Epidemiological investigation of Cryptosporidium in children with diarrhea in middle Inner Mongolia, China.

Cryptosporidia (Cryptosporidium) is a protozoan that is widely parasitic in the intestinal cells of humans and animals, and it is also an important zoonotic parasite. However, there is no epidemiological investigation on Cryptosporidium spp. infection in infants with diarrhea of Inner Mongolia, the largest livestock region in China. To investigate the prevalence of Cryptosporidium, 2435 fresh fecal samples were collected from children with diarrhea in Inner Mongolia Maternal and Child Health Care Hospital. Molecular characterization of Cryptosporidium was carried out based on its 18S rRNA and gp60 gene sequences. The overall prevalence was 12.85% (313/2435), and in Hohhot (12.15%), it was lower than that in the surrounding city (14.87%) (P < 0.05). Moreover, Cryptosporidium was detected in different seasons and sexes. Concerning the age of children with diarrhea, the prevalence of those age groups between 0 and 1 was obviously lower than others, and there were significant differences in the prevalence at different ages (P < 0.001). Analysis of the 18S rRNA gene sequence revealed that all the positive samples were Cryptosporidium parvum, and there were 5 subtypes (IIdA23G3, IIdA24G3, IIdA24G4, IIdA25G3, and IIdA25G4). To the best of our knowledge, the above subtypes have not been reported. Our results provide a relevant basis for control and education on food safety and foodborne illness prevention.

Identification of host protein ENO1 (alpha-enolase) interacting with Cryptosporidium parvum sporozoite surface protein, Cpgp40.

BACKGROUND: Cryptosporidium parvum is an apicomplexan zoonotic parasite causing the diarrheal illness cryptosporidiosis in humans and animals. To invade the host intestinal epithelial cells, parasitic proteins expressed on the surface of sporozoites interact with host cells to facilitate the formation of parasitophorous vacuole for the parasite to reside and develop. The gp40 of C. parvum, named Cpgp40 and located on the surface of sporozoites, was proven to participate in the process of host cell invasion. METHODS: We utilized the purified Cpgp40 as a bait to obtain host cell proteins interacting with Cpgp40 through the glutathione S-transferase (GST) pull-down method. In vitro analysis, through bimolecular fluorescence complementation assay (BiFC) and coimmunoprecipitation (Co-IP), confirmed the solid interaction between Cpgp40 and ENO1. In addition, by using protein mutation and parasite infection rate analysis, it was demonstrated that ENO1 plays an important role in the C. parvum invasion of HCT-8 cells. RESULTS: To illustrate the functional activity of Cpgp40 interacting with host cells, we identified the alpha-enolase protein (ENO1) from HCT-8 cells, which showed direct interaction with Cpgp40. The mRNA level of ENO1 gene was significantly decreased at 3 and 24 h after C. parvum infection. Antibodies and siRNA specific to ENO1 showed the ability to neutralize C. parvum infection in vitro, which indicated the participation of ENO1 during the parasite invasion of HCT-8 cells. In addition, we further demonstrated that ENO1 protein was involved in the regulation of cytoplasmic matrix of HCT-8 cells during C. parvum invasion. Functional study of the protein mutation illustrated that ENO1 was also required for the endogenous development of C. parvum. CONCLUSIONS: In this study, we utilized the purified Cpgp40 as a bait to obtain host cell proteins ENO1 interacting with Cpgp40. Functional studies illustrated that the host cell protein ENO1 was involved in the regulation of tight junction and adherent junction proteins during C. parvum invasion and was required for endogenous development of C. parvum.

Cryptosporidium parvum infection alters the intestinal mucosa transcriptome in neonatal calves: implications for immune function.

