Cryptosporidium Parvum Infection Research Articles

MiR-199a-3p regulates HCT-8 cell autophagy and apoptosis in response to Cryptosporidium parvum infection by targeting MTOR

The microRNAs (miRNAs) of their hosts play an important role in regulating both the innate and adaptive immune responses to Cryptosporidium parvum infection. The mechanisms of autophagy and apoptosis are important components of the defense system against C. parvum infection. In this study, we investigate the role of miRNA-199a-3p in regulating MTOR-mediated autophagy and apoptosis in HCT-8 cells induced by C. parvum. The expression of miR-199a-3p increased at 3, 6 and 12 hours postinfection (hpi) but decreased at 24 and 48 hpi. The upregulation of miR-199a-3p promoted autophagy and apoptosis and limited the parasite burden in HCT-8 cells after C. parvum infection. The downregulation of miR-199a-3p inhibited the autophagy and apoptosis induced by C. parvum and enhanced the parasite burden in HCT-8 cells. A luciferase reporter showed that MTOR was a target gene of miR-199a-3p. Suppressed expression of MTOR by small interfering RNA (siRNA) promoted autophagy and apoptosis and limited C. parvum burden in HCT-8 cells. Co-transfection with miR-199a-3p inhibitor or si-mTOR revealed that miR-199a-3p regulates autophagy and apoptosis in HCT-8 cells through MTOR, to resist C. parvum infection. In conclusion, intestinal epithelial cells defend against C. parvum infection by regulating their autophagy and apoptosis through the miR-199a-3p-MTOR axis.

Microbial Health Risks of Cryptosporidium parvum and Giardia lamblia in Tropical Coastal Water in Araromi, Nigeria.

Giardia and Cryptosporidium are enteric protozoa that can cause a variety of gastrointestinal diseases, especially in vulnerable people like children, the elderly, and those with impaired immune systems. In order to ascertain the microbiological quality of the recreational water from Araromi Beach in Ilaje Local Government Area, Ondo State, Nigeria. This risk assessment is of great significance to human health protection against waterborne diseases. The aim of this study was to determine the microbial quality of recreational water from Araromi Beach in Ilaje Local Government Area, Ondo State, Nigeria. Microscopic examination of Cryptosporidium and Giardia oocysts were done. Results revealed maximum occurrence of Cryptosporidium parvum (20 oocysts/100 mL) of water sample in the month of April and maximum occurrence of Giardia lamblia (300 cysts/100 mL) of water sample in the month of June. Additionally, according to Kolmogorov-Smirnov tests for normalcy Ho =0.05, Giardia lamblia and Cryptosporidium parvum were not regularly distributed in the water samples collected from the beach throughout the study period. The average likelihood of contracting Giardia lamblia and Cryptosporidium parvum infections after consuming 100 mL of beach water was 0.96 and 0.35, respectively. The risks of infection associated with Cryptosporidium parvum was lower than those associated with Giardia lamblia in water from the beach, but were both above the acceptable risk limit of 10-4. The results of this study indicate that Giardia and Cryptosporidium may represent serious health hazards to people who engage in aquatic activities. Adopting a comprehensive strategy that includes regular inspections, enhanced detection techniques, and the prevention of aquatic environment pollution may provide clean and safe recreational water for all, thereby safeguarding the public's health.

Effect of oxymatrine on Cryptosporidium parvum infection in mice based on the HMGB1-TLR2/TLR4-NF-κB pathway

Effect of oxymatrine on Cryptosporidium parvum infection in mice based on the HMGB1-TLR2/TLR4-NF-κB pathway

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.

Alteration of goat kids’ gut microbiota by Cryptosporidium parvum infection

Alteration of goat kids’ gut microbiota by Cryptosporidium parvum infection

Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection.

Cryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum, with a high prevalence in livestock (cattle, sheep, and goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and particularly of type 1 conventional dendritic cells (cDC1) among mononuclear phagocytes (MPs) in controlling the acute phase of C. parvum infection. These immune populations are well described in mice and humans, but their fine characterization in the intestine of young ruminants remained to be further explored. Immune cells of the small intestinal Peyer's patches and of the distal jejunum were isolated from naive lambs and calves at different ages. This was followed by their fine characterization by flow cytometry and transcriptomic analyses (q-RT-PCR and single cell RNAseq (lamb cells)). Newborn animals were infected with C. parvum, clinical signs and parasite burden were quantified, and isolated MP cells were characterized by flow cytometry in comparison with age matched control animals. Here, we identified one population of macrophages and three subsets of cDC (cDC1, cDC2, and a minor cDC subset with migratory properties) in the intestine of lamb and calf by phenotypic and targeted gene expression analyses. Unsupervised single-cell transcriptomic analysis confirmed the identification of these four intestinal MP subpopulations in lamb, while highlighting a deeper diversity of cell subsets among monocytic and dendritic cells. We demonstrated a weak proportion of cDC1 in the intestine of highly susceptible newborn lambs together with an increase of these cells within the first days of life and in response to the infection. Considering cDC1 importance for efficient parasite control in the mouse model, one may speculate that the cDC1/cDC2 ratio plays also a key role for the efficient control of C. parvum in young ruminants. In this study, we established the first fine characterization of intestinal MP subsets in young lambs and calves providing new insights for comparative immunology of the intestinal MP system across species and for future investigations on host-Cryptosporidium interactions in target species.

Analysis of gut microbiota with cryptosporidiosis based on fecal condition in neonatal dairy calves on a farm in Japan

Cryptosporidiosis is a major cause of diarrhea and is associated with high morbidity in calves. Changes in the gut microbiota exacerbate diarrhea caused by Cryptosporidium parvum (C. parvum) infection in neonatal and weaned calves. However, information on the gut microbiota of neonatal calves with C. parvum infection is scarce, and research into the microbiome of calves is essential for developing preventive and therapeutic interventions. This study aimed to elucidate the gut microbiota of neonatal calves with cryptosporidiosis. We collected 31 fecal samples from 31 neonatal calves on a dairy farm with or without C. parvum antigen [CP(+) or CP(-)] using a kit and analyzed the differences in the microbiota between diarrheal (D) and normal (N) fecal samples with C. parvum infection based on the fecal score. The analyses revealed the α diversity indexes of fecal microbiota in CP(+)-N samples were higher than that in CP(+)-D samples. Megasophaera spp. and other rumen microbes were identified, and significantly associated with CP(+)-N samples compared with CP(+)-D samples by LEfSe analysis. We conclude that the specific gut microbiota could characterize fecal microbiota in calves with neonatal cryptosporidiosis without clinical symptoms.

Detection of Cryptosporidium parvum by modified acid-fast stain among cancer patients in Thi-Qar province

Cryptosporidium parvum is a widespread opportunistic parasite belonging to the family Apicomplexa that completes its life cycle in a single host and is found frequently in immunocompromised persons, especially in patients with cancer. This research's objectives were to determine the prevalence of Cryptosporidium parvum infection among cancer patients in the Thi-Qar Province of Iraq, using modified Ziehl-Neelsen (MZN) stain. This study included 300 stool samples, 200 of which were for patients with cancer and 100 were for healthy people (the control group). Based on the modified acid-fast stain, the results showed the rate of infection with Cryptosporidium parvum among cancer patients was 54.0% (108/200) compared to 30.0% (30/100) in the control group, and there were statistically significant differences between them .And the percentage of infection among cancer patients in males was higher than in females, but there were no significant differences between them, as the percentage was 57.14% and 52.03%, respectively. As for the age groups Cryptosporidium parvum it affects all age groups in different proportions. The study showed that the highest infection rate among the age groups reached ?60 years at a rate of 64.44%, and the lowest infection rate among the age group ?20 years at a rate of 35.71%. As for the number doses of chemotherapy the results showed that the infection rate was higher in patients who received half and more than half of the doses of chemotherapy, where the percentage of those who received 16-20 doses (64.0%)

<i>Sero-Prevalence of Cryptosporidiosis</i> among People Living with HIV/AIDS in North-western Nigeria

