Diarrheal Disease In Young Children Research Articles

A Mathematical Model for Rotavirus Infection Incorporating Time Delay on the Effectiveness of Vaccination with Treatment

Rotavirus is the most common cause of severe diarrheal disease in young children globally attributing approximately 527,000 deaths of children under five each year. A Rotavirus vaccine was developed in 1998, however, it takes time for vaccine induced immunity to take place. The aim of the study was develop and analyze a mathematical model on rotavirus infection which incorporated a time delay on effectiveness of vaccination with treatment. The developed model was shown to be positively invariant and bounded.Conditions for stability of the equilibrium points is obtained and it is also shown that a bigger time delay would make the population not to be predictable. The findings of this study is useful to the government, ministry of Health stakeholders and policy developers and further provides baseline information for studies of this nature.

Transcriptional control of the Cryptosporidium life cycle.

The parasite Cryptosporidium is aleading agent of diarrhoeal disease in young children, and a cause and consequence of chronic malnutrition1,2. There are no vaccines and only limited treatment options3. The parasite infects enterocytes, in which it engages in asexual and sexual replication4, both of which are essential to continued infection and transmission. However, their molecular mechanisms remain largely unclear5. Here we use single-cell RNA sequencing to reveal the gene expression programme of the entire Cryptosporidium parvum life cycle in culture and in infected animals. Diverging from the prevailing model6, we find support for only three intracellular stages: asexual type-I meronts, male gamonts and female gametes. We reveal a highly organized program for the assembly of components at each stage. Dissecting the underlying regulatory network, we identify the transcription factor Myb-M as the earliest determinant of male fate, in an organism that lacks genetic sex determination. Conditional expression of this factor overrides the developmental program and induces widespread maleness, while conditional deletion ablates male development. Both have a profound impact on the infection. A large set of stage-specific genes now provides the opportunity to understand, engineer and disrupt parasite sex and life cycle progression to advance the development of vaccines and treatments.

Immunity to Cryptosporidium: insights into principles of enteric responses to infection.

Cryptosporidium parasites replicate within intestinal epithelial cells and are an important cause of diarrhoeal disease in young children and in patients with primary and acquired defects in T cell function. This Review of immune-mediated control of Cryptosporidium highlights advances in understanding how intestinal epithelial cells detect this infection, the induction of innate resistance and the processes required for activation of T cell responses that promote parasite control. The development of a genetic tool set to modify Cryptosporidium combined with tractable mouse models provide new opportunities to understand the principles that govern the interface between intestinal epithelial cells and the immune system that mediate resistance to enteric pathogens.

Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum.

The apicomplexan parasite Cryptosporidium is a leading global cause of diarrheal disease, and the infection poses a particularly grave threat to young children and those with weakened immune function. Infection occurs by ingestion of meiotic spores called oocysts, and transmission relies on fecal shedding of new oocysts. The entire life cycle thus occurs in a single host and features asexual as well as sexual forms of replication. Here, we identify and locus tag two Apetala 2-type (AP2) transcription factors and demonstrate that they are exclusively expressed in male and female gametes, respectively. To enable functional studies of essential genes in Cryptosporidium parvum, we develop and validate a small-molecule-inducible gene excision system, which we apply to the female factor AP2-F to achieve conditional gene knockout. Analyzing this mutant, we find the factor to be dispensable for asexual growth and early female fate determination in vitro but to be required for oocyst shedding in infected animals in vivo. Transcriptional analyses conducted in the presence or absence of AP2-F revealed that the factor controls the transcription of genes encoding crystalloid body proteins, which are exclusively expressed in female gametes. In C. parvum, the organelle is restricted to sporozoites, and its loss in other apicomplexan parasites leads to blocked transmission. Overall, our development of conditional gene ablation in C. parvum provides a robust method for genetic analysis in this parasite that enabled us to identify AP2-F as an essential regulator of transcription required for oocyst shedding and transmission. IMPORTANCE The parasite Cryptosporidium infects millions of people worldwide each year, leading to life-threatening diarrheal disease in young children and immunosuppressed individuals. There is no vaccine and only limited treatment. Transmission occurs via the fecal-oral route by an environmentally resilient spore-like oocyst. Infection takes place in the intestinal epithelium, where parasites initially propagate asexually before transitioning to male and female gametes, with sex leading to the formation of new oocysts. The essential role of sexual development for continuous infection and transmission makes it an attractive target for therapy and prevention. To study essential genes and potential drug targets across the life cycle, we established inducible gene excision for C. parvum. We determined that the female-specific transcription factor AP2-F is not required for asexual growth and early female development in vitro but is necessary for oocyst shedding in vivo. This work enhances the genetic tools available to study Cryptosporidium gene function.

