Point-of-Use Removal of <i>Cryptosporidium parvum</i> from Water: Independent Effects of Disinfection by Silver Nanoparticles and Silver Ions and by Physical Filtration in Ceramic Porous Media

Ceramic water filters (CWFs) impregnated with silver nanoparticles are a means of household-level water treatment. CWFs remove/deactivate microbial pathogens by employing two mechanisms: metallic disinfection and physical filtration. Herein we report on the independent effects of silver salt and nanoparticles on <i>Cryptosporidium parvum</i> and the removal of <i>C. parvum</i> by physical filtration in porous ceramic filter media. Using a murine (mouse) model, we observed that treatment of oocysts with silver nitrate and proteinate-capped silver nanoparticles resulted in decreased infection relative to untreated oocysts. Microscopy and excystation experiments were conducted to support the disinfection investigation. Heat and proteinate-capped silver-nanoparticle treatment of oocysts resulted in morphological modifications and decreased excystation rates of sporozoites. Subsequently, disk-shaped ceramic filters were produced to investigate the transport of <i>C. parvum</i>. Two factors were varied: sawdust size and clay-to-sawdust ratio. Five disks were prepared with combinations of 10, 16, and 20 mesh sawdust and sawdust percentage that ranged from 9 to 11%. <i>C. parvum</i> removal efficiencies ranged from 1.5 log (96.4%) to 2.1 log (99.2%). The 16-mesh/10% sawdust had the greatest mean reduction of 2.1-log (99.2%), though there was no statistically significant difference in removal efficiency. Based on our findings, physical filtration and silver nanoparticle disinfection likely contribute to treatment of <i>C. parvum</i> for silver impregnated ceramic water filters, although the contribution of physical filtration is likely greater than silver disinfection

The “vicious cycle" of enteropathogens, malnutrition, and impaired childhood development, and multifaceted opportunities for intervention.

<p>Figure adapted from Nutr Rev. 2008 September; 66(9): 487–505 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002125#pntd.0002125-Guerrant1" target="_blank">[15]</a>.</p

The Role of Peroxisome Proliferator-Activated Receptor γ in Immune Responses to Enteroaggregative <em>Escherichia coli</em> Infection

<div><p>Background</p><p>Enteroaggregative <i>Escherichia coli</i> (EAEC) is recognized as an emerging cause of persistent diarrhea and enteric disease worldwide. Mucosal immunity towards EAEC infections is incompletely understood due in part to the lack of appropriate animal models. This study presents a new mouse model and investigates the role of peroxisome proliferator-activated receptor gamma (PPARγ) in the modulation of host responses to EAEC in nourished and malnourished mice.</p> <p>Methods/Principal Findings</p><p>Wild-type and T cell-specific PPARγ null C57BL/6 mice were fed protein-deficient diets at weaning and challenged with 5×10⁹cfu EAEC strain JM221 to measure colonic gene expression and immune responses to EAEC. Antigen-specific responses to <i>E. coli</i> antigens were measured in nourished and malnourished mice following infection and demonstrated the immunosuppressive effects of malnutrition at the cellular level. At the molecular level, both pharmacological blockade and deletion of PPARγ in T cells resulted in upregulation of TGF-β, IL-6, IL-17 and anti-microbial peptides, enhanced Th17 responses, fewer colonic lesions, faster clearance of EAEC, and improved recovery. The beneficial effects of PPARγ blockade on weight loss and EAEC clearance were abrogated by neutralizing IL-17 <i>in vivo</i>.</p> <p>Conclusions</p><p>Our studies provide <i>in vivo</i> evidence supporting the beneficial role of mucosal innate and effector T cell responses on EAEC burden and suggest pharmacological blockade of PPARγ as a novel therapeutic intervention for EAEC infection.</p> </div

Immune responses during enteroaggregative Escherichia coli (EAEC) infection in peroxisome proliferator-activated receptor γ (PPARγ)-deficient mice associated with bacterial clearance.

<p>Antigen specific recall responses of spleenocytes from mice infected with EAEC were measured ex vivo using the lymphocyte blastogenesis test. EAEC JM221 whole cell and whole cell sonicate were used in parallel to two negative controls, <i>E. coli</i> HS and mutant EAEC Aff/I strains as well as one positive control, concanavalin A (ConA). Lymphocyte proliferation is expressed stimulation indexes which are calculated by dividing the counts per minute (CPM) of antigen-stimulated wells by the CPM of unstimulated wells (A). IL-17 expression was assessed in colonic lamina propria (B) and whole blood (C) CD4+ T cells by flow cytometry and in the colon by quantitative real time RT-PCR (D) 14 days PI. Mice per group: n = 10. Asterisks indicate values where differences are statistically significant (<i>p</i><0.05) while bars connect groups where comparisons are made.</p

Pharmacological blockade of peroxisome proliferator-activated receptor γ (PPARγ) associated with antimicrobial response and bacterial clearance.

