Diarrheal Disease In Humans Research Articles

Rotavirus infection markers in children with rotaviral gastroenteritis and their relation to disease severity

Abstract Introduction Although natural infection with rotavirus causes damage to the enteric tract resulting in diarrheal disease in humans and animals, recent studies evidenced the presence of serum rotavirus antigen/RNA in children with rotavirus diarrhea. Methods In this study, we investigated the markers of acute rotavirus infection (antigenemia, viremia and anti-rotavirus IgM antibody) in a group of 50 rotavirus infected children, using enzyme-linked immunosorbant assay and conventional polymerase chain reaction in stool and serum specimens. Results Rotavirus antigenemia and viremia were identified in 50% and 54% of acute-phase serum samples respectively. The mean level of rotavirus antigen in stools was greater than in serum. The rate of viremia was significantly higher in the serum of children with antigenemia than in those without (P Conclusion Accordingly, we can conclude that rotaviral antigenemia and viremia were common in children with rotaviral diarrhea, however, the impact of rotavirus antigenemia/viremia on clinical manifestations of infection is unknown.

Vasodilator-stimulated phosphoprotein restricts cell-to-cell spread of Shigella flexneri at the cell periphery.

Shigella spp. are intracellular bacterial pathogens that cause diarrhoeal disease in humans. Shigella utilize the host actin cytoskeleton to enter cells, move through the cytoplasm of cells and pass into adjacent cells. Ena/VASP family proteins are highly conserved proteins that participate in actin-dependent dynamic cellular processes. We tested whether Ena/VASP family members VASP (vasodilator-stimulated phosphoprotein), Mena (mammalian-enabled) or EVL (Ena-VASP-like) contribute to Shigella flexneri spread through cell monolayers. VASP and EVL restricted cell-to-cell spread without significantly altering actin-based motility, whereas Mena had no effect on these processes. Phosphorylation of VASP on Ser153, Ser235 and Thr274 regulated its subcellular distribution and function. VASP derivatives that lack the Ena/VASP homology 1 (EVH1) domain or contain a phosphoablative mutation of Ser153 were defective in restricting S. flexneri spread, indicating that the EVH1 domain and phosphorylation on Ser153 are required for this process. The EVH1 domain and Ser153 of VASP were required for VASP localization to focal adhesions, and localization of VASP to focal adhesions and/or the leading edge was required for restriction of spread. The contribution of the EVH1 domain was from both the donor and the recipient cell, whereas the contribution of Ser153 phosphorylation was only from the donor cell. Thus, unlike host proteins characterized in Shigella pathogenesis that promote bacterial spread, VASP and EVL function to limit it. The ability of VASP and EVL to limit spread highlights the critical role of focal adhesion complexes and/or the leading edge in bacterial passage between cells.

Prevalence and molecular characterization of Cryptosporidium in giant panda (Ailuropoda melanoleuca) in Sichuan province, China.

BackgroundCryptosporidium spp. have been extensively reported to cause significant diarrheal disease in humans and domestic animals. On the contrary, little information is available on the prevalence and characterization of Cryptosporidium in wild animals in China, especially in giant pandas. The aim of the present study was to detect Cryptosporidium infections and identify Cryptosporidium species at the molecular level in both captive and wild giant pandas in Sichuan province, China.FindingsUsing a PCR approach, we amplified and sequenced the 18S rRNA gene from 322 giant pandas fecal samples (122 from 122 captive individuals and 200 collected from four habitats) in Sichuan province, China. The Cryptosporidium species/genotypes were identified via a BLAST comparison against published Cryptosporidium sequences available in GenBank followed by phylogenetic analysis. The results revealed that both captive and wild giant pandas were infected with a single Cryptosporidium species, C. andersoni, at a prevalence of 15.6 % (19/122) and 0.5 % (1/200) in captive and wild giant pandas, respectively.ConclusionsThe present study revealed the existence of C. andersoni in both captive and wild giant panda fecal samples for the first time, and also provided useful fundamental data for further research on the molecular epidemiology and control of Cryptosporidium infection in giant pandas.

Molecular characterization of Cryptosporidium spp. and Giardia duodenalis from yaks in the central western region of China.

