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Sample records for porcine enteropathogenic escherichia

  1. Cloning and expression of the porcine attaching and effacing-associated (paa) gene of enteropathogenic Escherichia coli.

    PubMed

    Pereira, D A; Teixeira Florian, E C; Ono, M A; Rossi, C Nachi; Vidotto, O; Vidotto, M C

    2015-01-01

    Porcine enteropathogenic Escherichia coli (PEPEC) produce an outer membrane protein (intimin) called Paa (porcine attaching and effacing-associated), which is involved in the pathogenesis of E. coli in piglets with diarrhea. The paa gene of a PEPEC strain isolated in Paraná, Brazil, was amplified by polymerase chain reaction, sequenced, and cloned into the pTrcHisTOPO2 vector. The deduced amino acid sequence encoded by the paa gene of PEPEC from Paraná, Brazil, showed 99% homology to the sequences from other PEPEC strains. In this study, the overexpression of recombinant Paa (rPaa) using alternative induction strategies was attempted. The auto-induction protocol showed excellent results for rPaa protein production with 0.4% (w/v) lactose. The rPaa protein is insoluble and was purified with Triton X-100 wash as a total antigen. This method produced a relatively high yield of rPaa. rPaa was recognized by serum from pigs immunized with the PEPEC strain. These results suggest that rPaa could be included in the development of a vaccine against swine colibacillosis. PMID:26345788

  2. Emerging Enteropathogenic Escherichia coli Strains?

    PubMed Central

    Irino, Kinue; Girão, Dennys M.; Girão, Valéria B.C.; Guth, Beatriz E.C.; Vaz, Tânia M.I.; Moreira, Fabiana C.; Chinarelli, Silvia H.; Vieira, Mônica A.M.

    2004-01-01

    Escherichia coli strains of nonenteropathogenic serogroups carrying eae but lacking the enteropathogenic E. coli adherence factor plasmid and Shiga toxin DNA probe sequences were isolated from patients (children, adults, and AIDS patients) with and without diarrhea in Brazil. Although diverse in phenotype and genotype, some strains are potentially diarrheagenic. PMID:15504277

  3. Two distinct groups of porcine enteropathogenic Escherichia coli strains of serogroup O45 are revealed by comparative genomic hybridization and virulence gene microarray

    PubMed Central

    Bruant, Guillaume; Zhang, Yongxiang; Garneau, Philippe; Wong, Justin; Laing, Chad; Fairbrother, John M; Gannon, Victor PJ; Harel, Josée

    2009-01-01

    Background Porcine enteropathogenic Escherichia coli (PEPEC) strains of serogroup O45 cause post-weaning diarrhea and produce characteristic attaching and effacing (A/E) lesions. Most O45 PEPEC strains possess the locus of enterocyte effacement (LEE), encoding the virulence factors required for production of A/E lesions, and often possess the paa gene, which is thought to contribute to the early stages of PEPEC pathogenicity. In this study, nine O45 PEPEC strains and a rabbit enteropathogenic (REPEC) strain, known to produce A/E lesions in vivo, were characterized using an E. coli O157-E. coli K12 whole genome microarray and a virulence gene-specific microarray, and by PCR experiments. Results Based on their virulence gene profiles, the 10 strains were considered to be atypical EPEC. The differences in their genomes pointed to the identification of two distinct evolutionary groups of O45 PEPEC, Groups I and II, and provided evidence for a contribution of these genetic differences to their virulence in pigs. Group I included the REPEC strain and four O45 PEPEC strains known to induce severe A/E lesions in challenged pigs whereas Group II was composed of the five other O45 PEPEC strains, which induced less severe or no A/E lesions in challenged pigs. Significant differences between Groups I and II were found with respect to the presence or absence of 50 O-Islands (OIs) or S-loops and 13 K-islands (KIs) or K-loops, including the virulence-associated islands OI#1 (S-loop#1), OI#47 (S-loop#71), OI#57 (S-loop#85), OI#71 (S-loop#108), OI#115, OI#122, and OI#154 (S-loop#253). Conclusion We have genetically characterized a collection of O45 PEPEC strains and classified them into two distinct groups. The differences in their virulence gene and genomic island content may influence the pathogenicity of O45 PEPEC strains, and explain why Group I O45 PEPEC strains induced more severe A/E lesions in explants and challenged pigs than Group II strains. PMID:19709428

  4. Enteropathogenic Escherichia coli Serotypes and Endemic Diarrhea in Infants

    PubMed Central

    Toledo, M. Regina F.; Alvariza, M. do Carmo B.; Murahovschi, Jayme; Ramos, Sonia R. T. S.; Trabulsi, Luiz R.

    1983-01-01

    Enteropathogenic Escherichia coli serotypes were searched for in feces of 550 children with endemic diarrhea and in 129 controls, in São Paulo, in 1978 and 1979; serotypes O111ab:H−, O111ab:H2, and O119:H6 were significantly associated with diarrhea in children 0 to 5 months old and were the most frequent agents of diarrhea in this age group as compared with enterotoxigenic and enteroinvasive E. coli, Salmonella sp., Shigella sp., and Yersinia enterocolitica. It is concluded that various enteropathogenic E. coli serotypes may be agents of endemic infantile diarrhea. PMID:6339384

  5. Enteropathogenic Escherichia coli Prevalence in Laboratory Rabbits

    PubMed Central

    Swennes, Alton G.; Buckley, Ellen M.; Madden, Carolyn M.; Byrd, Charles P.; Donocoff, Rachel S.; Rodriguez, Loretta; Parry, Nicola M. A.; Fox, James G.

    2013-01-01

    Rabbit-origin enteropathogenic E. coli (EPEC) causes substantial diarrhea-associated morbidity and has zoonotic potential. A culture-based survey was undertaken to ascertain its prevalence. EPEC was isolated from 6/141 (4.3%) commercially-acquired laboratory rabbits. Three of these did not have diarrhea or EPEC-typical intestinal lesions; they instead had background plasmacytic intestinal inflammation. Asymptomatically infected rabbits may function as EPEC reservoirs. PMID:23391439

  6. Differential effects of clathrin and actin inhibitors on internalization of Escherichia coli and Salmonella choleraesuis in porcine jejunal Peyer's patches

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peyer’s patches constitute both an inductive immune site and an enteropathogen invasion route. Peyer’s patch mucosae from porcine jejunum were mounted in Ussing chambers, and either Salmonella choleraesuis vaccine strain SC-54 or non-pathogenic rodent and porcine Escherichia coli strains contacted ...

  7. Enteropathogenicity and antimicrobial susceptibility of new Escherichia spp.

    PubMed

    Chaudhury, A; Nath, G; Tikoo, A; Sanyal, S C

    1999-06-01

    To determine the mechanism of enteropathogenicity of the newly described Escherichia species, a total of 50 clinical isolates of Escherichia spp. from diarrhoeal stools were studied. Twelve isolates (24%) were found to be E. vulneris, 6 (12%) E. fergusonii, 2 (4%) E. hermannii, and the rest 30 (60%) were E. coli. Most isolates of the new species were resistant to ampicillin, tetracycline, and co-trimoxazole, but were susceptible to cephalosporins and aminoglycosides. The representative strains of all the new species produced significant fluid accumulation in the rat ileal loops both by live cells and their culture filtrates. E. vulneris, isolated from stools, showed maximum fluid accumulation. Thus, it can be inferred that these species are diarrhoeagenic, but their roles on extra-intestinal infections remain to be determined. PMID:10897892

  8. Enteropathogenic Escherichia coli (EPEC) infection in association with acute gastroenteritis in 7 dogs from Saskatchewan.

    PubMed

    Kjaergaard, Astrid B; Carr, Anthony P; Gaunt, M Casey

    2016-09-01

    Seven dogs diagnosed with enteropathogenic Escherichia coli (EPEC) infection in association with acute gastroenteritis are described. Disease severity ranged from mild in adults to fatal disease in young dogs. Enteropathogenic E. coli infection should be considered as a possible differential diagnosis in dogs with diarrhea. PMID:27587889

  9. Enteropathogenic Escherichia coli in raw and cooked food.

    PubMed

    Norazah, A; Rahizan, I; Zainuldin, T; Rohani, M Y; Kamel, A G

    1998-03-01

    A total of 402 Escherichia coli isolates were obtained from a variety of food samples and screened for enteropathogenic E. coli (EPEC). Screening was carried out using 15 specific monovalent antisera from Murex Diagnostic Limited. A total of 19 E. coli isolates were serotyped as EPEC. The EPEC strains were shown to belong to 8 serotypes. Eight out of 19 EPEC strains belonged to serotype 018C:K77 (B21). Seventeen out of 19 of the EPEC strains were isolated from cooked food. The presence of E. coli in cooked food is an indicator of fecal contamination and a sign of unhygienic food handling. The presence of EPEC in food could be a potential source of food-borne outbreak. Hygiene training for every food-handler is a necessity. PMID:9740276

  10. relA enhances the adherence of enteropathogenic Escherichia coli.

    PubMed

    Spira, Beny; Ferreira, Gerson Moura; de Almeida, Luiz Gustavo

    2014-01-01

    Enteropathogenic Escherichia coli (EPEC) is a known causative agent of diarrhea in children. In the process of colonization of the small intestine, EPEC synthesizes two types of adhesins, the bundle-forming pilus (BFP) and intimin. The BFP pilus is an adhesin associated with the initial stages of adherence of EPEC to epithelial cells, while the outer membrane protein intimin carries out the intimate adherence that takes place at the third stage of infection. BFP is encoded by the bfp operon located in plasmid EAF, present only in typical EPEC isolates, while eae, the gene that encodes intimin is situated in the LEE, a chromosomal pathogenicity island. Transcription of bfp and eae is regulated by the products of the perABC operon, also present in plasmid EAF. Here we show that deletion of relA, that encodes a guanosine penta and tetraphosphate synthetase impairs EPEC adherence to epithelial cells in vitro. In the absence of relA, the transcription of the regulatory operon perABC is reduced, resulting in lower levels of BFP and intimin. Bacterial adherence, BFP and intimin synthesis and perABC expression are restored upon complementation with the wild-type relA allele. PMID:24643076

  11. Enteropathogenic Escherichia coli Infection Triggers Host Phospholipid Metabolism Perturbations

    PubMed Central

    Wu, Y.; Lau, B.; Smith, S.; Troyan, K.; Barnett Foster, D. E.

    2004-01-01

    Enteropathogenic Escherichia coli (EPEC) specifically recognizes phosphatidylethanolamine (PE) on the outer leaflet of host epithelial cells. EPEC also induces apoptosis in epithelial cells, which results in increased levels of outer leaflet PE and increased bacterial binding. Consequently, it is of interest to investigate whether EPEC infection perturbs host cell phospholipid metabolism and whether the changes play a role in the apoptotic signaling. Our findings indicate that EPEC infection results in a significant increase in the epithelial cell PE level and a corresponding decrease in the phosphatidylcholine (PC) level. PE synthesis via both the de novo pathway and the serine decarboxylation pathway was enhanced, and de novo synthesis of phosphatidylcholine via CDP-choline was reduced. The changes were transitory, and the maximum change was noted after 4 to 5 h of infection. Addition of exogenous PC or CDP-choline to epithelial cells prior to infection abrogated EPEC-induced apoptosis, suggesting that EPEC infection inhibits the CTP-phosphocholine cytidylyltransferase step in PC synthesis, which is reportedly inhibited during nonmicrobially induced apoptosis. On the other hand, incorporation of exogenous PE by the host cells enhanced EPEC-induced apoptosis and necrosis without increasing bacterial adhesion. This is the first report that pathogen-induced apoptosis is associated with significant changes in PE and PC metabolism, and the results suggest that EPEC adhesion to a host membrane phospholipid plays a role in disruption of host phospholipid metabolism. PMID:15557596

  12. Transcriptional Inhibitor of Virulence Factors in Enteropathogenic Escherichia coli

    PubMed Central

    Gauthier, Annick; Robertson, Marilyn L.; Lowden, Michael; Ibarra, J. Antonio; Puente, José Luis; Finlay, B. Brett

    2005-01-01

    The type III secretion system (TTSS) is a key virulence mechanism of many important gram-negative bacterial pathogens. The TTSS is conserved among different bacterial pathogens, and mutations and deletions to the system significantly decrease virulence, making the TTSS an important potential therapeutic target. We have developed a high-throughput assay to search for inhibitors of the TTSS. We screened a commercial library of 20,000 small molecules for their ability to inhibit type III secretion by enteropathogenic Escherichia coli (EPEC). After discarding compounds that had no effect on secretion, inhibited bacterial growth, and/or caused degradation of EPEC-secreted proteins, the search was focused on a class of compounds that, while not direct inhibitors of type III secretion, inhibit expression of TTSS-related genes and other genes involved in virulence. This class of compounds does not affect bacterial viability or motility, indicating that it is not significantly affecting the expression of essential genes and is specific to virulence-associated genes. Transcriptional fusion assays confirmed that virulence-associated promoters were more sensitive to inhibition by this class of compounds. Overall, we have identified a class of compounds that can be used as a tool to probe the mechanism(s) that regulates virulence gene expression in EPEC. PMID:16189086

  13. Mutual Enhancement of Virulence by Enterotoxigenic and Enteropathogenic Escherichia coli

    PubMed Central

    Crane, John K.; Choudhari, Shilpa S.; Naeher, Tonniele M.; Duffey, Michael E.

    2006-01-01

    Enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) are common causes of diarrhea in children in developing countries. Dual infections with both pathogens have been noted fairly frequently in studies of diarrhea around the world. In previous laboratory work, we noted that cholera toxin and forskolin markedly potentiated EPEC-induced ATP release from the host cell, and this potentiated release was found to be mediated by the cystic fibrosis transmembrane conductance regulator. In this study, we examined whether the ETEC heat-labile toxin (LT) or the heat-stable toxin (STa, also known as ST) potentiated EPEC-induced ATP release. We found that crude ETEC culture filtrates, as well as purified ETEC toxins, did potentiate EPEC-induced ATP release in cultured T84 cells. Coinfection of T84 cells with live ETEC plus EPEC bacteria also resulted in enhanced ATP release compared to EPEC alone. In Ussing chamber studies of chloride secretion, adenine nucleotides released from the host by EPEC also significantly enhanced the chloride secretory responses that were triggered by crude ETEC filtrates, purified STa, and the peptide hormone guanylin. In addition, adenosine and LT had additive or synergistic effects in inducing vacuole formation in T84 cells. Therefore, ETEC toxins and EPEC-induced damage to the host cell both enhance the virulence of the other type of E. coli. Our in vitro data demonstrate a molecular basis for a microbial interaction, which could result in increased severity of disease in vivo in individuals who are coinfected with ETEC and EPEC. PMID:16495521

  14. Effect of zinc in enteropathogenic Escherichia coli infection.

    PubMed

    Crane, John K; Naeher, Tonniele M; Shulgina, Irina; Zhu, Chengru; Boedeker, Edgar C

    2007-12-01

    Enteropathogenic Escherichia coli (EPEC) infection triggers the release of ATP from host intestinal cells, and the ATP is broken down to ADP, AMP, and adenosine in the lumen of the intestine. Ecto-5'-nucleotidase (CD73) is the main enzyme responsible for the conversion of 5'-AMP to adenosine, which triggers fluid secretion from host intestinal cells and also has growth-promoting effects on EPEC bacteria. In a recent study, we examined the role of the host enzyme CD73 in EPEC infection by testing the effect of ecto-5'-nucleotidase inhibitors. Zinc was a less potent inhibitor of ecto-5'-nucleotidase in vitro than the nucleotide analog alpha,beta-methylene-ADP, but in vivo, zinc was much more efficacious in preventing EPEC-induced fluid secretion in rabbit ileal loops than alpha,beta-methylene-ADP. This discrepancy between the in vitro and in vivo potencies of the two inhibitors prompted us to search for potential targets of zinc other than ecto-5'-nucleotidase. Zinc, at concentrations that produced little or no inhibition of EPEC growth, caused a decrease in the expression of EPEC protein virulence factors, such as bundle-forming pilus (BFP), EPEC secreted protein A, and other EPEC secreted proteins, and reduced EPEC adherence to cells in tissue culture. The effects of zinc were not mimicked by other transition metals, such as manganese, iron, copper, or nickel, and the effects were not reversed by an excess of iron. Quantitative real-time PCR showed that zinc reduced the abundance of the RNAs encoded by the bfp gene, by the plasmid-encoded regulator (per) gene, by the locus for the enterocyte effacement (LEE)-encoded regulator (ler) gene, and by several of the esp genes. In vivo, zinc reduced EPEC-induced fluid secretion into ligated rabbit ileal loops, decreased the adherence of EPEC to rabbit ileum, and reduced histopathological damage such as villus blunting. Some of the beneficial effects of zinc on EPEC infection appear to be due to the action of the metal on

  15. Lack of virulence factors in Escherichia coli strains of enteropathogenic serogroups isolated from water.

    PubMed Central

    Valentini, S R; Gomes, T A; Falcão, D P

    1992-01-01

    Thirty-eight Escherichia coli strains belonging to 14 human enteropathogenic serogroups were isolated from 33 of 208 water samples studied. No virulence factor or virulence-related gene sequences were found in any of the 38 strains analyzed. The results point out the importance of detecting specific virulence factors before incriminating water as a source of human diarrhea. PMID:1539989

  16. Antibiotics in the treatment of gastroenteritis caused by enteropathogenic Escherichia coli.

    PubMed

    Thorén, A; Wolde-Mariam, T; Stintzing, G; Wadström, T; Habte, D

    1980-01-01

    The role of antibiotics in treating endemic infantile diarrhea caused by enteropathogenic Escherichia coli has not been determined. In a controlled study of 49 patients, one group received mecillinam and another group received trimethoprim-sulfamethoxazole. A third group served as control subjects. Serotype O111:B4 dominated. Treatment, as evaluated clinically on the third day, resulted in cure for 79% of those receiving mecillinam, 73% of those receiving trimethoprim-sulfamethoxazole, and 7% of the control subjects. Bacteriologic cure was confirmed in 53%, 53%, and 0, respectively. The statistically significant difference between antibiotic-treated patients and control subjects (P less than 0.001) indicated that antibiotics are an important supplement in the treatment of endemic severe diarrhea caused by enteropathogenic E. coli. PMID:6245145

  17. Enteropathogenic Escherichia coli serotype O111:HNT isolated from preterm neonates in Nairobi, Kenya.

    PubMed Central

    Senerwa, D; Olsvik, O; Mutanda, L N; Lindqvist, K J; Gathuma, J M; Fossum, K; Wachsmuth, K

    1989-01-01

    This investigation was initiated as a consequence of several cases of diarrhea in a nursery ward for preterm babies in Nairobi, Kenya. Ten lactose-positive colonies were isolated from the stools of each of 30 neonates, regardless of whether they had diarrhea; 229 strains were identified as Escherichia coli and 65 strains were identified as Klebsiella pneumoniae. Six strains were lost during laboratory handling. No other bacterial, viral, or parasitic enteropathogens were identified. Using synthetic alkaline phosphatase-labeled probes, the bacterial isolates were found to be negative for the presence of genes coding for heat-stable and heat-labile enterotoxins. Seventy-eight E. coli strains isolated from a total of 13 neonates possessed the E. coli enteropathogenic adhesion factor (EAF) gene, as demonstrated by the use of a cloned radiolabeled DNA fragment probe. These strains possessed similar plasmid profiles constituting a core plasmid profile, and while all adhered to HeLa cells, none produced Vero cell cytotoxins. The EAF gene was located on a 65-megadalton plasmid. Serotyping showed the strains to be of serogroup O111 and serotype H nontypable, a well known enteropathogenic type. Five neonates died during the outbreak, and the fatality rate was 30.7% (4 of 13) for neonates infected with EAF-positive E. coli strains compared with 7.7% (1 of 13) for neonates from whom only EAF-negative E. coli strains were isolated. K. pneumoniae only was isolated from five neonates. Images PMID:2568996

  18. Modulation of the Inflammasome Signaling Pathway by Enteropathogenic and Enterohemorrhagic Escherichia coli.

    PubMed

    Yen, Hilo; Karino, Masaki; Tobe, Toru

    2016-01-01

    Innate immunity is an essential component in the protection of a host against pathogens. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) are known to modulate the innate immune responses of infected cells. The interference is dependent on their type III secretion system (T3SS) and T3SS-dependent effector proteins. Furthermore, these cytosolically injected effectors have been demonstrated to engage multiple immune signaling pathways, including the IFN/STAT, MAPK, NF-κB, and inflammasome pathways. In this review, recent work describing the interaction between EPEC/EHEC and the inflammasome pathway will be discussed. PMID:27617233

  19. Isolation of atypical enteropathogenic Escherichia coli from chicken and chicken-derived products.

    PubMed

    Alonso, M Z; Sanz, M E; Irino, K; Krüger, A; Lucchesi, P M A; Padola, N L

    2016-04-01

    Atypical enteropathogenic Escherichia coli (EPEC) strains from chicken and chicken-derived products were isolated and characterised. The strains presented a wide variety of serotypes, some have been reported in other animal species (O2:H40, O5:H40) and in children with diarrhoea (O8:H-). Most of the strains carried intimin β. The results indicate that chicken and chicken products are important sources of atypical EPEC strains that could be associated with human disease, and highlight the need to improve hygiene practices in chicken slaughtering and meat handling. PMID:26810335

  20. Modulation of the Inflammasome Signaling Pathway by Enteropathogenic and Enterohemorrhagic Escherichia coli

    PubMed Central

    Yen, Hilo; Karino, Masaki; Tobe, Toru

    2016-01-01

    Innate immunity is an essential component in the protection of a host against pathogens. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) are known to modulate the innate immune responses of infected cells. The interference is dependent on their type III secretion system (T3SS) and T3SS-dependent effector proteins. Furthermore, these cytosolically injected effectors have been demonstrated to engage multiple immune signaling pathways, including the IFN/STAT, MAPK, NF-κB, and inflammasome pathways. In this review, recent work describing the interaction between EPEC/EHEC and the inflammasome pathway will be discussed. PMID:27617233

  1. Diarrhea, bacteremia and multiorgan dysfunction due to an extraintestinal pathogenic Escherichia coli strain with enteropathogenic E. coli genes

    PubMed Central

    Kessler, Robert; Nisa, Shahista; Hazen, Tracy H.; Horneman, Amy; Amoroso, Anthony; Rasko, David A.; Donnenberg, Michael S.

    2015-01-01

    A 55-year-old man with well-controlled HIV had severe diarrhea for 3 weeks and developed multiorgan dysfunction and bacteremia due to Escherichia coli. The genome of the patient's isolate had features characteristic of extraintestinal pathogenic E. coli and genes distantly related to those defining enteropathogenic E. coli. PMID:26410828

  2. Diarrhea, bacteremia and multiorgan dysfunction due to an extraintestinal pathogenic Escherichia coli strain with enteropathogenic E. coli genes.

    PubMed

    Kessler, Robert; Nisa, Shahista; Hazen, Tracy H; Horneman, Amy; Amoroso, Anthony; Rasko, David A; Donnenberg, Michael S

    2015-11-01

    A 55-year-old man with well-controlled HIV had severe diarrhea for 3 weeks and developed multiorgan dysfunction and bacteremia due to Escherichia coli. The genome of the patient's isolate had features characteristic of extraintestinal pathogenic E. coli and genes distantly related to those defining enteropathogenic E. coli. PMID:26410828

  3. Modulation of host cell signalling by enteropathogenic and Shiga toxin-producing Escherichia coli.

    PubMed

    Kresse, A U; Guzmán, C A; Ebel, F

    2001-09-01

    The majority of Escherichia coli strains are harmless symbionts in the intestinal tract. However, there are several pathogenic forms, which are responsible for various diseases in humans and live stock. In this review we discuss the interactions between Shiga toxin-producing E. coli and enteropathogenic E. coli and their target host cells, describing their strategies to activate specific cellular signalling pathways which lead to subversion of critical physiological functions. We mainly concentrate on those pathogenic mechanisms that are dependent on a functional type III secretion system, but we also briefly discuss additional factors that contribute to the specific pathogenic profiles of Shiga toxin-producing E. coli and enreropathogenic E. coli. PMID:11680788

  4. Role of F1C fimbriae, flagella, and secreted bacterial components in the inhibitory effect of probiotic Escherichia coli Nissle 1917 on atypical enteropathogenic E. coli infection.

