Cryptosporidium Parvum Infection Research Articles

Haptoglobin and serum amyloid-A concentrations and their relationship with oocyst count in neonatal lambs experimentally infected with Cryptosporidium parvum

This study aimed to evaluate the acute phase response (APR) through haptoglobin (Hp) and serum amyloid A (SAA) concentrations in serum and to examine the correlation between these acute phase proteins (APPs) and oocyst shedding using experimental Cryptosporidium parvum (C. parvum) infection model in neonatal lambs. Twenty lambs were divided into two equal groups: group CON remained uninfected as negative control and lambs of the group EXP were inoculated orally with 1×106C. parvum oocysts. Blood and faecal samples were obtained from both groups before colostrum intake and prior to inoculation (day-1), and at 2, 6, 13, and 20days post-inoculation (dpi). The serum concentrations of SAA increased following the experimental infection of lambs with C. parvum, the difference being statistically significant from pre-inoculation levels at 2 dpi, while significant increases in serum concentration of Hp were observed at 2 and 6 dpi. At the same occasions, serum concentrations of both APPs were significantly higher in the C. parvum-infected lambs compared to the healthy control lambs. A moderate positive correlation (rho=0.67; p< 0.001) was observed between serum Hp concentration and oocyst count (OPG),whereas the serum SAA concentration didn’t significantly correlate with OPG (rho=0.18; p>0.05). In conclusion, the results of the study shed some light on APR due to C. parvum infection in neonatal lambs.

Delivery of parasite Cdg7_Flc_0990 RNA transcript into intestinal epithelial cells during Cryptosporidium parvum infection suppresses host cell gene transcription through epigenetic mechanisms.

Cryptosporidial infection causes dysregulated transcription of host genes key to intestinal epithelial homeostasis, but the underlying mechanisms remain obscure. Previous studies demonstrate that several Cryptosporidium parvum (C.parvum) RNA transcripts are selectively delivered into epithelial cells during host cell invasion and may modulate gene transcription in infected cells. We report here that C.parvum infection suppresses the transcription of LRP5, SLC7A8, and IL33 genes in infected intestinal epithelium. Trans-suppression of these genes in infected host cells is associated with promoter enrichment of suppressive epigenetic markers (i.e., H3K9me3). Cdg7_FLc_0990, a C.parvum RNA that has previously demonstrated to be delivered into the nuclei of infected epithelial cells, is recruited to the promoter regions of LRP5, SLC7A8, and IL33 genes. Cdg7_FLc_0990 appears to be recruited to their promoter regions together with G9a, a histone methyltransferase for H3K9 methylation. The PR domain zinc finger protein 1, a G9a-interacting protein, is required for the assembly of Cdg7_FLc_0990 to the G9a complex and gene-specific enrichment of H3K9 methylation. Our data demonstrate that cryptosporidial infection induces epigenetic histone methylations in infected cells through nuclear transfer of parasite Cdg7_Flc_0990 RNA transcript, resulting in transcriptional suppression of the LRP5, SLC7A8, and IL33 genes.

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum.

Cryptosporidium spp. are apicomplexan parasites of global importance that cause human diarrheal disease. In vitro culture models that may be used to study this parasite and that have physiological relevance to in vivo infection remain suboptimal. Thus, the pathogenesis of cryptosporidiosis remains poorly characterized, and interventions for the disease are limited. In this study, we evaluated the potential of a novel bioengineered three-dimensional (3D) human intestinal tissue model (which we developed previously) to support long-term infection by Cryptosporidium parvum Infection was assessed by immunofluorescence assays and confocal and scanning electron microscopy and quantified by quantitative reverse transcription-PCR. We found that C. parvum infected and developed in this tissue model for at least 17 days, the extent of the study time used in the present study. Contents from infected scaffolds could be transferred to fresh scaffolds to establish new infections for at least three rounds. Asexual and sexual stages and the formation of new oocysts were observed during the course of infection. Additionally, we observed ablation, blunting, or distortion of microvilli in infected epithelial cells. Ultimately, a 3D model system capable of supporting continuous Cryptosporidium infection will be a useful tool for the study of host-parasite interactions, identification of putative drug targets, screening of potential interventions, and propagation of genetically modified parasites.