One of the leading causes of infectious diarrhea in newborn calves is the apicomplexan protozoan Cryptosporidium parvum (C. parvum). However, little is known about its immunopathogenesis. Using next generation sequencing, this study investigated the immune transcriptional response to C. parvum infection in neonatal calves. Neonatal male Holstein-Friesian calves were either orally infected (N = 5) or not (CTRL group, N = 5) with C. parvum oocysts (gp60 subtype IIaA15G2R1) at day 1 of life and slaughtered on day 7 after infection. Total RNA was extracted from the jejunal mucosa for short read. Differentially expressed genes (DEGs) between infected and CTRL groups were assessed using DESeq2 at a false discovery rate < 0.05. Infection did not affect plasma immunohematological parameters, including neutrophil, lymphocyte, monocyte, leucocyte, thrombocyte, and erythrocyte counts as well as hematocrit and hemoglobin concentration on day 7 post infection. The immune-related DEGs were selected according to the UniProt immune system process database and were used for gene ontology (GO) and pathway enrichment analysis using Cytoscape (v3.9.1). Based on GO analysis, DEGs annotated to mucosal immunity, recognizing and presenting antigens, chemotaxis of neutrophils, eosinophils, natural killer cells, B and T cells mediated by signaling pathways including toll like receptors, interleukins, tumor necrosis factor, T cell receptor, and NF-KB were upregulated, while markers of macrophages chemotaxis and cytosolic pattern recognition were downregulated. This study provides a holistic snapshot of immune-related pathways induced by C. parvum in calves, including novel and detailed feedback and feedforward regulatory mechanisms establishing the crosstalk between innate and adaptive immune response in neonate calves, which could be utilized further to develop new therapeutic strategies.

Genomic and virulence analysis of in vitro cultured Cryptosporidium parvum.

Recent advances in the in vitro cultivation of Cryptosporidium parvum using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (>20 months) in vitro culture on virulence-factors, genome conservation, and in vivo pathogenicity of the host by in vitro cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as in vivo passaged parasites.

Sequence introgression from exogenous lineages underlies genomic and biological differences among Cryptosporidium parvum IOWA lines.

The IOWA strain of Cryptosporidium parvum is widely used in studies of the biology and detection of the waterborne pathogens Cryptosporidium spp. While several lines of the strain have been sequenced, IOWA-II, the only reference of the original subtype (IIaA15G2R1), exhibits significant assembly errors. Here we generated a fully assembled genome of IOWA-CDC of this subtype using PacBio and Illumina technologies. In comparative analyses of seven IOWA lines maintained in different laboratories (including two sequenced in this study) and 56 field isolates, IOWA lines (IIaA17G2R1) with less virulence had mixed genomes closely related to IOWA-CDC but with multiple sequence introgressions from IOWA-II and unknown lineages. In addition, the IOWA-IIaA17G2R1 lines showed unique nucleotide substitutions and loss of a gene associated with host infectivity, which were not observed in other isolates analyzed. These genomic differences among IOWA lines could be the genetic determinants of phenotypic traits in C. parvum. These data provide a new reference for comparative genomic analyses of Cryptosporidium spp. and rich targets for the development of advanced source tracking tools.

Detection and molecular characterization of Cryptosporidium spp. in dairy calves in Lisbon and Tagus Valley, Portugal.

Cryptosporidium is a protozoan parasite with worldwide distribution, infecting a wide range of hosts with some zoonotic species. Calves have been identified as one of the most common reservoirs of this parasite. However, little is known about the genetics of Cryptosporidium in calves in Portugal. This study aimed to molecularly characterize infections of Cryptosporidium in pre-weaned calves from the Lisbon and Tagus Valley (LTV) in Portugal. Fifty-two samples were collected from calves from eight dairy and two beef farms in LTV, Portugal. Cryptosporidium oocysts were detected by Modified Ziehl-Neelsen staining (MZN) and direct immunofluorescent assay (DFA). MZN and DFA revealed the presence of Cryptosporidium oocysts in 40.4% (21/52) and 67.3% (35/52) samples, respectively. Positive samples were analyzed by PCR-RFLP of the 18 s rRNA gene for species identification. DNA amplification of the 18S rRNA gene was successful for 88.6% (31/35) of samples. Cryptosporidium parvum was identified in 96.8% (30/31) of the samples, and from one sample Cryptosporidium bovis was identified. Cryptosporidium parvum positive samples were subtyped by sequencing the PCR product of a partial fragment of the 60 kDa glycoprotein (gp60) gene. Subtype analysis of the C. parvum isolates revealed that all isolates belonged to subtype family IIa. Four subtypes were recognized within this subtype family, including the hyper-transmissible IIaA15G2R1 subtype that is the most frequently reported worldwide (27/30), IIaA14G2R1 (1/30), IIaA16G2R1 (1/30) and IIaA19G2R1 (1/30). To our knowledge, this is the first report of C. bovis, and C. parvum subtypes IIaA14G2R1 and IIaA19G2R1 in cattle in LTV, Portugal. The presence of the zoonotic C. parvum subtype in this study suggests that pre-weaned calves are likely to be a significant reservoir of zoonotic C. parvum, highlighting the importance of animal-to-human infection transmission risk. Further molecular studies are required to better understand the epidemiology of cryptosporidiosis in Portugal.