BackgroundCryptosporidium spp. and Giardia duodenalis are important causes of diarrheal diseases in humans and animals worldwide, and there is an increased interest in the role of animals in the mechanical transmission of these protozoa. To examine the role of yaks in this process, we examined the occurrence and genotypes of Cryptosporidium and G. duodenalis in yaks in western China.ResultsA total of 545 fecal specimens were collected from yaks from nine different counties in the central western region of China. The prevalence for Cryptosporidium spp. and G. duodenalis were 4.0 % (22/545) and 6.0 % (16/545), respectively. Mixed infections of Cryptosporidium and G. duodenalis were also detected in four specimens. The prevalence of both protozoa differed significantly between some age groups, with higher rates of infection in animals < 1 year old. Sequence analysis of the small subunit rRNA (SSU rRNA) gene of the Cryptosporidium isolates identified the species as C. parvum (n = 12), C. bovis (n = 6), C. ryanae (n = 3), and C. ubiquitum (n = 1). Genotyping based on 60-kDa glycoprotein (gp60) gene from five C. parvum isolates identified all as IId with three isolates identified as IIdA15G1, one as IIdA18G1, and one as IIdA19G1. One C. ubiquitum isolate was identified as subtype VIIa. Amongst the G. duodenalis isolates, 16 were identified as assemblage E at the SSU rRNA gene. Four novel glutamate dehydrogenase (gdh) subtypes and two triosephosphate isomerase (tpi) subtypes were found amongst the G. duodenalis assemblage E isolates.ConclusionsThe presence of C. parvum subtype IIdA15G1, IIdA18G1, and IIdA19G1 isolates further confirms the dominance of the C. parvum IId subtypes in China. These findings also indicate that yaks may be a source of zoonotic Cryptosporidium infection, and this is the first report of G. duodenalis in yaks. The data presented here provides the basis for further genotyping or subtyping studies of G. duodenalis in yaks.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0446-0) contains supplementary material, which is available to authorized users.

Draft Genome Sequence of Enterotoxigenic Escherichia coli Strain E24377A, Obtained from a Tribal Drinking Water Source in India.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease in humans and animals. Its dissemination can occur through water sources contaminated by it. Here, we report for the first time the draft genome sequence of ETEC strain E24377A, obtained from a tribal drinking water source in India.

Molecular detection identified a type six secretion system in Campylobacter jejuni from various sources but not from human cases.

To determine the presence of the T6SS in Campylobacter jejuni from diverse sources. The recently identified type VI secretion system (T6SS) is a bacterial injection machinery that plays a role in virulence, symbiosis, bacterial interactions and environmental stress responses. This system has been recently discovered in the major enteric pathogen Camp.jejuni. In this study, we used multiplex PCR (mPCR), based on conserved genetic markers of the T6SS, to screen 366 Pakistani Camp.jejuni isolates from humans, poultry, cattle, wildlife or waste-water sources. We identified the T6SS in isolates from all of these sources except humans. The overall prevalence of the T6SS among the isolates was 17/366 (4·6%) and the T6SS positive isolates clustered into four different groups. Transcription of the T6SS genes, determined using RT-PCR, was observed in bacteria cultured at 37 or 42°C but not in 37°C cultures adjusted to pH3. Campylobacter jejuni isolates harbouring T6SS markers genes were identified in livestock and non-livestock sources but in this study we did not identify human diarrhoeal isolates which possessed the T6SS. We demonstrated down-regulation of T6SS in an acidic environment. This study questions the role of the T6SS in human diarrhoeal disease. Moreover this study did not identify a clear association of Camp.jejuni isolates harbouring T6SS with any of the niches tested. Our study highlights the need to establish the role of the T6SS in environmental survival or virulence.

Campylobacter jejuni acquire new host-derived CRISPR spacers when in association with bacteriophages harboring a CRISPR-like Cas4 protein.

Campylobacter jejuni is a worldwide cause of human diarrhoeal disease. Clustered Repetitively Interspaced Palindromic Repeats (CRISPRs) and associated proteins allow Bacteria and Archaea to evade bacteriophage and plasmid infection. Type II CRISPR systems are found in association with combinations of genes encoding the CRISPR-associated Cas1, Cas2, Cas4 or Csn2, and Cas9 proteins. C. jejuni possesses a minimal subtype II-C CRISPR system containing cas1, cas2, and cas9 genes whilst cas4 is notably absent. Cas4 proteins possess 5′-3′ exonuclease activity to create recombinogenic-ends for spacer acquisition. Here we report a conserved Cas4-like protein in Campylobacter bacteriophages that creates a novel split arrangement between the bacteriophage and host that represents a new twist in the bacteriophage/host co-evolutionary arms race. The continuous association of bacteriophage and host in the carrier state life cycle of C. jejuni provided an opportunity to study spacer acquisition in this species. Remarkably all the spacer sequences observed were of host origin. We hypothesize that Campylobacter bacteriophages can use Cas4-like protein to activate spacer acquisition to use host DNA as an effective decoy to bacteriophage DNA. Bacteria that acquire self-spacers and escape phage infection must overcome CRISPR-mediated autoimmunity either by loss of the interference functions leaving them susceptible to foreign DNA incursion or tolerate changes in gene regulation.