Reducing gut effects from Cryptosporidium parvum infection in dairy calves through prophylactic glucagon-like peptide 2 therapy or feeding of an artificial sweetener

Glucagon-like peptide 2 (GLP-2) therapy was shown previously to reduce inflammation-related gut damage from coccidiosis in dairy calves, and feeding of artificial sweetener stimulates GLP-2 secretion from intestinal L cells. The purpose of this study was to determine whether GLP-2 treatment or artificial sweetener feeding beginning 1 wk before an experimental inoculation with the coccidian parasite Cryptosporidium parvum can reduce infection-related intestinal damage in Holstein bull calves. Newborn calves were assigned to 1 of 4 treatment groups of 6 calves each, including noninfected control calves injected s.c. every 12 h with control buffer (CON), infected control calves injected s.c. every 12 h with control buffer (INF), infected calves injected s.c. every 12 h with 50 µg/kg of body weight of GLP-2 (GLP2), and infected calves injected s.c. every 12 h with control buffer and supplemented in the diet with Sucram (Pancosma, Geneva, Switzerland) at 400 mg/kg of dry matter of milk replacer (SUC). Treatments were initiated on d 1, and calves in INF, GLP2, and SUC were orally dosed on d 8 with 12,500 C. parvum oocysts. Fecal scores were recorded daily, plasma was collected on d 1, 8, 12, 15, and 18 to evaluate markers of inflammation, and fecal samples were collected on d 1, 8, and every other day thereafter to determine the presence of oocysts. Calves were euthanized on d 18 for collection of intestinal tissues and histological and gene expression analyses. Relative to CON, calves in INF exhibited an increase in diarrhea severity, increased plasma serum amyloid A concentration on d 15 and 18, reduced intestinal villus height, increased villus apoptosis and crypt cell proliferation, and increased intestinal mRNA expression of MARVELD2 and GPX2. However, calves in SUC and GLP2 had reduced diarrhea severity and fecal C. parvum oocyst shedding, reduced plasma serum amyloid A concentration on d 15 and 18, and, depending on the intestinal segment, increased villus height, reduced crypt cell proliferation, and reduced mRNA expression of MARVELD2, GPX2, and other tight junction proteins relative to INF. Lastly, GLP2 and SUC exhibited increased intestinal mass-to-length ratio and decreased length-to-empty body weight ratio relative to INF. Our findings suggest that GLP-2 and Sucram treatments administered before a low-level C. parvum exposure may contribute to fewer effects on intestinal integrity, morphology, and inflammation in response to infection, and shorter, denser intestines.

Alterations in oxidative stress parameters and its associated correlation with clinical disease on experimental Cryptosporidium parvum infection in Swiss albino mice.

The present study was conducted to evaluate the changes in oxidative stress parameters on experimental infection with Cryptosporidium parvum in Swiss albino mice. The mice were divided into four groups viz., group I-IV, each group comprising of 15 mice. Group I mice served as healthy control. In Group II mice, C. parvum oocysts @ 104/os were administered, mice of group III were given dexamethasone @ 30µg/ml in drinking water whereas group IV mice were given dexamethasone @ 30µg/ml along with C. parvum oocysts @ 104/os. Significant changes were seen in oxidative stress parameters which included significant increase in LPO and decrease in levels of SOD, CAT and GSH in liver and intestine in group IV mice at 10th DPI when compared to others indicating an important role played by free radical induced oxidative stress in the development of C. parvum infection in mice which was clinically characterized by loss of body condition, profuse bloody diarrhoea and peak oocyst shedding intensity occurring at 10th DPI.

Cryptosporidium parvumincreases intestinal permeability through interaction with epithelial cells and IL-1β and TNFα released by inflammatory monocytes

Intestinal epithelial cells form a single layer separating the intestinal lumen containing nutriments and microbiota from the underlying sterile tissue and therefore play a key role in maintaining homeostasis. We investigated the factors contributing to the alteration of the epithelial barrier function during Cryptosporidium parvum infection. Infected polarized epithelial cell monolayers exhibit a drop in transepithelial resistance associated with a delocalization of E-cadherin and β-catenin from their intercellular area of contact, the adherens junction complex. In neonatal mice infected by C. parvum, the increased permeability is correlated with parasite development and with an important recruitment of Ly6c+ inflammatory monocytes to the subepithelial space. TNFα and IL-1β produced by inflammatory monocytes play a key role in the loss of barrier function. Our findings demonstrate for the first time that both the parasite and inflammatory monocytes contribute to the loss of intestinal barrier function during cryptosporidiosis.

