Impacts of a highly pathogenic ovine Eimeria ovinoidalis on the growth of Hu lambs.

The apicomplexan Eimeria ovinoidalis is distributed worldwide. It can cause clinical coccidiosis, which is one of the most pathogenic species in sheep, reducing growth rates and resulting in significant economic losses in the industry. Its principal clinical sign is profuse diarrhoea in young animals. In this study, we established a model of E. ovinoidalis infection in lambs to understand its pathogenicity and evaluate the gut microbiota and fecal metabolite profiles. Specifically, we observed a significant shift in the abundance of bacteria and disrupted metabolism in lambs. Especially during the peak period of excrete oocysts, it promoted the reproduction of some harmful bacteria in Proteobacteria and Actinobacteriota, and reduced the abundance of beneficial bacteria such as Lachnospiraceae and Rikenellaceae. In the later stage of the patent period, the abundance of harmful bacteria in the intestine decreased, the abundance of beneficial bacteria which could produce anti-inflammatory substances began to increase, and the abundance and diversity of intestinal flora also tended to parallel with the control group. Coccidia infection could lead to the increase of differential metabolites and metabolic pathways between infected and control group, but the difference decreased with time. During the peak period of excrete oocysts, although the antimicrobial metabolites such as Lividamine were up-regulated, the excess of these metabolites could still induce the production of endotoxin, while Butanoic acid and other anti-inflammatory metabolites decreased significantly. A metabolomics analysis showed that E. ovinoidalis infection altered metabolites and metabolic pathways, with biosynthesis of unsaturated fatty acids, Teichoic acid biosynthesis and Butanoate metabolism as the major disrupted metabolic pathways. Details of the gut microbiota and the metabolome after infection with E. ovinoidalis may aid in the discovery of specific diagnostic markers and help us understand the changes in parasite metabolic pathways.

Unveiling the presence and genotypic diversity of Giardia duodenalis on large-scale sheep farms: insights from the Henan and Ningxia Regions, China.

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 ß-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). However, the difference between the rates of environmental pollution in Henan (1.9%) and Ningxia (1.0%) was not significant (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.

Aflatoxin B1 as a complicit in intestinal damage caused by Eimeria ovinoidalis in lambs: Novel insights to reveal parasite-gut battle.

Mycotoxins, unavoidable contaminants in feed and feed ingredients, have the potential to influence the incidence and severity of various diseases upon ingestion. Sheep coccidiosis is an enteric disease caused by protozoa of Eimeria spp. However, the extent to which the presence of aflatoxin b1 (AFB1) synergistically exacerbates damage to intestinal health in lambs with Eimeria remains unclear. 50-day-old female lambs were randomly assigned to a 2 × 2 factorial arrangement of treatments for 15 days to assess the impact of AFB1 exposure on lambs with or without Eimeria (E.) ovinoidalis infection. Our findings reveal that AFB1 synergistically intensifies damage to intestinal health in lambs challenged by E. ovinoidalis. This is evidenced by disruptions to the intestinal microbiota and reductions in the production of short-chain fatty acids. AFB1 further aggravates damage to the cecal mechanical barrier. Additionally, AFB1 contributes to the entry of lipopolysaccharide into the bloodstream, activating the inflammatory response. Interestingly, AFB1 exposure history results in an early peak of oocyst excretion and a decreased number of oocyst excretion in E. ovinoidalis infected lambs. This may be closely linked to the destruction of the intestinal epithelial cell structure and its apoptosis, as indicated by a decreased ratio of Bcl-2 to Bax and increased caspase-3 levels. Mechanistically, proteomics analysis identified mitochondrial dysfunction (inhibition of the oxidative phosphorylation pathway) as the primary factor intensifying intestinal epithelial cell destruction caused by coccidia, exacerbated by AFB1 through the inhibiting the conversion of NADH to NAD+ in the cecum of lambs via down-regulation of the PGC-1α/NRF1/TFAM pathway. Overall, these results offer novel insights into the AFB1 complicity in accelerating intestinal damage caused by E. ovinoidalis in lambs. Targeting the mitochondrial oxidative phosphorylation pathway of the intestine may represent a new therapeutic strategy against the detrimental effects of mycotoxin and coccidia.

miR-181d targets BCL2 to regulate HCT-8 cell apoptosis and parasite burden in response to Cryptosporidium parvum infection via the intrinsic apoptosis pathway.

Cryptosporidium parvum is an important zoonotic pathogen that is studied worldwide. MicroRNAs (miRNAs) act as post-transcriptional regulators and may play a key role in modulating host epithelial responses following Cryptosporidium infection. Our previous study has shown that C. parvum downregulates the expression of miR-181d through the p50-dependent TLRs/NF-κB pathway. However, the mechanism by which miR-181d regulates host cells in response to C. parvum infection remains unclear. The present study found that miR-181d downregulation inhibited cell apoptosis and increased parasite burden in HCT-8 cells after C. parvum infection. Bioinformatics analysis and luciferase reporter assays have shown that BCL2 was a target gene for miR-181d. Moreover, BCL2 overexpression and miR-181d downregulation had similar results. To further investigate the mechanism by which miR-181d regulated HCT-8 cell apoptosis during C. parvum infection, the expression of molecules involved in the intrinsic apoptosis pathway was detected. Bax, caspase-9, and caspase-3 expression was decreased at 4, 8, 12, and 24 hpi and upregulated at 36 and 48 hpi. Interfering with the expression of miR-181d or BCL2 significantly affected the expression of molecules in the intrinsic apoptosis pathway. These data indicated that miR-181d targeted BCL2 to regulate HCT-8 cell apoptosis and parasite burden in response to C. parvum infection via the intrinsic apoptosis pathway. These results allowed us to further understand the regulatory mechanisms of host miRNAs during Cryptosporidium infection, and provided a theoretical foundation for the design and development of anti-cryptosporidiosis drugs.