    PubMed

    Kleta, Sylvia; Nordhoff, Marcel; Tedin, Karsten; Wieler, Lothar H; Kolenda, Rafal; Oswald, Sibylle; Oelschlaeger, Tobias A; Bleiss, Wilfried; Schierack, Peter

    2014-05-01

    Enteropathogenic Escherichia coli (EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probiotic E. coli strain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While both in vitro and in vivo studies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenic E. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenic E. coli (aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression. PMID:24549324

  5. Role of F1C Fimbriae, Flagella, and Secreted Bacterial Components in the Inhibitory Effect of Probiotic Escherichia coli Nissle 1917 on Atypical Enteropathogenic E. coli Infection

    PubMed Central

    Kleta, Sylvia; Nordhoff, Marcel; Tedin, Karsten; Wieler, Lothar H.; Kolenda, Rafal; Oswald, Sibylle; Oelschlaeger, Tobias A.; Bleiß, Wilfried

    2014-01-01

    Enteropathogenic Escherichia coli (EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probiotic E. coli strain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While both in vitro and in vivo studies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenic E. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenic E. coli (aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression. PMID:24549324

  6. Phage biocontrol of enteropathogenic and shiga toxin-producing Escherichia coli in meat products.

    PubMed

    Tomat, David; Migliore, Leonel; Aquili, Virginia; Quiberoni, Andrea; Balagué, Claudia

    2013-01-01

    Ten bacteriophages were isolated from faeces and their lytic effects assayed on 103 pathogenic and non-pathogenic Enterobacteriaceae. Two phages (DT1 and DT6) were selected based on their host ranges, and their lytic effects on pathogenic E. coli strains inoculated on pieces of beef were determined. We evaluated the reductions of viable cells of Escherichia coli O157:H7 and non-O157 Shiga toxigenic E. coli strains on meat after exposure to DT6 at 5 and 24°C for 3, 6, and 24 h and the effect of both phages against an enteropathogenic E. coli strain. Significant viable cell reductions, compared to controls without phages, at both temperatures were observed, with the greatest decrease taking place within the first hours of the assays. Reductions were also influenced by phage concentration, being the highest concentrations, 1.7 × 10(10) plaque forming units per milliliter (PFU/mL) for DT1 and 1.4 × 10(10) PFU/mL for DT6, the most effective. When enteropathogenic E. coli and Shiga toxigenic E. coli (O157:H7) strains were tested, we obtained viable cell reductions of 0.67 log (p = 0.01) and 0.77 log (p = 0.01) after 3 h incubation and 0.80 log (p = 0.01) and 1.15 log (p = 0.001) after 6 h. In contrast, all nonpathogenic E. coli strains as well as other enterobacteria tested were resistant. In addition, phage cocktail was evaluated on two strains and further reductions were observed. However, E. coli bacteriophage insensitive mutants (BIMs) emerged in meat assays. BIMs isolated from meat along with those isolated by using the secondary culture method were tested to evaluate resistance phenotype stability and reversion. They presented low emergence frequencies (6.5 × 10(-7)-1.8 × 10(-6)) and variable stability and reversion. Results indicate that isolated phages were stable on storage, negative for all the virulence factors assayed, presented lytic activity for different E. coli virotypes and could be useful in reducing Shiga toxigenic E. coli and enteropathogenic E

  7. Phage biocontrol of enteropathogenic and shiga toxin-producing Escherichia coli in meat products

    PubMed Central

    Tomat, David; Migliore, Leonel; Aquili, Virginia; Quiberoni, Andrea; Balagué, Claudia

    2013-01-01

    Ten bacteriophages were isolated from faeces and their lytic effects assayed on 103 pathogenic and non-pathogenic Enterobacteriaceae. Two phages (DT1 and DT6) were selected based on their host ranges, and their lytic effects on pathogenic E. coli strains inoculated on pieces of beef were determined. We evaluated the reductions of viable cells of Escherichia coli O157:H7 and non-O157 Shiga toxigenic E. coli strains on meat after exposure to DT6 at 5 and 24°C for 3, 6, and 24 h and the effect of both phages against an enteropathogenic E. coli strain. Significant viable cell reductions, compared to controls without phages, at both temperatures were observed, with the greatest decrease taking place within the first hours of the assays. Reductions were also influenced by phage concentration, being the highest concentrations, 1.7 × 1010 plaque forming units per milliliter (PFU/mL) for DT1 and 1.4 × 1010 PFU/mL for DT6, the most effective. When enteropathogenic E. coli and Shiga toxigenic E. coli (O157:H7) strains were tested, we obtained viable cell reductions of 0.67 log (p = 0.01) and 0.77 log (p = 0.01) after 3 h incubation and 0.80 log (p = 0.01) and 1.15 log (p = 0.001) after 6 h. In contrast, all nonpathogenic E. coli strains as well as other enterobacteria tested were resistant. In addition, phage cocktail was evaluated on two strains and further reductions were observed. However, E. coli bacteriophage insensitive mutants (BIMs) emerged in meat assays. BIMs isolated from meat along with those isolated by using the secondary culture method were tested to evaluate resistance phenotype stability and reversion. They presented low emergence frequencies (6.5 × 10−7–1.8 × 10−6) and variable stability and reversion. Results indicate that isolated phages were stable on storage, negative for all the virulence factors assayed, presented lytic activity for different E. coli virotypes and could be useful in reducing Shiga toxigenic E. coli and enteropathogenic E

  8. Flagellar Cap Protein FliD Mediates Adherence of Atypical Enteropathogenic Escherichia coli to Enterocyte Microvilli.

    PubMed

    Sampaio, Suely C F; Luiz, Wilson B; Vieira, Mônica A M; Ferreira, Rita C C; Garcia, Bruna G; Sinigaglia-Coimbra, Rita; Sampaio, Jorge L M; Ferreira, Luís C S; Gomes, Tânia A T

    2016-04-01

    The expression of flagella correlates with different aspects of bacterial pathogenicity, ranging from adherence to host cells to activation of inflammatory responses by the innate immune system. In the present study, we investigated the role of flagella in the adherence of an atypical enteropathogenic Escherichia coli (aEPEC) strain (serotype O51:H40) to human enterocytes. Accordingly, isogenic mutants deficient in flagellin (FliC), the flagellar structural subunit; the flagellar cap protein (FliD); or the MotAB proteins, involved in the control of flagellar motion, were generated and tested for binding to differentiated Caco-2 cells. Binding of the aEPEC strain to enterocytes was significantly impaired in strains with the fliCa nd fliD genes deleted, both of which could not form flagella on the bacterial surface. A nonmotile but flagellated MotAB mutant also showed impaired adhesion to Caco-2 cells. In accordance with these observations, adhesion of a EPEC strain 1711-4 to Caco-2 cells was drastically reduced after the treatment of Caco-2 cells with purified FliD. In addition, incubation of a EPEC bacteria with specific anti-FliD serum impaired binding to Caco-2 cells. Finally, incubation of Caco-2 cells with purified FliD, followed by immunolabeling, showed that the protein was specifically bound to the microvillus tips of differentiated Caco-2 cells. The a EPEC FliD or anti-FliD serum also reduced the adherence of prototype typical enteropathogenic, enterohemorrhagic, and enterotoxigenic E. coli strains to Caco-2 cells. In conclusion, our findings further strengthened the role of flagella in the adherence of a EPEC to human enterocytes and disclosed the relevant structural and functional involvement of FliD in the adhesion process. PMID:26831466

  9. Enterotoxigenic Escherichia coli and other enteropathogens in paediatric diarrhoea in Addis Ababa.

    PubMed

    Stintzing, G; Möllby, R; Habte, D

    1982-03-01

    This study was performed during two weeks among 86 paediatric outpatients of poor socio-economic background. A control group comprised 60 healthy children. Enterotoxigenic Escherichia coli (ETEC) was the most common diarrhoeal agent isolated (26%). Strains of ETEC producing heat-labile (LT) only or LT and heat-stable (ST) enterotoxin were isolated from 11% each and ETEC producing ST only from 4% of the patients. ETEC was also found not infrequently among controls (10%). ETEC with O-antigens 78, 6 and 8 were shown to harbour colonization factors. Enterotoxigenic bacteria were found as contaminants in 5 of 24 feeding bottles investigated. Enteropathogenic Escherichia coli (EPEC) and Shigella species were isolated from 8% each and rotavirus from 24% of the patients. Twelve patients infected with ETEC only were compared to 66 patients not infected with ETEC. Patients infected with ETEC had a relatively mild disease and it was not possible by clinical findings to distinguish those patients infected with ETEC, LT and/or ST producing, carrying or not carrying colonization factors from those infected with other agents. This study underlines the need for extended studies of the clinical significance of ETEC infection in developing countries. PMID:6753473

  10. Isolation of atypical enteropathogenic and shiga toxin encoding Escherichia coli strains from poultry in Tehran, Iran

    PubMed Central

    Doregiraee, Fatemeh; Alebouyeh, Masoud; Nayeri Fasaei, Bahar; Charkhkar, Saeed; Tajedin, Elahe; Zali, Mohammad Reza

    2016-01-01

    Aim: The purpose of this study was to investigate the prevalence of enteropathogenic Escherichia coli (EPEC) and shiga toxin producing E. coli (STEC) strains in healthy broilers in Iran. Background: STEC and EPEC strains as diarrheagenic E. coli are among the most prevalent causative agents in acute diarrhea. Domestic animals, mainly cattle and sheep, have been implicated as the principal reservoirs of these pathotypes; however their prevalence among the broilers is varied among different countries. Patients and methods: A total of 500 cloacal swab samples from broilers of five different poultry houses (A-E) were collected to investigate the presence of stx1, stx2, hly, eae, and bfp virulence genes among the E. coli isolates by polymerase chain reaction. The shiga toxin encoding strains were evaluated serologically to detect their interaction with a commercial antiserum against O157 antigen. Results: Out of the 500 collected samples, 444 E. coli strains were isolated. Three strains (0.67%) presented at least one of the studied virulence genes (stx2, hly and eae), two strains were identified as STEC (stx2+, O157:nonH7) and one as an atypical EPEC strains (eae+ bfp-). Conclusion: The study established the presence of STEC and atypical EPEC in healthy broilers in Iran. Poultry might serve as vectors for transmission of pathogenic E. coli to human populations. PMID:26744615

  11. Lactobacillus reuteri Inhibition of Enteropathogenic Escherichia coli Adherence to Human Intestinal Epithelium

    PubMed Central

    Walsham, Alistair D. S.; MacKenzie, Donald A.; Cook, Vivienne; Wemyss-Holden, Simon; Hews, Claire L.; Juge, Nathalie; Schüller, Stephanie

    2016-01-01

    Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrheal infant death in developing countries, and probiotic bacteria have been shown to provide health benefits in gastrointestinal infections. In this study, we have investigated the influence of the gut symbiont Lactobacillus reuteri on EPEC adherence to the human intestinal epithelium. Different host cell model systems including non-mucus-producing HT-29 and mucus-producing LS174T intestinal epithelial cell lines as well as human small intestinal biopsies were used. Adherence of L. reuteri to HT-29 cells was strain-specific, and the mucus-binding proteins CmbA and MUB increased binding to both HT-29 and LS174T cells. L. reuteri ATCC PTA 6475 and ATCC 53608 significantly inhibited EPEC binding to HT-29 but not LS174T cells. While pre-incubation of LS174T cells with ATCC PTA 6475 did not affect EPEC attaching/effacing (A/E) lesion formation, it increased the size of EPEC microcolonies. ATCC PTA 6475 and ATCC 53608 binding to the mucus layer resulted in decreased EPEC adherence to small intestinal biopsy epithelium. Our findings show that L. reuteri reduction of EPEC adhesion is strain-specific and has the potential to target either the epithelium or the mucus layer, providing further rationale for the selection of probiotic strains. PMID:26973622

  12. Examination of Feces from Food Handlers for Salmonellae, Shigellae, Enteropathogenic Escherichia coli, and Clostridium perfringens

    PubMed Central

    Hall, Herbert E.; Hauser, George H.

    1966-01-01

    Duplicate fecal specimens from food handlers were collected in Louisiana. One set of specimens was examined immediately for salmonellae and shigellae by the Central Laboratory of the Louisiana State Board of Health in New Orleans; the other set was shipped to the Food Microbiology Unit at the Robert A. Taft Sanitary Engineering Center in Cincinnati, Ohio, where it was examined for enteropathogenic Escherichia coli (EEC) and Clostridium perfringens. A total of 219 specimens were examined by both laboratories. None yielded salmonellae or shigellae; 171 (78.1%) yielded C. perfringens; 175 (79.9%) yielded E. coli; and 14 (6.4%) yielded EEC. The 14 isolates of EEC were distributed among eight serotypes; one specimen yielded two serotypes. Multiple isolations of C. perfringens strains (two to four) were made from 64 (37.4%) of the specimens, and a total of 244 strains were isolated and studied for identifying characteristics. Of the total, only 87 (35.5%) could be identified serologically by a battery of 67 antisera; only 4 (1.6%) possessed the characteristics of the English “food-poisoning type.” The hemolytic activity on agar containing horse, ox, or sheep blood showed that 140 (57.1%) were “hemolytic,” 81 (33.1%) were “nonhemolytic,” and 23 (9.8%) gave varied results. Only 12 (4.9%) of the strains produced spores that resisted boiling for 30 min or more. PMID:16349698

  13. Biological Activities of Uric Acid in Infection Due to Enteropathogenic and Shiga-Toxigenic Escherichia coli.

    PubMed

    Crane, John K; Broome, Jacqueline E; Lis, Agnieszka

    2016-04-01

    In previous work, we identified xanthine oxidase (XO) as an important enzyme in the interaction between the host and enteropathogenic Escherichia coli(EPEC) and Shiga-toxigenic E. coli(STEC). Many of the biological effects of XO were due to the hydrogen peroxide produced by the enzyme. We wondered, however, if uric acid generated by XO also had biological effects in the gastrointestinal tract. Uric acid triggered inflammatory responses in the gut, including increased submucosal edema and release of extracellular DNA from host cells. While uric acid alone was unable to trigger a chloride secretory response in intestinal monolayers, it did potentiate the secretory response to cyclic AMP agonists. Uric acid crystals were formed in vivo in the lumen of the gut in response to EPEC and STEC infections. While trying to visualize uric acid crystals formed during EPEC and STEC infections, we noticed that uric acid crystals became enmeshed in the neutrophilic extracellular traps (NETs) produced from host cells in response to bacteria in cultured cell systems and in the intestine in vivo Uric acid levels in the gut lumen increased in response to exogenous DNA, and these increases were enhanced by the actions of DNase I. Interestingly, addition of DNase I reduced the numbers of EPEC bacteria recovered after a 20-h infection and protected against EPEC-induced histologic damage. PMID:26787720

  14. Adhesion and its role in the virulence of enteropathogenic Escherichia coli.

    PubMed Central

    Law, D

    1994-01-01

    Enteropathogenic Escherichia coli (EPEC) organisms are an important cause of diarrheal disease in young children. The virulence of EPEC is a multifactorial process and involves a number of distinct stages. Initial adherence to intestinal mucosa is mediated by fimbriae which bring about a distinct form of adhesion, localized adhesion. Intimate adhesion of the bacterium to the eukaryotic membrane occurs, resulting in the activation of signal transduction pathways. Microvilli are disrupted and effaced from the apical membrane which then cups around the organism to form pedestal structures, the attaching and effacing lesion. Diarrhea may be produced by alteration of the permeability of the apical membrane and also through a malabsorption mechanism. The pathways involved in the production of the attaching and effacing lesion are described. EPEC organisms were originally thought to belong to a number of distinct serogroups; it is now apparent that many isolates belonging to these serogroups are not pathogenic or belong to other pathogenic groups of E. coli. In addition, isolates falling outside of these serogroups are considered to be true EPEC. The definition of EPEC based on serotyping is inaccurate and should be replaced by methods that specifically detect the virulence properties of EPEC. Images PMID:8055465

  15. Host cell death due to enteropathogenic Escherichia coli has features of apoptosis.

    PubMed

    Crane, J K; Majumdar, S; Pickhardt, D F

    1999-05-01

    Enteropathogenic Escherichia coli (EPEC) is a cause of prolonged watery diarrhea in children in developing countries. The ability of EPEC to kill host cells was investigated in vitro in assays using two human cultured cell lines, HeLa (cervical) and T84 (colonic). EPEC killed epithelial cells as assessed by permeability to the vital dyes trypan blue and propidium iodide. In addition, EPEC triggered changes in the host cell, suggesting apoptosis as the mode of death; such changes included early expression of phosphatidylserine on the host cell surface and internucleosomal cleavage of host cell DNA. Genistein, an inhibitor of tyrosine kinases, and wortmannin, an inhibitor of host phosphatidylinositol 3-kinase, markedly increased EPEC-induced cell death and enhanced the features of apoptosis. EPEC-induced cell death was contact dependent and required adherence of live bacteria to the host cell. A quantitative assay for EPEC-induced cell death was developed by using the propidium iodide uptake method adapted to a fluorescence plate reader. With EPEC, the rate and extent of host cell death were less that what has been reported for Salmonella, Shigella, and Yersinia, three other genera of enteric bacteria known to cause apoptosis. However, rapid apoptosis of the host cell may not favor the pathogenic strategy of EPEC, a mucosa-adhering, noninvasive pathogen. PMID:10225923

  16. Dynamics of the Type III Secretion System Activity of Enteropathogenic Escherichia coli

    PubMed Central

    Mills, Erez; Baruch, Kobi; Aviv, Gili; Nitzan, Mor; Rosenshine, Ilan

    2013-01-01

    ABSTRACT Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics. PMID:23900171

  17. Age-Dependent Susceptibility to Enteropathogenic Escherichia coli (EPEC) Infection in Mice.

    PubMed

    Dupont, Aline; Sommer, Felix; Zhang, Kaiyi; Repnik, Urska; Basic, Marijana; Bleich, André; Kühnel, Mark; Bäckhed, Fredrik; Litvak, Yael; Fulde, Marcus; Rosenshine, Ilan; Hornef, Mathias W

    2016-05-01

    Enteropathogenic Escherichia coli (EPEC) represents a major causative agent of infant diarrhea associated with significant morbidity and mortality in developing countries. Although studied extensively in vitro, the investigation of the host-pathogen interaction in vivo has been hampered by the lack of a suitable small animal model. Using RT-PCR and global transcriptome analysis, high throughput 16S rDNA sequencing as well as immunofluorescence and electron microscopy, we characterize the EPEC-host interaction following oral challenge of newborn mice. Spontaneous colonization of the small intestine and colon of neonate mice that lasted until weaning was observed. Intimate attachment to the epithelial plasma membrane and microcolony formation were visualized only in the presence of a functional bundle forming pili (BFP) and type III secretion system (T3SS). Similarly, a T3SS-dependent EPEC-induced innate immune response, mediated via MyD88, TLR5 and TLR9 led to the induction of a distinct set of genes in infected intestinal epithelial cells. Infection-induced alterations of the microbiota composition remained restricted to the postnatal period. Although EPEC colonized the adult intestine in the absence of a competing microbiota, no microcolonies were observed at the small intestinal epithelium. Here, we introduce the first suitable mouse infection model and describe an age-dependent, virulence factor-dependent attachment of EPEC to enterocytes in vivo. PMID:27159323

  18. The serine protease Pic as a virulence factor of atypical enteropathogenic Escherichia coli.

    PubMed

    Abreu, Afonso G; Abe, Cecilia M; Nunes, Kamila O; Moraes, Claudia T P; Chavez-Dueñas, Lucia; Navarro-Garcia, Fernando; Barbosa, Angela S; Piazza, Roxane M F; Elias, Waldir P

    2016-01-01

    Autotransporter proteins (AT) are associated with bacterial virulence attributes. Originally identified in enteroaggregative Escherichia coli (EAEC), Shigella flexneri 2a and uropathogenic E. coli, the serine protease Pic is one of these AT. We have previously detected one atypical enteropathogenic E. coli strain (BA589) carrying the pic gene. In the present study, we characterized the biological activities of Pic produced by BA589 both in vitro and in vivo. Contrarily to other Pic-producers bacteria, pic in BA589 is located on a high molecular weight plasmid. PicBA589 was able to agglutinate rabbit erythrocytes, cleave mucin and degrade complement system molecules. BA589 was able to colonize mice intestines, and an intense mucus production was observed. The BA589Δpic mutant lost the capacity to colonize as well as the above-mentioned in vitro activities. Thus, Pic represents an additional virulence factor in aEPEC strain BA589, associated with adherence, colonization and evasion from the innate immune system. PMID:26963626

  19. Lactobacillus reuteri Inhibition of Enteropathogenic Escherichia coli Adherence to Human Intestinal Epithelium.

    PubMed

    Walsham, Alistair D S; MacKenzie, Donald A; Cook, Vivienne; Wemyss-Holden, Simon; Hews, Claire L; Juge, Nathalie; Schüller, Stephanie

    2016-01-01

    Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrheal infant death in developing countries, and probiotic bacteria have been shown to provide health benefits in gastrointestinal infections. In this study, we have investigated the influence of the gut symbiont Lactobacillus reuteri on EPEC adherence to the human intestinal epithelium. Different host cell model systems including non-mucus-producing HT-29 and mucus-producing LS174T intestinal epithelial cell lines as well as human small intestinal biopsies were used. Adherence of L. reuteri to HT-29 cells was strain-specific, and the mucus-binding proteins CmbA and MUB increased binding to both HT-29 and LS174T cells. L. reuteri ATCC PTA 6475 and ATCC 53608 significantly inhibited EPEC binding to HT-29 but not LS174T cells. While pre-incubation of LS174T cells with ATCC PTA 6475 did not affect EPEC attaching/effacing (A/E) lesion formation, it increased the size of EPEC microcolonies. ATCC PTA 6475 and ATCC 53608 binding to the mucus layer resulted in decreased EPEC adherence to small intestinal biopsy epithelium. Our findings show that L. reuteri reduction of EPEC adhesion is strain-specific and has the potential to target either the epithelium or the mucus layer, providing further rationale for the selection of probiotic strains. PMID:26973622

  20. Age-Dependent Susceptibility to Enteropathogenic Escherichia coli (EPEC) Infection in Mice

    PubMed Central

    Dupont, Aline; Sommer, Felix; Zhang, Kaiyi; Repnik, Urska; Basic, Marijana; Bleich, André; Kühnel, Mark; Bäckhed, Fredrik; Litvak, Yael; Fulde, Marcus; Rosenshine, Ilan; Hornef, Mathias W.

    2016-01-01

    Enteropathogenic Escherichia coli (EPEC) represents a major causative agent of infant diarrhea associated with significant morbidity and mortality in developing countries. Although studied extensively in vitro, the investigation of the host-pathogen interaction in vivo has been hampered by the lack of a suitable small animal model. Using RT-PCR and global transcriptome analysis, high throughput 16S rDNA sequencing as well as immunofluorescence and electron microscopy, we characterize the EPEC-host interaction following oral challenge of newborn mice. Spontaneous colonization of the small intestine and colon of neonate mice that lasted until weaning was observed. Intimate attachment to the epithelial plasma membrane and microcolony formation were visualized only in the presence of a functional bundle forming pili (BFP) and type III secretion system (T3SS). Similarly, a T3SS-dependent EPEC-induced innate immune response, mediated via MyD88, TLR5 and TLR9 led to the induction of a distinct set of genes in infected intestinal epithelial cells. Infection-induced alterations of the microbiota composition remained restricted to the postnatal period. Although EPEC colonized the adult intestine in the absence of a competing microbiota, no microcolonies were observed at the small intestinal epithelium. Here, we introduce the first suitable mouse infection model and describe an age-dependent, virulence factor-dependent attachment of EPEC to enterocytes in vivo. PMID:27159323

  1. RegR virulence regulon of rabbit-specific enteropathogenic Escherichia coli strain E22.

    PubMed

    Srikhanta, Yogitha N; Hocking, Dianna M; Praszkier, Judyta; Wakefield, Matthew J; Robins-Browne, Roy M; Yang, Ji; Tauschek, Marija

    2013-04-01

    AraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli, enteroaggregative E. coli, and Citrobacter rodentium. Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, of C. rodentium. Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target, sefA. Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression of sefA by binding to a region upstream of the sefA promoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22. PMID:23340312

  2. Products of enteropathogenic Escherichia coli inhibit lymphocyte activation and lymphokine production.

    PubMed Central

    Klapproth, J M; Donnenberg, M S; Abraham, J M; Mobley, H L; James, S P

    1995-01-01

    The aim of this study was to determine whether products of enteric bacteria are able to regulate lymphocyte activation and cytokine production. Whole bacteria and bacterial lysates from different strains of Escherichia coli were tested for their ability to inhibit cytokine production by peripheral blood mononuclear cells as determined by reverse transcription-PCR, Northern (RNA) blotting of cellular RNA, or enzyme-linked immunosorbent assay for cytokine protein. Lysates from two pathogenic strains of E. coli, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli, inhibited mitogen-stimulated expression of interleukin-2 (IL-2), IL-4, IL-5, and gamma interferon. IL-1 beta, IL-6, IL-8, IL-10, IL-12, and Rantes mRNA expression was not affected. The inhibitory activity was dose dependent, protease and heat sensitive, nondialyzable, and not due to cellular toxicity. The inhibitory activity remained in EPEC strains having mutations in known virulence factors. Nonpathogenic E. coli HB101 transformed with a 22-kb cosmid clone derived from EPEC chromosomal DNA expressed the inhibitory activity. Thus, certain strains of pathogenic E. coli express a protein or proteins encoded by chromosomal genes that selectively inhibit lymphocyte activation and lymphokine production. Therefore, immunosuppressive factors produced by pathogenic bacteria could be important in modifying gastrointestinal immune responses in enteric bacterial infections or gastrointestinal autoimmune diseases. PMID:7768605

  3. Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis.

    PubMed Central

    Sohel, I; Puente, J L; Ramer, S W; Bieber, D; Wu, C Y; Schoolnik, G K

    1996-01-01

    Sequence flanking the bfpA locus on the enteroadherent factor plasmid of the enteropathogenic Escherichia coli (EPEC) strain B171-8 (O111:NM) was obtained to identify genes that might be required for bundle-forming pilus (BFP) biosynthesis. Deletion experiments led to the identification of a contiguous cluster of at least 12 open reading frames, including bfpA, that could direct the synthesis of a morphologically normal BFP filament. Within the bfp gene cluster, we identified open reading frames that share homology with other type IV pilus accessory genes and with genes required for transformation competence and protein secretion. Immediately upstream of the bfp gene cluster, we identified a potential replication origin including genes that are predicted to encode proteins homologous with replicase and resolvase. Restriction fragment length polymorphism analysis of DNA from six additional EPEC serotypes showed that the organization of the bfp gene cluster and its juxtaposition with a potential plasmid origin of replication are highly conserved features of the EPEC biotype. PMID:8626330

  4. Ler Is a Negative Autoregulator of the LEE1 Operon in Enteropathogenic Escherichia coli

    PubMed Central

    Berdichevsky, Tatiana; Friedberg, Devorah; Nadler, Chen; Rokney, Assaf; Oppenheim, Amos; Rosenshine, Ilan

    2005-01-01

    Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. Essential for colonization of the host intestine is the LEE pathogenicity island, which comprises a cluster of operons encoding a type III secretion system and related proteins. The LEE1 operon encodes Ler, which positively regulates many EPEC virulence genes in the LEE region and elsewhere in the chromosome. We found that Ler acts as a specific autorepressor of LEE1 transcription. We further show that Ler specifically binds upstream of the LEE1 operon in vivo and in vitro. A comparison of the Ler affinities to different DNA regions suggests that the autoregulation mechanism limits the steady-state level of Ler to concentrations that are just sufficient for activation of the LEE2 and LEE3 promoters and probably other LEE promoters. This mechanism may reflect the need of EPEC to balance maximizing the colonization efficiency by increasing the expression of the virulence genes and minimizing the immune response of the host by limiting their expression. In addition, we found that the autoregulation mechanism reduces the cell-to-cell variability in the levels of LEE1 expression. Our findings point to a new negative regulatory circuit that suppresses the noise and optimizes the expression levels of ler and other LEE1 genes. PMID:15601719

  5. Recognition of Enteropathogenic Escherichia coli Virulence Determinants by Human Colostrum and Serum Antibodies

    PubMed Central

    Parissi-Crivelli, Aurora; Parissi-Crivelli, Joaquín M.; Girón, Jorge A.