Effects of Enterococcus faecalis CECT 7121 on Cryptosporidium parvum infection in mice.

Cryptosporidium is an opportunistic protozoan parasite of humans and animals worldwide and causes diarrheal disease that is typically self-limiting in immunocompetent hosts but often life threatening to immunocompromised individuals. However, there is a lack of completely efficient therapy available. Probiotics have attracted the attention as potential antiparasite compounds against protozoa involved in intestinal infections. This study investigated the effects of administration of probiotic Enterococcus faecalis CECT 7121 on Cryptosporidium parvum infection in immunosuppressed mice. Effects on C. parvum infection at the intestinal mucosa were studied and scored at each portion of the gut. It was demonstrated that Ef CECT 7121 interfered with C. parvum infection when both probiotic and parasite were present in the same intestinal location suggesting that Ef CECT 7121 supplementation can alleviate the negative effects of C. parvum infection.

Oregano Essential Oil and Carvacrol reduce Cryptosporidium parvum infectivity of HCT‐8 Cells

Lipid‐based nutrition supplements (LNS) are commonly used to treat moderate acute undernutrition in children. However, existing LNS products mainly focus on providing nutrients and do not address other immediate causes of undernutrition, such as parasitic infection and gut inflammation. Enteric parasites are known to reduce nutrient digestion and absorption, cause chronic gut inflammation, iron deficiency anemia, protein‐energy malnutrition, and reduce growth and cognitive development in children. Oregano essential oil (OEO) is known to have anti‐bacterial and anti‐fungal activity, however its anti‐parasitic properties are poorly understood. This study systemically investigated the effect of OEO and its main bioactive, carvacrol (CV), on prevention of Cryptosporidium parvum infectivity of HCT‐8 (human colon carcinoma) cells in vitro, for potential incorporation of these bioactives into LNS products. HCT‐8 cells were seeded (1 × 106) in 96‐well microtiter plates until confluency (2 days). HCT‐8 cell viability at various concentrations of either OEO or CV was measured using a Live/Dead cell imaging kit. Confluent HCT‐8 cell monolayers were seeded with C. parvum oocytes (1 × 104) in two modalities: 1) 4 h co‐culture of C. parvum oocysts, cells and several dosages (0 μg/mL to 250 μg/mL) of CV or OEO in complete medium, followed by PBS washing and replacement with bioactive‐free media; and 2) 4 h co‐culture of C. parvum oocysts and cells alone, followed by PBS washing and treatment with several dosages (0 μg/mL to 250 μg/mL) of CV or OEO. In either modality, plates were incubated at 37°C and 5% CO2, and after 2‐day incubation, cells and C. parvum were fixed with methanol: acetic acid solution (9:1 v/v). Infectivity was assessed via immunofluorescence detection using C. parvum‐specific antibody staining (Sporo‐Glo™) and phase contrast/fluorescent microscopy. Deformation of cell monolayers and loss of cell viability was found at 50 μg/mL CV, whereas no loss in cell viability was observed in cells treated with OEO at dosages as high as 125 μg/mL (containing similar concentrations of CV). In modality 1 experiments, cells simultaneously treated with C. parvum and various dosages of OEO or CV presented similar amounts of infection after 4 h incubation as those cells treated with C. parvum alone. In modality 2 experiments, after 2‐day treatment with bioactives, a 50% reduction in C. parvum infectivity was observed at 25 μg/mL of CV and at 125 μg/mL of OEO. C. parvum is one of the most resilient enteric parasites and prevalent in most of the developing world. Incorporation of OEO or CV into LNS formulas may provide double benefit to at risk populations afflicted by undernutrition and gut infection living in low‐income settings.Support or Funding InformationIndian Council of Agricultural Research (ICAR), India

Lymphocytic Interstitial Pneumonitis: An Unusual Presentation of X-Linked Hyper Ig M Syndrome.