Effect of a single dose of Saccharomyces cerevisiae var. boulardii on the occurrence of porcine neonatal diarrhoea

Piglet neonatal diarrhoea is an important issue in modern pig production and is linked to increased mortality and poor growth rates, affecting long-term pig health, increasing use of medication and cost of production. Saccharomyces cerevisiae var. boulardii (SB) is a probiotic yeast with documented clinical efficacy in the prevention and treatment of diarrhoeal diseases in humans. The objectives of the current study were to evaluate the effect of SB on occurrence and severity of neonatal diarrhoea in piglets, mortality and growth rate. Forty-six litters (606 piglets) were randomly allocated to a control or SB treatment (n=23 per treatment). Within 24 h of farrowing, piglets assigned to the SB treatment received a single oral dose of a paste containing 3.3×109 CFU of SB CNCM I-1079. Piglets from the control litters received a placebo paste. Piglet weight, mortality and diarrhoea were recorded up to day 7 of age. It was shown that numbers of diarrhoea days were significantly correlated with increased mortality rate and reduced weight gain (P<0.05). SB treatment had no effect on growth or mortality in diarrhoeic litters. However, SB-supplemented litters had significantly lower faecal scores, indicating firmer faeces (P<0.01) and fewer numbers of diarrhoeic days (P<0.01) during the 1st week of life. Reduction in the number of diarrhoeic litters compared with the control group was observed following the probiotic administration (P<0.05). These results highlight the detrimental effects of neonatal diarrhoea on pre-weaning performance and suggest that SB, by reducing diarrhoea duration and severity, has the potential of improving enteric health in the early stages of life in pigs.

The prevalence of Giardia infection in dogs and cats, a systematic review and meta-analysis of prevalence studies from stool samples

Giardia has a wide range of host species and is a common cause of diarrhoeal disease in humans and animals. Companion animals are able to transmit a range of zoonotic diseases to their owners including giardiasis, but the size of this risk is not well known. The aim of this study was to analyse giardiasis prevalence rates in dogs and cats worldwide using a systematic search approach. Meta-analysis enabled to describe associations between Giardia prevalence and various confounding factors. Pooled prevalence rates were 15.2% (95% CI 13.8–16.7%) for dogs and 12% (95% CI 9.2–15.3%) for cats. However, there was very high heterogeneity between studies. Meta-regression showed that the diagnostic method used had a major impact on reported prevalence with studies using ELISA, IFA and PCR reporting prevalence rates between 2.6 and 3.7 times greater than studies using microscopy. Conditional negative binomial regression found that symptomatic animals had higher prevalence rates ratios (PRR) than asymptomatic animals 1.61 (95% CI 1.33–1.94) in dogs and 1.94 (95% CI 1.47–2.56) in cats. Giardia was much more prevalent in young animals. For cats >6 months, PRR=0.47 (0.42–0.53) and in dogs of the same age group PRR=0.36 (0.32–0.41). Additionally, dogs kept as pets were less likely to be positive (PRR=0.56 (0.41–0.77)) but any difference in cats was not significant. Faecal excretion of Giardia is common in dogs and slightly less so in cats. However, the exact rates depend on the diagnostic method used, the age and origin of the animal. What risk such endemic colonisation poses to human health is still unclear as it will depend not only on prevalence rates but also on what assemblages are excreted and how people interact with their pets.