Analysis of Basic Environmental Health Facilities Associated with Risk Factors of Diarrhea Among Toddlers

Diarrheal disease in young children (toddlers) seems increasingly common in tropical countries, especially in dirty and dense urban areas. Diarrhea can last several days and cause the body to lose the fluids needed for survival. This study aimed to determine the sanitation factors that influence the incidence of diarrhea. Methods: This study used a case-control design involving 100 respondents. All respondents were divided into two groups: the case class (toddlers who had diarrhea) and the control class (toddlers who did not experience diarrhea). Results and Discussion: This showed that four sub-variables of sanitation (waste, drainage conditions, domestic wastewater management, and water source safety) had a significant relationship with the incidence of diarrhea (p-value 0.05). Domestic wastewater was the most influencing factor and securing clean water and drinking water sources, with a Nagelkerke R Square value of 0.952. There was an indication that the independent variable (sanitation) affected the dependent variable (diarrhea incidence) by 95.2%. Conclusion: The sub-variable of domestic wastewater management and the sub-variable of the safety of clean water and drinking water sources were the most affecting the incidence of diarrhea. The clean water source factor was 12 times riskier. It means children who did not get it will have 12 times the risk of getting diarrhea. Meanwhile, in the aspect of domestic wastewater management, children who did not meet the requirements for domestic wastewater have a risk of 8.13 times the incidence of diarrhea.

The enteric pathogen Cryptosporidium parvum exports proteins into the cytosol of the infected host cell.

The parasite Cryptosporidium is responsible for diarrheal disease in young children causing death, malnutrition, and growth delay. Cryptosporidium invades enterocytes where it develops in a unique intracellular niche. Infected cells exhibit profound changes in morphology, physiology, and transcriptional activity. How the parasite effects these changes is poorly understood. We explored the localization of highly polymorphic proteins and found members of the Cryptosporidium parvum MEDLE protein family to be translocated into the cytosol of infected cells. All intracellular life stages engage in this export, which occurs after completion of invasion. Mutational studies defined an N-terminal host-targeting motif and demonstrated proteolytic processing at a specific leucine residue. Direct expression of MEDLE2 in mammalian cells triggered an ER stress response, which was also observed during infection. Taken together, our studies reveal the presence of a Cryptosporidium secretion system capable of delivering parasite proteins into the infected enterocyte.

Insulinase-like Protease 1 Contributes to Macrogamont Formation in Cryptosporidium parvum.

The apicomplexan parasite Cryptosporidium parvum contains an expanded family of 22 insulinase-like proteases (INS), a feature that contrasts with their otherwise streamlined genome. Here, we examined the function of INS1, which is most similar to the human insulinase protease that cleaves a variety of small peptide substrates. INS1 is an M16A clan member and contains a signal peptide, an N-terminal domain with the HXXEH active site, followed by three inactive domains. Unlike previously studied C. parvum INS proteins that are expressed in sporozoites and during merogony, INS1 was expressed exclusively in macrogamonts, where it was localized in small cytoplasmic vesicles. Although INS1 did not colocalize with the oocyst wall protein recognized by the antibody OW50, immune-electron microscopy indicated that INS1 resides in small vesicles in the secretory system. Notably, these small INS1-positive vesicles were often in close proximity to large OW50-positive vacuoles resembling wall-forming bodies, which contain precursors for oocyst wall formation. Genetic deletion of INS1, or replacement with an active-site mutant, resulted in lower formation of macrogamonts in vitro and reduced oocyst shedding in vivo Our findings reveal that INS1 functions in the formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice.IMPORTANCE Cryptosporidiosis is a debilitating diarrheal disease in young children in developing countries. The absence of effective treatments or vaccines makes this infection very difficult to manage in susceptible populations. Although the oral dose of oocysts needed to cause infection is low, infected individuals shed very high numbers of oocysts, readily contaminating the environment. Our studies demonstrate that the protease INS1 is important for formation of female sexual stages and that in its absence, parasites produce fewer oocysts and are attenuated in immunocompromised mice. These findings suggest that mutants lacking INS1, or related proteases, are useful for further characterizing the pathway that leads to macrogamont maturation and oocyst wall formation.