Lymphocytic Interstitial Pneumonitis: An Unusual Presentation of X-Linked Hyper Ig M Syndrome.

Prevalence of Cryptosporidium parvum infection in adult with acute diarrhea referred to clinical laboratories in Jahrom by Nested PCR, 2014

Prevalence of Cryptosporidium parvum infection in adult with acute diarrhea referred to clinical laboratories in Jahrom by Nested PCR, 2014

Health benefits of Il-10 egg yolk antibodies administered to milk-fed dairy calves

Cryptosporidium parvum infection in calves causes villous atrophy, resulting in reduced surface area and absorptive capacity of the small intestine. Prevention and treatment of C. parvum infections are hindered by a lack of approved, efficacious products and inadequate colostral immunity. An integral component of infection is the dampening of the cell-mediated response by inducing anti-inflammatory cytokines. In calves, ileal intraepithelial lymphocytes express interleukin-10 (Il-10) when infected with C. parvum. Interleukin-10 decreases production of interferon-gamma, which is a vital Thl-associated cytokine. Studies have shown that Il-10 knockout mice are resistant to C. parvum infection. Oral administration of chicken egg yolk antibodies as a means of controlling enteric disease in calves is of interest to the dairy industry. Egg yolk Il-10 antibodies have been shown to survive gastrointestinal transit, are accessible in the intestinal lumen, and remain locally active, without persistence of systemic residues. Therefore, the objective of this trial was to investigate the effect of feeding Il-10 antibodies on calf health and performance after natural C. parvum infection in pre-weaned Holstein dairy calves.

The Occurrence of Cryptosporidium parvum in Dairy Calves and the Influence of Management Practices

Cryptosporidium species infections are associated with the clinical manifestation of diarrhea, which can possibly generate economic losses associated with morbidity and mortality depending on the type of property management used. Additionally, animals with diarrhea eliminate large quantities of oocysts via the feces, thus contributing to the dispersion of oocysts in the environment and favoring the infection of susceptible hosts. The study aimed to determine the rate of Cryptosporidium parvum infection in 79 calves under one year of age at a dairy farm in southern Rio de Janeiro state, Brazil, in association with the cattle raising management practices. During the study period, two different management practices (A and B) were adopted. Under practice A, 21.9% of the samples (7/32) were Cryptosporidium spp. positive according to microscopic diagnosis. After switching to practice B, the percentage of infected animals decreased to 8.5% (4/47). Although diarrhea was reported in 13.9% (11/79) of the calves studied, there was no correlation with Cryptosporidium spp. infection (p>0.05). Cryptosporidium parvum, which is considered to have high zoonotic potential, was identified in this study. This fact is of great importance with regard to public health because of the environmental contamination of the area surrounding the property, which increases the risk to both human and animal populations. Additionally, the implementation of some changes in farm management practices contributed to a decrease in the percentage of infected animals, thereby reducing the dissemination of oocysts in the environment.

Cryptosporidium parvum infection among Egyptian school children.

The present study determined cryptosporidiosis among 120 randomly chosen school children aged 4-16 years. Medical sheets were filled out on each child. The fresh stool samples were examined by using Sheather's sugar flotation stained with modified Ziehl Neelsen stain. Blood samples were examined by ELISA and IFA techniques. The results revealed that, the prevalence rate was 13.51% with a peak among the age group (5-10) and. significant relation between males and females. There was a significant relation between infection and low socio-economic level in rural area. Also, a significant relation was obtained between the infection and the presence of animal contact. Watery and loose diarrhea was more significant among infected children. Positive stool samples were among 37 (30.8 %), while ELISA and IFA detected 30 (25%) and 33 (27.5%) respectively. The validity test of ELISA declared sensitivity and specificity with 93.3% and 90% while IFA declared 90.9% and 91.1% respectively.