Background: Cryptosporidium species are obligate, intracellular, protozoan parasites belonging to the phylum Apicomplexa, which is a substantial threat to HIV/AIDS-infected individuals with an estimated risk of infection of around 52% in developing countries.
 Aim: The study was aimed at determining the Sero-Prevalence of Cryptosporidiosis among people living with HIV/AIDS in North-western Nigeria. 
 Methods: Structured questionnaires were administered to 90 participants who were selected by inclusion criteria sampling technique with a total of 90 blood samples collected from them. Serological analysis of the blood samples was carried out using the Human Cryptosporidium parvum (CP) ELISA detection test Kit. (Melsin Medical Co., Limited, China LOT NUMBER: 20191023), and CD4 cell count was performed.
 Results: Out of the 100 participants, 19(19%) were found to be positive for Cryptosporidium parvum and 81(81%) were negative with an overall prevalence of 19%. There was no statistical significance between Cryptosporidium infection and those who used pit latrine (p=0.347), eating outside (p=0.494), reared animals (p=0.838) and the type of water source (p=0.641). The association between Cryptosporidium parvum and the CD4+ count of the participants was determined using a statistically insignificant chi-square test (p= 0.409). The higher the CD4+ count, the lesser the risk for Cryptosporidium parvum infection.
 Conclusion: This study reveals an overall Sero-Prevalence of 19% of C. parvum among HIV/AIDS patients and there is no association between CD4+ count and infection of Cryptosporidium parvum.

Specific pathway abundances in the neonatal calf faecal microbiome are associated with susceptibility to Cryptosporidium parvum infection: a metagenomic analysis

BackgroundCryptosporidium parvum is the main cause of calf scour worldwide. With limited therapeutic options and research compared to other Apicomplexa, it is important to understand the parasites’ biology and interactions with the host and microbiome in order to develop novel strategies against this infection. The age-dependent nature of symptomatic cryptosporidiosis suggests a link to the undeveloped immune response, the immature intestinal epithelium, and its associated microbiota. This led us to hypothesise that specific features of the early life microbiome could predict calf susceptibility to C. parvum infection.ResultsIn this study, a single faecal swab sample was collected from each calf within the first week of life in a cohort of 346 animals. All 346 calves were subsequently monitored for clinical signs of cryptosporidiosis, and calves that developed diarrhoea were tested for Rotavirus, Coronavirus, E. coli F5 (K99) and C. parvum by lateral flow test (LFT). A retrospective case–control approach was taken whereby a subset of healthy calves (Control group; n = 33) and calves that went on to develop clinical signs of infectious diarrhoea and test positive for C. parvum infection via LFT (Cryptosporidium-positive group; n = 32) were selected from this cohort, five of which were excluded due to low DNA quality. A metagenomic analysis was conducted on the faecal microbiomes of the control group (n = 30) and the Cryptosporidium-positive group (n = 30) prior to infection, to determine features predictive of cryptosporidiosis. Taxonomic analysis showed no significant differences in alpha diversity, beta diversity, and taxa relative abundance between controls and Cryptosporidium-positive groups. Analysis of functional potential showed pathways related to isoprenoid precursor, haem and purine biosynthesis were significantly higher in abundance in calves that later tested positive for C. parvum (q ≤ 0.25). These pathways are either absent or streamlined in the C. parvum parasites. Though the de novo production of isoprenoid precursors, haem and purines are absent, C. parvum has been shown to encode enzymes that catalyse the downstream reactions of these pathway metabolites, indicating that C. parvum may scavenge those products from an external source.ConclusionsThe host has previously been put forward as the source of essential metabolites, but our study suggests that C. parvum may also be able to harness specific metabolic pathways of the microbiota in order to survive and replicate. This finding is important as components of these microbial pathways could be exploited as potential therapeutic targets for the prevention or mitigation of cryptosporidiosis in bovine neonates.

Comparative proteomics reveals Cryptosporidium parvum infection disrupts cellular barriers

