Effects of Backpacker Use, Pack Stock Trail Use, and Pack Stock Grazing on Water-Quality Indicators, Including Nutrients, E. coli, Hormones, and Pharmaceuticals, in Yosemite National Park, USA.

We investigated how visitor-use affects water quality in wilderness in Yosemite National Park. During the summers of 2012-2014, we collected and analyzed surface-water samples for water-quality indicators, including fecal indicator bacteria Escherichia coli, nutrients (nitrogen, phosphorus, carbon), suspended sediment concentration, pharmaceuticals, and hormones. Samples were collected upstream and downstream from different types of visitor use at weekly to biweekly intervals and during summer storms. We conducted a park-wide synoptic sampling campaign during summer 2014, and sampled upstream and downstream from meadows to evaluate the mitigating effect of meadows on water quality. At pack stock stream crossings, Escherichia coli concentrations were greater downstream from crossings than upstream (median downstream increase in Escherichia coli of three colony forming units 100 mL-1), with the greatest increases occurring during storms (median downstream increase in Escherichia coli of 32 CFU 100 mL-1). At backpacker use sites, hormones, and pharmaceuticals (e.g., insect repellent) were detected at downstream sites, and Escherichia coli concentrations were greater at downstream sites (median downstream increase in Escherichia coli of 1 CFU 100 mL-1). Differences in water quality downstream vs. upstream from meadows grazed by pack stock were not detectable for most water-quality indicators, however, Escherichia coli concentrations decreased downstream, suggesting entrapment and die-off of fecal indicator bacteria in meadows. Our results indicate that under current-use levels pack stock trail use and backpacker use are associated with detectable, but relatively minor, effects on water quality, which are most pronounced during storms.

Cryptosporidium Priming Is More Effective than Vaccine for Protection against Cryptosporidiosis in a Murine Protein Malnutrition Model.

Cryptosporidium is a major cause of severe diarrhea, especially in malnourished children. Using a murine model of C. parvum oocyst challenge that recapitulates clinical features of severe cryptosporidiosis during malnutrition, we interrogated the effect of protein malnutrition (PM) on primary and secondary responses to C. parvum challenge, and tested the differential ability of mucosal priming strategies to overcome the PM-induced susceptibility. We determined that while PM fundamentally alters systemic and mucosal primary immune responses to Cryptosporidium, priming with C. parvum (106 oocysts) provides robust protective immunity against re-challenge despite ongoing PM. C. parvum priming restores mucosal Th1-type effectors (CD3+CD8+CD103+ T-cells) and cytokines (IFNγ, and IL12p40) that otherwise decrease with ongoing PM. Vaccination strategies with Cryptosporidium antigens expressed in the S. Typhi vector 908htr, however, do not enhance Th1-type responses to C. parvum challenge during PM, even though vaccination strongly boosts immunity in challenged fully nourished hosts. Remote non-specific exposures to the attenuated S. Typhi vector alone or the TLR9 agonist CpG ODN-1668 can partially attenuate C. parvum severity during PM, but neither as effectively as viable C. parvum priming. We conclude that although PM interferes with basal and vaccine-boosted immune responses to C. parvum, sustained reductions in disease severity are possible through mucosal activators of host defenses, and specifically C. parvum priming can elicit impressively robust Th1-type protective immunity despite ongoing protein malnutrition. These findings add insight into potential correlates of Cryptosporidium immunity and future vaccine strategies in malnourished children.

Effects of physiological and/or disease status on the response of postpartum dairy cows to synchronization of estrus using an intravaginal progesterone device.