    2000-01-01

    Human colostra and sera collected from Mexican mothers and their children at birth and 6 months thereafter were studied for the presence of antibodies against the bundle-forming pilus and several chromosomal virulence gene products (intimin and secreted proteins EspA and EspB) of enteropathogenic Escherichia coli (EPEC). Among 21 colostrum samples studied, 76, 71.5, 57, and 47% of them contained immunoglobulin A (IgA) antibodies against EspA, intimin, EspB, and BfpA, respectively. Interestingly, there was a difference in IgG response to EPEC antigens between the sera from neonates and sera from the same children 6 months later. While the number of neonates reacting to Esps and intimin diminished when they reached 6 months of age, those reacting with BfpA increased from 9 to 71%. Intimin from an enterohemorrhagic E. coli strain was also recognized by most of the samples reacting with EPEC intimin. These data suggest that Bfp and Esps elicit an antibody response during the early days of life of neonates and support the value of breast-feeding in areas of the world where bacterial diarrheal infections are endemic. PMID:10878066

  6. Detection of typical and atypical enteropathogenic Escherichia coli (EPEC) in Iranian children with and without diarrhoea.

    PubMed

    Alikhani, M Yousef; Mirsalehian, Akbar; Aslani, M Mehdi

    2006-09-01

    The present study was performed to investigate the contribution of typical and atypical enteropathogenic Escherichia coli (EPEC) as a cause of infectious diarrhoea among children less than 10 years old in Iran. During the summer months, 247 specimens from children with diarrhoea and 1108 from asymptomatic children were analysed for the presence of EPEC and other bacterial pathogens. Potential enteric pathogens were identified in 140 cases of children with diarrhoea (56.7%). EPEC was the most frequently identified agent (111 cases), followed by Shiga toxin-producing E. coli (13), Shigella (9), Salmonella (6) and Aeromonas sp. (1). EPEC isolates were examined for the presence of eaeA, bfpA and stx genes by PCR. EPEC isolates were classified as typical (eaeA+ bfpA+) or atypical (eaeA+ bfpA-). Typical EPEC was diagnosed in 35 cases (11.8%), compared with 8 (0.4%) in the asymptomatic group (P<0.05). Atypical EPEC strains were isolated from 23 cases (9.3%), compared with 13 (1.2%) of the healthy control group (P<0.05). In conclusion, the data suggest that typical and atypical EPEC are an important cause of diarrhoea in Iranian children. PMID:16914644

  7. Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins.

    PubMed

    Freeman, N L; Zurawski, D V; Chowrashi, P; Ayoob, J C; Huang, L; Mittal, B; Sanger, J M; Sanger, J W

    2000-12-01

    When enteropathogenic Escherichia coli (EPEC) attach and infect host cells, they induce a cytoskeletal rearrangement and the formation of cytoplasmic columns of actin filaments called pedestals. The attached EPEC and pedestals move over the surface of the host cell in an actin-dependent reaction [Sanger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the membrane of host cells, where it binds the EPEC outer membrane protein, intimin [Kenny et al., 1997: Cell 91:511-520], suggests Tir serves two functions: tethering the bacteria to the host cell and providing a direct connection to the host's cytoskeleton. The sequence of Tir predicts a protein of 56.8 kD with three domains separated by two predicted trans-membrane spanning regions. A GST-fusion protein of the N-terminal 233 amino acids of Tir (Tir1) binds to alpha-actinin, talin, and vinculin from cell extracts. GST-Tir1 also coprecipitates purified forms of alpha-actinin, talin, and vinculin while GST alone does not bind these three focal adhesion proteins. Biotinylated probes of these three proteins also bound Tir1 cleaved from GST. Similar associations of alpha-actinin, talin, and vinculin were also detected with the C-terminus of Tir, i.e., Tir3, the last 217 amino acids. Antibody staining of EPEC-infected cultured cells reveals the presence of focal adhesion proteins beneath the attached bacteria. Our experiments support a model in which the cytoplasmic domains of Tir recruit a number of focal adhesion proteins that can bind actin filaments to form pedestals. Since pedestals also contain villin, tropomyosin and myosin II [Sanger et al., 1996: Cell Motil. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure sharing properties of both focal adhesions and microvilli. PMID:11093251

  8. Enteropathogenic Escherichia coli inhibits ileal sodium-dependent bile acid transporter ASBT.

    PubMed

    Annaba, Fadi; Sarwar, Zaheer; Gill, Ravinder K; Ghosh, Amit; Saksena, Seema; Borthakur, Alip; Hecht, Gail A; Dudeja, Pradeep K; Alrefai, Waddah A

    2012-05-15

    Apical sodium-dependent bile acid transporter (ASBT) is responsible for the absorption of bile acids from the intestine. A decrease in ASBT function and expression has been implicated in diarrhea associated with intestinal inflammation. Whether infection with pathogenic microorganisms such as the enteropathogenic Escherichia coli (EPEC) affect ASBT activity is not known. EPEC is a food-borne enteric pathogen that translocates bacterial effector molecules via type three secretion system (TTSS) into host cells and is a major cause of infantile diarrhea. We investigated the effects of EPEC infection on ileal ASBT function utilizing human intestinal Caco2 cells and HEK-293 cells stably transfected with ASBT-V5 fusion protein (2BT cells). ASBT activity was significantly inhibited following 60 min infection with EPEC but not with nonpathogenic E. coli. Mutations in bacterial escN, espA, espB, and espD, the genes encoding for the elements of bacterial TTSS, ablated EPEC inhibitory effect on ASBT function. Furthermore, mutation in the bacterial BFP gene encoding for bundle-forming pili abrogated the inhibition of ASBT by EPEC, indicating the essential role for bacterial aggregation and the early attachment. The inhibition by EPEC was associated with a significant decrease in the V(max) of the transporter and a reduction in the level of ASBT on the plasma membrane. The inhibition of ASBT by EPEC was blocked in the presence of protein tyrosine phosphatase inhibitors. Our studies provide novel evidence for the alterations in the activity of ASBT by EPEC infection and suggest a possible effect for EPEC in influencing intestinal bile acid homeostasis. PMID:22403793

  9. Ecto-5′-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli

    PubMed Central

    Shulgina, Irina; Naeher, Tonniele M.

    2007-01-01

    Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5′-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5′-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5′-AMP. EPEC infection triggered a release of ecto-5′-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5′-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5′-nucleotidase was susceptible to inhibition by zinc acetate and by α,β-methylene-adenosine diphosphate (α,β-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5′-AMP. In addition, α,β-methylene-ADP and zinc blocked the ability of 5′-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5′-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5′-nucleotidase, such as α,β-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection. PMID:18404437

  10. Survey and Experimental Infection of Enteropathogenic Escherichia coli in Common Marmosets (Callithrix jacchus).

    PubMed

    Hayashimoto, Nobuhito; Inoue, Takashi; Morita, Hanako; Yasuda, Masahiko; Ueno, Masami; Kawai, Kenji; Itoh, Toshio

    2016-01-01

    Common marmosets (Callithrix jacchus) are frequently used for biomedical research but can be afflicted with diarrhea-a serious and potentially lethal health problem. Enteropathogenic Escherichia coli (EPEC) is thought to be the causative pathogen of hemorrhagic typhlocolitis in common marmosets, but the actual incidence of the disease and the relationship between EPEC and hematochezia are unknown. This study investigated the prevalence of EPEC infection in common marmosets and the association between EPEC and hematochezia. A total of 230 stool or rectal swab samples were collected from 230 common marmosets (98 clinically healthy, 85 diarrhea, and 47 bloody stool samples) and tested by culture-based detection and PCR amplification of VT1, VT2, LT, ST, eae, and bfp genes. Healthy animals were divided into three groups (n = 4 each for high and low concentration groups and n = 2 as negative control), and those in the experimental groups were perorally inoculated with a 2-ml of suspension of EPEC R811 strain adjusted to 5 × 108 (high concentration) and 5 × 104 (low concentration) CFU/ ml. Two animals in each group were examined 3 and 14 days post-inoculation (DPI). EPEC was detected in 10 of 98 clinically healthy samples (10.2%), 17 of 85 diarrhea samples (20%), and all 47 bloody stool samples (100%), with a significant difference detected between presence of EPEC and sample status (P < 0.01). Acute hematochezia was observed in all animals of the high-concentration group but not in other groups at 1 or 2 DPI. A histopathological examination revealed the attachment of gram-negative bacilli to epithelial apical membranes and desquamated epithelial cells in the cecum of animals in the high-concentration group at 3 DPI. These findings suggest that EPEC is a causative agent of hemorrhagic typhlocolitis in common marmosets. PMID:27501144

  11. Survey and Experimental Infection of Enteropathogenic Escherichia coli in Common Marmosets (Callithrix jacchus)

    PubMed Central

    Hayashimoto, Nobuhito; Inoue, Takashi; Morita, Hanako; Yasuda, Masahiko; Ueno, Masami; Kawai, Kenji; Itoh, Toshio

    2016-01-01

    Common marmosets (Callithrix jacchus) are frequently used for biomedical research but can be afflicted with diarrhea—a serious and potentially lethal health problem. Enteropathogenic Escherichia coli (EPEC) is thought to be the causative pathogen of hemorrhagic typhlocolitis in common marmosets, but the actual incidence of the disease and the relationship between EPEC and hematochezia are unknown. This study investigated the prevalence of EPEC infection in common marmosets and the association between EPEC and hematochezia. A total of 230 stool or rectal swab samples were collected from 230 common marmosets (98 clinically healthy, 85 diarrhea, and 47 bloody stool samples) and tested by culture-based detection and PCR amplification of VT1, VT2, LT, ST, eae, and bfp genes. Healthy animals were divided into three groups (n = 4 each for high and low concentration groups and n = 2 as negative control), and those in the experimental groups were perorally inoculated with a 2-ml of suspension of EPEC R811 strain adjusted to 5 × 108 (high concentration) and 5 × 104 (low concentration) CFU/ ml. Two animals in each group were examined 3 and 14 days post-inoculation (DPI). EPEC was detected in 10 of 98 clinically healthy samples (10.2%), 17 of 85 diarrhea samples (20%), and all 47 bloody stool samples (100%), with a significant difference detected between presence of EPEC and sample status (P < 0.01). Acute hematochezia was observed in all animals of the high-concentration group but not in other groups at 1 or 2 DPI. A histopathological examination revealed the attachment of gram-negative bacilli to epithelial apical membranes and desquamated epithelial cells in the cecum of animals in the high-concentration group at 3 DPI. These findings suggest that EPEC is a causative agent of hemorrhagic typhlocolitis in common marmosets. PMID:27501144

  12. Molecular Characterization of Human Atypical Sorbitol-Fermenting Enteropathogenic Escherichia coli O157 Reveals High Diversity.

    PubMed

    Kossow, Annelene; Zhang, Wenlan; Bielaszewska, Martina; Rhode, Sophie; Hansen, Kevin; Fruth, Angelika; Rüter, Christian; Karch, Helge; Mellmann, Alexander

    2016-05-01

    Alongside the well-characterized enterohemorrhagic Escherichia coli (EHEC) O157:H7, serogroup O157 comprises sorbitol-fermenting typical and atypical enteropathogenic E. coli (EPEC/aEPEC) strains that carry the intimin-encoding gene eae but not Shiga toxin-encoding genes (stx). Since little is known about these pathogens, we characterized 30 clinical isolates from patients with hemolytic uremic syndrome (HUS) or uncomplicated diarrhea with respect to their flagellin gene (fliC) type and multilocus sequence type (MLST). Moreover, we applied whole-genome sequencing (WGS) to determine the phylogenetic relationship with other eae-positive EHEC serotypes and the composition of the rfbO157 region. fliC typing resulted in five fliC types (H7, H16, H34, H39, and H45). Isolates of each fliC type shared a unique ST. In comparison to the 42 HUS-associated E. coli (HUSEC) strains, only the stx-negative isolates with fliCH7 shared their ST with EHEC O157:H7/H(-) strains. With the exception of one O157:H(-) fliCH16 isolate, HUS was exclusively associated with fliCH7. WGS corroborated the separation of the fliCH7 isolates, which were closely related to the EHEC O157:H7/H(-) isolates, and the diverse group of isolates exhibiting different fliC types, indicating independent evolution of the different serotypes. This was also supported by the heterogeneity within the rfbO157 region that exhibited extensive recombinations. The genotypic subtypes and distribution of clinical symptoms suggested that the stx-negative O157 strains with fliCH7 were originally EHEC strains that lost stx The remaining isolates form a distinct and diverse group of atypical EPEC isolates that do not possess the full spectrum of virulence genes, underlining the importance of identifying the H antigen for clinical risk assessment. PMID:26984976

  13. Enteropathogenic Escherichia coli Outbreak and its Incubation Period: Is it Short or Long?

    PubMed Central

    Lee, Dong-Woo; Gwack, Jin; Youn, Seun-Ki

    2012-01-01

    Objectives The aim of this study is to determine the incubation period of enteropathogenic Escherichia coli (EPEC), which creates several outbreaks in a year in South Korea. Methods We reviewed all water and food-borne outbreaks data reported to the Korea Centers for Disease Control and Prevention (KCDC) from 2009 to 2010 and determined their characteristics. Through this process, we can presume the incubation period of EPEC among outbreaks in South Korea. Results A total of 497 water and food-borne outbreaks were reported to KCDC and 66 (13.28%) are defined as E coli-origin outbreaks. EPEC was the most common subtype of E coli, being confirmed as a causative organism in 26 outbreaks. Overall attack rate was 15.85% (range 0.9–100). The subjects were eight outbreaks that have a clear history of single exposure and we can estimate the incubation time of EPEC as minimum 0.5 hours to maximum 34.0 hours with a mean 12.9 hours (range 4.5–24.0). The cases of those cannot completely rule out the chance of multiple exposure from same source or place have minimum 1.0 hour, to a maximum of 195.5 hours and a mean 30.5 (range 22.7–61.0) hours of incubation period. Conclusions This serial analysis suggests that EPEC has actually shorter mean incubation period as much as 12 hours. When this period is longer than 1 day or over, then the epidemiologic investigator should consider the chance of repeated or continuous exposure by making it clear whether there is any chance of any other exposure in common. PMID:24159486

  14. Tir Is Essential for the Recruitment of Tks5 to Enteropathogenic Escherichia coli Pedestals

    PubMed Central

    Jensen, Helene H.; Pedersen, Hans N.; Stenkjær, Eva; Pedersen, Gitte A.; Login, Frédéric H.; Nejsum, Lene N.

    2015-01-01

    Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that infects the epithelial lining of the small intestine and causes diarrhea. Upon attachment to the intestinal epithelium, EPEC uses a Type III Secretion System to inject its own high affinity receptor Translocated intimin receptor (Tir) into the host cell. Tir facilitates tight adhesion and recruitment of actin-regulating proteins leading to formation of an actin pedestal beneath the infecting bacterium. The pedestal has several similarities with podosomes, which are basolateral actin-rich extensions found in some migrating animal cells. Formation of podosomes is dependent upon the early podosome-specific scavenger protein Tks5, which is involved in actin recruitment. Although Tks5 is expressed in epithelial cells, and podosomes and EPEC pedestals share many components in their structure and mechanism of formation, the potential role of Tks5 in EPEC infections has not been studied. The aim of this study was to determine the subcellular localization of Tks5 in epithelial cells and to investigate if Tks5 is recruited to the EPEC pedestal. In an epithelial MDCK cell line stably expressing Tks5-EGFP, Tks5 localized to actin bundles. Upon infection, EPEC recruited Tks5-EGFP. Tir, but not Tir phosphorylation was essential for the recruitment. Time-lapse microscopy revealed that Tks5-EGFP was recruited instantly upon EPEC attachment to host cells, simultaneously with actin and N-WASp. EPEC infection of cells expressing a ΔPX-Tks5 deletion version of Tks5 showed that EPEC was able to both infect and form pedestals when the PX domain was deleted from Tks5. Future investigations will clarify the role of Tks5 in EPEC infection and pedestal formation. PMID:26536015

  15. Tir Is Essential for the Recruitment of Tks5 to Enteropathogenic Escherichia coli Pedestals.

    PubMed

    Jensen, Helene H; Pedersen, Hans N; Stenkjær, Eva; Pedersen, Gitte A; Login, Frédéric H; Nejsum, Lene N

    2015-01-01

    Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that infects the epithelial lining of the small intestine and causes diarrhea. Upon attachment to the intestinal epithelium, EPEC uses a Type III Secretion System to inject its own high affinity receptor Translocated intimin receptor (Tir) into the host cell. Tir facilitates tight adhesion and recruitment of actin-regulating proteins leading to formation of an actin pedestal beneath the infecting bacterium. The pedestal has several similarities with podosomes, which are basolateral actin-rich extensions found in some migrating animal cells. Formation of podosomes is dependent upon the early podosome-specific scavenger protein Tks5, which is involved in actin recruitment. Although Tks5 is expressed in epithelial cells, and podosomes and EPEC pedestals share many components in their structure and mechanism of formation, the potential role of Tks5 in EPEC infections has not been studied. The aim of this study was to determine the subcellular localization of Tks5 in epithelial cells and to investigate if Tks5 is recruited to the EPEC pedestal. In an epithelial MDCK cell line stably expressing Tks5-EGFP, Tks5 localized to actin bundles. Upon infection, EPEC recruited Tks5-EGFP. Tir, but not Tir phosphorylation was essential for the recruitment. Time-lapse microscopy revealed that Tks5-EGFP was recruited instantly upon EPEC attachment to host cells, simultaneously with actin and N-WASp. EPEC infection of cells expressing a ΔPX-Tks5 deletion version of Tks5 showed that EPEC was able to both infect and form pedestals when the PX domain was deleted from Tks5. Future investigations will clarify the role of Tks5 in EPEC infection and pedestal formation. PMID:26536015

  16. Enteropathogenic Escherichia coli Uses NleA to Inhibit NLRP3 Inflammasome Activation

    PubMed Central

    Yen, Hilo; Sugimoto, Nakaba; Tobe, Toru

    2015-01-01

    Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are related strains capable of inducing severe gastrointestinal disease. For optimal infection, these pathogens actively modulate cellular functions through the deployment of effector proteins in a type three secretion system (T3SS)-dependent manner. In response to enteric pathogen invasion, the Nod-like receptor pyrin domain containing (NLRP) inflammasome has been increasingly recognized as an important cytoplasmic sensor against microbial infection by activating caspase-1 and releasing IL-1β. EPEC and EHEC are known to elicit inflammasome activation in macrophages and epithelial cells; however, whether the pathogens actively counteract such innate immune responses is unknown. Using a series of compound effector-gene deletion strains of EPEC, we screened and identified NleA, which could subdue host IL-1β secretion. It was found that the reduction is not because of blocked NF-κB activity; instead, the reduction results from inhibited caspase-1 activation by NleA. Immunostaining of human macrophage-like cells following infection revealed limited formation of inflammasome foci with constituents of total caspase-1, ASC and NLRP3 in the presence of NleA. Pulldown of PMA-induced differentiated THP-1 lysate with purified MBP-NleA reveals that NLRP3 is a target of NleA. The interaction was verified by an immunoprecipitation assay and direct interaction assay in which purified MBP-NleA and GST-NLRP3 were used. We further showed that the effector interacts with regions of NLRP3 containing the PYD and LRR domains. Additionally, NleA was found to associate with non-ubiquitinated and ubiquitinated NLRP3 and to interrupt de-ubiquitination of NLRP3, which is a required process for inflammasome activation. Cumulatively, our findings provide the first example of EPEC-mediated suppression of inflammasome activity in which NieA plays a novel role in controlling the host immune response through targeting of

  17. Antibody against the Carboxyl Terminus of Intimin α Reduces Enteropathogenic Escherichia coli Adherence to Tissue Culture Cells and Subsequent Induction of Actin Polymerization

    PubMed Central

    Carvalho, Humberto M.; Teel, Louise D.; Kokai-Kun, John F.; O'Brien, Alison D.

    2005-01-01

    The C-terminal third of intimin binds to its translocated receptor (Tir) to promote attaching and effacing lesion formation during infection with enteropathogenic Escherichia coli (EPEC). We observed that the adherence of EPEC strains to HEp-2 cells was reduced and that actin polymerization was blocked by antibody raised against the C-terminal third of intimin α. PMID:15784601

  18. Colonization of neonates in a nursery ward with enteropathogenic Escherichia coli and correlation to the clinical histories of the children.

    PubMed Central

    Senerwa, D; Olsvik, O; Mutanda, L N; Gathuma, J M; Wachsmuth, K

    1989-01-01

    Stool samples were examined from 30 preterm neonates admitted to a nursery ward; 16 neonates had diarrhea, 12 constituted an age-matched control group without diarrhea, and 2 had an unknown history regarding diarrhea. Variable numbers of enteropathogenic Escherichia coli serotype O111:HNT strains possessing the gene coding for the enteroadherence factor (EAF) were found in stool samples from 13 of the neonates. No other microbiological enteropathogen was found. A total of 294 strains (9 or 10 from each neonate, comprising 229 E. coli and 65 Klebsiella pneumoniae strains) were characterized with respect to plasmid content and grouped into 37 plasmid profile groups. Diarrhea was found not to be correlated with any specific plasmid profile or with the presence of the EAF-positive strains but rather with the number of strains with one specific plasmid profile or with the number of EAF-positive strains (of the 9 or 10 strains) isolated from each stool sample. All the neonates who died had diarrhea (5 died of 16 with diarrhea); all five of the neonates who died possessed strains with one specific plasmid profile group, and EAF-positive strains were isolated from four of them. Of the seven neonates from whom seven or more EAF-positive isolates were isolated, three died, compared with only one of five of those from whom only a few (1 to 3 of 10) EAF-positive strains were isolated. Both plasmid profiling and genetic probing with the EAF probe were found to be good alternatives when serotyping is not available for identification of O111:HNT enteropathogenic E. coli strains. PMID:2681255

  19. Prevalence of enteropathogenic and shiga toxin-producing Escherichia coli among children with and without diarrhoea in Iran.

    PubMed

    Alikhani, M Yousef; Mirsalehian, Akbar; Fatollahzadeh, Bahram; Pourshafie, Mohammad R; Aslani, M Mehdi

    2007-03-01

    The aim of the study was to determine the rates of detection of enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) strains among children in two randomly-selected populations in Iran. In total, 1,292 randomly-selected faecal samples from children aged less than 10 years were screened for EPEC and STEC. Of the 1,292 cases participated in the study, 184 had diarrhoea, and 1,108 were healthy/asymptomatic children. The conventional culture method and slide agglutination with 12 different commercial EPEC antisera were used for the detection of EPEC. The colony sweep polymyxin-B extraction method, non-sorbitol fermentation (NSF) phenotype, and slide agglutination with O157: H7 antisera were used for the screening and detection of STEC. Of EPEC belonging to 11 different serogroups, 0111 and 0127 were most commonly found in 36.4% of the diarrhoeal cases and 7.2% of the asymptomatic children. A significant association (p<0.05) was found between isolation of EPEC and diarrhoea. 8.7% of the diarrhoeal cases and 2% of children without diarrhoea were infected with STEC, but none of the isolates belonged to the 0157:H7 serotype. A significant association (p<0.05) was found between STEC and diarrhoeal cases. Based on these findings, it can be concluded that different EPEC serogroups may be agents of endemic infantile diarrhoea, and STEC strains are an important enteropathogen among young children. PMID:17615908

  20. Comparative analysis of virulence determinants, antibiotic susceptibility patterns and serogrouping of atypical enteropathogenic Escherichia coli versus typical enteropathogenic E. coli in India.

    PubMed

    Malvi, Supriya; Appannanavar, Suma; Mohan, Balvinder; Kaur, Harsimran; Gautam, Neha; Bharti, Bhavneet; Kumar, Yashwant; Taneja, Neelam

    2015-10-01

    The epidemiology of enteropathogenic Escherichia coli (EPEC) and the significance of isolation of atypical EPEC (aEPEC) in childhood diarrhoea have not been well studied in an Indian context. A comparative study was undertaken to investigate virulence determinants, antibiotic susceptibility patterns and serogrouping of typical EPEC (tEPEC) versus aEPEC causing diarrhoea in children. A total of 400 prospective and 500 retrospective E. coli isolates were included. PCR was performed for eae, bfpA, efa, nleB, nleE, cdt, ehxA and paa genes. The Clinical and Laboratory Standards Institute's disc diffusion test was used to determine the antimicrobial susceptibility. Phenotypic screening of extended spectrum β-lactamases (ESBLs), AmpC and Klebsiella pneumoniae carbapenemase (KPC) production, and molecular detection of bla(NDM-1), bla(VIM), bla(CTX-M-15), bla(IMP) and bla(KPC) were performed. aEPEC (57.6 %) were more common as compared with tEPEC (42.3 %). The occurrence of virulence genes was observed to be three times higher in aEPEC as compared with tEPEC, efa1 (14.7 % of aEPEC, 4 % of tEPEC) being the most common. Most of the isolates did not belong to the classical EPEC O-serogroups. The highest resistance was observed against amoxicillin (93.22 %) followed by quinolones (83 %), cephalosporins (37.28 %), cotrimoxazole (35.59 %) and carbapenems (30.5 %). Overall equal numbers of aEPEC (41.17 %) and tEPEC (40 %) were observed to be multidrug-resistant. Fifteen EPEC strains demonstrated presence of ESBLs, five produced AmpC and four each produced metallo-β-lactamases and KPC-type carbapenemases; eight, seven and one isolate(s) each were positive for bla(VIM), bla(CTX-M-15) and bla(NDM-1), respectively. Here, to the best of our knowledge, we report for the first time on carbapenem resistance and the presence of bla(NDM-1) and bla(CTX-M-15) in EPEC isolates from India. PMID:26233663

  1. Genetic Diversity of Intimin Gene of Atypical Enteropathogenic Escherichia coli Isolated from Human, Animals and Raw Meats in China.