<p>Enteroaggregative <i>Escherichia coli</i> (EAEC) burden in colon was assessed by quantitative real time RT-PCR using bacterial DNA isolated from feces of infected mice treated with PPARγ antagonist GW9662 (n = 9) or left untreated (n = 9). Data is presented as CFU/mg of tissue. S100A8 and S100A9 gene expression was analyzed in colonic tissue from C57BL/6 malnourished mice at day 5 days PI (n = 10) using quantitative real-time RT-PCR (B and C). S100 proteins are presented as values normalized to β-actin. Asterisks indicate values where differences are statistically significant (<i>p</i><0.05).</p

Gene expression suggests a T helper 17 response in mice when peroxisome proliferator-activated receptor γ (PPARγ) is antagonized.

<p>Gene expression data from colonic tissue of malnourished C57BL/6 mice was analyzed using quantitative real-time RT-PCR and reported as values normalized to β-actin. IL-6, IL-1β, MCP-1, CCL20, and CXCL1 were quantified at day 5PI (mice per group: n = 10) (A–E) while IL-6, TGF-β, and IL-17 were quantified 14 days PI (n = 10) (F–H). Asterisks indicate values where differences are statistically significant (<i>p</i><0.05) while bars connect groups where comparisons are made.</p

Neutralization of IL-17 abrogates the beneficial effects of GW9662 on weight loss and bacterial burden.

<p>Growth retardation in infected wild type mice is expressed as percent growth from day 0 after challenge (A). Enteroaggregative <i>Escherichia coli</i> (EAEC) burden in the colon was assessed by quantitative real time RT-PCR using bacterial DNA isolated from feces of infected mice treated with 1 µM PPARγ antagonist GW9662 (n = 3), 50 µg anti-IL17 and 1 µM GW9662 combined (n = 3) or untreated (n = 3). Asterisks indicate values where differences are statistically significant (<i>p</i><0.05), NS signifies no significant difference, and bars are present to indicate significance between groups.</p

Early beneficial effects of PPARγ deficiency in T cells during enteroaggregative Escherichia coli (EAEC) challenge.

<p>Growth retardation in wild type (A) and T cell specific PPARγ deficient mice (B) is expressed as percent growth from day 0 after challenge. Gene expression for IL-6 and TNF-α in colonic tissue of malnourished C57BL/6 and PPARγ CD4cre+ mice was analyzed using quantitative real-time RT-PCR on day 5 PI (C). Representative photomicrographs of colonic specimens of infected mice at 5 or 14 days PI in infected wild type mice (D,E,I,J), infected mice lacking PPARγ expression in T cells (F,G,K,L), and uninfected controls (H,M). The top panel corresponds to nourished mice whereas the bottom panel corresponds to malnourished mice. Original magnification 200×. Boxes and arrows are areas where an amplified image (400×) is provided to emphasize examples of leukocyte infiltration, mucosal thickening, goblet cell hyperplasia, and vasodilation. Mice per group: n = 8. Asterisks indicate values where differences are statistically significant (<i>p</i><0.05).</p

Results Interpretation: Meta-analysis of the output from the regression of standardized cognitive z-score onto LAZ and diarrhea prevalence as continuous variables (model 1).^a.

a<p>. We only report the pooled coefficients for variables with like definitions across study sites (i.e. diarrhea prevalence, LAZ, sex). Variables treated differently across study sites were not pooled (i.e. SES, age at cognitive assessment, maternal education).</p

Results of country-level linear regression models to determine the association of diarrhea on cognition.^a^,^b^,^c^,^d^,^e.

a<p>. For each data set, standardized cognitive Z-score served as the outcome of linear regression.</p>b<p>. All four regression analyses controlled for diarrhea prevalence, stunting and sex in the same way. Diarrhea prevalence was coded as the percentage of days/periods during which diarrhea was reported; LAZ was treated as a continuous z-score; sex was a categorical variable (0 = male, 1 = female).</p>c<p>. SES was controlled for differently in each country given the data available from each site: 1) Philippines: controlled for log household income and ownership of assets; 2) Brazil: controlled for monthly income and the number of rooms in the household per person; 3) Peru: controlled for log household income; and 4) Guatemala: controlled for a multi-component SES score (through factor analysis) for the household as a continuous variable and residence in one of four villages.</p>d<p>. Age (in months) at cognitive evaluation was treated as a continuous variable in Philippines, Brazil, and Peru.. In Guatemala, the children were 4 years of age at cognitive assessment, and therefore age was controlled for by the addition of categorical indicator variables for birth year to the regression model.</p>e<p>. In Philippines, Peru and Guatemala, maternal education was defined as the average number of years enrolled in school. In Brazil, maternal education was a categorical variable representing the percentage of mothers that had not completed primary school.</p