Progesterone treatments are used to increase submission rates in postpartum dairy cows; however, in many cases the protocol is used as a blanket therapy for all cows without regard for physiological or disease state. The objective of this study was to identify the physiological or disease classes of cows that respond well (or not) to synchronization of estrus via progesterone. Dairy cows (n = 402) were monitored peri and postpartum to establish their physiological or disease status. Animals were classified as having negative energy balance, clinical lameness, uterine infection (UI), anovulatory anestrus, high somatic cell counts, and healthy (H). Blood samples were collected at five different time points and analyzed for metabolites. All animals received an 8-day controlled internal drug release protocol, which included GnRH at insertion and PGF2α the day before removal. Response to the protocol was determined by visual observation of estrus synchronization. Conception rate was determined by ultrasonography between Days 32 and 35 after artificial insemination. Animals without UI were 1.9 times more likely to respond and two times more likely to be confirmed pregnant than those with UI. There was no relationship between negative energy balance and clinical lameness in the visual estrous response, but both conditions were associated with reduced conception rates. Dairy cows in anovulatory anestrus responded successfully to the protocol in both estrous response and conception rates. High glutathione peroxidase concentrations had a positive effect on conception rates, whereas high non-esterified fatty acids and beta-hydroxybutyrate had a negative effect on the estrous response. In conclusion, disease and physiological states of dairy cows determined the response to progesterone-based synchronization. The more disease or physiological problems the cows had, the lower the estrous response and conception rates; cows with these problems were not ideal candidates for synchronization. Both anestrus and healthy dairy cows were good responders to progesterone-based synchronization.

Apolipoprotein E plays a key role against cryptosporidial infection in transgenic undernourished mice.

Apolipoliprotein E (apoE), a critical targeting protein in lipid homeostasis, has been found to have immunoinflammatory effects on murine models of infection and malnutrition. The effects of apoE in undernourished and Cryptosporidium parvum-infected mice have not been investigated. In order to study the role of apoE in a model of C. parvum infection, we used the following C57BL6J mouse genetic strains: APOE-deficient, wild-type controls, and APOE targeted replacement (TR) mice expressing human APOE genes (E3/3; E4/4). Experimental mice were orally infected with 10(7)-unexcysted-C. parvum oocysts between post-natal days 34-35 followed by malnutrition induced with a low-protein diet. Mice were euthanized seven days after C. parvum-challenge to investigate ileal morphology, cytokines, and cationic arginine transporter (CAT-1), arginase 1, Toll-like receptor 9 (TLR9), and inducible nitric oxide synthase (iNOS) expression. In addition, we analyzed stool oocyst shedding by qRT-PCR and serum lipids. APOE4/4-TR mice had better weight gains after infection plus malnutrition compared with APOE3/3-TR and wild-type mice. APOE4/4-TR and APOE knockout mice had lower oocyst shedding, however the latter exhibited with villus blunting and higher ileal pro-inflammatory cytokines and iNOS transcripts. APOE4/4-TR mice had increased ileal CAT-1, arginase-1, and TLR9 transcripts relative to APOE knockout. Although with anti-parasitic effects, APOE deficiency exacerbates intestinal inflammatory responses and mucosal damage in undernourished and C. parvum-infected mice. In addition, the human APOE4 gene was found to be protective against the compounded insult of Cryptosporidium infection plus malnutrition, thus extending our previous findings of the protection against diarrhea in APOE4 children. Altogether our findings suggest that apoE plays a key role in the intestinal restitution and immunoinflammatory responses with Cryptosporidium infection and malnutrition.

Correction: Characterization of the Th Profile of the Bovine Endometrium during the Oestrous Cycle and Early Pregnancy.

[This corrects the article DOI: 10.1371/journal.pone.0075571.].

Characterization of the Th profile of the bovine endometrium during the oestrous cycle and early pregnancy.