Articles of Significant Interest in This Issue

Rotaviruses are a leading cause of severe diarrheal disease in young children throughout the world. Using reverse genetics technologies, Philip and Patton (e00959-20) found that the rotavirus RNA genome is remarkably flexible and can accommodate additional coding sequence sufficient to allow expression of separate foreign proteins. Among the recombinant rotaviruses that were engineered are those that produce fluorescent reporters, enabling the study of rotavirus biology and pathogenesis by live cell imaging. Given the capacity of rotavirus to encode foreign proteins, the potential exists for developing rotavirus vaccines as vector systems against other enteric and mucosal diseases.

1660. Protective Effect of Living with Animals on Diarrheal Illness Among Children Younger Than Five in Sub-Saharan Africa and South Asia

BackgroundDiarrheal disease is one of the top three causes of death in children <5 years old, with the majority occurring in sub-Saharan Africa and South Asia. Animals are known reservoirs of diarrheal pathogens. While previous studies have investigated companion animals and livestock as contributors to human diarrheal disease, few have investigated this association in sub-Saharan Africa and/or South Asia among children younger than 5 years old. The aim of this study was to determine the relationship between animals living in or near the home and diarrheal disease in this population.MethodsWe conducted a secondary analysis of the Global Enteric Multicenter Study (GEMS), a case–control study that described the epidemiology of and evaluated the risk factors for diarrheal illness in children <5 years old living in sub-Saharan Africa and South Asia. Children with moderate-to-severe diarrhea (MSD) were matched to community controls without diarrhea. Associations between the predictor of animals present in a child’s living environment and the outcomes of diarrhea and diarrheal pathogens were assessed using multivariable conditional logistic regression with alpha = 0.05. Known risk factors for MSD were assessed for confounding.ResultsFrom December 1, 2007 to March 3, 2011, there were 9,439 cases with MSD and 13,128 controls enrolled in GEMS. 87% of children had at least 1 animal present at their home. When controlling for exclusive breastfeeding, access to an improved water source, access to a private improved sanitation facility, and the number of children <5 living in the home, children were less likely to have MSD if there was at least one animal present at their home (aOR 0.66, CI95 0.59 – 0.74). Children living with an animal were also 15% less likely to be colonized or infected with a viral diarrheal pathogen (aOR 0.85, CI95 0.72–0.99), when controlling for exclusive breastfeeding. There were no significant differences in the presence of bacterial or protozoal pathogens in the stool of children with and without animals present at their home.ConclusionIn contrast to prior research, having an animal present at the home does not appear to increase the risk of childhood diarrhea. Furthermore, the presence of animals may have a protective effect against viral diarrheal disease in young children. Additional research is needed to gain greater understanding of this relationship.Disclosures All authors: No reported disclosures.

Rotavirus Species B Encodes a Functional Fusion-Associated Small Transmembrane Protein.

Rotavirus is an important cause of diarrheal disease in young mammals. Rotavirus species A (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Rotavirus species B (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytium formation in cultured human cells. Based on sequence alignment, NSP1-1 proteins from species B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other genera of the Reoviridae family. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multicycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis.IMPORTANCE While species A rotavirus is commonly associated with diarrheal disease in young children, species B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between species A and B rotaviruses is the NSP1 gene, which encodes two proteins for species B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. These comparisons suggest that there is a fatty acid modification at the amino terminus of the protein, and our results show that an intact amino terminus is required for NSP1-1-mediated fusion. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances species A rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB host species tropism, immune evasion, and pathogenesis.