Antimicrobial Susceptibility and Molecular Typing ofSalmonellaAgona Isolated from Humans and Other Sources

Salmonella enterica serotype Agona (Salmonella Agona) has been among the top 10 serotypes that cause human diarrheal diseases in China. A total of 95 Salmonella Agona (67 from humans, and 28 from animals, food of animal origins, and environmental sources) recovered in Shanghai, China from 2005 to 2011 were subjected to antimicrobial susceptibility testing and molecular subtyping using pulsed-field gel electrophoresis (PFGE). Approximately 68.4% of the Salmonella Agona isolates were pansusceptible to 15 antimicrobial agents tested, and 4 isolates (4.21%) were resistant to at least 3 antimicrobials. PFGE analysis resulted in 41 unique patterns, of which 4 major PFGE patterns (X3, X4, X5, and X6) were grouped together at 96.1% similarity. Isolates of the four patterns included those from food (pork, beef, and chicken) and humans. Our findings showed that the same clones of Salmonella Agona were recovered from human patients and food, and that food of animal origin was potentially a major vehicle of Salmonella Agona in human salmonellosis in Shanghai.

Immunogenicity of a fusion protein comprising coli surface antigen 3 and labile B subunit of enterotoxigenic Escherichia coli.

Background: Enterotoxigenic Escherichia coli (ETEC) strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate. Methods: A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit (LTB) was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated. Results: Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GM1 binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells. Conclusion: The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation.

Interaction between Campylobacter and intestinal epithelial cells leads to a different proinflammatory response in human and porcine host

Campylobacter jejuni and Campylobacter coli are recognized as the leading causes of human diarrheal disease throughout the development world. Unlike human beings, gastrointestinal tract of pigs are frequently colonized by Campylobacter to a high level in a commensal manner. The aim of this study was to identify the differences underlying the divergent outcome following Campylobacter challenge in porcine versus human host. In order to address this, a comparative in vitro infection model was combined with microscopy, gentamicin protection assay, ELISA and quantitative PCR techniques. Invasion assays revealed that Campylobacter invaded human cells up to 10-fold more than porcine cells (p<0.05). In addition, gene expression of proinflammatory genes encoding for IL1α, IL6, IL8, CXCL2 and CCL20 were strongly up-regulated by Campylobacter in human epithelial cell at early times of infection, whereas a very reduced cytokine gene expression was detected in porcine epithelial cells. These data indicate that Campylobacter fails to invade porcine cells compared to human cells, and this leads to a lack of proinflammatory response induction, probably due to its pathogenic or commensal behavior in human and porcine host, respectively.

Proteomic evidences for rex regulation of metabolism in toxin-producing Bacillus cereus ATCC 14579.

The facultative anaerobe, Bacillus cereus, causes diarrheal diseases in humans. Its ability to deal with oxygen availability is recognized to be critical for pathogenesis. The B. cereus genome comprises a gene encoding a protein with high similarities to the redox regulator, Rex, which is a central regulator of anaerobic metabolism in Bacillus subtilis and other Gram-positive bacteria. Here, we showed that B. cereus rex is monocistronic and down-regulated in the absence of oxygen. The protein encoded by rex is an authentic Rex transcriptional factor since its DNA binding activity depends on the NADH/NAD+ ratio. Rex deletion compromised the ability of B. cereus to cope with external oxidative stress under anaerobiosis while increasing B. cereus resistance against such stress under aerobiosis. The deletion of rex affects anaerobic fermentative and aerobic respiratory metabolism of B. cereus by decreasing and increasing, respectively, the carbon flux through the NADH-recycling lactate pathway. We compared both the cellular proteome and exoproteome of the wild-type and Δrex cells using a high throughput shotgun label-free quantitation approach and identified proteins that are under control of Rex-mediated regulation. Proteomics data have been deposited to the ProteomeXchange with identifier PXD000886. The data suggest that Rex regulates both the cross-talk between metabolic pathways that produce NADH and NADPH and toxinogenesis, especially in oxic conditions.

Draft Genome Sequence of Enterotoxigenic Escherichia coli Strain W25K.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease in humans and newly weaned pigs. Here, we report the draft genome sequence of ETEC strain W25K, which causes diarrhea in piglets.

COMPARACIÓN DE MÉTODOS CONVENCIONALES Y MOLECULARES PARA LA DETECCIÓN DE Giardia lamblia EN HECES HUMANAS

Resumen es: Los protozoarios del genero Giardia representan algunos de los parasitos humanos mas comunes en el mundo y estan entre los principales causantes de infec...

Zebrafish as a Natural Host Model for Vibrio cholerae Colonization and Transmission

The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is associated with several varieties of aquatic life, including insect egg masses, shellfish, and vertebrate fish. Here we describe a novel animal model for V. cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish intestinal tract after exposure in water with no manipulation of the animal required. Colonization occurs in close contact with the intestinal epithelium and mimics colonization observed in mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naive fish, which then become colonized. Striking differences in colonization between V. cholerae classical and El Tor biotypes were apparent. The zebrafish natural habitat in Asia heavily overlaps areas where cholera is endemic, suggesting that zebrafish and V. cholerae evolved in close contact with each other. Thus, the zebrafish provides a natural host model for the study of V. cholerae colonization, transmission, and environmental survival.

