Cryptosporidium Parvum Infection Research Articles

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

Assessment of the Immune Response of Clinically Infected Calves to Cryptosporidium parvum Infection

Cryptosporidium parvum (C. parvum) infection is one of the main causes of diarrhea in calves. The current study assessed the role of blood biomarkers (acute-phase proteins (APPs), procalcitonin, neopterin, cytokines, and oxidative stress in the pathogenesis, diagnosis, and prognosis of calves naturally infected with C. parvum. Fifty-seven calves, aged from 10 to 45 days, were detected positive for C. parvum and were allotted into the diseased group; twenty healthy calves were selected as a control group. Serum amyloid A, haptoglobin, cytokines, neopterin, procalcitonin, and stress biomarkers were tested in the diseased and healthy groups. The serum levels of APPs, cytokines, neopterin, procalcitonin, and malondialdehyde increased, whereas antioxidant levels were significantly decreased in diseased calves compared to the healthy group. Moreover, all examined biomarkers were significantly altered towards normal values in infected calves following different treatment protocols. All biomarkers examined were highly effective in discriminating between C. parvum-infected calves and healthy individuals. Furthermore, the area under the curve (AUC) showed that all tested parameters had a higher degree of prognostic accuracy in predicting the treatment response of calves naturally infected with C. parvum. Our data suggest the usefulness of the examined biomarkers in the immune pathogenesis of the C. parvum infection in calves, contributing to diagnosis and treatment efficacy.

RNA-Based Therapy for Cryptosporidium parvum Infection: Proof-of-Concept Studies.

Cryptosporidium is a leading cause of moderate-to-severe diarrhea in children, which is one of the major causes of death in children under 5 years old. Nitazoxanide is the only FDA-approved treatment for cryptosporidiosis. However, it has limited efficacy in immunosuppressed patients and malnourished children. Therefore, it is urgent to develop novel therapies against this parasite. RNA interference-mediated therapies are emerging as novel approaches for the treatment of infectious diseases. We have developed a novel method to silence essential genes in Cryptosporidium using single-stranded RNA (ssRNA)/Argonaute (Ago) complexes. In this work we conducted proof-of-concept studies to test the anticryptosporidial activity of these complexes by silencing Cryptosporidium parvum nucleoside diphosphate kinase (NDK) using in vitro and in vivo models. We demonstrated that a 3-day treatment with anti-sense NDK ssRNA/Ago decreased parasite burden by ~98% on infected cells. In vivo studies showed that ssRNA/Ago complexes encapsulated in lipid nanoparticles can be delivered onto intestinal epithelial cells of mice treated orally. In addition a cryptosporidiosis-mouse model showed that treatment with NDK ssRNA/Ago complexes reduced oocyst shedding in 4/5 SCID/beige mice during the acute phase of the infection. Our findings highlight the potential use of antisense RNA-based therapy as an alternative approach to cryptosporidiosis treatment.

Age and episode-associated occurrence of Cryptosporidium species and subtypes in a birth-cohort of dairy calves.

The role of species-specific immunity in infection patterns of Cryptosporidium spp. in humans and farm animals is not well understood. In the present study, the dynamics of Cryptosporidium infections in a natural cryptosporidiosis model was examined using genotyping, subtyping and whole genome sequencing tools. In a cross-sectional survey of Cryptosporidium spp. in 934 dairy cattle on one farm, marked age-associated differences in the distribution of Cryptosporidium species and C. bovis subtypes were observed. In a closely followed longitudinal birth cohort study of 81 calves over a 9-month period, shedding of C. parvum oocysts by the IIdA19G1 subtype started at 4 days, peaked at 2weeks and ended mostly by 4weeks. In contrast, the shedding of C. bovis oocysts started at 2weeks, peaked initially at 6weeks and had a second wave during 15th to 23rd weeks. For C. ryanae, calves had mostly only one episode of infection by one subtype, with accumulative infection increasing much slower than C. parvum and C. bovis. Overall, the accumulative infection rates and mean duration of oocyst shedding for calves in the cohort were 97.4% (76/78) and 2.3weeks, 100.0% (80/80) and 3.9weeks, and 78.7% (63/80) and 3.2weeks for C. parvum, C. bovis and C. ryanae, respectively. The oocyst shedding intensity was much lower in C. bovis and C. ryanae infections compared with C. parvum infection, and in the second episode of C. bovis infection compared with the first episode. The two episodes of C. bovis infections were caused by different genome types that differed mostly in nine genes. Cryptosporidium parvum infection was associated with the occurrence of watery diarrhoea. Data from the natural history study of cryptosporidiosis indicate that despite the existence of acquired immunity against homologous pathogens, neonatal animals experience waves of Cryptosporidium infections by different species and genome types.

Management of calves in commercial dairy farms in Mecklenburg-Western Pomerania, Germany and its impact on calf mortality and prevalence of rotavirus and Cryptosporidium parvum infections in pre-weaned calves

In a cross-sectional study, impact of management in dairy farms on calf mortality rates and prevalence of rotavirus and Cryptosporidium parvum in feces of calves was investigated. Sixty-two commercial dairy herds in Mecklenburg-Western Pomerania, Germany, were stratified selected in 2019. We performed in-person interviews and fecal specimens in samples of all-female calves of age 7 up to 21 days. Management data were documented on farm level. A Multiscreen Ag-ELISA was performed to determine rotavirus and Cryptosporidium parvum. Associations between two calf mortality rates, detection of C. parvum and rotavirus, and predictors were examined with GLM models. In farms with routine vaccination against respiratory diseases, 31-days mortality rate was 4.2% +/-1.26 compared to 7.6% +/-0.97 (p = 0.040) on non-vaccinating farms. Six-months mortality was lower in farms that continued feeding milk to calves during periods of diarrhea compared to farms that did not (6.9% +/-0.8 vs. 12.4% +/-2.3). In case of a routine shifting of calves from the calving box into calf boxes less C. parvum was detected compared to an individual moving of calves (33.3% +/-2.6 vs. 19.6% +/-5.3; p = 0.024). Our model confirms a positive association between occurrence of aqueous feces and frequency of detection of C. parvum (45.4% +/-23.6 vs. 21.4% +/-18.7; p < 0.001). Frequency of detection of rotavirus was lower in farms that reported a defined amount of applicated colostrum per calf than in farms that presented a range of colostrum instead of a defined amount. This study indicates the potential for mitigation of risk factors for mortality in calves.

Curcumin mitigates Cryptosporidium parvum infection through modulation of gut microbiota and innate immune-related genes in immunosuppressed neonatal mice

Cryptosporidium parvum is a major cause of diarrheal disease in immature or weakened immune systems, mainly in infants and young children in resource-poor settings. Despite its high prevalence, fully effective and safe drugs for the treatment of C. parvum infections remain scarce, and there is no vaccine. Meanwhile, curcumin has shown protective effects against C. parvum infections. However, the mechanisms of action and relationship to the gut microbiota and innate immune responses are unclear. Immunosuppressed neonatal mice were infected with oocysts of C. parvum and either untreated or treated with a normal diet, curcumin or paromomycin. We found that curcumin stopped C. parvum oocysts shedding in the feces of infected immunosuppressed neonatal mice, prevented epithelial damage, and villi degeneration, as well as prevented recurrence of infection. Curcumin supplementation increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes and Proteobacteria in mice infected with C. parvum as shown by 16S rRNA gene sequencing analysis. The relative abundance of Lactobacillus, Bacteroides, Akkermansia, Desulfovibrio, Prevotella, and Helicobacter was significantly associated with C. parvum infection inhibited by curcumin. Curcumin significantly (P < 0.01) suppressed IFN-γ and IL -18 gene expression levels in immunosuppressed neonatal C. parvum-infected mice. We demonstrate that the therapeutic effects curcumin are associated with alterations in the gut microbiota and innate immune-related genes, which may be linked to the anti-Cryptosporidium mechanisms of curcumin.

Chitosan Protects Immunosuppressed Mice Against Cryptosporidium parvum Infection Through TLR4/STAT1 Signaling Pathways and Gut Microbiota Modulation.

Cryptosporidium parvum infection is very common in infants, immunocompromised patients, or in young ruminants, and chitosan supplementation exhibits beneficial effects against the infection caused by C. parvum. This study investigated whether chitosan supplementation modulates the gut microbiota and mediates the TLR4/STAT1 signaling pathways and related cytokines to attenuate C. parvum infection in immunosuppressed mice. Immunosuppressed C57BL/6 mice were divided into five treatment groups. The unchallenged mice received a basal diet (control), and three groups of mice challenged with 1 × 106 C. parvum received a basal diet, a diet supplemented with 50 mg/kg/day paromomycin, and 1 mg/kg/day chitosan, and unchallenged mice treated with 1 mg/kg/day chitosan. Chitosan supplementation regulated serum biochemical indices and significantly (p < 0.01) reduced C. parvum oocyst excretion in infected mice treated with chitosan compared with the infected mice that received no treatment. Chitosan-fed infected mice showed significantly (p < 0.01) decreased mRNA expression levels of interferon-gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) compared to infected mice that received no treatment. Chitosan significantly inhibited TLR4 and upregulated STAT1 protein expression (p < 0.01) in C. parvum-infected mice. 16S rRNA sequencing analysis revealed that chitosan supplementation increased the relative abundance of Bacteroidetes/Bacteroides, while that of Proteobacteria, Tenericutes, Defferribacteres, and Firmicutes decreased (p < 0.05). Overall, the findings revealed that chitosan supplementation can ameliorate C. parvum infection by remodeling the composition of the gut microbiota of mice, leading to mediated STAT1/TLR4 up- and downregulation and decreased production of IFN-γ and TNF-α, and these changes resulted in better resolution and control of C. parvum infection.

Detection of Cryptosporidiosis in Dogs of Veterinary Clinics in Surabaya City Using Acid-Fast Staining and PCR

The need for maintaining pets, such as dogs, is increasing along with the human population. When individuals keep dogs as their pets, they must be aware of disease transmission from dogs. One of the disease agents transmitted from pets to their owners is Cryptosporidium spp. causing cryptosporidiosis. The aim of the present study was to detect Cryptosporidium spp. infection in dogs through a fecal examination using the acid-fast staining method (Ziehl Neelsen) confirmed with the molecular examination of Polymerase Chain Reaction (PCR). Detection of Cryptosporidium sp. in feces of dogs was set up by using an acid-fast staining method. Positive results of the acid-fast staining were further confirmed using PCR. Polymerase Chain Reaction used primary AB210854 specific to the Cryptosporidium canis and S139-S141 genes which were specific primary for the Cryptosporidium parvum gene. Results of the acid-fast staining showed that 80% of the samples (40 samples from total samples) were infected with Cryptosporidium spp. Further detection using PCR showed that four samples were positive for Cryptosporidium canis infection, and two samples showed positive results of Cryptosporidium parvum infection. Dog samples were mostly infected with Cryptosporidium spp. including Cryptosporidium canis and Cryptosporidium parvum through a fecal examination using acid-fast staining and PCR. Keywords: Acid-fast staining, Cryptosporidium spp., Dogs, PCR

Persistent Cryptosporidium parvum Infection Leads to the Development of the Tumor Microenvironment in an Experimental Mouse Model: Results of a Microarray Approach.

Cryptosporidium spp. are enteric protozoa parasites that infect a variety of vertebrate hosts. These parasites are capable of inducing life-threatening gastrointestinal disease in immunocompromised individuals. With the rising epidemiological evidence of the occurrence of Cryptosporidium infections in humans with digestive cancer, the tumorigenic potential of the parasite has been speculated. In this regard, Cryptosporidium parvum has been reported to induce digestive adenocarcinoma in a rodent model of chronic cryptosporidiosis. However, the processes by which the parasite could induce this carcinogenesis are still unknown. Therefore, the transcriptomes of C. parvum infected ileo-cecal regions of mice developing tumors were analyzed in the current study. For the first time, downregulation of the expression of α-defensin, an anti-microbial target of the parasite in response to C. parvum infection was observed in the transformed tissues. This phenomenon has been speculated to be the result of resistance of C. parvum to the host defense through the upregulated expression of interferon γ-stimulated genes. The inflammatory response generated as result of attenuated expression of anti-microbial peptides highlights the role of immune evasion in the C. parvum-induced tumorigenesis. The study has also succeeded in the characterization of the tumor microenvironment (TME) which is characterized by the presence of cancer associated fibroblasts, myeloid-derived suppressor cells, tumor-associated macrophages and extracellular matrix components. Identification of immune suppressor cells and accumulation of pro-inflammatory mediators speculates that chronic inflammation induced by persistent C. parvum infection assists in development of an immunosuppressive tumor microenvironment.

Prophylactic and therapeutic efficacy of clinoptilolite against Cryptosporidium parvum in experimentally challenged neonatal lambs

This study was designed to test the prophylactic and therapeutic efficacy of clinoptilolite against Cryptosporidium (C.) parvum infection in lambs. Within the first day of life, three groups of 10 lambs were each inoculated with 1 × 106 oocysts of C. parvum. The prophylactic (PROP) group received orally clinoptilolite supplemented at a rate of 3% to the colostrum within the first day of life before inoculation and then to milk replacer for two weeks. The therapeutic (TREA) group was supplemented with the same rate, route and duration of clinoptilolite, starting from the day of the first appearance of oocysts in the faeces. The positive control group (pCON) was left untreated and fed only the basal diet. Disease development and clinoptilolite efficacy were assessed daily by evaluating oocyst per gram of faeces (OPG) counts, faecal consistent score (FCS), and clinical health score (CHS) from day -1 to 20 days post inoculation. A significantly (p < 0.001) lower OPG value was found in the PROP and TREA groups in comparison to the pCON group. The FCS and CHS were decreased in the PROP (p < 0.01 and p < 0.001) and TREA (p < 0.05 and p < 0.001) groups compared to the pCON group, respectively. The percentage efficacy of clinoptilolite was calculated to be 97.4 % in the PROP group and 91.6 % in the TREA group.In conclusion, this study proved for the first time that clinoptilolite has promising prophylactic and therapeutic activities against C. parvum in experimentally infected lambs.

Correlation between Cryptosporidium parvum and Helicobacter Pylori Infections in Gastrointestinal Patients at Wasit Province

The present study aimed to identify the relationship between Cryptosporidium parvum parasite andHelicobacter pylori in patients at Wasit province and its environs. One hundred stool samples were collected(male and female) from suspected diarrheal patients of parasitic and bacterial infection during the periodOctober 2020 to April 2021 who attended to Al-Karamah Teaching Hospital at Wasit province and GeneralHospital of Martyr Fairuz at Hay district.The investigation of the oocysts of the Cryptosporidium parvum parasite was carried out by staining withModified Ziehl Neelson method and also detecting of Helicobacter pylori by using dipstick. The results ofour study revealed that 55( 55%) was positive for C. parvum and 48 (48%) was positive for Helicobacterpylori and the co- infections between the parasite and bacteria reached to 32( 32%). The age group(1-20years) showed the highest11 (11%) prevalence rate while the lowest prevalence was in patient with agegroup (21-40 years) reached to 6(6%) interplay infections between the parasite and bacteria .

Autophagy - related 16 - like 1 single nucleotide gene polymorphism increases the risk and severity of Cryptosporidium parvum infection

Cryptosporidium parvum (C. parvum) is an intracellular parasite of the intestinal cells. It causes cryptosporidiosis that can be fatal in immunosuppressed individuals. Autophagy is a process to eliminate intracellular microbes. The autophagy-related 16 – like 1 (ATG16L1) gene encodes proteins involved in the autophagy pathway. Single nucleotide polymorphism (SNP) in this gene increases the invasion and survival of the intracellular microbes. This study aimed to assess whether SNP in the ATG16L1 gene influences the risk and severity of cryptosporidiosis. Group I: cases with C. parvum infection (C. parvum, n = 40) and group II: healthy control (HC, n = 120) were included. Genotyping of the ATG16L1 gene was done for all participants to determine the polymorphism status as AA, GG, or AG genotype. A significant association between C. parvum infection and ATG16L1 genotypes was detected. C. parvum group had a significantly higher frequency of GG genotype and G allele when compared to HC group. The genotypes (AG + GG) and G allele had 2.428 and 2.13 folds risk of C. parvum infection when compared to the AA genotype and the A allele. Patients with the AG + GG genotype had statistically significant higher Cryptosporidium oocyst counts in stool, higher infection intensity, more frequency of vomiting and dehydration, longer disease duration, and more recurrence. The GG or AG genotypes were independent risk factors in the disease severity (p- value = 0.013). In conclusion, ATG16L1 SNP increased the risk and severity of cryptosporidiosis. Thus, individuals with such SNP can benefit from autophagy up-regulating approaches in decreasing the risk and controlling C. parvum infection.

Dual transcriptomics to decipher the interferon-gamma independent responses to intestinal Cryptosporidium parvum infection.

Abstract Background: This study characterizes the IFNγ independent host immune response against C. parvum to discover new targets to halt the disease. We used transcriptomic analysis of ileum and cecum intestinal sections of IFNγ-deleted mice that were treated with Soluble Toxoplasma gondii antigen (STAg) or PBS upon C. parvum infection. Results: STAg treatment reduced the oocyst shedding in C. parvum infected IFNγ deleted mice. To understand the IFNγ independent mechanism of STAg treatment against C. parvum, we performed 12 RNAseq comparisons between tissues, treatments, and infection status for Mus musculus and C. parvum. In the ileum, regardless of STAg or PBS treatment, more than 100 genes had a significant differential expression. Within STAg treated mice, a total of 129 genes for ileum and 78 for the cecum were classified as differentially expressed. We found in high abundance at least 28 genes related to IFN type I response in infected mice treated with STAg, among them, the oligoadenylate synthetase and Schlafen family gene members. For C. parvum transcriptome analysis, we observed in the ileum high abundance of the oocyst wall protein 1 and some mucins; while in the cecum, the RRM domain containing-protein and Tryptophan synthase beta chain transcripts were the more abundant transcripts. Conclusions: STAg treatment and C. parvum infection induced a type I interferon response that would be the cause of the reduction of oocyst shedding in STAG treated mice in an IFNy independent manner. While the depth of sequence did not allow us to observe in detail differential expression of genes in C. parvum, we found differences in parasite gene expression between the ileum and the cecum.

ROLE OF APOPTOSIS IN EXPERIMENTAL CRYPTOSPORIDIUM PARVUM INFECTED ALBINO MICE

Cryptosporidium is one of the commonest opportunistic parasites. But, its chemotherapeuticoptions are limited, with controversy about the role of apoptosis in cryptosporidiosis. Caspasesare key molecules of apoptosis. The study assessed the role of apoptosis in cryptosporidiosisby studying the relationship between the effects of phenyl vinyl sulfone (PVS), nitazoxanide(NTZ) and combined therapy on Cryptosporidium parvum infection and caspase 3immunostaining. Ninety female laboratory bred Swiss Albino mice were divided into twomajor groups; immunocompetent and immunosuppressed. Both groups were divided into thefollowing subgroups, for each one respectively: negative control (1 & 6), infected control (2& 7), infected treated with PVS (3 & 8), infected treated with NTZ (4 & 9) and infected treatedcombined (5 & 10). Sacrification were done on 18th day post infection.Histopathological study of ileum was done and endogenous developmental stages werecounted. Also, caspase 3 immunohistochemical staining was carried out on ileal sections.Combined therapy in groups 5 &10 showed the highest reduction in mean number of endogenousdevelopmental stages as in G10 (P <0.05). Caspase expression in PVS treated groups (3& 8) showed significant decrease when compared to their control groups (P < 0.05). Whilethere were significant increase in caspase expression in groups treated with NTZ (4 & 9)when compared to their contorls (P < 0.05). Caspase 3 epithelial overexpression was evidentin infected immunocompetent G2 than immunosuppressed G7 (P<0.05).

Dendritic cell TLR4 induces Th1-type immune response against Cryptosporidium parvum infection.

The objective of this study was to investigate the mechanism of Toll-like receptor (TLR4)- mediated dendritic cell (DC) immune against Cryptosporidium parvum infection. C. parvum sporozoites were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester. Murine bone marrow-derived DCs were isolated, and divided into TLR4 antibody blocking (TAB; infected with 2 × 105 labeled sporozoites and 0.5 μg TLR4 blocking antibody), TLR4 antibody unblocking (TAU; infected with 2 × 105 labeled sporozoites), and blank control (BC; with 1.5 mL Roswell Park Memorial Institute 1640 medium) groups. The adhesion of Cryptosporidium sporozoites to DCs and CD11c+ levels were examined by fluorescence microscopy and flow cytometry. Male KM mice were orally injected with C. parvum. The proliferation of T lymphocytes in spleen, expression of cytokines in peripheral blood, and TLR4 distribution features in different organs were further determined by immunohistochemistry. A significantly higher expression of CD11c+ and higher C. parvum sporozoite adhesion were found in the TAU group compared with other groups. The expression of CD4+CD8- /CD8+CD4- in the spleen were obviously differences between the TAB and TAU groups. The expression of TLR4, interleukin IL-4, IL-12, IL-18 and IFN-γ improved in the TAU group compared with TAB group. Higher expression of TLR4 was detected in the lymph nodes of mice in the TAU group, with pathological changes in the small intestine. Hence, TLR4 could mediate DCs to recognize C. parvum, inducing Th1 immune reaction to control C. parvum infection.

Molecular Epidemiology of Human Cryptosporidiosis in Low- and Middle-Income Countries.

Cryptosporidiosis is one of the most important causes of moderate to severe diarrhea and diarrhea-related mortality in children under 2 years of age in low- and middle-income countries. In recent decades, genotyping and subtyping tools have been used in epidemiological studies of human cryptosporidiosis. Results of these studies suggest that higher genetic diversity of Cryptosporidium spp. is present in humans in these countries at both species and subtype levels and that anthroponotic transmission plays a major role in human cryptosporidiosis. Cryptosporidium hominis is the most common Cryptosporidium species in humans in almost all the low- and middle-income countries examined, with five subtype families (namely, Ia, Ib, Id, Ie, and If) being commonly found in most regions. In addition, most Cryptosporidium parvum infections in these areas are caused by the anthroponotic IIc subtype family rather than the zoonotic IIa subtype family. There is geographic segregation in Cryptosporidium hominis subtypes, as revealed by multilocus subtyping. Concurrent and sequential infections with different Cryptosporidium species and subtypes are common, as immunity against reinfection and cross protection against different Cryptosporidium species are partial. Differences in clinical presentations have been observed among Cryptosporidium species and C. hominis subtypes. These observations suggest that WASH (water, sanitation, and hygiene)-based interventions should be implemented to prevent and control human cryptosporidiosis in low- and middle-income countries.

Cryptosporidiosis outbreak caused by Cryptosporidium parvum subtype IIdA20G1 in neonatal calves.

Cryptosporidium parvum is a major zoonotic pathogen responsible for outbreaks of severe diarrhoea in humans and calves. Almost all investigations of cryptosporidiosis outbreaks caused by C. parvum have focused on its IIa subtype family in industrialized nations. From December 2018 to April 2019, approximately 200 neonatal calves on a large cattle farm in Hebei Province, China, were diagnosed with watery diarrhoea and over 40 died. To investigate the cause of the outbreak, faecal samples were taken during and after the outbreak from neonatal calves of ≤4weeks of age (n=40 and n=56) and older calves of 4-24weeks of age (n=79 and n=38). A total of 18 faecal samples collected from ill calves at the peak of the outbreak were analysed for four common enteric pathogens using an enzymatic immunoassay (EIA). In addition, 75 samples from neonatal calves were tested for rotavirus by EIA. All samples were analysed for Cryptosporidium spp. using PCR and sequencing techniques. Of the initial 18 samples from sick calves, ten were positive for C. parvum, five for rotavirus, and one for coronavirus. The overall prevalence of rotavirus in neonatal calves was 20.0% (15/75), with no significant differences during and after the outbreak. In contrast, Cryptosporidium parvum infections were significantly higher during the outbreak (60.0%, 24/40) than after the outbreak (30.4%, 17/56; p=.004). Cryptosporidium parvum infection was associated with the presence of watery diarrhoea in neonatal calves (OR=11.19), while no association was observed between C. bovis infection and diarrhoea. All C. parvum isolates were identified as subtype IIdA20G1. This is one of the few reports of outbreaks of severe diarrhoea caused by C. parvum IId subtypes in calves. More attention should be directed towards the dissemination of C. parvum in China.