    PubMed

    Xu, Yanmei; Bai, Xiangning; Zhao, Ailan; Zhang, Wang; Ba, Pengbin; Liu, Kai; Jin, Yujuan; Wang, Hong; Guo, Qiusheng; Sun, Hui; Xu, Jianguo; Xiong, Yanwen

    2016-01-01

    Atypical enteropathogenic Escherichia coli (aEPEC) is considered to be an emerging enteropathogen that is more prevalent than typical EPEC in developing and developed countries. The major adherence factor, intimin, an outer membrane protein encoded by eae, plays a pivotal role in the pathogenesis of aEPEC. This study investigated the distribution and polymorphisms of intimin subtypes of 143 aEPEC strains from diarrheal patients, healthy carriers, animals, and raw meats in China. These aEPEC strains belonged to more than 71 different serotypes, which comprised 52 O serogroups and 24 H types. Sixty-eight different eae genotypes and 19 intimin subtypes were detected. Eighteen, eight, seven, and five intimin subtypes were identified from 86 diarrheal patients, 14 healthy carriers, 19 animals, and 24 raw meats strains, respectively. Intimin β1 was the most prevalent subtype in strains from diarrheal patients (34.88%) and animals (47.37%). There was a statistically significant difference in the distribution of eae-β1 between diarrheal patients and healthy carriers (P = 0.004). Intimin-θ was more predominant among raw meat strains (50%) than among diarrheal patients strains (12.79%, P = 0.0003), healthy carrier strains (7.14%, P = 0.007), or animal strains (15.79%, P = 0.020). The two predominant subtypes (eae-β1 and eae-θ) had considerable polymorphisms with no significant differences among the four sources. PFGE analysis revealed 119 distinct patterns and the strains were clustered into 11 groups with similarity indices ranging from 63% to 100%. These results suggest that in China, aEPEC strains from different sources are highly heterogeneous. Animals and raw meats are important sources of genetically diverse intimin-harboring aEPEC, which might serve as important transmission vehicles of these bacteria. PMID:27031337

  2. Genetic Diversity of Intimin Gene of Atypical Enteropathogenic Escherichia coli Isolated from Human, Animals and Raw Meats in China

    PubMed Central

    Xu, Yanmei; Bai, Xiangning; Zhao, Ailan; Zhang, Wang; Ba, Pengbin; Liu, Kai; Jin, Yujuan; Wang, Hong; Guo, Qiusheng; Sun, Hui; Xu, Jianguo; Xiong, Yanwen

    2016-01-01

    Atypical enteropathogenic Escherichia coli (aEPEC) is considered to be an emerging enteropathogen that is more prevalent than typical EPEC in developing and developed countries. The major adherence factor, intimin, an outer membrane protein encoded by eae, plays a pivotal role in the pathogenesis of aEPEC. This study investigated the distribution and polymorphisms of intimin subtypes of 143 aEPEC strains from diarrheal patients, healthy carriers, animals, and raw meats in China. These aEPEC strains belonged to more than 71 different serotypes, which comprised 52 O serogroups and 24 H types. Sixty-eight different eae genotypes and 19 intimin subtypes were detected. Eighteen, eight, seven, and five intimin subtypes were identified from 86 diarrheal patients, 14 healthy carriers, 19 animals, and 24 raw meats strains, respectively. Intimin β1 was the most prevalent subtype in strains from diarrheal patients (34.88%) and animals (47.37%). There was a statistically significant difference in the distribution of eae-β1 between diarrheal patients and healthy carriers (P = 0.004). Intimin-θ was more predominant among raw meat strains (50%) than among diarrheal patients strains (12.79%, P = 0.0003), healthy carrier strains (7.14%, P = 0.007), or animal strains (15.79%, P = 0.020). The two predominant subtypes (eae-β1 and eae-θ) had considerable polymorphisms with no significant differences among the four sources. PFGE analysis revealed 119 distinct patterns and the strains were clustered into 11 groups with similarity indices ranging from 63% to 100%. These results suggest that in China, aEPEC strains from different sources are highly heterogeneous. Animals and raw meats are important sources of genetically diverse intimin-harboring aEPEC, which might serve as important transmission vehicles of these bacteria. PMID:27031337

  3. CsrA and TnaB coregulate tryptophanase activity to promote exotoxin-induced killing of Caenorhabditis elegans by enteropathogenic Escherichia coli.

    PubMed

    Bhatt, Shantanu; Anyanful, Akwasi; Kalman, Daniel

    2011-09-01

    Enteropathogenic Escherichia coli(EPEC) requires the tnaA-encoded enzyme tryptophanase and its substrate tryptophan to synthesize diffusible exotoxins that kill the nematode Caenorhabditis elegans. Here, we demonstrate that the RNA-binding protein CsrA and the tryptophan permease TnaB coregulate tryptophanase activity, through mutually exclusive pathways, to stimulate toxin-mediated paralysis and killing of C. elegans. PMID:21705596

  4. Thiophenone Attenuates Enteropathogenic Escherichia coli O103:H2 Virulence by Interfering with AI-2 Signaling.

    PubMed

    Witsø, Ingun Lund; Valen Rukke, Håkon; Benneche, Tore; Aamdal Scheie, Anne

    2016-01-01

    Interference with bacterial quorum sensing communication provides an anti-virulence strategy to control pathogenic bacteria. Here, using the Enteropathogenic E. coli (EPEC) O103:H2, we showed for the first time that thiophenone TF101 reduced expression of lsrB; the gene encoding the AI-2 receptor. Combined results of transcriptional and phenotypic analyses suggested that TF101 interfere with AI-2 signalling, possibly by competing with AI-2 for binding to LsrB. This is supported by in silico docking prediction of thiophenone TF101 in the LsrB pocket. Transcriptional analyses furthermore showed that thiophenone TF101 interfered with expression of the virulence genes eae and fimH. In addition, TF101 reduced AI-2 induced E. coli adhesion to colorectal adenocarcinoma cells. TF101, on the other hand, did not affect epinephrine or norepinephrine enhanced E. coli adhesion. Overall, our results showed that thiophenone TF101 interfered with virulence expression in E. coli O103:H2, suggestedly by interfering with AI-2 mediated quorum sensing. We thus conclude that thiophenone TF101 might represent a promising future anti-virulence agent in the fight against pathogenic E. coli. PMID:27309855

  5. Thiophenone Attenuates Enteropathogenic Escherichia coli O103:H2 Virulence by Interfering with AI-2 Signaling

    PubMed Central

    Valen Rukke, Håkon; Benneche, Tore; Aamdal Scheie, Anne

    2016-01-01

    Interference with bacterial quorum sensing communication provides an anti-virulence strategy to control pathogenic bacteria. Here, using the Enteropathogenic E. coli (EPEC) O103:H2, we showed for the first time that thiophenone TF101 reduced expression of lsrB; the gene encoding the AI-2 receptor. Combined results of transcriptional and phenotypic analyses suggested that TF101 interfere with AI-2 signalling, possibly by competing with AI-2 for binding to LsrB. This is supported by in silico docking prediction of thiophenone TF101 in the LsrB pocket. Transcriptional analyses furthermore showed that thiophenone TF101 interfered with expression of the virulence genes eae and fimH. In addition, TF101 reduced AI-2 induced E. coli adhesion to colorectal adenocarcinoma cells. TF101, on the other hand, did not affect epinephrine or norepinephrine enhanced E. coli adhesion. Overall, our results showed that thiophenone TF101 interfered with virulence expression in E. coli O103:H2, suggestedly by interfering with AI-2 mediated quorum sensing. We thus conclude that thiophenone TF101 might represent a promising future anti-virulence agent in the fight against pathogenic E. coli. PMID:27309855

  6. Expression of enteropathogenic Escherichia coli map is significantly different than that of other type III secreted effectors in vivo.

    PubMed

    Nguyen, Mai; Rizvi, Jason; Hecht, Gail

    2015-01-01

    The enteropathogenic Escherichia coli (EPEC) locus of enterocyte effacement (LEE)-encoded effectors EspF and Map are multifunctional and have an impact on the tight junction barrier while the non-LEE-encoded proteins NleH1 and NleH2 possess significant anti-inflammatory activity. In order to address the temporal expression of these important genes in vivo, their promoters were cloned upstream of the luxCDABE operon, and luciferase expression was measured in EPEC-infected mice by bioluminescence using an in vivo imaging system (IVIS). Bioluminescent images of living mice, of excised whole intestines, and of whole intestines longitudinally opened and washed were assessed. The majority of bioluminescent bacteria localized in the cecum by 3 h postinfection, indicating that the cecum is not only a major colonization site of EPEC but also a site of EPEC effector gene expression in mice. espF, nleH1, and nleH2 were abundantly expressed over the course of infection. In contrast, map expression was suppressed at 2 days postinfection, and at 4 days postinfection it was totally abolished. After 2 to 4 days postinfection, when map is suppressed, EPEC colonization is significantly reduced, indicating that map may be one of the factors required to maintain EPEC colonization. This was confirmed in a competitive colonization study and in two models of chronic infection, repeated exposure to ketamine and Citrobacter rodentium infection. Our data suggest that map expression contributes to the maintenance of EPEC colonization. PMID:25312947

  7. Characterization of the pathogenome and phylogenomic classification of enteropathogenic Escherichia coli of the O157:non-H7 serotypes

    PubMed Central

    Sanjar, Fatemeh; Rusconi, Brigida; Hazen, Tracy H.; Koenig, Sara S.K.; Mammel, Mark K.; Feng, Peter C.H.; Rasko, David A.; Eppinger, Mark

    2015-01-01

    Escherichia coli of the O157 serogroup are comprised of a diverse collection of more than 100 O157:non-H7 serotypes that are found in the environment, animal reservoir and infected patients and some have been linked to severe outbreaks of human disease. Among these, the enteropathogenic E. coli O157:non-H7 serotypes carry virulence factors that are hallmarks of enterohemorrhagic E. coli, such as causing attaching and effacing lesions during human gastrointestinal tract infections. Given the shared virulence gene pool between O157:H7 and O157:non-H7 serotypes, our objective was to examine the prevalence of virulence traits of O157:non-H7 serotypes within and across their H-serotype and when compared to other E. coli pathovars. We sequenced six O157:non-H7 genomes complemented by four genomes from public repositories in an effort to determine their virulence state and genetic relatedness to the highly pathogenic enterohemorrhagic O157:H7 lineage and its ancestral O55:H7 serotype. Whole-genome-based phylogenomic analysis and molecular typing is indicative of a non-monophyletic origin of the heterogeneous O157:non-H7 serotypes that are only distantly related to the O157:H7 serotype. The availability of multiple genomes enables robust phylogenomic placement of these strains into their evolutionary context, and the assessment of the pathogenic potential of the O157:non-H7 strains in causing human disease. PMID:25962987

  8. Sensitive and specific detection of enteropathogenic and enterohemorrhagic Escherichia coli using recombinant anti-intimin antibody by immunofluorescence assay.

    PubMed

    Caravelli, Andressa; Luz, Daniela E; Andrade, Fernanda B; Moraes, Claudia T P; Maranhão, Andrea Q; Piazza, Roxane M F

    2013-12-01

    The main and common virulence factor expressed by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is intimin, a 94-kDa outer membrane protein, which is a product of the eae gene, and, thus, an excellent target for the detection of these pathogens. Among the methods for detection of virulence factor expression, immunoassays can be considered the first alternative to either animal use or in vitro culture cells assays, for which polyclonal and/or monoclonal antibodies are raised. In the present work, we evaluated the sensitivity and specificity of an intimin recombinant antibody (scFv-intimin) using immunofluorescence assay. The scFv-intimin detected typical EPEC, atypical EPEC, and EHEC isolates (100% sensitivity) with no detection of eae- isolates (100% specificity). Thus, immunofluorescence is an effective and rapid method, and scFv-intimin, an excellent tool for the diagnosis of diarrhea caused by EPEC and EHEC and also can be employed in case-control epidemiological surveys. PMID:24095642

  9. Enteropathogenic Escherichia coli protein secretion is induced in response to conditions similar to those in the gastrointestinal tract.

    PubMed Central

    Kenny, B; Abe, A; Stein, M; Finlay, B B

    1997-01-01

    The pathogenicity of enteropathogenic Escherichia coli (EPEC) is associated with the expression and secretion of specific bacterial factors. EspB is one such secreted protein which is required to trigger host signaling pathways resulting in effacement of microvilli and cytoskeletal rearrangements. These events presumably contribute to the ensuing diarrhea associated with EPEC infections. EPEC encounters several environmental changes and stimuli during its passage from the external environment into the host gastrointestinal tract. In this paper we show that the secretion of EspB is subject to environmental regulation, and maximal secretion occurs under conditions reminiscent of those in the gastrointestinal tract. Thus, secretion is maximal at 37 degrees C, pH 7, and physiological osmolarity. In addition, maximal secretion requires the presence of sodium bicarbonate and calcium and is stimulated by millimolar concentrations of Fe(NO3)3. The secretion of the four other EPEC-secreted proteins appears to be modulated in a manner similar to that of EspB. Our results also show that secretion is not dependent on CO2, as originally reported by Haigh et al. (FEMS Microbiol. Lett. 129: 63-67, 1995), but that CO2 more likely acts as a component of the medium buffering system, since CO2 dependence was abolished by the use of alternative buffers. PMID:9199427

  10. The RpoE Stress Response Pathway Mediates Reduction of the Virulence of Enteropathogenic Escherichia coli by Zinc

    PubMed Central

    Xue, Yuan; Osborn, Jossef; Panchal, Anand

    2015-01-01

    Zinc supplements are an effective clinical treatment for infantile diarrheal disease caused by enteric pathogens. Previous studies demonstrated that zinc acts on enteropathogenic Escherichia coli (EPEC) bacteria directly to suppress several virulence-related genes at a concentration that can be achieved by oral delivery of dietary zinc supplements. Our in vitro studies showed that a micromolar concentration of zinc induced the envelope stress response and suppressed virulence in EPEC, providing a possible mechanistic explanation for zinc's therapeutic action. In this report, we investigated the molecular and physiological changes in EPEC induced by zinc. We found that micromolar concentrations of zinc reduced the bacterial growth rate without affecting viability. We observed increased membrane permeability caused by zinc. Zinc upregulated the RpoE-dependent envelope stress response pathway and suppressed EPEC virulence gene expression. RpoE alone was sufficient to inhibit virulence factor expression and to attenuate attaching and effacing lesion formation on human host cells. By mutational analysis we demonstrate that the DNA-binding motif of RpoE is necessary for suppression of the LEE1, but not the LEE4, operon. Predictably, inhibition of the RpoE-mediated envelope stress response in combination with micromolar concentrations of zinc reduced EPEC viability. In conclusion, zinc induces the RpoE and stress response pathways in EPEC, and the alternate sigma factor RpoE downregulates EPEC LEE and non-LEE virulence genes by multiple mechanisms. PMID:25819956

  11. Detection of Escherichia coli Enteropathogens by Multiplex Polymerase Chain Reaction from Children's Diarrheal Stools in Two Caribbean–Colombian Cities

    PubMed Central

    Arzuza, Octavio; Urbina, Delfina; Bai, Jing; Guerra, Julio; Montes, Oscar; Puello, Marta; Mendoza, Ketty; Castro, Gregorio Y.

    2010-01-01

    Abstract Acute diarrheal disease is a leading cause of childhood morbidity and mortality in the developing world and Escherichia coli intestinal pathogens are important causative agents. Information on the epidemiology of E. coli intestinal pathogens and their association with diarrheal disease is limited because no diagnostic testing is available in countries with limited resources. To evaluate the prevalence of E. coli intestinal pathogens in a Caribbean–Colombian region, E. coli clinical isolates from children with diarrhea were analyzed by a recently reported two-reaction multiplex polymerase chain reaction (Gomez-Duarte et al., Diagn Microbiol Infect Dis 2009;63:1–9). The phylogenetic group from all E. coli isolates was also typed by a single-reaction multiplex polymerase chain reaction. We found that among 139 E. coli strains analyzed, 20 (14.4%) corresponded to E. coli diarrheagenic pathotypes. Enterotoxigenic, shiga-toxin–producing, enteroaggregative, diffuse adherent, and enteropathogenic E. coli pathotypes were detected, and most of them belonged to the phylogenetic groups A and B1, known to be associated with intestinal pathogens. This is the first report on the molecular characterization of E. coli diarrheogenic isolates in Colombia and the first report on the potential role of E. coli in childhood diarrhea in this geographic area. PMID:19839760

  12. The Cpx envelope stress response both facilitates and inhibits elaboration of the enteropathogenic Escherichia coli bundle-forming pilus

    PubMed Central

    Vogt, Stefanie L; Nevesinjac, Anna Z; Humphries, Romney M; Donnenberg, Michael S; Armstrong, Glen D; Raivio, Tracy L

    2010-01-01

    The Cpx envelope stress response is induced by the misfolding of periplasmic proteins and restores envelope homeostasis by upregulating several periplasmic protein folding and degrading factors. The Cpx response also regulates the expression of a variety of envelope-spanning protein complexes, including flagella, secretion systems and pili, which play an important role in pathogenesis. In a previous study, we inactivated the Cpx response in enteropathogenic Escherichia coli (EPEC), a causative agent of infant diarrhoea, and observed decreased expression of its major adhesin, the bundle-forming pilus (BFP). Here, we examined the mechanism underlying this BFP expression defect, and found that this phenotype can be attributed to insufficient expression of periplasmic folding factors, such as DsbA, DegP and CpxP. Hence, a low level of Cpx pathway activity promotes BFP synthesis by upregulating factors important for folding of BFP component proteins. Conversely, we found that full induction of the Cpx response inhibits BFP expression, mainly by repressing transcription of the bfp gene cluster. In combination with a previous report examining EPEC type III secretion, our results demonstrate that the Cpx response co-ordinates the repression of cell-surface structures during periods of envelope stress. PMID:20444097

  13. Molecular Evolution of Typical Enteropathogenic Escherichia coli: Clonal Analysis by Multilocus Sequence Typing and Virulence Gene Allelic Profiling▿ †

    PubMed Central

    Lacher, David W.; Steinsland, Hans; Blank, T. Eric; Donnenberg, Michael S.; Whittam, Thomas S.

    2007-01-01

    Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Typical EPEC isolates are differentiated from other types of pathogenic E. coli by two distinctive phenotypes, attaching effacement and localized adherence. The genes specifying these phenotypes are found on the locus of enterocyte effacement (LEE) and the EPEC adherence factor (EAF) plasmid. To describe how typical EPEC has evolved, we characterized a diverse collection of strains by multilocus sequence typing (MLST) and performed restriction fragment length polymorphism (RFLP) analysis of three virulence genes (eae, bfpA, and perA) to assess allelic variation. Among 129 strains representing 20 O-serogroups, 21 clonal genotypes were identified using MLST. RFLP analysis resolved nine eae, nine bfpA, and four perA alleles. Each bfpA allele was associated with only one perA allele class, suggesting that recombination has not played a large role in shuffling the bfpA and perA loci between separate EAF plasmids. The distribution of eae alleles among typical EPEC strains is more concordant with the clonal relationships than the distribution of the EAF plasmid types. These results provide further support for the hypothesis that the EPEC pathotype has evolved multiple times within E. coli through separate acquisitions of the LEE island and EAF plasmid. PMID:17098897

  14. Substrate recognition by the zinc metalloprotease effector NleC from enteropathogenic Escherichia coli.

    PubMed

    Giogha, Cristina; Lung, Tania Wong Fok; Mühlen, Sabrina; Pearson, Jaclyn S; Hartland, Elizabeth L

    2015-12-01

    Upon infection of epithelial cells, enteropathogenic Escherichia coli suppresses host cell inflammatory signalling in a type III secretion system (T3SS) dependent manner. Two key T3SS effector proteins involved in this response are NleE and NleC. NleC is a zinc metalloprotease effector that degrades the p65 subunit of NF-κB. Although the site of p65 cleavage by NleC is now well described, other areas of interaction have not been precisely defined. Here we constructed overlapping truncations of p65 to identify regions required for NleC cleavage. We determined that NleC cleaved both p65 and p50 within the Rel homology domain (RHD) and that two motifs, E22IIE25 and P177VLS180 , within the RHD of p65 were important for recognition and binding by NleC. Alanine substitution of one or both of these motifs protected p65 from binding and degradation by NleC. The E22IIE25 and P177VLS180 motifs were located within the structurally distinct N-terminal subdomain of the RHD involved in DNA binding by p65 on adjacent, parallel strands. Although these motifs have not been recognized previously, both were needed for the correct localization and function of p65. In summary, this work has identified two regions of p65 within the RHD needed for binding and cleavage by NleC and provides further insight into the molecular basis of substrate recognition by a T3SS effector. PMID:26096513

  15. Designed Coiled-Coil Peptides Inhibit the Type Three Secretion System of Enteropathogenic Escherichia coli

    PubMed Central

    Larzábal, Mariano; Mercado, Elsa C.; Vilte, Daniel A.; Salazar-González, Hector; Cataldi, Angel; Navarro-Garcia, Fernando

    2010-01-01

    Background Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are two categories of E. coli strains associated with human disease. A major virulence factor of both pathotypes is the expression of a type three secretion system (TTSS), responsible for their ability to adhere to gut mucosa causing a characteristic attaching and effacing lesion (A/E). The TTSS translocates effector proteins directly into the host cell that subvert mammalian cell biochemistry. Methods/Principal Findings We examined synthetic peptides designed to inhibit the TTSS. CoilA and CoilB peptides, both representing coiled-coil regions of the translocator protein EspA, and CoilD peptide, corresponding to a coiled–coil region of the needle protein EscF, were effective in inhibiting the TTSS dependent hemolysis of red blood cells by the EPEC E2348/69 strain. CoilA and CoilB peptides also reduced the formation of actin pedestals by the same strain in HEp-2 cells and impaired the TTSS-mediated protein translocation into the epithelial cell. Interestingly, CoilA and CoilB were able to block EspA assembly, destabilizing the TTSS and thereby Tir translocation. This blockage of EspA polymerization by CoilA or CoilB peptides, also inhibited the correct delivery of EspB and EspD as detected by immunoblotting. Interestingly, electron microscopy of bacteria incubated with the CoilA peptide showed a reduction of the length of EspA filaments. Conclusions Our data indicate that coiled-coil peptides can prevent the assembly and thus the functionality of the TTSS apparatus and suggest that these peptides could provide an attractive tool to block EPEC and EHEC pathogenesis. PMID:20140230

  16. Estimating the prevalence of potential enteropathogenic Escherichia coli and intimin gene diversity in a human community by monitoring sanitary sewage.

    PubMed

    Yang, Kun; Pagaling, Eulyn; Yan, Tao

    2014-01-01

    Presently, the understanding of bacterial enteric diseases in the community and their virulence factors relies almost exclusively on clinical disease reporting and examination of clinical pathogen isolates. This study aimed to investigate the feasibility of an alternative approach that monitors potential enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) prevalence and intimin gene (eae) diversity in a community by directly quantifying and characterizing target virulence genes in the sanitary sewage. The quantitative PCR (qPCR) quantification of the eae, stx1, and stx2 genes in sanitary sewage samples collected over a 13-month period detected eae in all 13 monthly sewage samples at significantly higher abundance (93 to 7,240 calibrator cell equivalents [CCE]/100 ml) than stx1 and stx2, which were detected sporadically. The prevalence level of potential EPEC in the sanitary sewage was estimated by calculating the ratio of eae to uidA, which averaged 1.0% (σ = 0.4%) over the 13-month period. Cloning and sequencing of the eae gene directly from the sewage samples covered the majority of the eae diversity in the sewage and detected 17 unique eae alleles belonging to 14 subtypes. Among them, eae-β2 was identified to be the most prevalent subtype in the sewage, with the highest detection frequency in the clone libraries (41.2%) and within the different sampling months (85.7%). Additionally, sewage and environmental E. coli isolates were also obtained and used to determine the detection frequencies of the virulence genes as well as eae genetic diversity for comparison. PMID:24141131

  17. Quantitative Real-time Polymerase Chain Reaction for Enteropathogenic Escherichia coli: A Tool for Investigation of Asymptomatic Versus Symptomatic Infections

    PubMed Central

    Barletta, Francesca; Mercado, Erik; Ruiz, Joaquim; Ecker, Lucie; Lopez, Giovanni; Mispireta, Monica; Gil, Ana I.; Lanata, Claudio F.; Cleary, Thomas G.