Despite extensive research in the area of cow fertility, the extent to which the maternal immune system is modulated during pregnancy in cattle remains unclear. Therefore, the objective of the current study was to characterize the presence and response profile of B, T-helper (LTh), T- cytotoxic (LTc), gamma delta-T (γδT) and natural killer (NK) lymphocytes in terms of cell number, distribution and cytokine expression in bovine endometrial tissue to pregnancy. Endometrial tissue samples were collected from beef heifers on Days 5, 7, 13 and 16 of the estrous cycle or pregnancy. Samples were analysed by immunofluorescence to identify the presence and abundance of B-B7 (B-cells), CD4 (LTh), CD8 (LTc), γδT cell receptor (TCR) and CD335/NKp46 (NK cells) -positive immune cells. Quantitative real time PCR (QPCR) was carried out to analyse mRNA relative abundance of FOXP3 (a marker of regulatory T (Treg) cells) and a panel of immune factors, including MHC-I, LIF, Interleukins 1, 2, 6, 8, 10, 11,12A, IFNa and IFNG. Results indicate that B-B7+ cells are quite populous in bovine endometrial tissue, CD4+ and CD8+ -cells are present in moderate numbers and γδTCR+ and CD335+ cells are present in low numbers. Pregnancy affected the total number and distribution pattern of the NK cell population, with the most significant variation observed on Day 16 of pregnancy. Neither B lymphocytes nor T lymphocyte subsets were regulated temporally during the oestrous cycle or by pregnancy prior to implantation. mRNA transcript abundance of the immune factors LIF, IL1b, IL8 and IL12A, IFNa and IFNG, expression was regulated temporally during the estrous cycle and LIF, IL1b, IL-10, IL11, IL12A were also temporally regulated during pregnancy. In conclusion, the endometrial immune profile of the oestrous cycle favours a Th2 environment in anticipation of pregnancy and the presence of an embryo acts to fine tune this environment.

The micronutrient zinc inhibits EAEC strain 042 adherence, biofilm formation, virulence gene expression, and epithelial cytokine responses benefiting the infected host.

Enteroaggregative Escherichia coli (EAEC) is a major pathogen worldwide, associated with diarrheal disease in both children and adults, suggesting the need for new preventive and therapeutic treatments. We investigated the role of the micronutrient zinc in the pathogenesis of an E. coli strain associated with human disease. A variety of bacterial characteristics-growth in vitro, biofilm formation, adherence to IEC-6 epithelial cells, gene expression of putative EAEC virulence factors as well as EAEC-induced cytokine expression by HCT-8 cells-were quantified. At concentrations (≤ 0.05 mM) that did not alter EAEC growth (strain 042) but that are physiologic in serum, zinc markedly decreased the organism's ability to form biofilm (P<0.001), adhere to IEC-6 epithelial cells (P<0.01), and express putative EAEC virulence factors (aggR, aap, aatA, virK) (P<0.03). After exposure of the organism to zinc, the effect on virulence factor generation was prolonged (> 3 h). Further, EAEC-induced IL-8 mRNA and protein secretion by HCT-8 epithelial cells were significantly reduced by 0.05 mM zinc (P<0.03). Using an in vivo murine model of diet-induced zinc-deficiency, oral zinc supplementation (0.4 µg/mouse daily) administered after EAEC challenge (10 (10) CFU/mouse) significantly abrogated growth shortfalls (by>90%; P<0.01); furthermore, stool shedding was reduced (days 9-11) but tissue burden of organisms in the intestine was unchanged. These findings suggest several potential mechanisms whereby physiological levels of zinc alter pathogenetic events in the bacterium (reducing biofilm formation, adherence to epithelium, virulence factor expression) as well as the bacterium's effect on the epithelium (cytokine response to exposure to EAEC) to alter EAEC pathogenesis in vitro and in vivo. These effects may help explain and extend the benefits of zinc in childhood diarrhea and malnutrition.

Persistent G. lamblia impairs growth in a murine malnutrition model.