Multiple pathways for glucose phosphate transport and utilization support growth of Cryptosporidium parvum.

Cryptosporidium parvum is an obligate intracellular parasite with a highly reduced mitochondrion that lacks the tricarboxylic acid cycle and the ability to generate ATP, making the parasite reliant on glycolysis. Genetic ablation experiments demonstrated that neither of the two putative glucose transporters CpGT1 and CpGT2 were essential for growth. Surprisingly, hexokinase was also dispensable for parasite growth while the downstream enzyme aldolase was required, suggesting the parasite has an alternative way of obtaining phosphorylated hexose. Complementation studies in E. coli support a role for direct transport of glucose-6-phosphate from the host cell by the parasite transporters CpGT1 and CpGT2, thus bypassing a requirement for hexokinase. Additionally, the parasite obtains phosphorylated glucose from amylopectin stores that are released by the action of the essential enzyme glycogen phosphorylase. Collectively, these findings reveal that C. parvum relies on multiple pathways to obtain phosphorylated glucose both for glycolysis and to restore carbohydrate reserves.

Genetic crosses within and between species of Cryptosporidium.

Parasites and their hosts are engaged in reciprocal coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium, genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach, multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing Cryptosporidium parvum, a parasite of cattle and humans, and Cryptosporidium tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity.

MiR-4521 affects the propagation of Cryptosporidium parvum in HCT-8 cells through targeting foxm1 by regulating cell apoptosis.

Cryptosporidium parvum could regulate the expression of microRNAs of epithelial cells to facilitate its intracellular propagation. MiR-4521 has been reported to play an important role during the development and progression of tumors and infectious diseases by regulating cell proliferation, apoptosis, and autophagy. However, the implication of miR-4521 during C. parvum infection was still unknown. In this study, the expression of miR-4521 was found to be upregulated in HCT-8 cells infected with C. parvum from 8 h post-infection (pi) to 48 hpi, and its upregulation would be related with the TLR/NF-κB signal pathway during C. parvum infection. One potential target of miR-4521, foxm1, was down-regulated in HCT-8 cells from 24 hpi to 48 hpi, and the expression of foxm1 was negatively regulated by miR-4521. The target relationship between miR-4521 and foxm1 was further validated by using dual luciferase reporter assay. Further studies showed that miR-4521 promoted the propagation of C. parvum in HCT-8 cells through targeting foxm1 by regulating BCL2-mediating cell apoptosis. These results contribute to further understanding of the regulatory mechanisms of host miRNAs during Cryptosporidium infection.

Gene expression profile of HCT-8 cells following single or co-infections with Cryptosporidium parvum and bovine coronavirus.

Among the causative agents of neonatal diarrhoea in calves, two of the most prevalent are bovine coronavirus (BCoV) and the intracellular parasite Cryptosporidium parvum. Although several studies indicate that co-infections are associated with greater symptom severity, the host-pathogen interplay remains unresolved. Here, our main objective was to investigate the modulation of the transcriptome of HCT-8 cells during single and co-infections with BCoV and C. parvum. For this, HCT-8 cells were inoculated with (1) BCoV alone, (2) C. parvum alone, (3) BCoV and C. parvum simultaneously. After 24 and 72 h, cells were harvested and analyzed using high-throughput RNA sequencing. Following differential expression analysis, over 6000 differentially expressed genes (DEGs) were identified in virus-infected and co-exposed cells at 72 hpi, whereas only 52 DEGs were found in C. parvum-infected cells at the same time point. Pathway (KEGG) and gene ontology (GO) analysis showed that DEGs in the virus-infected and co-exposed cells were mostly associated with immune pathways (such as NF-κB, TNF-α or, IL-17), apoptosis and regulation of transcription, with a more limited effect exerted by C. parvum. Although the modulation observed in the co-infection was apparently dominated by the virus, over 800 DEGs were uniquely expressed in co-exposed cells at 72 hpi. Our findings provide insights on possible biomarkers associated with co-infection, which could be further explored using in vivo models.

[Investigation of the Effect of Pasteurization on the Viability of Cryptosporidium parvum in Cow's Milk by Propidium Monoazide qPCR]. / Pastörizasyonun Inek Sütündeki Cryptosporidium parvum Canliligi Üzerine Etkisinin Propidium Monoazide qPCR ile Arastirilmasi.