    2011-01-01

    Background. Enteropathogenic Escherichia coli (EPEC) strains are pediatric pathogens commonly isolated from both healthy and sick children with diarrhea in areas of endemicity. The aim of this study was to compare the bacterial load of EPEC isolated from stool samples from children with and without diarrhea to determine whether bacterial load might be a useful tool for further study of this phenomenon. Methods. EPEC was detected by polymerase chain reaction (PCR) of colonies isolated on MacConkey plates from 53 diarrheal and 90 healthy children aged <2 years. DNA was isolated from stool samples by cetyltrimethylammonium bromide extraction. To standardize quantification by quantitative real-time PCR (qRT-PCR), the correlation between fluorescence threshold cycle and copy number of the intimin gene of EPEC E2348/69 was determined. Results. The detection limit of qRT-PCR was 5 bacteria/mg stool. The geometric mean load in diarrhea was 299 bacteria/mg (95% confidence interval [CI], 77–1164 bacteria/mg), compared with 29 bacteria/mg (95% CI, 10–87 bacteria/mg) in control subjects (P = .016). Bacterial load was significantly higher in children with diarrhea than in control subjects among children <12 months of age (178 vs 5 bacteria/mg; P = .006) and among children with EPEC as the sole pathogen (463 vs 24 bacteria/mg; P = .006). Conclusions. EPEC load measured by qRT-PCR is higher in diarrheal than in healthy children. qRT-PCR may be useful to study the relationship between disease and colonization in settings of endemicity. PMID:22028433

  18. Transcriptional Regulation of the orf19 Gene and the tir-cesT-eae Operon of Enteropathogenic Escherichia coli

    PubMed Central

    Sánchez-SanMartín, Claudia; Bustamante, Víctor H.; Calva, Edmundo; Puente, José Luis

    2001-01-01

    To establish an intimate interaction with the host epithelial cell surface, enteropathogenic Escherichia coli (EPEC) produces Tir, a bacterial protein that upon translocation and insertion into the epithelial cell membrane constitutes the receptor for intimin. The tir gene is encoded by the locus for enterocyte effacement (LEE), where it is flanked upstream by orf19 and downstream by the cesT and eae genes. With the use of a series of cat transcriptional fusions and primer extension analysis, we confirmed that tir, cesT, and eae form the LEE5 operon, which is under the control of a promoter located upstream from tir, and found that the orf19 gene is transcribed as a monocistronic unit. We also demonstrated that the LEE-encoded regulator Ler was required for efficient activation of both the tir and the orf19 promoters and that a sequence motif located between positions −204 and −157 was needed for the Ler-dependent activation of the tir operon. Sequence elements located between positions −204 and −97 were determined to be required for the differential negative modulatory effects exerted by unknown regulatory factors under specific growth conditions. Upon deletion of the upstream sequences, the tir promoter was fully active even in the absence of Ler, indicating that tir expression is subject to a repression mechanism that is counteracted by this regulatory protein. However, its full activation was still repressed by growth in rich medium or at 25°C, suggesting that negative regulation also occurs at or downstream of the promoter. Expression of orf19, but not of the tir operon, became Ler independent in an hns mutant strain, suggesting that Ler overcomes the repression exerted by H-NS (histone-like nucleoid structuring protein) on this gene. PMID:11292802

  19. Characterization of enteropathogenic and Shiga toxin-producing Escherichia coli in cattle and deer in a shared agroecosystem

    PubMed Central

    Singh, Pallavi; Sha, Qiong; Lacher, David W.; Del Valle, Jacquelyn; Mosci, Rebekah E.; Moore, Jennifer A.; Scribner, Kim T.; Manning, Shannon D.

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. Cattle are suggested to be an important reservoir for STEC; however, these pathogens have also been isolated from other livestock and wildlife. In this study we sought to investigate transmission of STEC, enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) between cattle and white-tailed deer in a shared agroecosystem. Cattle feces were collected from 100 animals in a Michigan dairy farm in July 2012, while 163 deer fecal samples were collected during two sampling periods (March and June). The locations of deer fecal pellets were recorded via geographic information system mapping and microsatellite multi-locus genotyping was used to link the fecal samples to individual deer at both time points. Following subculture to sorbitol MacConkey agar and STEC CHROMagar, the pathogens were characterized by serotyping, stx profiling, and PCR-based fingerprinting; multilocus sequence typing (MLST) was performed on a subset. STEC and EHEC were cultured from 12 to 16% of cattle, respectively, and EPEC was found in 36%. Deer were significantly less likely to have a pathogen in March vs. June where the frequency of STEC, EHEC, and EPEC was 1, 6, and 22%, respectively. PCR fingerprinting and MLST clustered the cattle- and deer-derived strains together in a phylogenetic tree. Two STEC strains recovered from both animal species shared MLST and fingerprinting profiles, thereby providing evidence of interspecies transmission and highlighting the importance of wildlife species in pathogen shedding dynamics and persistence in the environment and cattle herds. PMID:25883908

  20. Estimating the Prevalence of Potential Enteropathogenic Escherichia coli and Intimin Gene Diversity in a Human Community by Monitoring Sanitary Sewage

    PubMed Central

    Yang, Kun; Pagaling, Eulyn

    2014-01-01

    Presently, the understanding of bacterial enteric diseases in the community and their virulence factors relies almost exclusively on clinical disease reporting and examination of clinical pathogen isolates. This study aimed to investigate the feasibility of an alternative approach that monitors potential enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) prevalence and intimin gene (eae) diversity in a community by directly quantifying and characterizing target virulence genes in the sanitary sewage. The quantitative PCR (qPCR) quantification of the eae, stx1, and stx2 genes in sanitary sewage samples collected over a 13-month period detected eae in all 13 monthly sewage samples at significantly higher abundance (93 to 7,240 calibrator cell equivalents [CCE]/100 ml) than stx1 and stx2, which were detected sporadically. The prevalence level of potential EPEC in the sanitary sewage was estimated by calculating the ratio of eae to uidA, which averaged 1.0% (σ = 0.4%) over the 13-month period. Cloning and sequencing of the eae gene directly from the sewage samples covered the majority of the eae diversity in the sewage and detected 17 unique eae alleles belonging to 14 subtypes. Among them, eae-β2 was identified to be the most prevalent subtype in the sewage, with the highest detection frequency in the clone libraries (41.2%) and within the different sampling months (85.7%). Additionally, sewage and environmental E. coli isolates were also obtained and used to determine the detection frequencies of the virulence genes as well as eae genetic diversity for comparison. PMID:24141131

  1. Occurrence and Characterization of Enteropathogenic Escherichia coli (EPEC) in Retail Ready-to-Eat Foods in China.

    PubMed

    Zhang, Shuhong; Wu, Qingping; Zhang, Jumei; Zhu, Xuemei

    2016-01-01

    Enteropathogenic Escherichia coli (EPEC) is an important foodborne pathogen that potentially causes infant and adult diarrhea. The occurrence and characteristics of EPEC in retail ready-to-eat (RTE) foods have not been thoroughly investigated in China. This study aimed to investigate EPEC occurrence in retail RTE foods sold in the markets of China and to characterize the isolated EPEC by serotyping, virulence gene analyses, antibiotic susceptibility test, and molecular typing based on enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). From May 2012 to April 2013, 459 RTE food samples were collected from retail markets in 24 cities of China. E. coli in general, and EPEC specifically, were detected in 144 (31.4%) and 39 (8.5%) samples, respectively. Cold vegetable in sauce was the food type most frequently contaminated with EPEC (18.6%). Of 39 EPEC isolates, 38 were atypical EPEC (eae+) and 1 was typical EPEC (eae+bfpA+) by multiplex PCR assays. The virulence genes espA, espB, tir, and iha were detected in 12, 9, 2, and 1 of 39 isolates, respectively, while genes toxB, etpD, katP, and saa were not detected. O-antigen serotyping results showed that among 28 typeable isolates, the most common serotype was O119, followed by O26, O111, and O128. Many isolates were resistant to tetracycline (64.1%; 25/39), ampicillin (48.7%; 19/39), and trimethoprim/sulfamethoxazole (48.7%; 19/39). ERIC-PCR indicated high genetic diversity in EPEC strains, which classified 42 strains (39 isolates and 3 reference strains) into 32 different profiles with a discrimination index of 0.981. The findings of this study highlight the need for close surveillance of the RTE foods at the level of production, packaging, and storage to minimize risks of foodborne disease. PMID:26575469

  2. [Epidemic of gastroenteritis in Noumea (New Caledonia) caused by an enterotoxinogenic strain of Escherichia coli (0l26:B16) believed to be enteropathogenic].

    PubMed

    Germani, Y; Amat, F; Brethes, B; Begaud, E; Plassart, H

    1985-01-01

    A strain of enteropathogenic Escherichia coli 0126:B16 has been isolated in fifteen children and one adult during a severe outbreak. One infant is dead. The strain produced heat-stable enterotoxin, attach to rabbit enterocytes but did not have colonization factor antigen CFA/I or CFA/II. Its hemagglutination type was the same that the E. coli H10407, CFA/I+. It presented a resistance at eight antibiotics and, with the loss of enterotoxigenicity, there was a loss of resistance at ampicillin and of the capacity to attach to enterocytes. PMID:3906346

  3. Neem (Azadirachta indica A. Juss) Oil to Tackle Enteropathogenic Escherichia coli

    PubMed Central

    Del Serrone, Paola; Nicoletti, Marcello

    2015-01-01

    Neem (Azadirachta indica A. Juss) oil (NO) was assayed against forty-eight isolates of Escherichia coli by standardised disc diffusion test and microdilution test. By molecular biology characterization, fourteen isolates resulted in diarrheagenic E. coli with sixteen primer pairs that specifically amplify unique sequences of virulence genes and of 16S rRNA. The NO showed biological activity against all isolates. The bacterial growth inhibition zone by disc diffusion method (100 µL NO) ranged between 9.50 ± 0.70 and 30.00 ± 1.00 mm. The antibacterial activity was furthermore determined at lower NO concentrations (1 : 10–1 : 10,000). The percent of growth reduction ranged between 23.71 ± 1.00 and 99.70 ± 1.53. The highest bacterial growth reduction was 1 : 10 NO concentration with 50 µL of bacterial suspension (ca. 1 × 106 CFU/mL). There is significant difference between the antibacterial activities against pathogenic and nonpathogenic E. coli, as well as NO and ciprofloxacin activities. Viable cells after the different NO concentration treatments were checked by molecular biology assay using PMA dye. On the basis of the obtained results, NO counteracts E. coli and also influences the virulence of E. coli viable cells after NO treatment. The NO metabolomic composition was obtained using fingerprint HPTLC. PMID:26064900

  4. Neem (Azadirachta indica A. Juss) Oil to Tackle Enteropathogenic Escherichia coli.

    PubMed

    Del Serrone, Paola; Toniolo, Chiara; Nicoletti, Marcello

    2015-01-01

    Neem (Azadirachta indica A. Juss) oil (NO) was assayed against forty-eight isolates of Escherichia coli by standardised disc diffusion test and microdilution test. By molecular biology characterization, fourteen isolates resulted in diarrheagenic E. coli with sixteen primer pairs that specifically amplify unique sequences of virulence genes and of 16S rRNA. The NO showed biological activity against all isolates. The bacterial growth inhibition zone by disc diffusion method (100 µL NO) ranged between 9.50 ± 0.70 and 30.00 ± 1.00 mm. The antibacterial activity was furthermore determined at lower NO concentrations (1 : 10-1 : 10,000). The percent of growth reduction ranged between 23.71 ± 1.00 and 99.70 ± 1.53. The highest bacterial growth reduction was 1 : 10 NO concentration with 50 µL of bacterial suspension (ca. 1 × 10(6) CFU/mL). There is significant difference between the antibacterial activities against pathogenic and nonpathogenic E. coli, as well as NO and ciprofloxacin activities. Viable cells after the different NO concentration treatments were checked by molecular biology assay using PMA dye. On the basis of the obtained results, NO counteracts E. coli and also influences the virulence of E. coli viable cells after NO treatment. The NO metabolomic composition was obtained using fingerprint HPTLC. PMID:26064900

  5. Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli*

    PubMed Central

    Fan, Haitian; Hahm, Joseph; Diggs, Stephen; Perry, J. Jefferson P.; Blaha, Gregor

    2015-01-01

    The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3′, 5′-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes to small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. This molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response. PMID:26163516

  6. SepD/SepL-Dependent Secretion Signals of the Type III Secretion System Translocator Proteins in Enteropathogenic Escherichia coli

    PubMed Central

    Deng, Wanyin; Yu, Hong B.; Li, Yuling

    2015-01-01

    ABSTRACT The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by the sepD and sepL mutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals. IMPORTANCE Many bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into

  7. Mutagenesis and Functional Analysis of the Bacterial Arginine Glycosyltransferase Effector NleB1 from Enteropathogenic Escherichia coli.

    PubMed

    Wong Fok Lung, Tania; Giogha, Cristina; Creuzburg, Kristina; Ong, Sze Ying; Pollock, Georgina L; Zhang, Ying; Fung, Ka Yee; Pearson, Jaclyn S; Hartland, Elizabeth L

    2016-05-01

    Enteropathogenic Escherichia coli (EPEC) interferes with host cell signaling by injecting virulence effector proteins into enterocytes via a type III secretion system (T3SS). NleB1 is a novel T3SS glycosyltransferase effector from EPEC that transfers a single N-acetylglucosamine (GlcNAc) moiety in an N-glycosidic linkage to Arg(117) of the Fas-associated death domain protein (FADD). GlcNAcylation of FADD prevents the assembly of the canonical death-inducing signaling complex and inhibits Fas ligand (FasL)-induced cell death. Apart from the DXD catalytic motif of NleB1, little is known about other functional sites in the enzyme. In the present study, members of a library of 22 random transposon-based, in-frame, linker insertion mutants of NleB1 were tested for their ability to block caspase-8 activation in response to FasL during EPEC infection. Immunoblot analysis of caspase-8 cleavage showed that 17 mutant derivatives of NleB1, including the catalytic DXD mutant, did not inhibit caspase-8 activation. Regions of interest around the insertion sites with multiple or single amino acid substitutions were examined further. Coimmunoprecipitation studies of 34 site-directed mutants showed that the NleB1 derivatives with the E253A, Y219A, and PILN(63-66)AAAA (in which the PILN motif from residues 63 to 66 was changed to AAAA) mutations bound to but did not GlcNAcylate FADD. A further mutant derivative, the PDG(236-238)AAA mutant, did not bind to or GlcNAcylate FADD. Infection of mice with the EPEC-like mouse pathogen Citrobacter rodentium expressing NleBE253A and NleBY219A showed that these strains were attenuated, indicating the importance of residues E253 and Y219 in NleB1 virulence in vivo In summary, we identified new amino acid residues critical for NleB1 activity and confirmed that these are required for the virulence function of NleB1. PMID:26883593

  8. Phenotypic and genotypic characteristics associated with biofilm formation in clinical isolates of atypical enteropathogenic Escherichia coli (aEPEC) strains

    PubMed Central

    2014-01-01

    Background Biofilm formation by enteropathogenic Escherichia coli (EPEC) have been recently described in the prototype typical EPEC E2348/69 strain and in an atypical EPEC O55:H7 strain. In this study, we sought to evaluate biofilm formation in a collection of 126 atypical EPEC strains isolated from 92 diarrheic and 34 nondiarrheic children, belonging to different serotypes. The association of biofilm formation and adhesin-related genes were also investigated. Results Biofilm formation occurred in 37 (29%) strains of different serotypes, when the assays were performed at 26°C and 37°C for 24 h. Among these, four strains (A79, A87, A88, and A111) formed a stronger biofilm than did the others. The frequency of biofilm producers was higher among isolates from patients compared with isolates from controls (34.8% vs 14.7%; P = 0.029). An association was found between biofilm formation and expression of type 1 fimbriae and curli (P < 0.05). Unlike the previously described aEPEC O55:H7, one aEPEC O119:HND strain (A111) formed a strong biofilm and pellicle at the air-liquid interface, but did not express curli. Transposon mutagenesis was used to identify biofilm-deficient mutants. Transposon insertion sequences of six mutants revealed similarity with type 1 fimbriae (fimC, fimD, and fimH), diguanylate cyclase, ATP synthase F1, beta subunit (atpD), and the uncharacterized YjiC protein. All these mutants were deficient in biofilm formation ability. Conclusion This study showed that the ability to adhere to abiotic surfaces and form biofilm is present in an array of aEPEC strains. Moreover, it seems that the ability to form biofilms is associated with the presence of type 1 fimbriae and diguanylate cyclase. Characterization of additional biofilm formation mutants may reveal other mechanisms involved in biofilm formation and bring new insights into aEPEC adhesion and pathogenesis. PMID:25012525

  9. Is Shiga Toxin-Negative Escherichia coli O157:H7 Enteropathogenic or Enterohemorrhagic Escherichia coli? Comprehensive Molecular Analysis Using Whole-Genome Sequencing.

    PubMed

    Ferdous, Mithila; Zhou, Kai; Mellmann, Alexander; Morabito, Stefano; Croughs, Peter D; de Boer, Richard F; Kooistra-Smid, Anna M D; Rossen, John W A; Friedrich, Alexander W

    2015-11-01

    The ability of Escherichia coli O157:H7 to induce cellular damage leading to disease in humans is related to numerous virulence factors, most notably the stx gene, encoding Shiga toxin (Stx) and carried by a bacteriophage. Loss of the Stx-encoding bacteriophage may occur during infection or culturing of the strain. Here, we collected stx-positive and stx-negative variants of E. coli O157:H7/NM (nonmotile) isolates from patients with gastrointestinal complaints. Isolates were characterized by whole-genome sequencing (WGS), and their virulence properties and phylogenetic relationship were determined. Because of the presence of the eae gene but lack of the bfpA gene, the stx-negative isolates were considered atypical enteropathogenic E. coli (aEPEC). However, they had phenotypic characteristics similar to those of the Shiga toxin-producing E. coli (STEC) isolates and belonged to the same sequence type, ST11. Furthermore, EPEC and STEC isolates shared similar virulence genes, the locus of enterocyte effacement region, and plasmids. Core genome phylogenetic analysis using a gene-by-gene typing approach showed that the sorbitol-fermenting (SF) stx-negative isolates clustered together with an SF STEC isolate and that one non-sorbitol-fermenting (NSF) stx-negative isolate clustered together with NSF STEC isolates. Therefore, these stx-negative isolates were thought either to have lost the Stx phage or to be a progenitor of STEC O157:H7/NM. As detection of STEC infections is often based solely on the identification of the presence of stx genes, these may be misdiagnosed in routine laboratories. Therefore, an improved diagnostic approach is required to manage identification, strategies for treatment, and prevention of transmission of these potentially pathogenic strains. PMID:26311863

  10. Is Shiga Toxin-Negative Escherichia coli O157:H7 Enteropathogenic or Enterohemorrhagic Escherichia coli? Comprehensive Molecular Analysis Using Whole-Genome Sequencing

    PubMed Central

    Ferdous, Mithila; Zhou, Kai; Mellmann, Alexander; Morabito, Stefano; Croughs, Peter D.; de Boer, Richard F.; Kooistra-Smid, Anna M. D.; Friedrich, Alexander W.

    2015-01-01

    The ability of Escherichia coli O157:H7 to induce cellular damage leading to disease in humans is related to numerous virulence factors, most notably the stx gene, encoding Shiga toxin (Stx) and carried by a bacteriophage. Loss of the Stx-encoding bacteriophage may occur during infection or culturing of the strain. Here, we collected stx-positive and stx-negative variants of E. coli O157:H7/NM (nonmotile) isolates from patients with gastrointestinal complaints. Isolates were characterized by whole-genome sequencing (WGS), and their virulence properties and phylogenetic relationship were determined. Because of the presence of the eae gene but lack of the bfpA gene, the stx-negative isolates were considered atypical enteropathogenic E. coli (aEPEC). However, they had phenotypic characteristics similar to those of the Shiga toxin-producing E. coli (STEC) isolates and belonged to the same sequence type, ST11. Furthermore, EPEC and STEC isolates shared similar virulence genes, the locus of enterocyte effacement region, and plasmids. Core genome phylogenetic analysis using a gene-by-gene typing approach showed that the sorbitol-fermenting (SF) stx-negative isolates clustered together with an SF STEC isolate and that one non-sorbitol-fermenting (NSF) stx-negative isolate clustered together with NSF STEC isolates. Therefore, these stx-negative isolates were thought either to have lost the Stx phage or to be a progenitor of STEC O157:H7/NM. As detection of STEC infections is often based solely on the identification of the presence of stx genes, these may be misdiagnosed in routine laboratories. Therefore, an improved diagnostic approach is required to manage identification, strategies for treatment, and prevention of transmission of these potentially pathogenic strains. PMID:26311863

  11. Bowman-Birk inhibitor-like protein is secreted by sprouted pea seeds in response to induced colonization by enteropathogenic Escherichia coli.

    PubMed

    Anuradha, Ravi; Raveendran, Muthuraj; Babu, Subramanian

    2013-11-01

    The interaction between the clinical isolate of enteropathogenic Escherichia coli (EPEC) SBANU8 and pea sprouts was compared with avirulent K 12. E. coli. This was carried out by repeated co-incubation with pea sprouts for 5 days, and the protein profile of the culture supernatant was analyzed by single and two-dimensional electrophoresis. Mass spectrometry analysis led to the identification of two serine protease inhibitors including a Bowman-Birk-type protein secreted by pea sprouts in response to clinical isolate. Expression of the E. coli intimin gene involved in animal host colonization and virulence was studied by reverse transcription polymerase chain reaction. Expression of this gene was high in SBANU8 when co-incubated with pea sprouts. The present study gives baseline data on the molecular level interactions of EPEC and pea sprouts, which are needed to design the outbreak control strategies. PMID:23862737

  12. Cluster investigation of mixed O76:H19 Shiga toxin-producing Escherichia coli and atypical enteropathogenic E. coli infection in a Spanish household.

    PubMed

    Sánchez, S; Cenoz, M García; Martín, C; Beristain, X; Llorente, M T; Herrera-León, S

    2014-05-01

    A Spanish household was identified through a Public Health follow up on a Shiga toxin-producing Escherichia coli (STEC)-positive 14-month-old girl reporting bloody diarrhoea, with the four household members experiencing either symptomatic or asymptomatic STEC and/or atypical enteropathogenic E. coli (aEPEC) shedding. In total, two different O76:H19 STEC strains and six aEPEC strains belonging to multiple serotypes were isolated and characterized in the household during a 5-month period. Prolonged asymptomatic shedding of O76:H19 STEC and O51:H49 aEPEC was detected in two family members. Although there was no conclusive evidence, consumption of vegetables fertilized with sheep manure was the suspected source of infection. This study highlights the risk of cross-infections posed by prolonged asymptomatic carriage and close household contact between family members, and illustrates the importance of molecular epidemiology in understanding disease clusters. PMID:23906309

  13. The Flagella of an Atypical Enteropathogenic Escherichia coli Strain Are Required for Efficient Interaction with and Stimulation of Interleukin-8 Production by Enterocytes In Vitro▿

    PubMed Central

    Sampaio, Suely C. F.; Gomes, Tânia A. T.; Pichon, Christophe; du Merle, Laurence; Guadagnini, Stéphanie; Abe, Cecilia M.; Sampaio, Jorge L. M.; Le Bouguénec, Chantal

    2009-01-01

    The ability of some typical enteropathogenic Escherichia coli (EPEC) strains to adhere to, invade, and increase interleukin-8 (IL-8) production in intestinal epithelial cells in vitro has been demonstrated. However, few studies regarding these aspects have been performed with atypical EPEC (aEPEC) strains, which are emerging enteropathogens in Brazil. In this study, we evaluated a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, in regard to its ability to adhere to and invade Caco-2 and T84 cells and to elicit IL-8 production in Caco-2 cells. The role of flagella in aEPEC 1711-4 adhesion, invasion, and IL-8 production was investigated by performing the same experiments with an isogenic aEPEC mutant unable to produce flagellin (FliC), the flagellum protein subunit. We demonstrated that this mutant (fliC mutant) had a marked decrease in the ability to adhere to T84 cells and invade both T84 and Caco-2 cells in gentamicin protection assays and by transmission electron microscopy. In addition, the aEPEC 1711-4 fliC mutant had a reduced ability to stimulate IL-8 production by Caco-2 cells in early (3-h) but not in late (24-h) infections. Our findings demonstrate that flagella of aEPEC 1711-4 are required for efficient adhesion, invasion, and early but not late IL-8 production in intestinal epithelial cells in vitro. PMID:19620340

  14. Locus of enterocyte effacement: a pathogenicity island involved in the virulence of enteropathogenic and enterohemorragic Escherichia coli subjected to a complex network of gene regulation.

    PubMed

    Franzin, Fernanda M; Sircili, Marcelo P

    2015-01-01

    The locus of enterocyte effacement (LEE) is a 35.6 kb pathogenicity island inserted in the genome of some bacteria such as enteropathogenic Escherichia coli, enterohemorrhagic E.coli, Citrobacter rodentium, and Escherichia albertii. LEE comprises the genes responsible for causing attaching and effacing lesions, a characteristic lesion that involves intimate adherence of bacteria to enterocytes, a signaling cascade leading to brush border and microvilli destruction, and loss of ions, causing severe diarrhea. It is composed of 41 open reading frames and five major operons encoding a type three system apparatus, secreted proteins, an adhesin, called intimin, and its receptor called translocated intimin receptor (Tir). LEE is subjected to various levels of regulation, including transcriptional and posttranscriptional regulators located both inside and outside of the pathogenicity island. Several molecules were described being related to feedback inhibition, transcriptional activation, and transcriptional repression. These molecules are involved in a complex network of regulation, including mechanisms such as quorum sensing and temporal control of LEE genes transcription and translation. In this mini review we have detailed the complex network that regulates transcription and expression of genes involved in this kind of lesion. PMID:25710006

  15. Phenotypic and genetic features of enteropathogenic Escherichia coli isolates from diarrheal children in the Ribeirão Preto metropolitan area, São Paulo State, Brazil.

    PubMed

    Pitondo-Silva, André; Nakazato, Gerson; Falcão, Juliana P; Irino, Kinue; Martinez, Roberto; Darini, Ana Lúcia C; Hernandes, Rodrigo Tavanelli

    2015-02-01

    This study was designed to characterize a collection of 60 enteropathogenic Escherichia coli (EPEC) isolates from diarrheic feces of patients in the Ribeirão Preto metropolitan area regarding different phenotypic and molecular features. We examined antibiotic resistance profiles, occurrence of virulence factors-encoding genes, intimin subtypes and the correlation of serotypes among typical (tEPEC) and atypical (aEPEC) EPEC isolates. The results demonstrated that atypical EPEC was more heterogeneous than typical EPEC concerning the characteristics investigated and 45.2% do not belong to classical EPEC serogroups. Intimin subtype β was the most frequent among the EPEC isolates (46.7%), being detected in both tEPEC and aEPEC. The majority of aEPEC isolates presented localized adherence-like (LAL) pattern to HEp-2 cells, although aEPEC isolates displaying diffuse adherence (DA) or non-adherent were also detected. High prevalence of antimicrobial resistance was found for ampicillin, cephalothin, sulfonamide and tetracycline. In general, tEPEC isolates were more resistant to the antimicrobials tested than aEPEC isolates. PMID:25257819

  16. Invasion of differentiated intestinal Caco-2 cells is a sporadic property among atypical enteropathogenic Escherichia coli strains carrying common intimin subtypes.