Cryptosporidium infection in rural Gambian children: Epidemiology and risk factors.

BackgroundCryptosporidium is a major pathogen associated with diarrheal disease in young children. We studied Cryptosporidium diarrhea in children enrolled in the Global Enteric Multicenter Study (GEMS) in rural Gambia.MethodsWe recruited children <5 years of age with moderate-to-severe diarrhea (MSD) for 3 years (2008–2010), and children with either MSD or less severe diarrhea (LSD) for one year (November 2011-November 2012) at sentinel health centers. One or more randomly selected controls were matched to each case. Stool samples were tested to identify Cryptosporidium by immunoassay. A subset of randomly selected case-controls pairs were tested for Cryptosporidium species. We investigated the epidemiology of, and evaluated possible risk factors for, Cryptosporidium-positive diarrhea.ResultsWe enrolled 1938 cases (1381 MSD, 557 LSD) and 2969 matched controls; 231/1929 (12.0%) of diarrhea cases and 141/2962 (4.8%) of controls were positive for Cryptosporidium. Most Cryptosporidium diarrhea cases (85.7%, 198/231) were aged 6–23 months, and most (81.4%, 188/231) occurred during the rainy season. Cryptosporidium hominis (C. hominis) was the predominant (82.6%) species. We found associations between increased risk of Cryptosporidium-positive MSD or LSD, or both, with consumption of stored drinking water and certain animals living in the compound—cow, cat (MSD only) and rodents (LSD only). Larger households, fowl living in the compound, and the presence of Giardia infection were associated with decreased risk of Cryptosporidium MSD and LSD.ConclusionCryptosporidium-positive diarrhea is prevalent in this setting, especially at 6–23 months of age. The preponderance of Cryptosporidium infection in the rainy season and increased risk of Cryptosporidium-positive diarrhea with consumption of stored drinking water suggest water-borne transmission. Further investigation is needed to clarify the role of animals and contamination of stored drinking water in Cryptosporidium transmission.

Prevalence and clinical profile of rotavirus A infection among diarrhoeal children and phylogenetic analysis with vaccine strains in Chengdu, West China, 2009-2014.

Rotaviruses are the most common cause of severe diarrhoeal disease in young children. However, little is known about the epidemiological and clinical profile of rotavirus A (RVA) in diarrhoeal children or the efficacy of Lanzhou lamb rotavirus vaccine (LLR) in Chengdu, China. This study aimed to determine the prevalence and clinical profile of RVA in diarrhoeal children and provide gene analysis information for RVA vaccination programmes. A total of 1121 faecal samples were collected from outpatient children with diarrhoea between 2009 and 2014. RT-PCR was performed to detect RVA infection and other gastroenteritis viruses. VP4 and VP7 genes of 13 RVA strains were sequenced to compare their similarity with vaccine strains. The overall RVA infection rate was 17.48%. G1 (54.72%) and G3 (18.87%) were the predominant G genotypes; P[8] (72.36%) and P[4] (11.38%) were the main P genotypes. Sixteen genotypes were identified; G1P[8] (57.33%) and G9P[8] (12.00%) were the most prevalent. The proportion of coinfection with RVA and other gastroenteritis viruses was 18.88%. RVA was mostly detected in winter and in diarrhoeal children 1-2 years of age. The genotypes of Rotarix and RotaTeq vaccines were consistent with RVA strains prevalent in Sichuan and shared high identity. RVA was one of the major aetiological agents of diarrhoeal children in Chengdu. Genotype distribution differed within each year and the gene analysis implied low efficacy of LLR. Continuous epidemiological monitoring of RVA is essential for the national vaccination programme.

