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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2022. The Author(s).)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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.

Cryptosporidium spp., Giardia duodenalis, Cyclospora cayetanensis, and Enterocytozoon bieneusi are known etiological agents of self-limiting diarrhea, chronic disorders, and severe debilitating illnesses in humans, particularly children and patients with immunodeficiency diseases. To assess the pathogen carriage status of raw vegetables and fruits and the potential transmission routes of the aforementioned parasites in Henan province, China, a total of 1099 vegetables and fruits samples (21 items) were purchased and collected from agricultural farms or open markets. Cryptosporidium spp., E. bieneusi, C. cayetanensis and G. duodenalis were screened by employing polymerase chain reaction (PCR) amplification of species-specific genes. Three kinds of human pathogenic agent (E. bieneusi, C. cayetanensis and C. parvum) were identified on the surfaces of the vegetables and fruits (3.7%, 41/1099). E. bieneusi was found in 3.5% (38/1099) of the samples, whereas C. cayetanensis and C. parvum were only identified in two (0.2%) and one (0.1%) of the vegetable and fruit samples, respectively. No G. duodenalis contamination was detected in the present study. In total, 12 different E. bieneusi ITS genotypes (eight known and four novel) were detected, of which the ten (EbpA, CM8, CHG19, EbpC, CTS3, Henan-IV, and CHV1 to CHV4) that occurred in 20 samples (20/38, 52.6%) clustered into the previously described high potential zoonotic group 1 in the phylogenetic analysis. The remaining two known genotypes (BEB8 and CD6) detected in 18 samples (18/38, 47.4%) belonged to group 2. That C. cayetanensis, C. parvum and some E. bieneusi genotypes have been reported in humans, highlights the possible risk of foodborne related disease outbreaks., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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.

Extensive genetic variation is observed within the genus Cryptosporidium and the distribution of Cryptosporidium species/genotypes in humans and animals appears to vary by geography and host species. To better understand the genetic diversity of Cryptosporidium spp. in horses and donkeys, we characterized five horse-derived and 82 donkey-derived Cryptosporidium isolates from five provinces or autonomous regions (Sichuan, Gansu, Henan, Inner Mongolia and Shandong) in China at the species/genotype and subtype levels. Three Cryptosporidium species/genotypes were identified based on the analysis of the SSU rRNA gene, including Cryptosporidium parvum (n=22), the Cryptosporidium horse genotype (n=4), and Cryptosporidium hominis (n=61). The identification of C. hominis was confirmed by sequence analysis of the HSP70 and actin genes. Subtyping using sequence analysis of the 60kDa glycoprotein gene identified 21 C. parvum isolates as subtype IIdA19G1, the four horse genotype isolates as subtypes VIaA15G4 (n=2) and VIaA11G3 (n=2), and the 61 C. hominis isolates as IkA16G1 (n=59) and IkA16 (n=2). The common finding of C. hominis reaffirms the heterogeneity of Cryptosporidium spp. in horses and donkeys and is possibly a reflection of endemic transmission of C. hominis in these animals. Data of the study suggest that horses and donkeys as companion animals may potentially transmit Cryptosporidium infections to humans., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

In this study, 111 Cryptosporidium parvum IId isolates from several species of animals in China, Sweden, and Egypt were subtyped by multilocus sequence typing (MLST). One to eleven subtypes were detected at each of the 12 microsatellite, minisatellite, and single nucleotide polymorphism (SNP) loci, forming 25 MLST subtypes. Host-adaptation and significant geographical segregation were both observed in the MLST subtypes. A clonal population structure was seen in C. parvum IId isolates from China and Sweden. Three ancestral lineages and the same RPGR sequence were shared by these isolates examined. Therefore, the present genetic observations including the higher nucleotide diversity of C. parvum IId GP60 sequences in Western Asia, as well as the unique distribution of IId subtypes (almost exclusively found in Asia, Europe, and Egypt) and in combination with the domestication history of cattle, sheep, and goats, indicated that C. parvum IId subtypes were probably dispersed from Western Asia to other geographical regions. More population genetic structure studies involving various C. parvum subtype families using high-resolution tools are needed to better elucidate the origin and dissemination of C. parvum in the world.

To identify Cryptosporidium species/genotypes in sheep in China and to elucidate the endemic transmission of cryptosporidiosis, a total of 1,701 fecal samples from five farms in four prefectures in Henan Province (central China) were examined. Eighty-two Cryptosporidium-positive samples were analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of the small subunit (SSU) rRNA gene and PCR analysis of the 60 kDa glycoprotein (gp60) gene, and 41 were further analyzed by DNA sequencing of the PCR products. The SSU rRNA-based PCR identified two Cryptosporidium species and one genotype, including the Cryptosporidium cervine genotype (74/82), Cryptosporidium andersoni (4/82), and Cryptosporidium xiaoi (4/82). The cervine genotype was found in all age groups, C. xiaoi in lambs, and C. andersoni in ewes. There were intragenetic differences in the SSU rRNA gene sequences of the Cryptosporidium cervine genotype and C. xiaoi. No Cryptosporidium parvum was detected by both SSU rRNA- and gp60-based PCR assays. These findings suggest that sheep are a potential source for zoonotic infections of the Cryptosporidium cervine genotype.

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. [ABSTRACT FROM AUTHOR]

Abstract: The Cryptosporidium in the small intestine of domestic mice (Mus musculus) was initially described as Cryptosporidium parvum. Recent genetic and biologic characterization of Cryptosporidium isolates indicate that domestic mice are infected with several morphologically indistinguishable intestinal Cryptosporidium parasites with different host specificities, including C. parvum sensu stricto, mouse genotype I, and mouse genotype II. In this study, the morphological, biological, and genetic characteristics of the Cryptosporidium mouse genotype I are described. As a full re-description of C. parvum was made in 1985 for isolates from calves and humans and the name C. parvum has been widely used for the parasite that is infectious to both ruminants and humans, the mouse genotype I is named as Cryptosporidium tyzzeri. Oocysts of the new species (4.64±0.05μm ×4.19±0.06μm, with a mean shape index of 1.11±0.02; n =69) are slightly smaller than those of the re-described C. parvum. The prepatent period was six and seven days, and the patent period was 24–28 and 28–29days in neonatal and adult mice, respectively. Oocysts were not infectious to lambs and calves. Light, transmission electron and scanning electron microscopy studies of the new species showed the presence of developmental stages in the microvillar brush border of the jejunum and ileum of experimentally infected mice, with the infection most intensive in the ileum. It had nucleotide sequences significantly different from C. parvum at the small subunit rRNA, 70kDa heat shock protein, oocyst wall protein, actin, and the 60kDa glycoprotein genes. Based on the morphological, genetic, and biological data and in compliance of established Cryptosporidium species naming criteria, this geographically widespread parasite is named as a new species in honor of Ernest Edward Tyzzer, who pioneered Cryptosporidium research. [Copyright &y& Elsevier]