    PubMed

    Pacheco, Veronica C R; Yamamoto, Denise; Abe, Cecilia M; Hernandes, Rodrigo T; Mora, Azucena; Blanco, Jorge; Gomes, Tânia A T

    2014-03-01

    Atypical enteropathogenic Escherichia coli (aEPEC) strains produce attaching-effacing (AE) lesions on enterocytes due to the interaction of the adhesin intimin with its translocated receptor. aEPEC strain 1551-2 was previously shown to invade HeLa and T84 cells by means of the uncommon intimin subtype omicron. Other aEPEC strains carrying uncommon intimin subtypes have also been shown to invade differentiated T84 intestinal cells. In this study, seven aEPEC strains carrying the most common EPEC intimin subtypes (alpha, beta, and gamma) were evaluated regarding the ability to invade differentiated intestinal Caco-2 cells. Although all strains adhered to and promoted AE lesions, the numbers of cell-associated bacteria varied significantly between the different strains regardless of the intimin subtype (P < 0.05). Gentamicin protection assay and transmission electron microscopy analyses showed that in comparison with the invasive strain 1551-2, only one strain (aEPEC EC423/03, intimin beta) was invasive (P = 0.05). Although both strains persisted intracellularly until 48 h, the number of viable bacteria of EC423/03 decreased, whereas that of 1551-2 increased significantly up to 24 h and then decreased. In conclusion, invasiveness is a sporadic property among aEPEC strains carrying some common intimin subtypes. PMID:24339197

  17. Late establishment of the attaching and effacing lesion caused by atypical enteropathogenic Escherichia coli depends on protein expression regulated by Per.

    PubMed

    Bueris, Vanessa; Huerta-Cantillo, Jazmín; Navarro-Garcia, Fernando; Ruiz, Renato M; Cianciarullo, Aurora M; Elias, Waldir P

    2015-01-01

    Enteropathogenic Escherichia coli (EPEC) is classified as typical (tEPEC) or atypical (aEPEC) based on the presence or absence of the E. coli adherence factor plasmid (pEAF), respectively. The hallmark of EPEC infection is the formation of the attaching and effacing (A/E) lesions on the gut mucosa. We compared the kinetics of A/E lesion formation induced by aEPEC and tEPEC. The examination of infected HEp-2 cells clearly demonstrated delayed A/E lesion formation by aEPEC in comparison to tEPEC. This delay was associated with the expression of locus of enterocyte effacement (LEE)-encoded virulence factors (i.e., intimin and EspD). Indeed, the insertion of a plasmid containing perABC, a transcriptional regulator of virulence factors involved in A/E formation, into aEPEC strains increased and accelerated the formation of A/E lesions. Interestingly, the enhanced expression and translocation of LEE-encoded proteins, such as those expressed in LEE5 (intimin) and LEE4 (EspD), in aEPEC (perABC) was independent of bacterial adhesion. The secretion kinetics of these two proteins representing LEE5 and LEE4 expression correlated with A/E lesion formation. We conclude that the lack of Per in the regulation network of virulence genes is one of the main factors that delay the establishment of A/E lesions induced by aEPEC strains. PMID:25385791

  18. Analysis of the Virulence of an Atypical Enteropathogenic Escherichia coli Strain In Vitro and In Vivo and the Influence of Type Three Secretion System

    PubMed Central

    Sampaio, Suely C. F.; Moreira, Fabiana C.; Liberatore, Ana M. A.; Vieira, Mônica A. M.; Knobl, Terezinha; Romão, Fabiano T.; Hernandes, Rodrigo T.; Ferreira, Claudete S. A.; Ferreira, Antônio P.; Felipe-Silva, Aloísio; Sinigaglia-Coimbra, Rita; Koh, Ivan H. J.; Gomes, Tania A. T.

    2014-01-01

    Atypical enteropathogenic Escherichia coli (aEPEC) inject various effectors into intestinal cells through a type three secretion system (T3SS), causing attaching and effacing (A/E) lesions. We investigated the role of T3SS in the ability of the aEPEC 1711-4 strain to interact with enterocytes in vitro (Caco-2 cells) and in vivo (rabbit ileal loops) and to translocate the rat intestinal mucosa in vivo. A T3SS isogenic mutant strain was constructed, which showed marked reduction in the ability to associate and invade but not to persist inside Caco-2 cells. After rabbit infection, only aEPEC 1711-4 was detected inside enterocytes at 8 and 24 hours pointing to a T3SS-dependent invasive potential in vivo. In contrast to aEPEC 1711-4, the T3SS-deficient strain no longer produced A/E lesions or induced macrophage infiltration. We also demonstrated that the ability of aEPEC 1711-4 to translocate through mesenteric lymph nodes to spleen and liver in a rat model depends on a functional T3SS, since a decreased number of T3SS mutant bacteria were recovered from extraintestinal sites. These findings indicate that the full virulence potential of aEPEC 1711-4 depends on a functional T3SS, which contributes to efficient adhesion/invasion in vitro and in vivo and to bacterial translocation to extraintestinal sites. PMID:24877131

  19. [Isolation of enteropathogenic Escherichia coli O157:H16 identified in a diarrhea case in a child and his household contacts in La Pampa Province, Argentina].

    PubMed

    Silveyra, Ivana M; Pereyra, Adriana M; Alvarez, María G; Villagran, Mariana D; Baroni, Andrea B; Deza, Natalia; Carbonari, Claudia C; Miliwebsky, Elizabeth; Rivas, Marta

    2015-01-01

    Enteropathogenic Escherichia coli (EPEC) is a major causative agent of acute diarrhea in children in developing countries. This pathotype is divided into typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence of the bfp virulence factor associated with adhesion, encoded in the pEAF plasmid. In the present study, the isolation of aEPEC O157:H16 from a bloody diarrhea case in a child and his household contacts (mother, father and sister) is described. The strain was characterized as E. coli O157:H16 eae-ɛ-positive, sorbitol fermenter with β-glucuronidase activity, susceptible to all antimicrobials tested, and negative for virulence factors stx1, stx2, ehxA and bfp. XbaI-PFGE performed on all isolates showed the AREXHX01.1040 macrorestriction pattern, with 100% similarity. These results highlight the importance of epidemiological surveillance of E. coli O157-associated diarrhea cases identified in children and their family contacts, as well as the incorporation of molecular techniques that allow the detection of the different E. coli pathotypes. PMID:26627113

  20. A host-specific virulence protein of Erwinia herbicola pv. gypsophilae is translocated into human epithelial cells by the Type III secretion system of enteropathogenic Escherichia coli.

    PubMed

    Valinsky, Lea; Nisan, Israel; Tu, Xuanlin; Nisan, Gal; Rosenshine, Ilan; Hanski, Emanuel; Barash, Isaac; Manulis, Shulamit

    2002-03-01

    summary HsvG is a virulence factor that determines the host specificity of Erwinia herbicola pathovars gypsophilae and betae on gypsophila. We used the calmodulin adenylate cyclase reporter (CyaA) to demonstrate that HsvG is secreted and translocated into HeLa cells by the type III secretion system (TTSS) of the enteropathogenic Escherichia coli (EPEC). A fusion of HsvG-CyaA containing 271 amino acids of the N-terminus of HsvG were introduced into a wild-type EPEC, espB mutant deficient in translocation and an escV mutant deficient in secretion. A significant secretion was detected in EPEC/HsvG-CyaA and its espB mutant, but not with the escV mutant. Translocation was only observed with the wild-type EPEC, and not with the other two mutants. To localize the secretion and translocation signals of HsvG, fusions containing 39, 11 and 3 amino acids of the N-terminus of HsvG were constructed and expressed in EPEC. A fusion containing the first 39 N-terminal amino acids of HsvG was secreted and translocated at significant level (31-35%) as compared to the original fusion. In contrast, fusions containing the 3 and 11 amino acids failed to be secreted and translocated. PMID:20569314

  1. Diarrheagenic Escherichia coli pathotypes investigation revealed atypical enteropathogenic E. coli as putative emerging diarrheal agents in children living in Botucatu, São Paulo State, Brazil.

    PubMed

    Dias, Regiane C B; Dos Santos, Bruna C; Dos Santos, Luis F; Vieira, Melissa A; Yamatogi, Ricardo S; Mondelli, Alessandro L; Sadatsune, Terue; Sforcin, José M; Gomes, Tânia A T; Hernandes, Rodrigo T

    2016-04-01

    The aim of the present study was to investigate the prevalence of Diarrheagenic Escherichia coli (DEC) pathotypes, a leading cause of diarrhea worldwide, among diarrheal and healthy children, up to 5 years of age, living in the city of Botucatu, São Paulo, Brazil. DEC, investigated by PCR detection of virulence factor-encoding genes associated with the distinct pathotypes, was isolated from 18.0% of the patients, and 19.0% of the controls, with enteroaggregative E. coli (EAEC), the most frequent pathotype, being detected in equal proportion between patients and controls (10.0%). Among the enteropathogenic E. coli (EPEC) isolates, only one isolate was able to produce the localized adherence pattern to HeLa cells, being thus the only typical EPEC identified. All the remaining EPEC were classified as atypical (aEPEC), and detected in 8.0% and 8.5% of the patients and controls, respectively. Regarding the serotypes, 26.5% of the analyzed EPEC isolates belonged to classical EPEC-serogroups, and the only two STEC found were serotyped as O26:H11 (patient) and O119:H7 (control). Antimicrobial susceptibility tests revealed that 43.6%, 29.5% and 2.6% of the DEC isolates were resistant to ampicillin, cotrimoxazole and gentamicin, respectively. Our data indicate that EAEC remains prevalent among children living in Botucatu, and revealed atypical EPEC as emerging putative diarrheal agents in this geographical region. PMID:26752102

  2. Monoclonal antibodies against all known variants of EspA: development of a simple diagnostic test for enteropathogenic Escherichia coli based on a key virulence factor

    PubMed Central

    Reissbrodt, Rolf; Kresse, Andreas; Pavankumar, Asalapuram; Sankaran, Krishnan; James, Roger; Jesudason, Mary; Anandan, Shalini; Prakasam, Agila; Balaji, Veeraraghavan; Dutta, Shanta; Dutta, Sanjucta; Ramamurthy, Thandavarayan; Fischer, Renate; Sander, Peter; Schaumann, Reiner; Navarro, Armando; Williams, Peter

    2014-01-01

    Enteropathogenic Escherichia coli (EPEC) are a major cause of infant diarrhoea in developing countries and a significant public health issue in industrialized countries. Currently there are no simple tests available for the diagnosis of EPEC. Serology of O-antigens is widely used routinely in many laboratories throughout the world, even though it has been known for many years to be an unreliable indicator of EPEC virulence. We have developed a simple, low-cost immunodiagnostic test based on the EspA filament, an essential virulence factor of EPEC and the related enterohaemorrhagic E. coli (EHEC). Using recombinant proteins of the five major variants of EspA as immunogens, we raised a panel of three monoclonal antibodies in mice that detects all variants of the native target in bacterial cultures. The antibodies proved suitable for application in sandwich-type assays, including ELISA and lateral flow immunoassays (LFI). Prototypes for both assays were specific for EPEC and EHEC strains when tested against a panel of control micro-organisms. We have also developed a simple, affordable culture medium, A/E medium, which optimizes expression of EspA allowing improved sensitivity of detection compared with standard Dulbecco’s modified Eagle’s medium. Together these reagents form the basis of robust, informative tests for EPEC for use especially in developing countries but also for routine screening in any clinical laboratory. PMID:25231626

  3. A Localized Adherence-Like Pattern as a Second Pattern of Adherence of Classic Enteropathogenic Escherichia coli to HEp-2 Cells That Is Associated with Infantile Diarrhea

    PubMed Central

    Scaletsky, Isabel C. A.; Pedroso, Margareth Z.; Oliva, Carlos A. G.; Carvalho, Rozane L. B.; Morais, Mauro B.; Fagundes-Neto, Ulysses

    1999-01-01

    Escherichia coli strains that cause nonbloody diarrhea in infants are known to present three distinct patterns of adherence to epithelial cells, namely, localized (LA), diffuse (DA), and aggregative (AA) adherence. Strains with LA (typical Enteropathogenic Escherichia coli [EPEC]) are well recognized as a cause of secretory diarrhea, but the role of strains with DA (DAEC) is controversial, and strains with AA (EAEC) have been more frequently related to persistent diarrhea whereas its relationship with acute diarrhea is not well defined. To determine the relationship of the different types of E. coli adherence patterns with acute diarrhea (lasting less than 14 days) and persistent diarrhea (lasting more than 14 days) in São Paulo, Brazil, we studied stool specimens from 40 infants under 1 year of age with diarrhea and 40 age-matched control infants without any gastrointestinal symptoms. Twenty-eight (35.0%) of eighty cases yielded adherent E. coli (HEp-2 cells). Strains with localized and aggregative adherence were associated with acute and persistent diarrhea. A total of 11.2% of the adherent strains were typical EPEC serotypes and hybridized with the enteroadherence factor probe; 5.0% were EAEC and hybridized with the EAEC probe. DAEC strains were isolated from 10.0% of patients and 7.5% of controls and did not hybridize with the two probes used (daaC and AIDA-I). Strains with a localized adherence-like pattern (atypical EPEC) were found significantly more frequently (P = 0.028) in cultures from children with diarrhea (17.5%) than in controls (2.5%). PMID:10377120

  4. Genome sequences and phylogenetic analysis of K88- and F18-positive porcine enterotoxigenic Escherichia coli.

    PubMed

    Shepard, Sara M; Danzeisen, Jessica L; Isaacson, Richard E; Seemann, Torsten; Achtman, Mark; Johnson, Timothy J

    2012-01-01

    Porcine enterotoxigenic Escherichia coli (ETEC) continues to result in major morbidity and mortality in the swine industry via postweaning diarrhea. The key virulence factors of ETEC strains, their serotypes, and their fimbrial components have been well studied. However, most studies to date have focused on plasmid-encoded traits related to colonization and toxin production, and the chromosomal backgrounds of these strains have been largely understudied. Here, we generated the genomic sequences of K88-positive and F18-positive porcine ETEC strains and examined the phylogenetic distribution of clinical porcine ETEC strains and their plasmid-associated genetic content. The genomes of porcine ETEC strains UMNK88 and UMNF18 were both found to contain remarkable plasmid complements containing known virulence factors, potential novel virulence factors, and antimicrobial resistance-associated elements. The chromosomes of these strains also possessed several unique genomic islands containing hypothetical genes with similarity to classical virulence factors, although phage-associated genomic islands dominated the accessory genomes of these strains. Phylogenetic analysis of 78 clinical isolates associated with neonatal and porcine diarrhea revealed that a limited subset of porcine ETEC lineages exist that generally contain common toxin and fimbrial profiles, with many of the isolates belonging to the ST10, ST23, and ST169 multilocus sequencing types. These lineages were generally distinct from existing human ETEC database isolates. Overall, most porcine ETEC strains appear to have emerged from a limited subset of E. coli lineages that either have an increased propensity to carry plasmid-encoded virulence factors or have the appropriate ETEC core genome required for virulence. PMID:22081385

  5. Occurrence of SHV, TEM and CTX-M β-Lactamase Genes Among Enteropathogenic Escherichia coli Strains Isolated From Children With Diarrhea

    PubMed Central

    Memariani, Mojtaba; Najar Peerayeh, Shahin; Zahraei Salehi, Taghi; Shokouhi Mostafavi, Seyyed Khalil

    2015-01-01

    Background: Antibiotic resistance is widespread among diarrheagenic Escherichia coli in developing countries, where the overuse of antibiotics is common. Information regarding β-lactamases, especially Extended-Spectrum β-Lactamases (ESBLs) in diarrheagenic pathogens should be considered in clinical management when an optimal treatment is needed. Objectives: The main objective of this study was to investigate the prevalence of blaCTX-M, blaSHV and blaTEM β-lactamase genes among enteropathogenic E. coli (EPEC) isolates in Tehran, Iran. Materials and Methods: Stool specimens were collected from children with diarrhea during a 17-month period from 2011 to 2013. Routine biochemical tests were performed for identification of E. coli isolates. The isolates were further examined by PCR for the presence of eae, stx1, stx2 and bfp genes. EPEC isolates have been screened for different β-lactamase genes. Genotyping EPEC isolates harboring blaCTX-M15 gene was performed through Multi-Locus VNTR Analysis (MLVA). Results: Of 42 EPEC, eight isolates carried the blaCTX-M1. None of the isolates carried blaCTX-M2 and blaCTX-M9. The blaCTX-M15 variant was identified in all of blaCTX-M1-positive isolates. Furthermore, blaSHV and blaTEM genes were detected in 40.5% (n = 17) and 19% (n = 8) of all EPEC isolates, respectively. No significant association was observed between the existence of bfp gene and presence of those β-lactamase genes (P > 0.05). MLVA analysis revealed high genetic diversity among blaCTX-M15-positive isolates. Conclusions: Our study emphasized the increasing role of ESBL genes, especially blaCTX-M15 in EPEC isolates. PMID:26034531

  6. Secretion of the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase by the LEE-encoded type III secretion system in enteropathogenic Escherichia coli.

    PubMed

    Aguilera, Laura; Ferreira, Elaine; Giménez, Rosa; Fernández, Francisco José; Taulés, Marta; Aguilar, Juan; Vega, M Cristina; Badia, Josefa; Baldomà, Laura

    2012-06-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein secreted by pathogens and involved in adhesion and/or virulence. Previously we reported that enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli secrete GAPDH into the culture medium. This bacterial protein binds human plasminogen and fibrinogen and remains associated with Caco-2 cells upon infection. In these pathogens, GAPDH secretion is not linked to outer membrane vesicles and depends on growth conditions, although the secretion mechanism is still unknown. EPEC is an attaching and effacing pathogen able to secrete and translocate multiple effector proteins into infected cells through a type III secretion system (T3SS). The secretion process is often dependent on a bacterial chaperone. The chaperone CesT displays broad substrate specificity and plays a central role in the recruitment of multiple type III effectors to the T3SS apparatus. Here we provide genetic evidences on GAPDH secretion through T3SS by EPEC grown in DMEM. Secretion of GAPDH is increased in ΔsepD mutants and abolished in mutants defective in the type III ATPase EscN. Complementation with escN gene restores GAPDH secretion. In addition, we prove by means of pull down experiments, overlay immunoblotting and biolayer interferometry a novel interaction between GAPDH and the chaperone CesT. This interaction, which is strong and slow dissociating, may stabilize a population of GAPDH molecules in a secretion competent-state and target them to the type III secretion apparatus. This is the first description of CesT interaction with a housekeeping protein and its export through T3SS. PMID:22433988

  7. Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenic Escherichia coli Tir effector.

    PubMed

    Nieto-Pelegrin, Elvira; Kenny, Brendan; Martinez-Quiles, Narcisa

    2014-01-01

    Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area. PMID:25482634

  8. Prevalence and behavior of multidrug-resistant shiga toxin-producing Escherichia coli, enteropathogenic E. coli and enterotoxigenic E. coli on coriander.

    PubMed

    Gómez-Aldapa, Carlos A; Segovia-Cruz, Jesús A; Cerna-Cortes, Jorge F; Rangel-Vargas, Esmeralda; Salas-Rangel, Laura P; Gutiérrez-Alcántara, Eduardo J; Castro-Rosas, Javier

    2016-10-01

    The prevalence and behavior of multidrug-resistant diarrheagenic Escherichia coli pathotypes on coriander was determined. One hundred coriander samples were collected from markets. Generic E. coli were determined using the most probable number procedure. Diarrheagenic E. coli pathotypes (DEPs) were identified using two multiplex polymerase chain reaction procedures. Susceptibility to sixteen antibiotics was tested for the isolated DEPs strains by standard test. The behavior of multidrug-resistant DEPs isolated from coriander was determined on coriander leaves and chopped coriander at 25°± 2 °C and 3°± 2 °C. Generic E. coli and DEPs were identified, respectively, in 43 and 7% of samples. Nine DEPs strains were isolated from positive coriander samples. The identified DEPs included Shiga toxin-producing E. coli (STEC, 4%) enterotoxigenic E. coli (ETEC, 2%) and enteropathogenic E. coli (EPEC, 1%). All isolated DEPs strains exhibited multi-resistance to antibiotics. On inoculated coriander leaves stored at 25°± 2 °C or 3°± 2 °C, no growth was observed for multidrug-resistant DEPs strains. However, multidrug-resistant DEPs strains grew in chopped coriander: after 24 h at 25° ± 2 °C, DEPs strains had grown to approximately 3 log CFU/g. However, at 3°± 2 °C the bacterial growth was inhibited. To the best of our knowledge, this is the first report of the presence and behavior of multidrug-resistant STEC, ETEC and EPEC on coriander and chopped coriander. PMID:27375249

  9. Characterization of two virulence proteins secreted by rabbit enteropathogenic Escherichia coli, EspA and EspB, whose maximal expression is sensitive to host body temperature.

    PubMed Central

    Abe, A; Kenny, B; Stein, M; Finlay, B B

    1997-01-01

    Enteropathogenic Escherichia coli (EPEC) and rabbit EPEC (RDEC-1) cause unique histopathological features on intestinal mucosa, including attaching/effacing (A/E) lesions. Due to the human specificity of EPEC, RDEC-1 has been used as an animal model to study EPEC pathogenesis. At least two of the previously identified EPEC-secreted proteins, EspA and EspB, are required for triggering host epithelial signal transduction pathways, intimate adherence, and A/E lesions. However, the functions of these secreted proteins and their roles in pathogenesis have not been characterized. To investigate the function of EspA and EspB in RDEC-1, the espA and espB genes were cloned and their sequences were compared to that of EPEC O127. The EspA proteins showed high similarity (88.5% identity), while EspB was heterogeneous in internal regions (69.8% identity). However, RDEC-1 EspB was identical to that of enterohemorrhagic E. coli serotype O26. Mutations in RDEC-1 espA and espB revealed that the corresponding RDEC-1 gene products are essential for triggering of host signal transduction pathways and invasion into HeLa cells. Complementation with plasmids containing EPEC espA or/and espB genes into RDEC-1 mutant strains demonstrated that they were functionally interchangeable, although the EPEC proteins mediated higher levels of invasion. Furthermore, maximal expression of RDEC-1 and EPEC-secreted proteins occurred at their respective host body temperatures, which may contribute to the lack of EPEC infectivity in rabbits. PMID:9284118

  10. Epidemiology, Antimicrobial Resistance and β-lactamase Genotypic Features of Enteropathogenic Escherichia coli Isolated from Children with Diarrhea in Southern China.

    PubMed

    Huang, Yong; Shan, Xue-feng; Deng, Haijun; Huang, Yu-jun; Mu, Xiao-ping; Huang, Ai-long; Long, Quan-xin

    2015-01-01

    The main objective of this study was to investigate the epidemiology, drug resistance and β-lactamase genotype distribution of enteropathogenic Escherichia coli (EPEC) isolated from pediatric patients with diarrhea in southern China. The prevalence of EPEC in children with diarrhea was 3.53%. The commonest serotypes were O55:K59 and O126:K71, and the typical EPEC were more prevalent than atypical EPEC (51 vs 7). Isolates from this region were most commonly found to be resistant to ampicillin and cotrimoxazole, followed by chloramphenicol, ceftriaxone, and ceftazidime. More than 96% of the strains were susceptible to cefoperazone/sulbactam and imipenem. The most common β-lactamase genotypes identified in 58 strains were blaCTX-M-1 (60.3%), blaTEM (56.9%), blaCTX-M-9 (27.6%), and blaSHV (15.5%). Among 58 isolates, 22 strains were found to harbor one β-lactamase gene, and the proportions of resistance to ampicillin, cotrimoxazole, chloramphenicol, ceftriaxone, and ceftazidime, were 81.8%, 63.6%, 40.9%, 18.2%, and 9.1%, respectively. A further 30 strains carrying multiple β-lactamase genes had increased resistance to the above antimicrobial agents (100%, 83.3%, 70.0%, 60.0%, and 30.0%, respectively). In contrast, antibiotic resistance in the last 6 strains without a detectable β-lactamase gene was substantially reduced. Drug resistance may be associated with the β-lactamase gene number, with a greater the number of β-lactamase genes resulting in higher antibiotic resistance. PMID:25672408

  11. RNA-Seq analysis of isolate- and growth phase-specific differences in the global transcriptomes of enteropathogenic Escherichia coli prototype isolates

    PubMed Central

    Hazen, Tracy H.; Daugherty, Sean C.; Shetty, Amol; Mahurkar, Anup A.; White, Owen; Kaper, James B.; Rasko, David A.

    2015-01-01

    Enteropathogenic Escherichia coli (EPEC) are a leading cause of diarrheal illness among infants in developing countries. E. coli isolates classified as typical EPEC are identified by the presence of the locus of enterocyte effacement (LEE) and the bundle-forming pilus (BFP), and absence of the Shiga-toxin genes, while the atypical EPEC also encode LEE but do not encode BFP or Shiga-toxin. Comparative genomic analyses have demonstrated that EPEC isolates belong to diverse evolutionary lineages and possess lineage- and isolate-specific genomic content. To investigate whether this genomic diversity results in significant differences in global gene expression, we used an RNA sequencing (RNA-Seq) approach to characterize the global transcriptomes of the prototype typical EPEC isolates E2348/69, B171, C581-05, and the prototype atypical EPEC isolate E110019. The global transcriptomes were characterized during laboratory growth in two different media and three different growth phases, as well as during adherence of the EPEC isolates to human cells using in vitro tissue culture assays. Comparison of the global transcriptomes during these conditions was used to identify isolate- and growth phase-specific differences in EPEC gene expression. These analyses resulted in the identification of genes that encode proteins involved in survival and metabolism that were coordinately expressed with virulence factors. These findings demonstrate there are isolate- and growth phase-specific differences in the global transcriptomes of EPEC prototype isolates, and highlight the utility of comparative transcriptomics for identifying additional factors that are directly or indirectly involved in EPEC pathogenesis. PMID:26124752

  12. EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    PubMed Central

    Serapio-Palacios, Antonio

    2016-01-01

    ABSTRACT Enteropathogenic Escherichia coli (EPEC) has the ability to antagonize host apoptosis during infection through promotion and inhibition of effectors injected by the type III secretion system (T3SS), but the total number of these effectors and the overall functional relationships between these effectors during infection are poorly understood. EspC produced by EPEC cleaves fodrin, paxillin, and focal adhesion kinase (FAK), which are also cleaved by caspases and calpains during apoptosis. Here we show the role of EspC in cell death induced by EPEC. EspC is involved in EPEC-mediated cell death and induces both apoptosis and necrosis in epithelial cells. EspC induces apoptosis through the mitochondrial apoptotic pathway by provoking (i) a decrease in the expression levels of antiapoptotic protein Bcl-2, (ii) translocation of the proapoptotic protein Bax from cytosol to mitochondria, (iii) cytochrome c release from mitochondria to the cytoplasm, (iv) loss of mitochondrial membrane potential, (v) caspase-9 activation, (vi) cleavage of procaspase-3 and (vii) an increase in caspase-3 activity, (viii) PARP proteolysis, and (ix) nuclear fragmentation and an increase in the sub-G1 population. Interestingly, EspC-induced apoptosis was triggered through a dual mechanism involving both independent and dependent functions of its EspC serine protease motif, the direct cleavage of procaspase-3 being dependent on this motif. This is the first report showing a shortcut for induction of apoptosis by the catalytic activity of an EPEC protein. Furthermore, this atypical intrinsic apoptosis appeared to induce necrosis through the activation of calpain and through the increase of intracellular calcium induced by EspC. Our data indicate that EspC plays a relevant role in cell death induced by EPEC. PMID:27329750

  13. The Serine Protease EspC from Enteropathogenic Escherichia coli Regulates Pore Formation and Cytotoxicity Mediated by the Type III Secretion System

    PubMed Central

    Guignot, Julie; Segura, Audrey; Tran Van Nhieu, Guy

    2015-01-01

    Type III secretion systems (T3SSs) are specialized macromolecular machines critical for bacterial virulence, and allowing the injection of bacterial effectors into host cells. The T3SS-dependent injection process requires the prior insertion of a protein complex, the translocon, into host cell membranes consisting of two-T3SS hydrophobic proteins, associated with pore-forming activity. In all described T3SS to date, a hydrophilic protein connects one hydrophobic component to the T3SS needle, presumably insuring the continuum between the hollow needle and the translocon. In the case of Enteropathogenic Escherichia coli (EPEC), the hydrophilic component EspA polymerizes into a filament connecting the T3SS needle to the translocon composed of the EspB and EspD hydrophobic proteins. Here, we identify EspA and EspD as targets of EspC, a serine protease autotransporter of Enterobacteriaceae (SPATE). We found that in vitro, EspC preferentially targets EspA associated with EspD, but was less efficient at proteolyzing EspA alone. Consistently, we found that EspC did not regulate EspA filaments at the surface of primed bacteria that was devoid of EspD, but controlled the levels of EspD and EspA secreted in vitro or upon cell contact. While still proficient for T3SS-mediated injection of bacterial effectors and cytoskeletal reorganization, an espC mutant showed increased levels of cell-associated EspA and EspD, as well as increased pore formation activity associated with cytotoxicity. EspP from enterohaemorrhagic E. coli (EHEC) also targeted translocator components and its activity was interchangeable with that of EspC, suggesting a common and important function of these SPATEs. These findings reveal a novel regulatory mechanism of T3SS-mediated pore formation and cytotoxicity control during EPEC/EHEC infection. PMID:26132339

  14. Bacterial aetiology of diarrhoea in young children: high prevalence of enteropathogenic Escherichia coli (EPEC) not belonging to the classical EPEC serogroups.

    PubMed

    Prère, M F; Bacrie, S Cohen; Baron, O; Fayet, O

    2006-12-01

    Diarrhoeal disease continues to be one of the most common causes of admittance in Children hospital emergency. The aim of the present study was to investigate the relative contribution of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) as a cause of infectious bacterial diarrhoea in children from the region of Toulouse. We analysed 280 samples of stools from 280 children (<2 years) with diarrhoea admitted in the "Hopital des Enfants" from January to August 2005. Classic pathogens (Salmonella, Campylobacter, Yersinia, Shigella, Aeromonas and Vibrio) were detected by standard culture methods. Enterotoxigenic Clostridium difficile were identified after culture by immuno-enzyme assay (IEA). Virulence genes of EPEC and EHEC were detected by using PCR. Shiga-toxin production of EHEC strains was confirmed with an IEA test. Potential enteric pathogens were identified in 55 patients. EPEC was the most frequently identified agent (30 patients), followed by Campylobacter (9 cases: 7 C. jejuni and 2 C. coli) and C. difficile (8 patients), then EHEC (5 patients) and Salmonella (3 patients). No Shigella, Yersinia, Aeromonas or other pathogenic bacteria were detected during this period in that class of children. EPEC not belonging to the classical EPEC serogroups were highly prevalent (24 versus 6). EHEC possessed different genotypes and serogroups: O26 (2 strains), O157 (2 strains) and one un-typable strain. This study demonstrates the importance of EPEC (55 % of positive cases) and of EHEC (more frequent than Salmonella) in the aetiology of diarrhoeal diseases of young children. We confirm the usefulness of the PCR methodology: it allows the detection of virulent E. coli and thus increases by two fold the diagnosis of bacterial diarrhoea. PMID:17030460

  15. The enteropathogenic Escherichia coli (EPEC) Map effector is imported into the mitochondrial matrix by the TOM/Hsp70 system and alters organelle morphology.

    PubMed

    Papatheodorou, Panagiotis; Domańska, Grazyna; Oxle, Marius; Mathieu, Johannes; Selchow, Olaf; Kenny, Brendan; Rassow, Joachim

    2006-04-01

    Enteropathogenic Escherichia coli (EPEC) is a human intestinal pathogen and a major cause of diarrhoea, particularly among infants in developing countries. EPEC target the Map and EspF multifunctional effector proteins to host mitochondria - organelles that play crucial roles in regulating cellular processes such as programmed cell death (apoptosis). While both molecules interfere with the organelles ability to maintain a membrane potential, EspF plays the predominant role and is responsible for triggering cell death. To learn more about the Map-mitochondria interaction, we studied Map localization to mitochondria with purified mitochondria (from mammalian and yeast cells) and within intact yeast. This revealed that (i) Map targeting is dependent on the predicted N-terminal mitochondrial targeting sequence, (ii) the N-terminal 44 residues are sufficient to target proteins to mitochondria and (iii) Map import involves the mitochondrial outer membrane translocase (Tom22 and Tom40), the mitochondrial membrane potential, and the matrix chaperone, mtHsp70. These results are consistent with Map import into the mitochondria matrix via the classical import mechanism. As all known, Map-associated phenotypes in mammalian cells are independent of mitochondrial targeting, this may indicate that import serves as a mechanism to remove Map from the cytoplasm thereby regulating cytoplasmic function. Intriguingly, Map, but not EspF, alters mitochondrial morphology with deletion analysis revealing important roles for residues 101-152. Changes in mitochondrial morphology have been linked to alterations in the ability of these organelles to regulate cellular processes providing a possible additional role for Map import into mitochondria. PMID:16548893

  16. Enteropathogenic Escherichia coli inhibits type I interferon- and RNase L-mediated host defense to disrupt intestinal epithelial cell barrier function.

    PubMed

    Long, Tiha M; Nisa, Shahista; Donnenberg, Michael S; Hassel, Bret A

    2014-07-01

    Enteropathogenic Escherichia coli (EPEC) primarily infects children in developing countries and causes diarrhea that can be deadly. EPEC pathogenesis occurs through type III secretion system (T3SS)-mediated injection of effectors into intestinal epithelial cells (IECs); these effectors alter actin dynamics, modulate the immune response, and disrupt tight junction (TJ) integrity. The resulting compromised barrier function and increased gastrointestinal (GI) permeability may be responsible for the clinical symptoms of infection. Type I interferon (IFN) mediates anti-inflammatory activities and serves essential functions in intestinal immunity and homeostasis; however, its role in the immune response to enteric pathogens, such as EPEC, and its impact on IEC barrier function have not been examined. Here, we report that IFN-β is induced following EPEC infection and regulates IEC TJ proteins to maintain barrier function. The EPEC T3SS effector NleD counteracts this protective activity by inhibiting IFN-β induction and enhancing tumor necrosis factor alpha to promote barrier disruption. The endoribonuclease RNase L is a key mediator of IFN induction and action that promotes TJ protein expression and IEC barrier integrity. EPEC infection inhibits RNase L in a T3SS-dependent manner, providing a mechanism by which EPEC evades IFN-induced antibacterial activities. This work identifies novel roles for IFN-β and RNase L in IEC barrier functions that are targeted by EPEC effectors to escape host defense mechanisms and promote virulence. The IFN-RNase L axis thus represents a potential therapeutic target for enteric infections and GI diseases involving compromised barrier function. PMID:24733098

  17. Detection and genetic analysis of the enteroaggregative Escherichia coli heat-stable enterotoxin (EAST1) gene in clinical isolates of enteropathogenic Escherichia coli (EPEC) strains

    PubMed Central

    2014-01-01

    Background The enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) encoded by astA gene has been found in enteropathogenic E. coli (EPEC) strains. However, it is not sufficient to simply probe strains with an astA gene probe due to the existence of astA mutants (type 1 and type 2 SHEAST) and EAST1 variants (EAST1 v1-4). In this study, 222 EPEC (70 typical and 152 atypical) isolates were tested for the presence of the astA gene sequence by PCR and sequencing. Results The astA gene was amplified from 54 strains, 11 typical and 43 atypical. Sequence analysis of the PCR products showed that 25 strains, 7 typical and 18 atypical, had an intact astA gene. A subgroup of 7 atypical strains had a variant type of the astA gene sequence, with four non-synonymous nucleotide substitutions. The remaining 22 strains had mutated astA gene with nucleotide deletions or substitutions in the first 8 codons. The RT-PCR results showed that the astA gene was transcribed only by the strains carrying either the intact or the variant type of the astA gene sequence. Southern blot analysis indicated that astA is located in EAF plasmid in typical strains, and in plasmids of similar size in atypical strains. Strains carrying intact astA genes were more frequently found in diarrheic children than in non-diarrheic children (p < 0.05). Conclusion In conclusion, our data suggest that the presence of an intact astA gene may represent an additional virulence determinant in both EPEC groups. PMID:24884767

  18. Development of a Multiplex PCR Assay for Detection of Shiga Toxin-Producing Escherichia coli, Enterohemorrhagic E. coli, and Enteropathogenic E. coli Strains

    PubMed Central

    Botkin, Douglas J.; Galli, Lucía; Sankarapani, Vinoth; Soler, Michael; Rivas, Marta; Torres, Alfredo G.

    2012-01-01

    Escherichia coli O157:H7 and other pathogenic E. coli strains are enteric pathogens associated with food safety threats and which remain a significant cause of morbidity and mortality worldwide. In the current study, we investigated whether enterohemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), and enteropathogenic E. coli (EPEC) strains can be rapidly and specifically differentiated with multiplex PCR (mPCR) utilizing selected biomarkers associated with each strain’s respective virulence genotype. Primers were designed to amplify multiple intimin (eae) and long polar fimbriae (lpfA) variants, the bundle-forming pilus gene bfpA, and the Shiga toxin-encoding genes stx1 and stx2. We demonstrated consistent amplification of genes specific to the prototype EHEC O157:H7 EDL933 (lpfA1-3, lpfA2-2, stx1, stx2, and eae-γ) and EPEC O127:H6 E2348/69 (eae-α, lpfA1-1, and bfpA) strains using the optimized mPCR protocol with purified genomic DNA (gDNA). A screen of gDNA from isolates in a diarrheagenic E. coli collection revealed that the mPCR assay was successful in predicting the correct pathotype of EPEC and EHEC clones grouped in the distinctive phylogenetic disease clusters EPEC1 and EHEC1, and was able to differentiate EHEC1 from EHEC2 clusters. The assay detection threshold was 2 × 104 CFU per PCR reaction for EHEC and EPEC. mPCR was also used to screen Argentinean clinical samples from hemolytic uremic syndrome and diarrheal patients, resulting in 91% sensitivity and 84% specificity when compared to established molecular diagnostic procedures. In conclusion, our mPCR methodology permitted differentiation of EPEC, STEC and EHEC strains from other pathogenic E. coli; therefore, the assay becomes an additional tool for rapid diagnosis of these organisms. PMID:22919600

  19. Inhibition of Adhesion of Enteropathogenic Escherichia coli to HEp-2 Cells by Binding of a Novel Peptide to EspB Protein.

    PubMed

    Li, Duoyun; Chen, Zhong; Cheng, Hang; Zheng, Jin-Xin; Pan, Wei-Guang; Yang, Wei-Zhi; Yu, Zhi-Jian; Deng, Qi-Wen

    2016-09-01

    Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea in developing countries. The translocator EspB is a key virulence factor in the process of the attaching and effacing effect of EPEC and plays a critical role in the pathogenesis of the bacteria. In this study, we aimed to select the peptides binding to EspB protein by phage display library and further investigate whether these peptides can decrease the extent of invasion and virulence of EPEC on host cells by targeting to EspB protein. The expression and purification of EspB protein from E. coli was demonstrated by Western blotting. The Ph.D. 12-mer peptide phage display library was used to screen the candidate peptides binding specifically to EspB protein. Furthermore, the affinity of these candidate peptides bound to EspB was identified by enzyme-linked immunosorbent assay (ELISA). Moreover, we investigated whether these screened peptides could decrease the adherence ratio of EPEC to HEp-2 cells with increasing concentration. Successful purification of EspB protein from pET21b-EspB-transformed E. coli was identified by Western blotting. Then, the candidate peptides including phages 6, 7, 8, and 12 were screened by the Ph.D. 12-mer peptide phage display library and ELISA test demonstrated that their affinity binding to EspB protein was high compared with the control. Functional analysis indicated that synthetic peptide-6 (YFPYSHTSPRQP) significantly decreased the adherence ratio of EPEC to HEp-2 cells with increasing concentration (P < 0.01). Peptide-6 (100 µg/mL) could lead to a 40 % decrease in the adherence ratio of EPEC to HEp-2 cells compared with control (P < 0.01). However, the other three peptides at different concentrations showed only a slight ability to block the adherence of EPEC to host cells. Our data provided a potential strategy to inhibit the adhesion of EPEC to epithelial cells by a candidate peptide targeted toward EspB protein. PMID:27246497

  20. Role of peripheral pooling in porcine Escherichia coli sepsis

    SciTech Connect

    Teule, G.J.; von Lingen, A.; Verwey von Vught, M.A.; Kester, A.D.; Mackaay, R.C.; Bezemer, P.D.; Heidenal, G.A.; Thijs, L.G.

    1984-01-01

    In anesthesized pigs the effects of E. coli (2 X 10(8)/kg) on hemodynamics and red cell distribution were studied. After injection of 99m-Tc red cells (15 mCi), regional radioactivity was followed during 3 hours. Gated bloodpool studies were performed to measure end-diastolic volumes (EDV). Escherichia coli E. coli was infused in 14 pigs, while 7 animals served as controls. E. coli resulted in an early increase in pulmonary arterial pressure. Systemic arterial pressure decreased gradually, while cardiac output did not change significantly. The gated studies revealed that especially left ventricular end-diastolic volume (LVEDV) declined, to 50% of the basal value. Regional radioactivity did not change over lungs, liver and abdomen. Splenic activity declined markedly. Over the hindlimb a significant increase (29 +/- 8%) was observed. It is concluded that E. coli infusion in pigs induces a hemodynamic pattern similar to human sepsis. The decrease in LVEDV is probably related to peripheral pooling and a change in right ventricle (RV) performance.

  1. Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane.

    PubMed

    Novakovic, Predrag; Huang, Yanyun Y; Lockerbie, Betty; Shahriar, Farshid; Kelly, John; Gordon, John R; Middleton, Dorothy M; Loewen, Matthew E; Kidney, Beverly A; Simko, Elemir

    2015-04-01

    F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization. PMID:25852227

  2. Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane

    PubMed Central

    Novakovic, Predrag; Huang, Yanyun Y.; Lockerbie, Betty; Shahriar, Farshid; Kelly, John; Gordon, John R.; Middleton, Dorothy M.; Loewen, Matthew E.; Kidney, Beverly A.; Simko, Elemir

    2015-01-01

    F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization. PMID:25852227

  3. Clay mineral type effect on bacterial enteropathogen survival in soil.

    PubMed

    Brennan, Fiona P; Moynihan, Emma; Griffiths, Bryan S; Hillier, Stephen; Owen, Jason; Pendlowski, Helen; Avery, Lisa M

    2014-01-15

    Enteropathogens released into the environment can represent a serious risk to public health. Soil clay content has long been known to have an important effect on enteropathogen survival in soil, generally enhancing survival. However, clay mineral composition in soils varies, and different clay minerals have specific physiochemical properties that would be expected to impact differentially on survival. This work investigated the effect of clay materials, with a predominance of a particular mineral type (montmorillonite, kaolinite, or illite), on the survival in soil microcosms over 96 days of Listeria monocytogenes, Salmonella Dublin, and Escherichia coli O157. Clay mineral addition was found to alter a number of physicochemical parameters in soil, including cation exchange capacity and surface area, and this was specific to the mineral type. Clay mineral addition enhanced enteropathogen survival in soil. The type of clay mineral was found to differentially affect enteropathogen survival and the effect was enteropathogen-specific. PMID:24035982

  4. BfpI, BfpJ, and BfpK Minor Pilins Are Important for the Function and Biogenesis of Bundle-Forming Pili Expressed by Enteropathogenic Escherichia coli

    PubMed Central

    Martinez de la Peña, Claudia F.; De Masi, Leon; Nisa, Shahista; Mulvey, George; Tong, Jesse; Donnenberg, Michael S.

    2015-01-01

    ABSTRACT Enteropathogenic Escherichia coli (EPEC) remains a significant cause of infant diarrheal illness and associated morbidity and mortality in developing countries. EPEC strains are characterized by their ability to colonize the small intestines of their hosts by a multistep program involving initial loose attachment to intestinal epithelial cells followed by an intimate adhesion phase. The initial loose interaction of typical EPEC with host intestinal cells is mediated by bundle-forming pili (BFP). BFP are type 4b pili (T4bP) based on structural and functional properties shared with T4bP expressed by other bacteria. The major structural subunit of BFP is called bundlin, a T4b pilin expressed from the bfpA gene in the BFP operon, which contains three additional genes that encode the pilin-like proteins BfpI, BfpJ, and BfpK. In this study, we show that, in the absence of the BFP retraction ATPase (BfpF), BfpI, BfpJ, and BfpK are dispensable for BFP biogenesis. We also demonstrate that these three minor pilins are incorporated along with bundlin into the BFP filament and contribute to its structural integrity and host cell adhesive properties. The results confirm that previous findings in T4aP systems can be extended to a model T4bP such as BFP. IMPORTANCE Bundle-forming pili contribute to the host colonization strategy of enteropathogenic Escherichia coli. The studies described here investigate the role for three minor pilin subunits in the structure and function of BFP in EPEC. The studies also suggest that these subunits could be antigens for vaccine development. PMID:26712935

  5. Efficient expression and purification of porcine circovirus type 2 virus-like particles in Escherichia coli.

    PubMed

    Wu, Pei-Ching; Chen, Tzu-Yu; Chi, Jiun-Ni; Chien, Maw-Sheng; Huang, Chienjin

    2016-02-20

    Porcine circovirus type 2 (PCV2) capsid (Cap) protein has been successfully used as a vaccine to control porcine circovirus associated disease (PCVAD). Most PCV2 subunit vaccines are recombinant Cap protein expressed in baculovirus/insect cell expression system, but using this eukaryotic system is laborious and expensive. In our previous study, full-length of PCV2Cap protein expressed in Escherichia coli formed virus-like particles (VLPs). This expression system has the advantages of being relatively simple and inexpensive. In this study, we constructed a recombinant plasmid containing the full-length codon-optimized cap (ORF2) gene to improve high-level expression of recombinant Cap protein (rCap) with no changed amino acids. The highly water-soluble rCap protein was purified by a single-column, high-throughput fractionation procedure based on size exclusion chromatography. Yield was 10mg per 200ml bacterial culture. The rCap protein self-assembled into VLPs of diameter 25-30nm that contained exogenous nucleic acids. The immunogenicity of PCV2 VLPs was analyzed by immunizing mice. VLP-immunized mice mounted specific immune responses to PCV2. Thus, expression of rCap in E. coli was feasible for large-scale production of PCV2 VLPs, which could potentially be used for a VLP-based PCV2 vaccine. PMID:26795354

  6. Porcine intestinal glycosphingolipids recognized by F6-fimbriated enterotoxigenic Escherichia coli.

    PubMed

    Madar Johansson, Miralda; Coddens, Annelies; Benktander, John; Cox, Eric; Teneberg, Susann

    2014-11-01

    One important virulence factor of enterotoxigenic Escherichia coli is their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. Here, the potential glycosphingolipid receptors of enterotoxigenic F6-fimbriated E. coli were examined by binding of purified F6 fimbriae, and F6-expressing bacteria, to glycosphingolipids on thin-layer chromatograms. When intestinal mucosal non-acid glycosphingolipids from single pigs were assayed for F6 binding capacity, a selective interaction with two glycosphingolipids was observed. The binding-active glycosphingolipids were isolated and characterized as lactotriaosylceramide (GlcNAcβ3Galβ4Glcβ1Cer) and lactotetraosylceramide (Galβ3GlcNAcβ3Galβ4Glcβ1Cer). Further binding assays using a panel of reference glycosphingolipids showed a specific interaction between the F6 fimbriae and a number of neolacto core chain (Galβ4GlcNAc) glycosphingolipids. In addition, an occasional binding of the F6 fimbriae to sulfatide, galactosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, isoglobotriaosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide was obtained. From the results we conclude that lactotriaosylceramide and lactotetraosylceramide are major porcine intestinal receptors for F6-fimbriated E. coli. PMID:25241919

  7. Differential gene expression profiling of porcine epithelial cells infected with three enterotoxigenic Escherichia coli strains

    PubMed Central

    2012-01-01

    Background Enterotoxigenic Escherichia coli (ETEC) is one of the most important pathogenic bacteria causing severe diarrhoea in human and pigs. In ETEC strains, the fimbrial types F4 and F18 are commonly found differently colonized within the small intestine and cause huge economic losses in the swine industry annually worldwide. To address the underlying mechanism, we performed a transcriptome study of porcine intestinal epithelial cells (IPEC-J2) with and without infection of three representative ETEC strains. Results A total 2443, 3493 and 867 differentially expressed genes were found in IPEC-J2 cells infected with F4ab ETEC (CF4ab), with F4ac ETEC (CF4ac) and with F18ac ETEC (CF18ac) compared to the cells without infection (control), respectively. The number of differentially expressed genes between CF4ab and CF4ac, CF4ab and CF18ac, and CF4ac and CF18ac were 77, 1446 and 1629, respectively. The gene ontology and pathway analysis showed that the differentially expressed genes in CF4abvs control are significantly involved in cell-cycle progress and amino acid metabolism, while the clustered terms of the differentially expressed genes in CF4acvs control comprise immune, inflammation and wounding response and apoptosis as well as cell cycle progress and proteolysis. Differentially expressed genes between CF18acvs control are mainly involved in cell-cycle progression and immune response. Furthermore, fundamental differences were observed in expression levels of immune-related genes among the three ETEC treatments, especially for the important pro-inflammatory molecules, including IL-6, IL-8, TNF-α, CCL20, CXCL2 etc. Conclusions The discovery in this study provides insights into the interaction of porcine intestinal epithelial cells with F4 ETECs and F18 ETEC, respectively. The genes induced by ETECs with F4 versus F18 fimbriae suggest why ETEC with F4 may be more virulent compared to F18 which seems to elicit milder effects. PMID:22823589

  8. Interleukin-8, CXCL1, and MicroRNA miR-146a Responses to Probiotic Escherichia coli Nissle 1917 and Enteropathogenic E. coli in Human Intestinal Epithelial T84 and Monocytic THP-1 Cells after Apical or Basolateral Infection.