Determinants of severe dehydration from diarrheal disease at hospital presentation: Evidence from 22 years of admissions in Bangladesh

BackgroundTo take advantage of emerging opportunities to reduce morbidity and mortality from diarrheal disease, we need to better understand the determinants of life-threatening severe dehydration (SD) in resource-poor settings.Methodology/findingsWe analyzed records of patients admitted with acute diarrheal disease over twenty-two years at the International Centre for Diarrhoeal Disease Research, Bangladesh (1993–2014). Patients presenting with and without SD were compared by multivariable logistic regression models, which included socio-demographic factors and pathogens isolated. Generalized additive models evaluated non-linearities between age or household income and SD. Among 55,956 admitted patients, 13,457 (24%) presented with SD. Vibrio cholerae was the most common pathogen isolated (12,405 patients; 22%), and had the strongest association with SD (AOR 4.77; 95% CI: 4.41–5.51); detection of multiple pathogens did not exacerbate SD risk. The highest proportion of severely dehydrated patients presented in a narrow window only 4–12 hours after symptom onset. Risk of presenting with SD increased sharply from zero to ten years of age and remained high throughout adolescence and adulthood. Adult women had a 38% increased odds (AOR 1.38; 95% CI: 1.30–1.46) of SD compared to adult men. The probability of SD increased sharply at low incomes. These findings were consistent across pathogens.Conclusions/significanceThere remain underappreciated populations vulnerable to life-threatening diarrheal disease that include adult women and the very poor. In addition to efforts that address diarrheal disease in young children, there is a need to develop interventions for these other high-risk populations that are accessible within 4 hours of symptom onset.

Functional and Antigen-Specific Serum Antibody Levels as Correlates of Protection against Shigellosis in a Controlled Human Challenge Study.

ABSTRACTShigella is an important cause of diarrheal disease in young children living in developing countries. No approved vaccines are available, and the development of vaccine candidates has been hindered by the lack of firm immunological correlates of protection, among other reasons. To address this gap in knowledge, we established quantitative assays to measure Shigella-specific serum bactericidal antibody (SBA) and opsonophagocytic killing antibody (OPKA) activities and investigated their potential association with protection against disease in humans. SBA, OPKA, and Ipa-, VirG (IscA)-, and Shigella flexneri 2a lipopolysaccharide-specific serum IgG titers were determined in adult volunteers who received Shigella vaccine candidate EcSf2a-2 and in unvaccinated controls, all of whom were challenged with virulent Shigella flexneri 2a. Prechallenge antibody titers were compared with disease severity after challenge. SBA and OPKA, as well as IpaB- and VirG-specific IgG, significantly correlated with reduced illness. SBA and OPKA assays were also used to evaluate the immunogenicity of leading live attenuated vaccine candidates Shigella CVD 1204 and CVD 1208S in humans. A single oral immunization with CVD 1204 or CVD 1208S resulted in SBA seroconversion rates of 71% and 47% and OPKA seroconversion rates of 57% and 35%, respectively. Higher functional antibody responses were induced by CVD 1204, which is consistent with its lower attenuation. This is the first demonstration of SBA, OPKA, and IpaB- and VirG-specific IgG levels as potential serological correlates of protection against shigellosis in humans. These results warrant further studies to establish their capacity to predict protective immunity and vaccine efficacy.

A genome-wide association meta-analysis of diarrhoeal disease in young children identifies FUT2 locus and provides plausible biological pathways.

More than a million childhood diarrhoeal episodes occur worldwide each year, and in developed countries a considerable part of them are caused by viral infections. In this study, we aimed to search for genetic variants associated with diarrhoeal disease in young children by meta-analyzing genome-wide association studies, and to elucidate plausible biological mechanisms. The study was conducted in the context of the Early Genetics and Lifecourse Epidemiology (EAGLE) consortium. Data about diarrhoeal disease in two time windows (around 1 year of age and around 2 years of age) was obtained via parental questionnaires, doctor interviews or medical records. Standard quality control and statistical tests were applied to the 1000 Genomes imputed genotypic data. The meta-analysis (N = 5758) followed by replication (N = 3784) identified a genome-wide significant association between rs8111874 and diarrhoea at age 1 year. Conditional analysis suggested that the causal variant could be rs601338 (W154X) in the FUT2 gene. Children with the A allele, which results in a truncated FUT2 protein, had lower risk of diarrhoea. FUT2 participates in the production of histo-blood group antigens and has previously been implicated in the susceptibility to infections, including Rotavirus and Norovirus Gene-set enrichment analysis suggested pathways related to the histo-blood group antigen production, and the regulation of ion transport and blood pressure. Among others, the gastrointestinal tract, and the immune and neuro-secretory systems were detected as relevant organs. In summary, this genome-wide association meta-analysis suggests the implication of the FUT2 gene in diarrhoeal disease in young children from the general population.