Cryptosporidium is a protozoan parasite capable of infecting humans and animals and is a leading cause of diarrheal disease and early childhood mortality. The molecular mechanisms underlying invasive infection and its pathogenesis remain largely unknown. To better understand the molecular mechanism of the interaction between C. parvum and host cells, we profiled the changes of host cells membrane proteins extracted using native membrane protein extraction kit between C. parvum-infected HCT-8 cells and the control group after C. parvum infected 6 h combined with quantitative Tandem Mass Tags (TMT) liquid chromatography-dual mass spectrometry proteomic analysis. Among the 4844 quantifiable proteins identified, the expression levels of 625 were upregulated, and those of 116 were downregulated at 6 h post-infection compared with controls (1.5-fold difference in abundance, p < 0.05). Enrichment analysis of the function, protein domain and Kyoto Encyclopedia of Genes and Genomes pathway of the differentially expressed proteins revealed that the differentially expressed proteins were mainly related to biological functions related to the cytoskeleton and cytoplasmic matrix. We also found that infection with C. parvum may destroy HCT-8 intercellular space adhesion. Six proteins were further verified using quantitative real-time reverse transcription polymerase chain reaction and western blotting. Through systematic analysis of proteomics related to HCT-8 cell membranes infected by C. parvum, we found many host membrane proteins that can serve as potential receptors in C. parvum adhesion or invasion. C. parvum infection destroyed host cell barrier function and caused extensive changes in host cytoskeleton proteins, providing a deeper understanding of the molecules and their functions involved in the host-C. parvum interaction. SignificanceThere is a lack of systematic research on the molecular mechanisms underlying the interaction of C. parvum with host cells. Changes of host cell membrane proteins after C. parvum infection may be used to examine the host cell receptors for parasite adhesion and invasion, and how the parasite interacts with these receptors. It is of great significance that host cells undergo membrane fusion to mediate invasion. Through proteomic studies on the host cell membrane after infection with HCT-8 cells by C. parvum, we observed disruption of the host cell cellular barrier function and widespread alteration of host cytoskeletal proteins caused by C. parvum infection, providing a deeper understanding of the molecules and their functions involved in host-C. parvum interaction.

A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum.

Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M's impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein-isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity.

Photoswitchable Inhibitors to Optically Control Specific Kinase Activity.

Potent and selective small-molecule inhibitors are valuable tools to elucidate the functions of protein kinases within complex signaling networks. Incorporation of a photoswitchable moiety into the inhibitor scaffold offers the opportunity to steer inhibitor potency with temporal precision, while the challenge of selective inhibition can often be addressed by employing a chemical genetic approach, termed the analog-sensitive method. Here, we combine the perks of these two approaches and report photoswitchable azopyrazoles to target calcium-dependent protein kinase 1 (CDPK1) from Toxoplasma gondii, a kinase naturally susceptible to analog-sensitive kinase inhibitors due to its glycine gatekeeper residue. The most promising azopyrazoles display favorable photochemical properties, thermal stability, and a substantial difference in IC50 values between both photostationary states. Consequently, the CDPK1 kinase reaction can be controlled dynamically and reversibly by applying light of different wavelengths. Inhibition of CDPK1 by the azopyrazoles drastically relies on the nature of the gatekeeper residue as a successive increase in gatekeeper size causes a concurrent loss of inhibitory activity. Furthermore, two photoswitchable inhibitors exhibit activity against T. gondii and Cryptosporidium parvum infection in a cell culture model, making them a promising addition to the toolbox for dissecting the role of CDPK1 in the infectious cycle with high temporal control. Overall, this work merges the benefits of the analog-sensitive approach and photopharmacology without compromising inhibitory potency and thus holds great promise for application to other protein kinases in the future.

Circular RNA ciRS-7 affects the propagation of Cryptosporidium parvum in HCT-8 cells via regulating miR-135a-5p/stat1 axis

Cryptosporidium spp. are protozoan parasites that mainly inhabit intestinal epithelial cells, causing diarrheal diseases in humans and a great number of animals. Cryptosporidium parvum is the most common zoonotic species, responsible for nearly 45% of human cryptosporidiosis worldwide. Understanding the interaction mechanisms between C. parvum and host gastrointestinal epithelial cells has significant implications to control cryptosporidiosis. One up-regulated circRNA ciRS-7 was found previously by our group to promote in vitro propagation of C. parvum in HCT-8 cells. In the present study, miR-135a-5p, was found to be a miRNA target of ciRS-7. Cryptosporidium parvum infection induced significantly down-regulation of miR-135a-5p and dramatic up-regulation of its potential target stat1 gene at mRNA and protein levels. Dual luciferase reporter assays validated the physical interactions between miR-135a-5p and stat1, and between ciRS-7 and miR-135a-5p. Further study revealed that ciRS-7 could sponge miR-135a-5p to positively regulate the protein levels of STAT1 and phosphorylated STAT1 (p-STAT1) and thus promote C. parvum propagation in HCT-8 cells. Our findings further reveal the mystery of regulatory roles of host circRNAs during Cryptosporidium infection, and provide a novel insight to develop strategies to control cryptosporidiosis.