Sa1776 Decreased SLC26A3 Expression and Function in Intestinal Epithelial Cells in Response to Cryptosporidium parvum Infection

Sa1776 Decreased SLC26A3 Expression and Function in Intestinal Epithelial Cells in Response to Cryptosporidium parvum Infection

Cryptosporidium parvum infection and associated risk factors in dairy calves in western France

This study was conducted to determine the prevalence and risk factors for Cryptosporidium infection in calf neonates on dairy farms in Normandy.Fecal samples were randomly collected between July 2010 and September 2011 from 968 calves (7–21 days old) on 97 farms. Up to 10 calves were selected and sampled per farm, and feces examined for oocysts by microscopy. C. parvum oocyst shedding was scored semi-quantitatively (0–5). A questionnaire about calf-level care and management was completed, and mortality rates were obtained from the French national registration database (BDNI). Bivariable and multivariable analyses of potential risk factors for C. parvum oocyst shedding were conducted using generalized estimating equation (GEE) models (family=Binomial).Overall, 402 out of 968 calves (41.5%) were positive for oocysts, and 25.1% of animals had a shedding score >2. Seven of the 97 farms (7%) were negative for oocysts in all fecal samples. At the time of collection, 375 calves (39%) had diarrhea, and its prevalence strongly correlated with the score for C. parvum oocyst shedding (p<0.0001). The mortality rate at 90 days was significantly greater for calves with high combined scores of diarrhea and shedding. Factors associated with the shedding of C. parvum were the Normande breed (odds ratio=1.49; 95% confidence interval (CI): 0.93–2.37), dispensing of colostrum using a bucket (odds ratio=1.37; 95% CI: 1.00–1.89), treatment with halofuginone (odds ratio=0.46; 95% CI: 0.19–1.15) and feeding with fermented milk (odds ratio=0.32; 95% CI: 0.17–0.63).C. parvum is widespread among calves under 21 days old in dairy herds of western France. Shedding of C. parvum is associated with a high incidence of diarrhea and increased risk of mortality in young calves. This study identified some associated calf-level factors, although further investigations are necessary to determine appropriate measures that farmers and veterinary practitioners should take to reduce the prevalence of C. parvum.

IL-18 cytokine levels modulate innate immune responses and cryptosporidiosis in mice.

IL-18 is known to play a key role limiting Cryptosporidium parvum infection. In this study, we show that IL-18 depletion in SCID mice significantly exacerbates C. parvum infection, whereas, treatment with recombinant IL-18 (rIL-18), significantly decreases the parasite load, as compared to controls. Increases in serum IFN-γ levels as well as the up-regulation of the antimicrobial peptides, cathelicidin antimicrobial peptide and beta defensin 3 (Defb3) were observed in the intestinal mucosa of mice treated with rIL-18. In addition, C. parvum infection significantly increased mRNA expression levels (>50 fold) of the alpha defensins, Defa3 and 5, respectively. Interestingly, we also found a decrease in mRNA expression of IL-33 (a recently identified cytokine in the same family as IL-18) in the small intestinal tissue from mice treated with rIL-18. In comparison, the respective genes were induced by IL-18 depletion. Our findings suggest that IL-18 can mediate its protective effects via different routes such as IFN-γ induction or by directly stimulating intestinal epithelial cells to increase antimicrobial activity.

Assessment of Serum Interleukin-2, -4 and C-reactive protein Levels in patients with Giardiasis and Cryptosporidiosis

Background: Giardia lamblia and Cryptosporidium parvum are recognized as important health problem in different countries. Humans may infect by two parasites at different age groups. But more common in countries with poor sanitation and unsafe water. Cellular and humoral response is engaged by the host to fight against parasitic infection, cytokines are produced by CD4 of payer's patches or generated from the mucosa lymphoid tissue as result of long duration antigen stimulation via trophozoites or cyst stage or others. &#x0D; Objectives: To evaluate the levels of IL-2, -4 and C- reactive protein in the sera of patients with Giardia lamblia and Cryptosporidium parvum.&#x0D; Materials and methods: A total of 142 patients with diarrhea (56 males, 86 females) and compared with 60 healthy looking controls (10 male, 50 female). Attended to the central teaching hospital for pediatric in Baghdad city - Iraq, between 1st April 2012 till 1st August 2013. The diagnosis was established based on direct microscopic examination of stool and Cer Test Crypto-Giardia card, is a one-step colored chromatographic immunoassay. The patients serum had been tested for IL-2, -4 by used Enzyme linked Immunosorbent Assay (ELISA), while the concentration of C- reactive protein was determined by auto chemistry analyzer.&#x0D; Results: The results of laboratory diagnosis revealed that 61 cases infected with Giardiasis, 31cases infected with Cryptosporidiosis and 50 cases infected with double infection. levels of IL-2, -4 and CRP in serum patients with Giardia lamblia and Cryptosporidium parvum was increase when comparing with the healthy looking controls, the statistical analysis shows no significant difference (p&lt;0.05) between infected groups.&#x0D; Conclusion: Giardia lamblia and Cryptosporidium parvum infections had significant effects on IL-2, -4 and CRP levels.

Direct ELISA aided coprological diagnosis of Cryptosporidium parvum infection in diarrheic neonatal calves in Mosul city, Iraq

This study was conducted for the detection of Cryptosporidium (C.) parvum infection in neonatal calves. A total of 220 fecal samples (diarrheic 110 and non-diarrheic 110) of neonatal calves were collected from Mosul city, Iraq over a period of 16 months from November 2010 to March 2012. The age of the calves ranged from 1 to 30 days. All the fecal samples were analyzed by capture direct ELISA. The infection was found in 29.0% (n=32/110) of the diarrheic calves. The infection was mostly prevalent (p<0.001) in the calves of three weeks of age. No C. parvum infection could be detected in the non-diarrheic animals.

Dendritic cells play a role in host susceptibility to Cryptosporidium parvum infection

Our previous studies have described dendritic cells (DCs) to be important sources of Th1 cytokines such as IL-12 and IL-2 in vitro, following stimulation with Cryptosporidium parvum antigens. We further established the role of DCs during cryptosporidiosis using a diphtheria toxin promoter regulated transgenic CD11c-DTR/EGFP mouse model. In vivo depletion of CD11c+ cells in CD11c-DTR-Tg mice significantly increased susceptibility to C. parvum infection. Adoptive transfer of unstimulated or antigen stimulated DCs into CD11c+ depleted CD11c-DTR-Tg mice resulted in an early decrease in parasite load at 4 days post infection. However, this response was transient since parasite load increased in mice engrafted with either unstimulated DCs or DCs stimulated with solubilized antigen by 6 days post infection. In contrast, in mice engrafted with DCs stimulated with live sporozoites, parasite load remained low during the entire period, suggesting the development of a more effective and sustained response. A corresponding increase in IFN-γ expression in T cells from spleen and mesenteric lymph nodes was also noted. Consistent with the in vivo engraftment study, DCs that are pulsed with live sporozoites in vitro and co-cultured with CD4+ and CD8+ T cells produced higher IFN-γ levels. Our study establishes the importance of DCs in susceptibility to infection by C. parvum and as important mediators of immune responses.

Amelioration of Cryptosporidium parvum Infection In Vitro and In Vivo by Targeting Parasite Fatty Acyl-Coenzyme A Synthetases

Cryptosporidium is emerging as 1 of the 4 leading diarrheal pathogens in children in developing countries. Its infections in patients with AIDS can be fatal, whereas fully effective treatments are unavailable. The major goal of this study is to explore parasite fatty acyl-coenzyme A synthetase (ACS) as a novel drug target. A colorimetric assay was developed to evaluate biochemical features and inhibitory kinetics of Cryptosporidium parvum ACSs using recombinant proteins. Anticryptosporidial efficacies of the ACS inhibitor triacsin C were evaluated both in vitro and in vivo. Cryptosporidium ACSs displayed substrate preference toward long-chain fatty acids. The activity of parasite ACSs could be specifically inhibited by triacsin C with the inhibition constant Ki in the nanomolar range. Triacsin C was highly effective against C. parvum growth in vitro (median inhibitory concentration, 136 nmol/L). Most importantly, triacsin C effectively reduced parasite oocyst production up to 88.1% with no apparent toxicity when administered to Cryptosporidium-infected interleukin 12 knockout mice at 8-15 mg/kg/d for 1 week. The findings of this study not only validated Cryptosporidium ACS (and related acyl-[acyl-carrier-protein]-ligases) as pharmacological targets but also indicate that triacsin C and analogues can be explored as potential new therapeutics against the virtually untreatable cryptosporidial infection in immunocompromised patients.