Portulaca oleracea exhibited anti-coccidian activity, fortified the gut microbiota of Hu lambs.

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.

Effects of three kinds of Chinese herbs on growth performance, oocysts output and gut microbiota in growing lambs with coccidiosis.

Coccidiosis is a protozoan intestinal disease that reduces the production of the sheep industry and causes large economic losses for sheep. Although chemically synthesised drugs are routinely employed to treat coccidiosis in sheep, the anticoccidial drug resistance and drug residues in edible meat have prompted an urgent search for alternatives. Herein, the anticoccidial properties of diclazuril, a conventional anticoccidial drug, and Allium sativum, Houttuynia cordata and Portulaca oleracea were assessed. Forty 45-day-old lambs naturally infected with Eimeria spp. were selected and randomly divided into five groups. The results showed that the sheep treated for coccidiosis had considerably decreased average daily gain (ADG) during both administration and withdrawal of the drug compared to the control group. Furthermore, at days 14, 21, 28 and 35, respectively, the three herbs and diclazuril had similar anticoccidial effects, with lower oocysts per gram (OPG) than the control group. On day 78, OPG in the three herbal groups was significantly lower than in the diclazuril group. In addition, the abundance and composition of the gut microbiota were changed in sheep treated with the three herbs and diclazuril compared to the untreated sheep. Moreover, some intestinal microorganisms have a correlation with OPG and ADG when using Spearman correlation analysis. In summary, our results suggest that all three herbs produce anticoccidial effects similar to diclazuril and modulate the balance of gut microbiota in growing lambs.

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.

Host specific Eimeria genus diagnosis and qPCR development in Ovis aries and Capra hircus.

The objective of the present study was to develop a real-time PCR (qPCR) technique for the diagnosis of Eimeria spp. in Ovis aries and Capra hircus. The qPCR technique was developed using SYBR Green, resulting in a PCR with high sensitivity, specificity, and reproducibility.

Effects of Radix dichroae extract supplementation on growth performance, oocysts output and gut microbiota in growing lambs with coccidiosis.

Coccidiosis is an intestinal protozoan disease of sheep, that causes substantial economic losses in the industry due to its intestinal protozoan origins. Many anti-protozoan drugs including ionophores, triazines, and sulfonamides have been widely used to treat sheep coccidiosis. Still, anticoccidial resistance and drug residues in edible tissues have prompted an urgent search for alternatives. In this study, the anti-coccidial effectiveness of the Radix dichroae extract was compared to that of the conventional anti-coccidial drug diclazuril. Here, eighteen 45-day-old lambs naturally-infected with Eimeria spp. were randomly allocated in three groups: control group, Radix dichroae extract group and diclazuril group. The results showed that the body weight gain (BWG) during the treatment and withdrawal periods was considerably improved in the coccidiosis-infected sheep treated with Radix dichroae extract and diclazuril compared to the control group, respectively. Additionally, the Radix dichroae extract and diclazuril had fewer oocysts per gram (OPG) than the control group, showing similar anti-coccidial effects on days 14, 21, 28, 35 and 78, respectively. Furthermore, Radix dichroae extract and diclazuril treatment altered the structure and composition of gut microbiota, promoting the relative abundance of Actinobacteriota, Firmicutes, Alistipes, and Bifidobacterium, while decreasing the abundance of Bacteroidota, Marinilaceae, Helicobacteraceae, and Prevotella. Moreover, Spearman's correlation analysis further revealed a correlation between the OPG and BWG and gut microorganisms. Collectively, the results indicated that Radix dichroae extract had similar anti-coccidial effects as diclazuril, and could regulate gut microbiota balance in growing lambs.

Evaluation of gut microbiota composition to screening for potential biomarker in AFB1-exposed sheep.

Aflatoxin B1 (AFB1) is an inevitable contaminant in animal feed and agricultural products, which seriously threatens the health of animals. However, there is currently no better diagnostic tool available than depending on clinical symptoms, pathophysiology, biochemical indicators, etc. Here, we profiled the fecal microbiomes of sheep exposed to and not exposed to AFB1 to identify potential non-invasive biomarkers of AFB1 intoxication by 16S rRNA gene sequencing technology, while measuring serum biochemical indexes. The results showed that the sheep exposed to AFB1 had significantly higher levels of the liver function indicators ALT (alanine transaminase) and AST (aspartate aminotransferase), and their microbial profiles were different from those of the CON (Control) group. In detail, the relative abundance of seven phyla and three genera were overrepresented in the AFB1 group from top 10 relative abundance. Importantly, we found that Prevotella and Bifidobacterium were significantly different in the CON and AFB1 groups (p = 0.032 and p = 0.021, respectively) based on linear discriminant analysis effect size (LEfSe) and random forest analysis. Additionally, the area under curve (AUC) of ALT was 1 (95% CI 1.00-1.00; p < 0.001) and that of Bifidobacterium was 0.95 (95% CI 0.81-1.00; p = 0.0275), suggesting that Bifidobacterium correlated with ALT (r = 0.783, p < 0.01) may be a potential biomarker for AFB1 exposure in sheep.

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.

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.

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.

MiR-942-5p targeting the IFI27 gene regulates HCT-8 cell apoptosis via a TRAIL-dependent pathway during the early phase of Cryptosporidium parvum infection.

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 miR­942­5p expression in HCT­8 cells via TLR2/TLR4­NF­κB 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.

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.