Giardia lamblia infections are nearly universal among children in low-income countries and are syndemic with the triumvirate of malnutrition, diarrhea, and developmental growth delays. Amidst the morass of early childhood enteropathogen exposures in these populations, G. lamblia­specific associations with persistent diarrhea, cognitive deficits, stunting, and nutrient deficiencies have demonstrated conflicting results, placing endemic pediatric giardiasis in a state of equipoise. Many infections in endemic settings appear to be asymptomatic/ subclinical, further contributing to uncertainty regarding a causal link between G. lamblia infection and developmental delay. We used G. lamblia H3 cyst infection in a weaned mouse model of malnutrition to demonstrate that persistent giardiasis leads to epithelial cell apoptosis and crypt hyperplasia. Infection was associated with a Th2-biased inflammatory response and impaired growth. Malnutrition accentuated the severity of these growth decrements. Faltering malnourished mice exhibited impaired compensatory responses following infection and demonstrated an absence of crypt hyperplasia and subsequently blunted villus architecture. Concomitantly, severe malnutrition prevented increases in B220+ cells in the lamina propria as well as mucosal Il4 and Il5 mRNA in response to infection. These findings add insight into the potential role of G. lamblia as a "stunting" pathogen and suggest that, similarly, malnourished children may be at increased risk of G. lamblia­ potentiated growth decrements.

The role of peroxisome proliferator-activated receptor γ in immune responses to enteroaggregative Escherichia coli infection.

BACKGROUND: Enteroaggregative Escherichia coli (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. METHODS/PRINCIPAL FINDINGS: Wild-type and T cell-specific PPARγ null C57BL/6 mice were fed protein-deficient diets at weaning and challenged with 5×10(9)cfu EAEC strain JM221 to measure colonic gene expression and immune responses to EAEC. Antigen-specific responses to E. coli 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-ß, 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 in vivo. CONCLUSIONS: Our studies provide in vivo 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.

Enteroaggregative Escherichia coli strain in a novel weaned mouse model: exacerbation by malnutrition, biofilm as a virulence factor and treatment by nitazoxanide.

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a common cause of diarrhoea in healthy, malnourished and immune-deficient adults and children. There is no reproducible non-neonatal animal model for longitudinal studies of disease mechanism or therapy. Using two strains of human-derived EAEC to challenge weaned C57BL/6 mice, we explored an in vivo model of EAEC infection in mice, in which disease was monitored quantitatively as the growth rate, stool shedding and tissue burden of organisms; nutritional status was varied, and a new class of therapeutics was assessed. A single oral challenge of EAEC strain 042 resulted in significant growth shortfalls (5-8 % of body weight in 12 days), persistent shedding of micro-organisms in stools [>10(3.2) c.f.u. (10 mg stool)(-1) for at least 14 days] and intestinal tissue burden [~10(3) c.f.u. (10 mg tissue)(-1) detectable up to 14 days post-challenge]. Moderate malnourishment of mice using a 'regional basic diet' containing 7 % protein and reduced fat and micronutrients heightened all parameters of infection. Nitazoxanide in subMIC doses, administered for 3 days at the time of EAEC challenge, lessened growth shortfalls (by >10 % of body weight), stool shedding [by 2-3 logs (10 mg stool)(-1)] and tissue burden of organisms (by >75 % in the jejunum and colon). Thus, weaned C57BL/6 mice challenged with EAEC is a convenient, readily inducible model of EAEC infection with three highly quantifiable outcomes in which disease severity is dependent on the nutritional status of the host, and which is modifiable in the presence of inhibitors of pyruvate ferredoxin oxidoreductase such as nitazoxanide.

Intranasal vaccination in mice with an attenuated Salmonella enterica Serovar 908htr A expressing Cp15 of Cryptosporidium: impact of malnutrition with preservation of cytokine secretion.