Subsidized Sachet Water to Reduce Diarrheal Disease in Young Children: A Feasibility Study in Accra, Ghana.

Use of drinking water sold in plastic bags (sachet water) is growing rapidly in west Africa. The impact on water consumption and child health remains unclear, and a debate on the taxation and regulation of sachet water is ongoing. This study assessed the feasibility of providing subsidized sachet water to low-income urban households in Accra and measured the resultant changes in water consumption. A total of 86 children, 6–36 months of age in neighborhoods lacking indoor piped water, were randomized to three study arms. The control group received education about diarrhea. The second arm received vouchers for 15 L/week/child of free water sachets (value: $0.63/week) plus education. The third arm received vouchers for the same water sachet volume at half price plus education. Water consumption was measured at baseline and followed for 4 months thereafter. At baseline, 66 of 81 children (82%) drank only sachet water. When given one voucher/child/week, households redeemed an average 0.94 vouchers/week/child in the free-sachet-voucher arm and 0.82 vouchers/week/child in the half-price arm. No change in water consumption was observed in the half-price arm, although the study was not powered to detect such differences. In the free-sachet-voucher arm, estimated sachet water consumption increased by 0.27 L/child/day (P = 0.03). The increase in sachet water consumption by children in the free-sachet-voucher arm shows that provision of fully subsidized water sachets might improve the quality of drinking water consumed by children. Further research is needed to quantify this and any related child health impacts.

Vitamin D3 supplementation alleviates rotavirus infection in pigs and IPEC-J2 cells via regulating the autophagy signaling pathway

Vitamin D had an anti-infection effect and benefited to the intestinal health. Autophagy signaling pathway was regulated by vitamin D3 to inhibit the infection of human immunodeficiency virus type-1. Rotavirus (RV) was a major cause of the severe diarrheal disease in young children and young animals. Although evidence suggested that vitamin D3 attenuates the negative effects of RV infection via the retinoic acid-inducible gene I signaling pathway, little is known of its antiviral effect whether through the regulation of autophagy. The present study was performed to investigate whether vitamin D3 alleviates RV infection in pig and porcine small intestinal epithelial cell line (IPEC-J2) models via regulating the autophagy signaling pathway. RV administration increased the Beclin 1 mRNA abundance in porcine jejunum and ileum. 5000 IU/kg dietary vitamin D3 supplementation greatly up-regulated LC3-II/LC3-I ratios and PR-39 mRNA expression under the condition of RV challenged. The viability of IPEC-J2 was significantly inhibited by RV infection. Incubation with 25-hydroxyvitamin D3 significantly decreased the concentrations of RV antigen and non-structural protein 4 (NSP4), and up-regulated the mRNA expression of Beclin 1 and PR-39 in the RV-infected IPEC-J2 cells. And then, based on the 25-hydroxyvitamin D3 treatment and RV infection, LC3-II mRNA expression in cells was inhibited by an autophagy inhibitor 3-methyladenine (3-MA). Bafilomycin A1 (Baf A1, a class of inhibitors of membrane ATPases, inhibits maturation of autophagic vacuoles) treatment numerically enhanced the LC3-II mRNA abundance, but had no effect on NSP4 concentration. Furthermore, 25-hydroxyvitamin D3 decreased the p62 mRNA expression and increased porcine cathelicidins (PMAP23, PG1-5 and PR-39) mRNA expression in the RV-infected cells. Taken together, these results indicated that vitamin D3 attenuates RV infection through regulating autophagic maturation and porcine cathelicidin genes expression.