Cryptosporidium parvum and gp60 genotype prevalence in dairy calves worldwide: a systematic review and meta-analysis

Cryptosporidium is a significant zoonotic pathogen that often occurs in dairy cattle. We conducted a systematic review and meta-analysis of the prevalence of Cryptosporidium parvum infection in dairy calves worldwide to help improve global animal husbandry and public policy implementation. Published articles were obtained from PubMed and Web of Science from January 1, 2000 to December 31, 2021. The prevalence of C. parvum infection in dairy calves was estimated using a random effects model, and the sources of heterogeneity were explored using meta-regression. In total, 118 datasets were included in the final quantitative analysis. The results showed that the global prevalence of C. parvum in dairy calves was 21.9% (7755/42,890; 95% confidence interval: 19.9–23.9%). C. parvum infection was high in pre-weaned dairy calves (24.9%, 6706/29,753) and diarrhea dairy calves (33.6%, 1637/6077). In countries with low dairy stocking density (<10 cows/farm), the prevalence of C. parvum in dairy calves was also relatively low (15.2%, 1960/16,584). Three subtype families [IIa (72.2%, 2293/3177), IId (27.4%, 872/3177), and IIl (0.4%, 12/3177)] were detected in dairy calves globally from selected studies. C. parvum IIa was the dominant zoonotic subtype. In the IIa subtype family of C. parvum, the proportions of subtypes from high to low (top nine) were IIaA15G2R1 (32.4%, 742/2293), IIaA18G3R1 (11.8%, 271/2293), IIaA13G2R1 (8.2%, 187/2293), IIaA16G1R1 (6.4%, 147/2293), IIaA20G1R1 (3.5%, 81/2293), IIaA16G3R1 (3.4%, 78/2293), IIaA17G2R1 (2.7%, 62/2293), IIaA18G1R1 (2.5%, 58/2293), and IIaA15G1R1 (2.4%, 56/2293). In the IId subtype family of C. parvum, the proportions of subtypes (top four) were IIdA19G1 (36.0%, 314/872), IIdA15G1 (27.3%, 238/872), IIdA20G1 (16.2%, 141/872), and IIdA14G1 (13.0%, 113/872). Furthermore, IId is commonly found in China (771/872). The study results indicated that the IIa subtype family is globally prevalent, while IId is found in Asia, Europe, and Africa and IIl is only found in Europe. Diarrhea in dairy calves is associated with C. parvum infection and a significantly higher prevalence is observed in diarrheic calves. Age and stock density are two significant risk factors in the prevalence of C. parvum in dairy calves. The prevention and control of this zoonosis in dairy calves should receive greater attention, especially in regions with a high degree of intensive dairy farming.

Comparison among Modified Acid-Fast Stain and Some Immunological Methods in Diagnosis of Cryptosporidium parvum in Kut City

A total of 100 children who attending to Al-Karamah Teaching Hospital at Kut city were suffered from watery diarrhoea and abdominal pain were examined. The present study was conducted from October 2011 to January 2012. A thirty-four stool samples in which Cryptosporidium parvum oocysts had been seen by modified acid –fast stain examination were investigated, positivity detected with the ImmunoCard method, and Direct Fluorescent Antibody( DFA ) method was found to be (30%), (28%) and (34%) respectively.Children aged one month to 144 months presenting with acute or persistent diarrhoea were selected randomly. prevalence of Cryptosporidium parvum infection was significantly higher (59%) in children under one year old compared to children between (7-12) years old (4%). The infective rate of Cryptosporidium parvum in males was higher 17% than females 13% . Also, there was no significant difference among children in gender.

Investigation of the prophylactic and therapeutic effectiveness of oral thyme extract in rats experimentally infected with cryptosporidium parvum.