Cow's milk, which is one of today's most important food sources, can be a reservoir for many pathogens that create a risk to public health. One of these pathogens is Cryptosporidium parvum. The oocysts of C.parvum, an obligate intracellular parasite, cause infection when ingested orally. The oocysts scattered around with the feces of infected cows or calves can contaminate raw milk and this is frequently seen in dairy farms. The aim of this study was to investigate the viability of C.parvum by propidium monoazide (PMA)-quantitative polymerase chain reaction (qPCR) method after heat treatment applied to contaminated raw cow's milk. For the study, 50 ml of unpasteurized cow's milk was contaminated with 5 X 105 C.parvum oocysts and portioned into 1.5 ml microcentrifuge tubes. Three groups, namely the control group, pasteurization group and boiling group were formed. No warming procedure was applied to the control group. In the pasteurization group, the milks in microcentrifuge tubes were poured into the wells of the dry block heater set to 71.7 °C and incubated for five seconds. At the end of the period, the milks were transferred to the wells of the cold metal tube, which was removed at -20 °C with the help of a micropipette, and incubated for five seconds. The milks in the boiling group were incubated for two minutes in a dry block heater set to 95 °C. After the heat treatment, the milks in microcentrifuge tubes were transferred to 10 ml centrifuge tubes, PBS was added to make the final volume 10 ml, and centrifuged at 4000 rpm for 20 minutes. After this process was repeated twice, 400 µl of PBS was added to the precipitate remaining at the bottom, and the precipitate was homogenized. One sample of each group was applied with PMA, while PMA was not applied to the other sample. PMA-applied samples were incubated for five minutes at room temperature and in the dark, and then exposed to UV light for five minutes in the device with cooling feature. The oocysts were collected by centrifugation at 5000 g for five minutes. After DNA isolation from oocysts, SYBR Green real time PCR (Rt-PCR) was performed using primers amplifying the COWP gene region. As a result of SYBR Green Rt-PCR, the mean Ct values of the control without PMA, pasteurization and boiling groups were determined as 25 ± 1.24, 23 ± 0.98 and 26 ± 1.03, respectively. While no peak was obtained in the boiling group after PMA application, the mean Ct values of the control and pasteurization groups were 28 ± 1.38 and 31 ± 1.46, respectively. As a result, it was concluded that live C.parvum cysts in milk could be detected by PMA-qPCR method and live oocysts could be found in pasteurized milk.

Genomic Heterogeneity of Cryptosporidium parvum Isolates From Children in Bangladesh: Implications for Parasite Biology and Human Infection.

Cryptosporidium species are a major cause of diarrhea and associated with growth failure. There is currently only limited knowledge of the parasite's genomic variability. We report a genomic analysis of Cryptosporidium parvum isolated from Bangladeshi infants and reanalysis of sequences from the United Kingdom. Human isolates from both locations shared 154 variants not present in the cattle-derived reference genome, suggesting host-specific adaptation of the parasite. Remarkably 34.6% of single-nucleotide polymorphisms unique to human isolates were nonsynonymous and 8.2% of these were in secreted proteins. Linkage disequilibrium decay indicated frequent recombination. The genetic diversity of C. parvum has potential implications for vaccine and therapeutic design. Clinical Trials Registration. NCT02764918.

Functional characterization of CpADF, an actin depolymerizing factor protein in Cryptosporidium parvum.

Cryptosporidium is a highly pathogenic water and food-borne zoonotic parasitic protozoan that causes severe diarrhea in humans and animals. Apicomplexan parasites invade host cells via a unique motility process called gliding, which relies on the parasite's microfilaments. Actin depolymerizing factor (ADF) is a fibrous-actin (F-actin) and globular actin (G-actin) binding protein essential for regulating the turnover of microfilaments. However, the role of ADF in Cryptosporidium parvum (C. parvum) remains unknown. In this study, we preliminarily characterized the biological functions of ADF in C. parvum (CpADF). The CpADF was a 135-aa protein encoded by cgd5_2800 gene containing an ADF-H domain. The expression of cgd5_2800 gene peaked at 12 h post-infection, and the CpADF was located in the cytoplasm of oocysts, middle region of sporozoites, and cytoplasm of merozoites. Neutralization efficiency of anti-CpADF serum was approximately 41.30%. Actin sedimentation assay revealed that CpADF depolymerized but did not undergo cosedimentation with F-actin and its ability of F-actin depolymerization was pH independent. These results provide a basis for further investigation of the roles of CpADF in the invasion of C. parvum.

First identification of Cryptosporidium parvum virus 1 (CSpV1) in various subtypes of Cryptosporidium parvum from diarrheic calves, lambs and goat kids from France.