Genetic characterization of human-pathogenic Cyclospora cayetanensis parasites from three endemic regions at the 18S ribosomal RNA locus

Cyclospora cayetanensis is an apicocomplexan parasite that infects the gastrointestinal tract and causes acute diarrheal disease in humans. In recent years, this human-pathogenic parasite has led to several foodborne outbreaks in the United States and Canada, mostly associated with imported produce. Understanding the biology and epidemiology of C. cayetanensis is difficult because little is known about its origin, possible zoonotic reservoirs, and genetic relationships with other coccidian parasites. Recently, we developed a 70kDa heat shock protein (HSP70) gene based nested PCR protocol for detection of C. cayetanensis parasite and sequenced the PCR products of 16 human isolates from Nepal, Mexico, and Peru. In this study, we have characterized the regions of 18S ribosomal RNA (rRNA) gene of 17 human C. cayetanensis isolates for molecular detection, and also to ascertain the genetic diversity of this parasite. The 18S rRNA primer sets were further tested by PCR amplification followed by nucleotide sequencing of the PCR amplified products of previously characterized C. cayetanensis isolates from three endemic regions at HSP70 locus. Although no genetic polymorphism was observed at the regions of HSP70 locus characterized in our previous study, the data analysis of this study revealed a minor genetic diversity at the 18S rRNA locus among the C. cayetanensis isolates. The 18S rRNA gene-based nested PCR protocol provides a useful genetic marker for the detection of C. cayetanensis parasite and confirms it as a genetically distinct species in genus Cyclospora. The results also supported lack of geographic segregation and existence of genetically homogeneous population for the C. cayetanensis parasites both at the HSP70 as well as at the18S rRNA loci.

Genome Sequence of Plesiomonas shigelloides Strain 302-73 (Serotype O1)

Plesiomonas shigelloides, the only species of the genus, is an emergent pathogenic bacterium associated with human diarrheal and extraintestinal disease. We present the whole-genome sequence analysis of the representative strain for the O1 serotype (strain 302-73), providing a tool for studying bacterial outbreaks, virulence factors, and accurate diagnostic methods.

RNA-Mediated Thermoregulation of Iron-Acquisition Genes in Shigella dysenteriae and Pathogenic Escherichia coli

The initiation, progression and transmission of most bacterial infections is dependent upon the ability of the invading pathogen to acquire iron from each of the varied environments encountered during the course of a natural infection. In total, 95% of iron within the human body is complexed within heme, making heme a potentially rich source of host-associated nutrient iron for invading bacteria. As heme is encountered only within the host, pathogenic bacteria often regulate synthesis of heme utilization factors such that production is maximal under host-associated environmental conditions. This study examines the regulated production of ShuA, an outer-membrane receptor required for the utilization of heme as a source of nutrient iron by Shigella dysenteriae, a pathogenic bacterium that causes severe diarrheal diseases in humans. Specifically, the impact of the distinct environmental temperatures encountered during infection within a host (37°C) and transmission between hosts (25°C) on shuA expression is investigated. We show that shuA expression is subject to temperature-dependent post-transcriptional regulation resulting in increased ShuA production at 37°C. The observed thermoregulation is mediated by nucleic acid sequences within the 5′ untranslated region. In addition, we have identified similar nucleotide sequences within the 5′ untranslated region of the orthologous chuA transcript of enteropathogenic E. coli and have demonstrated that it also functions to confer temperature-dependent post-transcriptional regulation. In both function and predicted structure, the regulatory element within the shuA and chuA 5′ untranslated regions closely resembles a FourU RNA thermometer, a zipper-like RNA structure that occludes the Shine-Dalgarno sequence at low temperatures. Increased production of ShuA and ChuA in response to the host body temperature allows for maximal production of these heme acquisition factors within the environment where S. dysenteriae and pathogenic E. coli strains would encounter heme, a host-specific iron source.

Ursodeoxycholic acid attenuates colonic epithelial secretory function

Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl(-) secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl(-) secretory responses to the Ca(2+) and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n = 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na(+)/K(+)-ATPase activity and basolateral K(+) channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg(-1)) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50-200 μm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea.