each of the four target Anaplasma species. Our study provides an effective, sensitive, specific, and accurate tool for the rapid differential clinical diagnosis and epidemiological surveillance of Anaplasma pathogens in sheep and goats.

Abstract Background Cryptosporidium spp. are protozoans that cause diarrheal illness in humans and animals, including birds, worldwide. The present study was aimed to revisit the infectivity and pathogenicity of C. avium, recently considered to be a valid avian-infecting species of Cryptosporidium, and foster further understanding of its biological characteristics. Results Results showed that no Cryptosporidium oocysts were detected in the feces of experimentally inoculated BALB/c mice, Mongolian gerbils, quail or budgerigars within 30 days post-infection (dpi). Oocysts were first detected in feces of 3-day-old and 40-day-old hens at 8 and 9 dpi, respectively. In ducks infected with C. avium, oocysts were first detected at 9 dpi. Oocysts of infected animals were studied using a nested-polymerase chain reaction (PCR) technique for the SSU rRNA gene, actin gene, HSP70 gene and Cryptosporidium oocyst wall protein gene (COWP) detection. Restriction fragment length polymorphism (RFLP), using SspI and VspI restriction enzymes, was carried out to genotype the species and obtained amplification products were sequenced. Cryptosporidium developmental stages were found in the longitudinal plica of the bursa fabricii (BF) of hens, with high levels observed in histological sections and scanning electron microscopy. No pathological changes were observed. Conclusions These findings indicate that the bursa fabricii may be the primary site of C. avium infection. More biological data are needed to support the establishment of new species and contribute to the taxonomy of Cryptosporidium.

Abstract Background Little is known about the prevalence and zoonotic potential of Cryptosporidium spp. and Giardia duodenalis in deer in China. In this study, 662 fecal samples were collected from 11 farms in Henan and Jilin Provinces between July 2013 and August 2014, and were screened for the presence of Cryptosporidium and G. duodenalis with genotyping and subtyping methods. Results Cryptosporidium spp. and G. duodenalis were detected in 6.80% (45/662) and 1.21% (5/662) of samples, respectively. Six Cryptosporidium species/genotypes were identified based on the small subunit ribosomal ribonucleic acid (SSU rRNA) gene: C. parvum (n = 11); C. andersoni (n = 5); C. ubiquitum (n = 3); C. muris (n = 1); C. suis-like (n = 1); and Cryptosporidium deer genotype (n = 24). When five of the 11 C. parvum isolates were subtyped by sequencing the 60 kDa glycoprotein (gp60) gene, zoonotic subtypes IIaA15G2R2 (n = 4) and IIdA19G1 (n = 1) were found. According to a subtype analysis, three C. ubiquitum isolates belonged to XIIa subtype 2. In contrast, only assemblage E was detected in the five Giardia-positive samples with small subunit ribosomal ribonucleic acid (SSU rRNA) gene sequencing. Conclusions To our knowledge, this is the first study to report C. andersoni, as well as C. parvum zoonotic subtypes IIaA15G2R2 and IIdA19G1 in cervids. These data, though limited, suggest that cervids may be a source of zoonotic Cryptosporidium and Giardia. Cervids in the present study are likely to be of low zoonotic potential to humans, and more molecular epidemiological studies are required to clarify the prevalence and public health significance of Cryptosporidium and G. duodenalis in cervids throughout China.

Abstract 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.

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.

Cryptosporidium is an enteric parasite that is transmitted via the faecal–oral route, water and food. Humans, wildlife and domestic livestock all potentially contribute Cryptosporidium to surface waters. Human encroachment into natural ecosystems has led to an increase in interactions between humans, domestic animals and wildlife populations. Increasing numbers of zoonotic diseases and spill over/back of zoonotic pathogens is a consequence of this anthropogenic disturbance. Drinking water catchments and water reservoir areas have been at the front line of this conflict as they can be easily contaminated by zoonotic waterborne pathogens. Therefore, the epidemiology of zoonotic species of Cryptosporidium in free-ranging and captive wildlife is of increasing importance. This review focuses on zoonotic Cryptosporidium species reported in global wildlife populations to date, and highlights their significance for public health and the water industry.

Toxoplasma gondii as a food-borne pathogen, the infection of it in food animals has relation with human toxoplasmosis, but the trends and epidemiological features of T. gondii infections in food animals are rarely studied in China. The aimed of this study was to assess the epidemiology and risks of T. gondii in sheep, goats, swines, chickens, yaks, cattle and humans from 2000 to 2017 and to explore prevention and control strategies. The overall seroprevalence of T. gondii infections in food animals is 23.7% (39,194/165,417, 95%CI, 23.49–23.90%), which is significantly higher than that in humans (8.2%, 95%CI, 8.06–8.39%, 8,502/103,383) (P < 0.0001). Compared the prevalence of T. gondii infections in animals and humans sampled from 2000 to 2010, it was significantly increased in the period 2011 to 2017 (P < 0.0001). Compared the food animals from non-Yangtze River, animals from regions of the Yangtze River have high seroprevalence rates for T. gondii (P < 0.0001). Furthermore, samples from the western to eastern regions of the Yellow River showed an increase in seroprevalence for T. gondii (P < 0.0001). It was speculated that T. gondii oocysts may be transmitted by water and annual precipitation possible help the oocyst spread and retain accessible for potential hosts. Effective prevention and control strategies are including water filtration or water boiling, inactivating oocysts from feline’s feces, monitoring birds and rodents. Chinese 1 (ToxoDB#9) is the predominant genotype in food animals from China.

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

To examine the occurrence and genotype distribution of Enterocytozoon bieneusi in cervids, 615 fecal samples were collected from red deer (Cervus elaphus) and sika deer (Cervus nippon) on 10 different farms in Henan and Jilin Province. Enterocytozoon bieneusi was identified and genotyped with a nested PCR analysis of the internal transcribed spacer (ITS) region of the rRNA genes, showing an average infection rate of 35.9% (221/615). In this study, 25 ITS genotypes were identified including seven known genotypes (BEB6, EbpC, EbpA, D, HLJDI, HLJD-IV, and COS-I) and 18 novel genotypes (designated JLD-I to JLD-XIV, HND-I to HND-IV). Among these, BEB6 (131/221, 59.3%) was the predominant genotype (P < 0.01), followed by HLJDI (18/221, 8.1%) and JLD-VIII (16/221, 7.2%). BEB6 has recently been detected in humans and nonhuman primates in China. The phylogenetic analysis showed that BEB6, HLJDI, HLJD-IV, COS-I, and 10 novel genotypes (JLD-VII to JLD-XIV, HND-III to HND-IV) clustered in group 2. Genotype D, EbpC, and EbpA, known to cause human microsporidiosis worldwide, clustered in group 1, the members of which have zoonotic potential, together with eight novel genotypes (JLD-I to JLD-VI, HND-I to HND-II). Therefore, deer may play a role in the transmission of E. bieneusi to humans.

Abstract Background Cryptosporidium and Giardia are important causes of diarrhea diseases in humans and animals worldwide, and both of them are transmitted by the fecal–oral route, either by direct contact or by the ingestion of contaminated food or water. The role of flies in the mechanical transmission of Cryptosporidium and Giardia has been receiving increasing attention. To date, no information is available in China about the occurrence of Cryptosporidium and Giardia in flies. We here investigated Cryptosporidium and Giardia in flies on dairy farms in Henan Province, China, at the genotype and subtype levels. Methods Eight hundred flies were randomly collected from two dairy farms from July 2010 to September 2010 and were divided evenly into 40 batches. The fly samples were screened for the presence of Cryptosporidium and Giardia with nested PCR. Cryptosporidium was genotyped and subtyped by analyzing the DNA sequences of small subunit rRNA (SSU rRNA) and 60-kDa glycoprotein (gp60) genes, respectively. The identity of Giardia was determined by sequence analyzing of the triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh), and β-giardin (bg) genes. Results Forty batches of flies had 10% of contamination with Cryptosporidium or Giardia, with a mixed infection of the two parasites in one batch of flies. The Cryptosporidium isolates were identified as C. parvum at the SSU rRNA locus, and all belonged to subtype IIdA19G1 at the gp60 locus. The Giardia isolates were all identified as assemblage E of G. duodenalis at the tpi, gdh, and bg loci. One novel subtype of assemblage E was identified based on the gdh and bg loci. Conclusions This is the first molecular study of Cryptosporidium and Giardia in flies identified at both genotype and subtype levels. SSU rRNA and gp60 sequences of C. parvum in flies was 100% homologous with those derived from humans, suggesting flies act as an epidemiological vector of zoonotic cryptosporidiosis. The variable PCR efficiencies observed in the analysis of Giardia at different loci suggest that we should use the multilocus genotyping tool in future studies to increase the detection rate, and importantly, to obtain more complete genetic information on Giardia isolates.

Cryptosporidium is one of the most common zoonotic waterborne parasitic diseases worldwide and represents a major public health concern of water utilities in developed nations. As animals in catchments can shed human-infectious Cryptosporidium oocysts, determining the potential role of animals in dissemination of zoonotic Cryptosporidium to drinking water sources is crucial. In the present study, a total of 952 animal faecal samples from four dominant species (kangaroos, rabbits, cattle and sheep) inhabiting Sydney's drinking water catchments were screened for the presence of Cryptosporidium using a quantitative PCR (qPCR) and positives sequenced at multiple loci. Cryptosporidium species were detected in 3.6% (21/576) of kangaroos, 7.0% (10/142) of cattle, 2.3% (3/128) of sheep and 13.2% (14/106) of rabbit samples screened. Sequence analysis of a region of the 18S rRNA locus identified C. macropodum and C. hominis in 4 and 17 isolates from kangaroos respectively, C. hominis and C. parvum in 6 and 4 isolates respectively each from cattle, C. ubiquitum in 3 isolates from sheep and C. cuniculus in 14 isolates from rabbits. All the Cryptosporidium species identified were zoonotic species with the exception of C. macropodum. Subtyping using the 5' half of gp60 identified C. hominis IbA10G2 (n = 12) and IdA15G1 (n = 2) in kangaroo faecal samples; C. hominis IbA10G2 (n = 4) and C. parvum IIaA18G3R1 (n = 4) in cattle faecal samples, C. ubiquitum subtype XIIa (n = 1) in sheep and C. cuniculus VbA23 (n = 9) in rabbits. Additional analysis of a subset of samples using primers targeting conserved regions of the MIC1 gene and the 3' end of gp60 suggests that the C. hominis detected in these animals represent substantial variants that failed to amplify as expected. The significance of this finding requires further investigation but might be reflective of the ability of this C. hominis variant to infect animals. The finding of zoonotic Cryptosporidium species in these animals may have important implications for the management of drinking water catchments to minimize risk to public health.

To determine prevalence of Cyclospora cayetanensis infection in Henan, China, we conducted a study of 11,554 hospital patients. Prevalence was 0.70% (95% confidence interval 0.70% ± 0.15%), with all age groups infected. Most cases were found in the summer. Minor sequence polymorphisms were observed in the 18S rRNA gene of 35 isolates characterized.

Giardia duodenalis is a common and widespread intestinal protozoan parasite of both humans and animals. Previous epidemiological and molecular studies have identified Giardia infections in different animals and humans, but only limited information is available about the occurrence and genotypes of Giardia in cattle in China. In this study, we determined the occurrence of giardiasis and genetically characterized G. duodenalis in dairy cattle in Henan Province, central China. The overall prevalence of G. duodenalis was 7.2% (128/1777) on microscopic analysis, with the highest infection rate (22.7%) in calves aged less than 1 month. G. duodenalis assemblages and subtypes were identified with multilocus genotyping based on the SSU rRNA, β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes. Two assemblages were detected in the successfully sequenced samples: assemblage A (n = 58), assemblage E (n = 21), with a mixed E and A assemblage (n = 2). Four novel subtypes of the gdh gene and seven of the bg gene were found among the G. duodenalis assemblage E isolates. Using the nomenclature for the multilocus genotype (MLG) model, nine novel multilocus genotypes E (MLGs E1-E9) and three MLGs A (a novel subtype AI, previously detected subtype AII-1, and a combination of both) were identified. MLG AII-1 identified in this study may be an important zoonotic subtype. The dairy cattle in Henan are a potential public health concern.

In this study, nine C. muris and 43 C. andersoni isolates from various animals in China were subtyped by a multilocus sequence typing (MLST) tool. DNA sequence analyses showed the presence of 1-2 subtypes of C. muris and 2-6 subtypes of C. andersoni at each of the four loci (MS1, MS2, MS3, and MS16), nine of which represented new subtypes. Altogether, two C. muris and 10 C. andersoni MLST subtypes were detected. Linkage disequilibrium analysis indicated although the overall population structure of the two parasites was clonal, the Chinese C. andersoni in cattle has an epidemic structure. Three and two clusters were produced in the C. muris and C. andersoni populations by Structure 2.3.3 analysis, with Chinese C. muris and C. andersoni substructures differing from other countries. Thus, this study suggested the prevalence of C. andersoni in China is not attributed to the introduction of dairy cattle. More studies involving more genetic loci and systematic sampling are needed to better elucidate the population genetic structure of C. muris and C. andersoni in the world and the genetic basis for the difference in host specificity among the two most common gastric parasites.