Cryptosporidium spp. remain a major cause of waterborne diarrhea and illness in developing countries and represent a significant burden to farmers worldwide. Cryptosporidium parvum virus 1 (CSpV1), of the genus Cryspovirus, was first reported to be present in the cytoplasm of C. parvum in 1997. Full-length genome sequences have been obtained from C. parvum from Iowa (Iowa), Kansas (KSU) and China. We aimed at characterizing the genome of CSpV1 from France and used sequence analysis from Cryptosporidium isolates to explore whether CSpV1 genome diversity varies over time, with geographical sampling location, C. parvum genetic diversity, or ruminant host species. A total of 123 fecal samples of cattle, sheep and goats were collected from 17 different French departments (57 diseased animal fecal samples and 66 healthy animal fecal samples). Subtyping analysis of the C. parvum isolates revealed the presence of two zoonotic subtype families IIa and IId. Sequence analysis of CSpV1 revealed that all CSpV1 from France, regardless of the subtype of C. parvum (IIaA15G2R1, IIaA17G2R1 and IIdA18G1R1) are more closely related to CSpV1 from Turkey, and cluster on a distinct branch from CSpV1 collected from C. parvum subtype IIaA15G2R1 from Asia and North America. We also found that samples collected on a given year or successive years in a given location are more likely to host the same subtype of C. parvum and the same CSpV1 strain. Yet, there is no distinct clustering of CSpV1 per French department or ruminants, probably due to trade, and transmission of C. parvum among host species. Our results point towards (i) a close association between CSpV1 movement and C. parvum movement, (ii) recent migrations of C. parvum among distantly located departments and (iii) incidental transmission of C. parvum between ruminants. All together, these results provide insightful information regarding CSpV1 evolution and suggest the virus might be used as an epidemiological tracer for C. parvum. Future studies need to investigate CSpV1's role in C. parvum virulence and on subtype ability to infect different species.

MiR-3976 regulates HCT-8 cell apoptosis and parasite burden by targeting BCL2A1 in response to Cryptosporidium parvum infection.

BACKGROUND: Cryptosporidium is second only to rotavirus as a cause of moderate-to-severe diarrhea in young children. There are currently no fully effective drug treatments or vaccines for cryptosporidiosis. MicroRNAs (miRNAs) are involved in regulating the innate immune response to Cryptosporidium parvum infection. In this study, we investigated the role and mechanism of miR-3976 in regulating HCT-8 cell apoptosis induced by C. parvum infection. METHODS: Expression levels of miR-3976 and C. parvum burden were estimated using real-time quantitative polymerase chain reaction (RT-qPCR) and cell apoptosis was detected by flow cytometry. The interaction between miR-3976 and B-cell lymphoma 2-related protein A1 (BCL2A1) was studied by luciferase reporter assay, RT-qPCR, and western blotting. RESULTS: Expression levels of miR-3976 were decreased at 8 and 12 h post-infection (hpi) but increased at 24 and 48 hpi. Upregulation of miR-3976 promoted cell apoptosis and inhibited the parasite burden in HCT-8 cells after C. parvum infection. Luciferase reporter assay indicated that BCL2A1 was a target gene of miR-3976. Co-transfection with miR-3976 and a BCL2A1 overexpression vector revealed that miR-3976 targeted BCL2A1 and suppressed cell apoptosis and promoted the parasite burden in HCT-8 cells. CONCLUSIONS: The present data indicated that miR-3976 regulated cell apoptosis and parasite burden in HCT-8 cells by targeting BCL2A1 following C. parvum infection. Future study should determine the role of miR-3976 in hosts' anti-C. parvum immunity in vivo.

Cryptosporidium parvum gp60 subtypes in diarrheic lambs and goat kids from Israel.

Cryptosporidium parvum is the second-most prevalent Cryptosporidium species that infects humans worldwide. In European countries, it is the most prevalent species in sheep, suggesting that these animals are a source of zoonotic infection. Preweaned lambs and goats are particularly susceptible to infection by the parasite and may suffer from severe diarrhea whilst excreting large quantities of infectious oocysts. Fifty fecal samples from preweaned lambs and goats with diarrhea from 35 farms across Israel, found to be Cryptosporidium-positive by microscopy, were tested by PCR and sequence analyses to determine the infective species and subtypes. Cryptosporidium parvum DNA was detected in most samples from both lambs and goats (46/50). Cryptosporidium xiaoi DNA was detected in three samples from kids, with co-infection detected in a single sample. Eleven different C. parvum subtypes were found, 10 in lambs and 5 in goats. All subtypes were from the IIa and IId subtype families, with subtypes IIdA20G1 and IIaA15G2R1 being the most prevalent and widespread. These subtypes were previously found in calves and humans in Israel and are considered the most prevalent C. parvum subtypes in small ruminants globally. These results underline the zoonotic potential of C. parvum from small ruminants and the high subtype diversity compared to previous reports from other Middle Eastern countries. In addition, this is the first report of C. xiaoi in Israel.