In this study, the prophylactic and therapeutic activities of thyme extract at different concentrations against experimental Cryptosporidium parvum infection in immunosuppressed rats were investigated. Thyme extract was prepared at four different concentrations (10%, 30%, 50%, and 100%) and administered as a single oral dose of 1 mL for evaluation of its prophylactic efficacy. Five consecutive days after infection was detected in all rats, therapeutic evaluations were also performed. According to the results obtained by daily counting of oocysts in stools, the prophylactic and therapeutic effects of thyme extract administration were significant in comparison to the control group (P˂0.01). Oocyst shedding continued in the control group at high numbers from the beginning to the end of the study, while oocyst counts in the prophylaxis groups remained low throughout the study. On the other hand, oocyst excretion rates were high in the therapeutic groups and decreased rapidly after thyme extract administration. At the end of the study, oocyst excretion had completely stopped for some rats administered thyme extract. There was no group in which oocyst shedding ceased for all rats. No significant differences were observed in the therapeutic or prophylaxis groups regarding the doses administered (P > 0.01). Renal and hepatic functions were monitored by measuring urea, creatinine, alanine transaminase, and aspartate transaminase levels ​​before and after thyme extract administration. As a result, it was concluded that oral thyme extract administration at the doses applied in this study is effective and safe in the prophylactic and therapeutic treatment of experimental cryptosporidiosis in rats.

Differential Response to the Course of Cryptosporidium parvum Infection and Its Impact on Epithelial Integrity in Differentiated versus Undifferentiated Human Intestinal Enteroids.

Cryptosporidium is a leading cause of diarrhea and death in young children and untreated AIDS patients and causes waterborne outbreaks. Pathogenic mechanisms underlying diarrhea and intestinal dysfunction are poorly understood. We previously developed stem-cell derived human intestinal enteroid (HIE) models for Cryptosporidium parvum which we used in this study to investigate the course of infection and its effect on intestinal epithelial integrity. By immunofluorescence and confocal microscopy, there was robust infection of undifferentiated and differentiated HIEs in two and three-dimensional (2D, 3D) models. Infection of differentiated HIEs in the 2D model was greater than that of undifferentiated HIEs but lasted only for 3 days, whereas infection persisted for 21 days and resulted in completion of the life cycle in undifferentiated HIEs. Infection of undifferentiated HIE monolayers suggest that C. parvum infects LGR5+ stem cells. Transepithelial electrical resistance measurement of HIEs in the 2D model revealed that infection resulted in decreased epithelial integrity which persisted in differentiated HIEs but recovered in undifferentiated HIEs. Compromised epithelial integrity was reflected in disorganization of the tight and adherens junctions as visualized using the markers ZO-1 and E-cadherin, respectively. Quantitation using the image analysis tools Tight Junction Organizational Rate and Intercellular Junction Organization Quantification, measurement of monolayer height, and RNA transcripts of both proteins by quantitative reverse transcription PCR confirmed that disruption persisted in differentiated HIEs but recovered in undifferentiated HIEs. These models, which more accurately recapitulate human infection, will be useful tools to dissect pathogenic mechanisms underlying diarrhea and intestinal dysfunction in cryptosporidiosis.

High infectivity and unique genomic sequence characteristics of Cryptosporidium parvum in China

Zoonotic Cryptosporidium parvum infections are mainly caused by IIa and IId subtypes. As most biological characterizations have been performed on IIa subtypes, the biological and genetic characteristics of IId subtypes in China are not clear. We evaluated the infection and genetic characteristics of IId isolates in interferon-γ-knockout mice using qPCR to quantify oocyst shedding, histological examination to monitor pathological changes and comparative genomic analyses to identify infectivity and virulence-associated differences. Compared with the reference IIa isolate, mice infected with the IId isolates had significantly higher and longer oocyst shedding and lower body weight gain. In addition, the four IId isolates examined differed significantly in infectivity (as indicated by the median infective dose), oocyst shedding duration, and pathogenicity. Comparative genomic analysis indicated that the IId isolates had three more subtelomeric genes than the reference IIa isolate and 5385-5548 nucleotide substitutions, with the hypervariable genes mostly in two blocks on chromosome 1. In contrast, the four IId isolates differed from each other by 77-1,452 nucleotides, with virulence-associated sequence differences mainly in nine genes within a 28-kb block on chromosome 6. These data indicate the newly emerged C. parvum IId subtypes in China have high animal infectivity and unique genomic characteristics.

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

BackgroundMicroRNAs (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.MethodsQuantitative real-time polymerase chain reaction, western blotting, flow cytometry, and immunofluorescence were used for analysis.ResultsForced 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.ConclusionsThese 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