Parasitology: CRISPR for Cryptosporidium.

Study of the diarrhoea-causing pathogen Cryptosporidium has been hindered by a lack of genetic-modification and culture tools. A description of genome editing and propagation methods for the parasite changes this picture. See Letter p.477 The protozoan parasite Cryptosporidium is a major cause of diarrhoeal disease in young children but until now it has been difficult to study and there is currently no vaccine and only a single drug (nitazoxanide) available to counter the infection. Here Boris Striepen and colleagues describe a robust genetic system for cryptosporidiosis. They genetically modify Cryptosporidium parvum by optimizing transfection of sporozoites using a CRISPR/Cas9 system, to generate stable transgenic lines suitable for in vitro and in vivo drug screening. Using this system they knockout the gene encoding thymidine kinase which increases susceptibility to trimethoprim, an antimalarial drug to which wild-type Cryptosporidium is resistant.

Complex epidemiology and zoonotic potential for Cryptosporidium suis in rural Madagascar

Cryptosporidium spp. is the most important parasitic diarrheal agent in the world, is among the top four causes of moderate-to-severe diarrheal disease in young children in developing nations, and is problematic as an opportunistic co-infection with HIV. In addition, Cryptosporidium is a persistent challenge for livestock production. Despite its zoonotic potential, few studies have examined the ecology and epidemiology of this pathogen in rural systems characterized by high rates of overlap among humans, domesticated animals, and wildlife. To improve our understanding of the zoonotic potential of Cryptosporidium species in the rural tropics, we screened humans, livestock, peridomestic rodents, and wildlife using PCR-RFLP and sequencing-based approaches to distinguish species of Cryptosporidium in rural southeastern Madagascar. Cryptosporidium of multiple species/genotypes were apparent in this study system. Interestingly, C. suis was the dominant species of Cryptosporidium in the region, infecting humans (n=1), cattle (n=18), pigs (n=3), and rodents (n=1). The broad species range of C. suis and the lack of common cattle Cryptosporidium species (Cryptosporidium parvum and Cryptosporidium andersoni) in this system are unique. This report represents the fifth confirmed case of C. suis infection in humans, and the first case in Africa. Few rural human and livestock populations have been screened for Cryptosporidium using genus-specific genotyping methods. Consequently, C. suis may be more widespread in human and cattle populations than previously believed.

CXCR2-Dependent Mucosal Neutrophil Influx Protects against Colitis-Associated Diarrhea Caused by an Attaching/Effacing Lesion-Forming Bacterial Pathogen

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrheal disease in young children, yet symptoms and duration are highly variable for unknown reasons. Citrobacter rodentium, a murine model pathogen that shares important functional features with EPEC, colonizes mice in colon and cecum and causes inflammation, but typically little or no diarrhea. We conducted genome-wide microarray studies to define mechanisms of host defense and disease in C. rodentium infection. A significant fraction of the genes most highly induced in the colon by infection encoded CXC chemokines, particularly CXCL1/2/5 and CXCL9/10, which are ligands for the chemokine receptors CXCR2 and CXCR3, respectively. CD11b(+) dendritic cells were the major producers of CXCL1, CXCL5, and CXCL9, while CXCL2 was mainly induced in macrophages. Infection of gene-targeted mice revealed that CXCR3 had a significant but modest role in defense against C. rodentium, whereas CXCR2 had a major and indispensable function. CXCR2 was required for normal mucosal influx of neutrophils, which act as direct antibacterial effectors. Moreover, CXCR2 loss led to severe diarrhea and failure to express critical components of normal ion and fluid transport, including ATPase beta(2)-subunit, CFTR, and DRA. The antidiarrheal functions were unique to CXCR2, since other immune defects leading to increased bacterial load and inflammation did not cause diarrhea. Thus, CXCR2-dependent processes, particularly mucosal neutrophil influx, not only contribute to host defense against C. rodentium, but provide protection against infection-associated diarrhea.