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. SIGNIFICANCE: There 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 48 h 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.

Cryptosporidium parvum hijacks a host's long noncoding RNA U90926 to evade intestinal epithelial cell-autonomous antiparasitic defense.

Cryptosporidium is a zoonotic apicomplexan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans. It is an important opportunistic pathogen in AIDS patients and a leading cause of infectious diarrhea and diarrheal-related death in children worldwide. The intestinal epithelial cells provide the first line of defense against Cryptosporidium infection and play a central role in activating and regulating the host's antiparasitic response. Increasing evidence suggests that long noncoding RNAs (lncRNAs) participate in host-pathogen interactions and play a regulatory role in the pathogenesis of diseases but the underlying molecular mechanisms are not fully understood. We previously identified a panel of host lncRNAs that are upregulated in murine intestinal epithelial cells following Cryptosporidium infection, including U90926. We demonstrate here that U90926 is acting in a pro-parasitic manner in regulating intestinal epithelial cell-autonomous antiparasitic defense. Inhibition of U90926 resulted in a decreased infection burden of the parasite while overexpression of U90926 showed an increase in infection burden in cultured murine intestinal epithelial cells. Induction of U90926 suppressed transcription of epithelial defense genes involved in controlling Cryptosporidium infection through epigenetic mechanisms. Specifically, transcription of Aebp1, which encodes the Aebp1 protein, a potent modulator of inflammation and NF-κB signaling, was suppressed by U90926. Gain- or loss-of-function of Aebp1 in the host's epithelial cells caused reciprocal alterations in the infection burden of the parasite. Interestingly, Cryptosporidium carries the Cryptosporidium virus 1 (CSpV1), a double-stranded (ds) RNA virus coding two dsRNA fragments, CSpV1-dsRdRp and CSpV1-dsCA. Both CSpV1-dsRdRp and CSpV1-dsCA can be delivered into infected cells as previously reported. We found that cells transfected with in vitro transcribed CSpV1-dsCA or CSpV1-dsRdRp displayed an increased level of U90926, suggesting that CSpV1 is involved in the upregulation of U90926 during Cryptosporidium infection. Our study highlights a new strategy by Cryptosporidium to hijack a host lncRNA to suppress epithelial cell-autonomous antiparasitic defense and allow for a robust infection.

Outbreak of severe diarrhea due to zoonotic Cryptosporidium parvum and C. xiaoi in goat kids in Chungcheongbuk-do, Korea.

Severe diarrhea was reported in goat kids in Chungcheongbuk-do, Korea, from 2021 to 2023, and Cryptosporidium infection was suspected. To confirm the cause of this outbreak, fecal samples were collected from goat farms where diarrhea had been reported and analyzed for Cryptosporidium infection using a molecular assay. A total of 65 fecal samples, including 37 from goats with diarrhea and 28 from goats without diarrhea, were collected from six goat farms. Forty-eight of the goats were kids (<2 months) and 17 were adults (>1 year). Cryptosporidium was identified in 53.8% (35/65) of total samples. Overall, 86.5% (32/37) of the diarrheic fecal samples tested positive; however, Cryptosporidium was not detected in any fecal sample from non-diarrheic adult goats. Therefore, cryptosporidiosis was significantly associated with diarrhea in goat kids, and adult goats were not responsible for transmission of Cryptosporidium to them. Phylogenetic analysis and molecular characterization revealed two Cryptosporidium species, namely, C. parvum (n = 28) and C. xiaoi (n = 7). In the C. parvum-positive samples, gp60 gene analysis revealed three zoonotic subtypes-IIaA18G3R1, IIdA15G1, and IIdA16G1. To the best of our knowledge, this study is the first to identify C. parvum IIaA18G3R1 and IIdA16G1 in goats, as well as the first to identify C. xiaoi in goats in Korea. These results suggest that goat kids play an important role as reservoir hosts for different Cryptosporidium species and that continuous monitoring with biosecurity measures is necessary to control cryptosporidiosis outbreaks.