Cryptosporidium is a protozoan parasite associated with acute and persistent diarrhea that, even in asymptomatic persons, can impair normal growth and potentially cognitive and physical development in young children. The recent availability of the complete gene sequence for Cryptosporidium hominis antigen Cp15 allows examination of innovative vaccine regimens involving intra-nasal antigen priming with live bacterial vectors applicable to human populations. We used a recently described weaned mouse model of cryptosporidiosis, where nourished and malnourished vaccinated mice receive the Cp15 antigen recombinant with cytolysinA on a Salmonella serovar Typhi CVD 908-htr A vector, followed by parenteral exposure to antigen with adjuvant. After challenge with Cryptosporidium oocysts via gavage, parameters of infection and disease (stool shedding of parasites, growth rates) were quantified, and serum/lymphoid tissue harvested to elucidate the Cp15-specific adaptive immune response. In vaccinated nourished mice, the regimen was highly immunogenic, with strong antigen-specific IL-6 and IFN-γ secretion and robust Cp15-specific immunoglobulin titers. In vaccinated malnourished mice, secretion of cytokines, particularly IFN-γ, and antigen-specific humoral immunity were generally undiminished despite protein deprivation and stunted growth. In contrast, after natural (oral) challenge with an identical inoculum of Cryptosporidium oocysts, cytokine and humoral responses to Cp15 were less than one-fourth those in vaccinated mice. Nevertheless, vaccination resulted in only transient reduction in stool shedding of parasites and was not otherwise protective against disease. Overall, immunogenicity for a C. hominis antigen was documented in mice, even in the setting of prolonged malnutrition, using an innovative vaccine regimen involving intra-nasal antigen priming with a live enteric bacterial vector, that has potential applicability to vulnerable human populations irrespective of nutritional status.

Modeling the role of peroxisome proliferator-activated receptor γ and microRNA-146 in mucosal immune responses to Clostridium difficile.

Clostridium difficile is an anaerobic bacterium that has re-emerged as a facultative pathogen and can cause nosocomial diarrhea, colitis or even death. Peroxisome proliferator-activated receptor (PPAR) γ has been implicated in the prevention of inflammation in autoimmune and infectious diseases; however, its role in the immunoregulatory mechanisms modulating host responses to C. difficile and its toxins remains largely unknown. To characterize the role of PPARγ in C. difficile-associated disease (CDAD), immunity and gut pathology, we used a mouse model of C. difficile infection in wild-type and T cell-specific PPARγ null mice. The loss of PPARγ in T cells increased disease activity and colonic inflammatory lesions following C. difficile infection. Colonic expression of IL-17 was upregulated and IL-10 downregulated in colons of T cell-specific PPARγ null mice. Also, both the loss of PPARγ in T cells and C. difficile infection favored Th17 responses in spleen and colonic lamina propria of mice with CDAD. MicroRNA (miRNA)-sequencing analysis and RT-PCR validation indicated that miR-146b was significantly overexpressed and nuclear receptor co-activator 4 (NCOA4) suppressed in colons of C. difficile-infected mice. We next developed a computational model that predicts the upregulation of miR-146b, downregulation of the PPARγ co-activator NCOA4, and PPARγ, leading to upregulation of IL-17. Oral treatment of C. difficile-infected mice with the PPARγ agonist pioglitazone ameliorated colitis and suppressed pro-inflammatory gene expression. In conclusion, our data indicates that miRNA-146b and PPARγ activation may be implicated in the regulation of Th17 responses and colitis in C. difficile-infected mice.

Pivotal role for monocytes/macrophages and dendritic cells in maternal immune response to the developing embryo in cattle.

In mammals, successful pregnancy is dependent in part on the adaptation or regulation of the maternal immune system to prevent the rejection of the embryonic semiallograft. A modification in Th cell function and secretion is a requirement for the establishment and maintenance of pregnancy. Although there is strong evidence from studies in humans and mice linking successful pregnancy with the predominance of Th2-type immunity, the situation in cattle remains unclear. This study describes the characterization of the immune response of the bovine maternal endometrium to the presence of a developing embryo, with specific emphasis on the macrophage and dendritic cell populations and associated factors, using quantitative real-time PCR, in situ hybridization, and immunohistochemistry. Furthermore, in vivo and in vitro models were developed to investigate the potential role of progesterone and interferon-tau (IFNT) in the regulation of these immune factors. There was a marked increase in the population of CD14(+) cells and CD172a-CD11c(+) cells in the endometrium in response to pregnancy, which was paralleled by increased mRNA expression of a number of non-Th-associated factors, including IL12B and IL15, and downregulation of IL18. In addition, we identified several novel IFNT- and progesterone-regulated factors, including IL12B, MCP1, MCP2, PTX3, RSAD2, and TNFA, whose regulation may be critical to pregnancy outcome. Our findings give center stage to non-Th cells, such as monocytes/macrophages and dendritic cells, in the bovine immune response to the semiallogenic embryo. In conclusion, we propose that in cattle, successful pregnancy establishment is associated with a dramatic regulation of the cytokine network, primarily by endometrial monocytes/macrophages and dendritic cells.

Effects of low progesterone on the endometrial transcriptome in cattle.

The objective of the present study was to determine how low progesterone (P4) affects the endometrial transcriptome, with specific emphasis on those changes that may impact conceptus elongation. Following estrous synchronization and detection (estrus = Day 0, n = 40), heifers were randomly assigned to a control group (n = 12) or a low P4 group (n = 28). Heifers in the low P4 group had consistently lower P4 concentrations compared to controls (P < 0.05). Microarray analysis of endometrial gene expression revealed low P4 altered the expression of 498 differentially expressed genes (DEGs; 215 up- and 283 down-regulated) on Day 7 and 351 DEGs (272 up- and 79 down-regulated) on Day 13. A similar number of temporal changes occurred between Day 7 and Day 13 in both groups (2212 in heifers with normal P4 compared with 2247 in heifers with low P4); of these DEGs, 1278 were common to both groups. Little overlap in the number of DEGs affected by high or low P4 was observed across days. Comparison of the temporal changes that occur during normal estrous cycle progression (i.e., from Day 7 to Day 13) to those affected by altered P4 found significant numbers of genes were modulated by elevated (4157) and decreased (809) P4 alone. Analysis of selected genes by quantitative real-time PCR and in situ hybridization revealed that expression of MEP1B, NID2, and PRSS23 increased on Day 13 compared to Day 7 (P < 0.05) and that the magnitude of increase was significantly diminished in heifers with low P4 compared to controls. MEP1B predominantly localized to the both the superficial and deep glandular epithelium (GE), NID2 localized to the deep GE, whereas PRSS23 localized only to the luminal epithelium. In conclusion, we have determined the global changes in the endometrial transcriptome induced by decreasing the output of P4 from the corpus luteum in vivo using a unique animal model. Placing these data into context with previous data in which P4 was supplemented or elevated after ovulation, we have identified a panel of genes that are truly regulated in the endometrium by circulating concentrations of P4 in vivo and that likely impact conceptus elongation.

Novel in vitro and in vivo models and potential new therapeutics to break the vicious cycle of Cryptosporidium infection and malnutrition.

BACKGROUND: Although several animal models of cryptosporidiosis have been reported, most involve genetically or pharmacologically immune-suppressed hosts. METHODS: We report challenge with excysted (in vitro and in vivo) and unexcysted (in vivo) Cryptosporidium parvum oocysts in human colonic adenocarcinoma (HCT-8) cells and weaned nourished and malnourished C57BL/6 mice, following outcomes of growth rate, stool shedding, and tissue burden. We tested treatment with an oligodeoxynucleotide containing unmethylated CpG motif (CpG-ODN) and alanyl-glutamine in vivo and in vitro. RESULTS: C. parvum-challenged mice showed prolonged weight loss (>10% over 4 days), robust stool shedding (>3 logs/d over 7 days), and epithelial infection in the ileum, cecum, and colon. Of 2 potential therapeutic compounds evaluated in the model, CpG-ODN reduced body weight loss (to <6% on days 3-7 after challenge), reduced shedding of organisms (by 25% on days 1 and 3 after challenge), and decreased the burden of parasites in the ileum. Alanyl-glutamine showed similar benefits. In vitro findings suggested that effects on the epithelial component of the mucosa probably likely responsible for beneficial effects seen in vivo. CONCLUSIONS: Weaned mice provide a convenient and reproducible model of cryptosporidial disease, including its vicious cycle with body weight loss and heavier infection with malnutrition, and this model may be useful in exploring innovative therapeutic solutions for this challenging infectious disease.

Cryptosporidium-malnutrition interactions: mucosal disruption, cytokines, and TLR signaling in a weaned murine model.

Cryptosporidiosis is a leading cause of persistent diarrhea in children in impoverished and developing countries and has both a short- and long-term impact on the growth and development of affected children. An animal model of cryptosporidial infection that mirrors closely the complex interaction between nutritional status and infection in children, particularly in vulnerable settings such as post-weaning and malnourishment, is needed to permit exploration of the pathogenic mechanisms involved. Weaned C57BL/6 mice received a protein-deficient (2%) diet for 3-12 days, then were infected with 5 × 10(7) excysted C. parvum oocyts, and followed for rate of growth, parasite stool shedding, and intestinal invasion/morphometry. Mice had about 20% reduction in weight gain over 12 days of malnutrition and an additional 20% weight loss after C. parvum challenge. Further, a significantly higher fecal C. parvum shedding was detected in malnourished infected mice compared to the nourished infected mice. Also, higher oocyst counts were found in ileum and colon tissue samples from malnourished infected mice, as well as a significant reduction in the villous height-crypt depth ratio in the ileum. Tissue Th1 cytokine concentrations in the ileum were significantly diminished by malnutrition and infection. mRNA for toll-like receptors 2 and 4 were diminished in malnourished infected mice. Treatment with nitazoxanide did not prevent weight loss or parasite stool shedding. These findings indicate that, in the weaned animal, malnutrition intensifies cryptosporidial infection, while cryptosporidial infection further impairs normal growth. Depressed TLR2 and 4 signaling and Th1 cytokine response may be important in the mechanisms underlying the vicious cycle of malnutrition and enteric infection.

Gene silencing in bovine zygotes: siRNA transfection versus microinjection.

The aim of this study was to compare gene silencing in bovine zygotes when small interfering RNAs (siRNAs) were introduced into bovine zygotes by microinjection or lipid-based transfection. In Experiment 1, E-cadherin siRNA was injected at 100 or 375 µM and compared with PBS-injected and non-injected controls. Embryos were then cultured in vitro for 7 days and periodically assessed for development. For transfection, zona-free zygotes were incubated in transfection medium with siRNA for 1h at 39°C and then cultured to Day 7. Injection of PBS or 375 µM E-cadherin siRNA resulted in a decrease in the number of embryos reaching the 8-cell stage (51.5% and 45.5%) or the blastocyst stage (39.0 and 32.5%) compared with non-injected controls (62.9 and 45.0%, respectively; P<0.05). Messenger RNA abundance was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 and 375 µM, respectively, compared with controls (P<0.05). Transfection with 100 nM E-cadherin siRNA decreased development to the 8-cell stage (20.3 versus 53.0%) and blastocyst stage (7.2 versus 18.2%) compared with controls (P<0.05). Messenger RNA relative abundance was not different between controls (non-transfected or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100 and 200 nM E-cadherin siRNA led to a 72 and 38% reduction, respectively, in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P<0.05).

Pediatric diarrhea in southern Ghana: etiology and association with intestinal inflammation and malnutrition.

Diarrhea is a major public health problem that affects the development of children. Anthropometric data were collected from 274 children with (N = 170) and without (N = 104) diarrhea. Stool specimens were analyzed by conventional culture, polymerase chain reaction for enteroaggregative Escherichia coli (EAEC), Shigella, Cryptosporidium, Entamoeba, and Giardia species, and by enzyme-linked immunosorbent assay for fecal lactoferrin levels. About 50% of the study population was mildly to severely malnourished. Fecal lactoferrin levels were higher in children with diarrhea (P = 0.019). Children who had EAEC infection, with or without diarrhea, had high mean lactoferrin levels regardless of nutritional status. The EAEC and Cryptosporidium were associated with diarrhea (P = 0.048 and 0.011, respectively), and malnourished children who had diarrhea were often co-infected with both Cryptosporidium and EAEC. In conclusion, the use of DNA-biomarkers revealed that EAEC and Cryptosporidium were common intestinal pathogens in Accra, and that elevated lactoferrin was associated with diarrhea in this group of children.