    PubMed

    Sabharwal, Harshana; Cichon, Christoph; Ölschläger, Tobias A; Sonnenborn, Ulrich; Schmidt, M Alexander

    2016-09-01

    Bacterium-host interactions in the gut proceed via directly contacted epithelial cells, the host's immune system, and a plethora of bacterial factors. Here we characterized and compared exemplary cytokine and microRNA (miRNA) responses of human epithelial and THP-1 cells toward the prototype enteropathogenic Escherichia coli (EPEC) strain E2348/69 (O127:H6) and the probiotic strain Escherichia coli Nissle 1917 (EcN) (O6:K5:H1). Human T84 and THP-1 cells were used as cell culture-based model systems for epithelial and monocytic cells. Polarized T84 monolayers were infected apically or basolaterally. Bacterial challenges from the basolateral side resulted in more pronounced cytokine and miRNA responses than those observed for apical side infections. Interestingly, the probiotic EcN also caused a pronounced transcriptional increase of proinflammatory CXCL1 and interleukin-8 (IL-8) levels when human T84 epithelial cells were infected from the basolateral side. miR-146a, which is known to regulate adaptor molecules in Toll-like receptor (TLR)/NF-κB signaling, was found to be differentially regulated in THP-1 cells between probiotic and pathogenic bacteria. To assess the roles of flagella and flagellin, we employed several flagellin mutants of EcN. EcN flagellin mutants induced reduced IL-8 as well as CXCL1 responses in T84 cells, suggesting that flagellin is an inducer of this cytokine response. Following infection with an EPEC type 3 secretion system (T3SS) mutant, we observed increased IL-8 and CXCL1 transcription in T84 and THP-1 cells compared to that in wild-type EPEC. This study emphasizes the differential induction of miR-146a by pathogenic and probiotic E. coli strains in epithelial and immune cells as well as a loss of probiotic properties in EcN interacting with cells from the basolateral side. PMID:27297392

  9. Interaction of porcine neutrophils with different strains of enterotoxigenic Escherichia coli.

    PubMed

    Ondrackova, Petra; Alexa, Pavel; Matiasovic, Jan; Volf, Jiri; Faldyna, Martin

    2012-11-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the most important causes of post-weaning diarrhea in piglets. Whilst serotype O149:F4 is frequently associated with hemorrhagic gastroenteritis, other serotypes have been found to be associated with mild or moderate enteritis. As neutrophils are recruited to sites of inflammation, the aim of this study was to ascertain whether or not there is any difference in the in vitro interaction between neutrophils and two different ETEC serotypes: O149:F4 and O147:F18. The association of bacteria with neutrophils was evaluated by flow cytometry. The respiratory burst was measured by the fluorescent probe dichlorofluorescein diacetate using flow cytometry and by L012-amplified chemiluminescence. The titers of antibodies against ETEC present in cultivation sera were assessed by agglutination. The viability of E. coli was ascertained by cultivation. It was found that the strains of O149 serotype were more frequently associated with neutrophils and induced a more intensive respiratory burst compared to the strains of O147 serotype. These differences might be due to the presence of different types of fimbriae on the surface of the strains tested and by the presence of anti-fimbrial antibodies in the porcine plasma. However, the intensive interaction between E. coli and the neutrophils and respiratory burst induced by the O149 strain did not lead to more efficient killing of the bacteria. It is suggested that a stronger respiratory burst may be an important factor causing severe clinical signs of post-weaning diarrhea in piglets. PMID:22704243

  10. Presence of Multidrug-Resistant Shiga Toxin-Producing Escherichia coli, Enteropathogenic E. coli and Enterotoxigenic E. coli, on Raw Nopalitos (Opuntia ficus-indica L.) and in Nopalitos Salads from Local Retail Markets in Mexico.

    PubMed

    Gómez-Aldapa, Carlos A; Cerna-Cortes, Jorge F; Rangel-Vargas, Esmeralda; Torres-Vitela, Mdel Refugio; Villarruel-López, Angelica; Gutiérrez-Alcántara, Eduardo J; Castro-Rosas, Javier

    2016-05-01

    The presence of multidrug-resistant pathogenic bacteria in food is a significant public health concern. Diarrheagenic Escherichia coli pathotypes (DEPs) are foodborne bacteria. In Mexico, DEPs have been associated with diarrheal illness. There is no information about the presence of multidrug-resistant DEPs on fresh vegetables and in cooked vegetable salads in Mexico. "Nopalitos" (Opuntia ficus-indica L.) is a Cactacea extensively used as a fresh green vegetable throughout Mexico. The presence of generic E. coli and multidrug-resistant DEPs on raw whole and cut nopalitos and in nopalitos salad samples was determined. One hundred raw whole nopalitos (without prickles) samples, 100 raw nopalitos cut into small square samples, and 100 cooked nopalitos salad samples were collected from markets. Generic E. coli was determined using the most probable number procedures. DEPs were identified using two multiplex polymerase chain reaction procedures. Susceptibility to 16 antibiotics was tested for the isolated DEP strains by standard test. Of the 100 whole nopalitos samples, 100 cut nopalitos samples, and 100 nopalitos salad samples, generic E. coli and DEPs were identified, respectively, in 80% and 10%, 74% and 10%, and 64% and 8%. Eighty-two DEP strains were isolated from positive nopalitos samples. The identified DEPs included Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC). All isolated strains exhibited resistance to at least six antibiotics. To the best of our knowledge, this is the first report of the presence of multidrug-resistant and antibiotic resistance profiles of STEC, ETEC, and EPEC on raw nopalitos and in nopalitos salads in Mexico. PMID:26954710

  11. Behavior of shiga toxin-producing Escherichia coli, enteroinvasive E. coli, enteropathogenic E. coli and enterotoxigenic E. coli strains on whole and sliced jalapeño and serrano peppers.

    PubMed

    Gómez-Aldapa, Carlos A; Rangel-Vargas, Esmeralda; Gordillo-Martínez, Alberto J; Castro-Rosas, Javier

    2014-06-01

    The behavior of enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC) and non-O157 shiga toxin-producing E. coli (non-O157-STEC) on whole and slices of jalapeño and serrano peppers as well as in blended sauce at 25 ± 2 °C and 3 ± 2 °C was investigated. Chili peppers were collected from markets of Pachuca city, Hidalgo, Mexico. On whole serrano and jalapeño stored at 25 ± 2 °C or 3 ± 2 °C, no growth was observed for EPEC, ETEC, EIEC and non-O157-STEC rifampicin resistant strains. After twelve days at 25 ± 2 °C, on serrano peppers all diarrheagenic E. coli pathotypes (DEP) strains had decreased by a total of approximately 3.7 log, whereas on jalapeño peppers the strains had decreased by approximately 2.8 log, and at 3 ± 2 °C they decreased to approximately 2.5 and 2.2 log respectively, on serrano and jalapeño. All E. coli pathotypes grew onto sliced chili peppers and in blended sauce: after 24 h at 25 ± 2 °C, all pathotypes had grown to approximately 3 and 4 log CFU on pepper slices and sauce, respectively. At 3 ± 2 °C the bacterial growth was inhibited. PMID:24549200

  12. Screening of extracts from natural feed ingredients for their ability to reduce enterotoxigenic Escherichia coli (ETEC) K88 adhesion to porcine intestinal epithelial cell-line IPEC-J2.

    PubMed

    González-Ortiz, G; Hermes, R G; Jiménez-Díaz, R; Pérez, J F; Martín-Orúe, S M

    2013-12-27

    Enterotoxigenic Escherichia coli (ETEC) K88 is the most prevalent enteropathogen in weaned piglets, with the ability to express fimbria F4 and specifically attach to intestinal receptors in the young piglet. The prevention of ETEC K88 adhesion to the epithelium by interfering in this fimbria-receptor recognition provides an alternative approach to prevent the initial stage of disease. The aim of this study is to screen, among different feed ingredients (FI), their ability to reduce ETEC K88 attachment to the porcine intestinal epithelial cell-line (IPEC-J2). The selected FI consisted of products of a vegetable or dairy origin, and microbial by-products, which could be suitable to be included in piglet's diet. Incubation of a mixture of each FI extract with the bacteria on IPEC-J2 monolayer was allowed. After washing with PBS to remove the non-adhered bacteria, the culture medium was added to grow the adhered bacteria and, simultaneously, to keep the cells alive. Then, the bacterial growth was monitored in a spectrophotometer reader for 12h. Casein glycomacropeptide (CGMP), locust bean (LB), exopolysaccharide (EPS) and wheat bran (WB) reduced the number of attached ETEC K88 to IPEC-J2, but no anti-adhesive effect was found for soybean hulls, sugar-beet pulp, locust gum, fructooligosaccharides, inulin, mushroom, mannanoligosaccharides or the fermented product from Aspergillus oryzae. The lineal analysis of dose responses demonstrated lineal activity (P<0.0001) for CGMP, LB, EPS and WB. These in vitro results suggest CGMP, LB, EPS and WB as good candidates to be included in piglet's diet with supported functional activity against colibacillosis. PMID:23992796

  13. Characterization of EspC, a 110-kilodalton protein secreted by enteropathogenic Escherichia coli which is homologous to members of the immunoglobulin A protease-like family of secreted proteins.

    PubMed Central

    Stein, M; Kenny, B; Stein, M A; Finlay, B B

    1996-01-01

    Enteropathogenic Escherichia coli (EPEC) secretes at least five proteins. Two of these proteins, EspA and EspB (previously called EaeB), activate signal transduction pathways in host epithelial cells. While the role of the other three proteins (39, 40, and 110 kDa) remains undetermined, secretion of all five proteins is under the control of perA, a known positive regulator of several EPEC virulence factors. On the basis of amino-terminal protein sequence data, we cloned and sequenced the gene which encodes the 110-kDa secreted protein and examined its possible role in EPEC signaling and interaction with epithelial cells. In accordance with the terminology used for espA and espB, we called this gene espC, for EPEC-secreted protein C. We found significant homology between the predicted EspC protein sequence and a family of immunoglobulin A (IgA) protease-like proteins which are widespread among pathogenic bacteria. Members of this protein family are found in avian pathogenic Escherichia coli (Tsh), Haemophilus influenzae (Hap), and Shigella flexneri (SepA). Although these proteins and EspC do not encode IgA protease activity, they have considerable homology with IgA protease from Neisseria gonorrhoeae and H. influenzae and appear to use a export system for secretion. We found that genes homologous to espC also exist in other pathogenic bacteria which cause attaching and effacing lesions, including Hafnia alvei biotype 19982, Citrobacter freundii biotype 4280, and rabbit diarrheagenic E. coli (RDEC-1). Although these strains secrete various proteins similar in molecular size to the proteins secreted by EPEC, we did not detect secretion of a 110-kDa protein by these strains. To examine the possible role of EspC in EPEC interactions with epithelial cells, we constructed a deletion mutant in espC by allelic exchange and characterized the mutant by standard tissue culture assays. We found that EspC is not necessary for mediating EPEC-induced signal transduction in He

  14. Bacterial enteropathogens in Malaysian shellfish.

    PubMed

    Jegathesan, M; Wah, L T; Soon, L E; Su Har, D; Boo Liat, L

    1976-06-01

    Three species of commonly eaten shellfish found in Malaysian coastal waters were examined for the presence of common bacterial enteropathogens. Vibrio parahaemolyticus, non-agglutinating vibrios, and various serotypes of enteropathogenic E. coli were isolated from a large proportion of them. Salmonella were isolated in two instances. High colony counts with evidence of faecal contamination indicated the strong possibility of pulltion being the cause for the presence of these enteropathogens. Methods of cooking and eating these shellfish enhance their likelihood of acting as vehicles of diarrhoeal disease. PMID:788266

  15. Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains.

    PubMed

    Wijemanne, Prageeth; Xing, Jun; Berberov, Emil M; Marx, David B; Francis, David H; Moxley, Rodney A

    2015-01-01

    Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and

  16. Relationship between Heat-Labile Enterotoxin Secretion Capacity and Virulence in Wild Type Porcine-Origin Enterotoxigenic Escherichia coli Strains

    PubMed Central

    Wijemanne, Prageeth; Xing, Jun; Berberov, Emil M.; Marx, David B.; Francis, David H.; Moxley, Rodney A.

    2015-01-01

    Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and

  17. In vitro activity of rifaximin against clinical isolates of Escherichia coli and other enteropathogenic bacteria isolated from travellers returning to the UK.

    PubMed

    Hopkins, Katie L; Mushtaq, Shazad; Richardson, Judith F; Doumith, Michel; de Pinna, Elizabeth; Cheasty, Tom; Wain, John; Livermore, David M; Woodford, Neil

    2014-05-01

    Rifaximin is licensed in the EU and USA for treating travellers' diarrhoea caused by non-invasive bacteria. Selection for resistance mechanisms of public health significance might occur if these are linked to rifamycin resistance. Rifaximin MICs were determined by agar dilution for 90 isolates each of Escherichia coli, Shigella spp., nontyphoidal Salmonella enterica, typhoidal S. enterica and Campylobacter spp., an additional 60 E. coli with CTX-M ESBLs isolated from patients with travellers' diarrhoea, and 30 non-diarrhoeal carbapenemase-producing E. coli. Comparators were rifampicin, ciprofloxacin, azithromycin, trimethoprim/sulfamethoxazole and doxycycline. Isolates with rifaximin MICs>32 mg/L were screened for arr genes, and critical rpoB regions were sequenced. Rifaximin was active at ≤32 mg/L against 436/450 (96.9%) diverse Enterobacteriaceae, whereas 81/90 (90%) Campylobacter spp. were resistant to rifaximin at ≥128 mg/L. Rifaximin MICs were ≥128 mg/L for two Shigella and five MDR E. coli producing NDM (n = 3), OXA-48 (n = 1) or CTX-M-15 (n = 1). Two of the five MDR E. coli had plasmids harbouring arr-2 together with bla(NDM), and two (one each with bla(NDM) and bla(CTX-M-15)) had His526Asn substitutions in RpoB. The rifamycin resistance mechanism remained undefined in one MDR E. coli isolate (with bla(OXA-48)) and the two Shigella isolates. Rifaximin showed good in vitro activity against diverse Enterobacteriaceae but was largely inactive against Campylobacter spp. Rifaximin has potential to co-select MDR E. coli in the gut flora, but much stronger associations were seen between ESBL and/or carbapenemase production and resistance to alternative treatments for travellers' diarrhoea, notably ciprofloxacin and azithromycin. PMID:24661532

  18. Escherichia coli expressing single-chain Fv on the cell surface as a potential prophylactic of porcine epidemic diarrhea virus.

    PubMed

    Pyo, Hyun-Mi; Kim, In-Joong; Kim, Seong-Hee; Kim, Hyun-Soo; Cho, Soo-Dong; Cho, In-Soo; Hyun, Bang-Hun

    2009-03-23

    Porcine epidemic diarrhea virus (PEDV) is a causative agent of severe diarrhea which leads to death in piglets. Because of the high mortality which is up to 100% in suckling piglets, PED is an important porcine disease in Korea. In this study, we developed a prophylactic candidate using single-chain Fvs to prevent the PEDV infection. ScFvs of mouse monoclonal antibody which was verified to neutralize PEDV was expressed in Escherichia coli expression system. After the confirmation of PEDV neutralizing activity of purified recombinant scFvs by VN test, scFvs were expressed on the surface of E. coli cells. The signal sequence and autotransporter beta domain of protease IgA (IgAP) of Neisseria gonorrhoeae were introduced to endow scFvs with the direction to the cell surface and the support as a transmembrane domain. 5x10(6)CFU of E. coli expressing scFvs against PEDV showed promising result of 94% foci reduction compared to wild type E. coli. This result demonstrated that E. coli expressing scFvs on the cell surface retained functional potency of parent antibody and therefore blocked PEDV infection into target cells in vitro. This in vitro assay result proposes the perspective of recombinant E. coli cells expressing scFvs as a novel prophylactic against PEDV infection. PMID:19428826

  19. Protection against human and porcine enterotoxigenic strains of Escherichia coli in rats immunized with a cross-linked toxoid vaccine.

    PubMed Central

    Klipstein, F A; Engert, R F; Clements, J D; Houghten, R A

    1983-01-01

    To compare their relative immunogenicities, we used synthetically produced Escherichia coli heat-stable toxin coupled to a protein carrier and the B subunit of porcine heat-labile toxin separately in graded dosages to immunize rats. Equivalent antigen unit dosages of each toxin raised approximately the same level of mucosal immunoglobulin A (IgA) antitoxin response and degree of protection against a challenge with respective heat-stable- or heat-labile-toxin-producing viable bacteria. Conjugation conditions were identified, therefore, which yielded a vaccine of these toxins, cross-linked by the carbodiimide reaction, that consisted of equal antigenic proportions of each toxin component as determined by enzyme-linked immunosorbent assay and expressed in antigen units. The dose-related response to immunization with this vaccine was the same as the response to its components given separately. The toxicity of the heat-stable toxin component was reduced greater than 600-fold. Immunization with optimal antigen unit dosages of the vaccine gave greater than or equal to sixfold increases in mucosal IgA antitoxin titers and provided significant (P less than 0.001) protection against challenge with heterologous serotypes of viable strains, of either human or porcine origin, that produce heat-stable or heat-labile toxin or both. PMID:6343245

  20. Correlating attachment behavior with cell properties for eight Porcine Escherichia coli Isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study we investigate how growth stage and depositional environment affect variability of cell properties and transport behavior of eight porcine E. coli isolates. We compared the surface properties and transport behavior for cells harvested at two different growth stages (exponential and sta...

  1. Correlating Transport Behavior with Cell Properties for Eight Porcine Escherichia coli Isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study we investigate how growth stage and depositional environment affect variability of cell properties and transport behavior of eight porcine E. coli isolates. We compared the surface properties and transport behavior for cells harvested at two different growth stages (exponential and sta...

  2. Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli

    PubMed Central

    2010-01-01

    Background Porcine circovirus 2 (PCV2) is a serious problem to the swine industry and can lead to significant negative impacts on profitability of pork production. Syndrome associated with PCV2 is known as porcine circovirus closely associated with post-weaning multisystemic wasting syndrome (PMWS). The capsid (Cap) protein of PCV2 is a major candidate antigen for development of recombinant vaccine and serological diagnostic method. The recombinant Cap protein has the ability to self-assemble into virus-like particles (VLPs) in vitro, it is particularly opportunity to develop the PV2 VLPs vaccine in Escherichia coli,(E.coli ), because where the cost of the vaccine must be weighed against the value of the vaccinated pig, when it was to extend use the VLPs vaccine of PCV2. Results In this report, a highly soluble Cap-tag protein expressed in E.coli was constructed with a p-SMK expression vector with a fusion tag of small ubiquitin-like modifiers (SUMO). The recombinant Cap was purified using Ni2+ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole native Cap protein was able to self-assemble into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size and shape that resembled the authentic Cap. The result could also be applied in the large-scale production of VLPs of PCV2 and could be used as a diagnostic antigen or a potential VLP vaccine against PCV2 infection in pigs. Conclusion we have, for the first time, utilized the SUMO fusion motif to successfully express the entire authentic Cap protein of PCV2 in E. coli. After the cleavage of the fusion motif, the nCap protein has the ability to self-assemble into VLPs, which can be used as as a potential vaccine to protect pigs from PCV2-infection. PMID:20646322

  3. Characterization of shiga toxin subtypes and virulence genes in Porcine shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a...

  4. Genetic relatedness and virulence properties of enteropathogenic Escherichia coli strains of serotype O119:H6 expressing localized adherence or localized and aggregative adherence-like patterns on HeLa cells.

    PubMed

    Garcia, Bruna G; Ooka, Tadasuke; Gotoh, Yasuhiro; Vieira, Mônica A M; Yamamoto, Denise; Ogura, Yoshitoshi; Girão, Dennys M; Sampaio, Suely C F; Melo, Alexis Bonfim; Irino, Kinue; Hayashi, Tetsuya; Gomes, Tânia A T

    2016-05-01

    Enteropathogenic Escherichia coli (EPEC) induce attaching and effacing (A/E) lesions in enterocytes and produce the bundle-forming pilus (BFP) contributing to the localized adherence (LA) pattern formation on HeLa cells. Enteroaggregative E. coli (EAEC) produce aggregative adherence (AA) on HeLa cells and form prominent biofilms. The ability to produce LA or AA is an important hallmark to classify fecal E. coli isolates as EPEC or EAEC, respectively. E. coli strains of serotype O119:H6 exhibit an LA+ phenotype and have been considered as comprising a clonal group of EPEC strains. However, we have recently identified O119:H6 EPEC strains that produce LA and an AA-like pattern concurrently (LA/AA-like+). In this study, we evaluated the relatedness of three LA/AA-like+ and three LA+ O119:H6 strains by comparing their virulence and genotypic properties. We first found that the LA/AA-like+ strains induced actin accumulation in HeLa cells (indicative of A/E lesions formation) and formed biofilms on abiotic surfaces more efficiently than the LA+ strains. MLST analysis showed that the six strains all belong to the ST28 complex. All strains carried multiple plasmids, but as plasmid profiles were highly variable, this cannot be used to differentiate LA/AA-like+ and LA+ strains. We further obtained their draft genome sequences and the complete sequences of four plasmids harbored by one LA/AA-like+ strain. Analysis of these sequences and comparison with 37 fully sequenced E. coli genomes revealed that both O119:H6 groups belong to the E. coli phylogroup B2 and are very closely related with only 58-67 SNPs found between LA/AA-like+ and LA+ strains. Search of the draft sequences of the six strains for adhesion-related genes known in EAEC and other E. coli pathotypes detected no genes specifically present in LA/AA-like+ strains. Unexpectedly however, we found that a large plasmid distinct from pEAF is responsible for the AA-like phenotype of the LA/AA-like+ strains. Although we

  5. Characterization of porcine intestinal receptors for the K88ac fimbrial adhesin of Escherichia coli as mucin-type sialoglycoproteins.

    PubMed Central

    Erickson, A K; Baker, D R; Bosworth, B T; Casey, T A; Benfield, D A; Francis, D H

    1994-01-01

    We have previously identified two K88ac adhesion receptors (210 and 240 kDa) which are present in membrane preparations from adhesive but not nonadhesive porcine intestinal brush border cells; these adhesin receptors are postulated to be important determinants of the susceptibility of pigs to K88ac+ enterotoxigenic Escherichia coli infections (A.K. Erickson, J.A. Willgohs, S.Y. McFarland, D.A. Benfield, and D.F. Francis, Infect. Immun. 60:983-988, 1992). We now describe a procedure for the purification of these two receptors. Receptors were solubilized from adhesive intestinal brush border vesicles using deoxycholate and were purified by gel filtration chromatography on Sepharose CL-4B and then by hydroxyapatite chromatography. Amino acid compositional analyses indicated that the two receptors have similar amino acid compositions. The most distinguishing characteristic of both receptors is a high percentage of threonine and proline residues. Neuraminidase treatment caused the K88ac adhesin receptors to migrate with a slower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, indicating that these receptors are sialoglycoproteins. Results from lectin-binding studies indicated that the receptors contain O-linked oligosaccharides composed of galactosyl (beta-1,3)N-acetylgalactosamine, alpha-linked fucose, galactosyl(beta-1,4)N-acetylglucosamine, sialic acid, galactose, and N-acetylgalactosamine. Collectively, these characteristics indicate that the K88ac adhesin receptors are mucin-type sialoglycoproteins. Images PMID:7960120

  6. Protective effects of Lactobacillus plantarum on epithelial barrier disruption caused by enterotoxigenic Escherichia coli in intestinal porcine epithelial cells.

    PubMed

    Wu, Yunpeng; Zhu, Cui; Chen, Zhuang; Chen, Zhongjian; Zhang, Weina; Ma, Xianyong; Wang, Li; Yang, Xuefen; Jiang, Zongyong

    2016-04-01

    Tight junctions (TJs) play an important role in maintaining the mucosal barrier function and gastrointestinal health of animals. Lactobacillus plantarum (L. plantarum) was reported to protect the intestinal barrier function of early-weaned piglets against enterotoxigenic Escherichia coli (ETEC) K88 challenge; however, the underlying cellular mechanism of this protection was unclear. Here, an established intestinal porcine epithelia cell (IPEC-J2) model was used to investigate the protective effects and related mechanisms of L. plantarum on epithelial barrier damages induced by ETEC K88. Epithelial permeability, expression of inflammatory cytokines, and abundance of TJ proteins, were determined. Pre-treatment with L. plantarum for 6h prevented the reduction in transepithelial electrical resistance (TEER) (P<0.05), inhibited the increased transcript abundances of interleukin-8 (IL-8) and tumor necrosis factor (TNF-α) (P<0.05), decreased expression of claudin-1, occludin and zonula occludens (ZO-1) (P<0.05) and protein expression of occludin (P<0.05) of IPEC-J2 cells caused by ETEC K88. Moreover, the mRNA expression of negative regulators of toll-like receptors (TLRs) [single Ig Il-1-related receptor (SIGIRR), B-cell CLL/lymphoma 3 (Bcl3), and mitogen-activated protein kinase phosphatase-1 (MKP-1)] in IPEC-J2 cells pre-treated with L. plantarum were higher (P<0.05) compared with those in cells just exposed to K88. Furthermore, L. plantarum was shown to regulate proteins of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. These results indicated that L. plantarum may improve epithelial barrier function by maintenance of TEER, inhibiting the reduction of TJ proteins, and reducing the expression of proinflammatory cytokines induced by ETEC K88, possibly through modulation of TLRs, NF-κB and MAPK pathways. PMID:27032504

  7. Production of soluble truncated spike protein of porcine epidemic diarrhea virus from inclusion bodies of Escherichia coli through refolding.

    PubMed

    Piao, Da-Chuan; Lee, Yoon-Seok; Bok, Jin-Duck; Cho, Chong-Su; Hong, Zhong-Shan; Kang, Sang-Kee; Choi, Yun-Jaie

    2016-10-01

    The emergence of highly pathogenic variant porcine epidemic diarrhea virus (PEDV) strains, from 2013 to 2014, in North American and Asian countries have greatly threatened global swine industry. Therefore, development of effective vaccines against PEDV variant strains is urgently needed. Recently, it has been reported that the N-terminal domain (NTD) of S1 domain of PEDV spike protein is responsible for binding to the 5-N-acetylneuraminic acid (Neu5Ac), a possible sugar co-receptor. Therefore, the NTD of S1 domain could be an attractive target for the development of subunit vaccines. In this study, the NTD spanning amino acid residues 25-229 (S25-229) of S1 domain of PEDV variant strain was expressed in Escherichia coli BL21 (DE3) in the form of inclusion bodies (IBs). S25-229 IBs were solubilized in 20 mM sodium acetate (pH 4.5) buffer containing 8 M urea and 1 mM dithiothreitol with 95% yield. Solubilized S25-229 IBs were refolded by 10-fold flash dilution and purified by one-step cation exchange chromatography with >95% purity and 20% yield. The CD spectrum of S25-229 showed the characteristic pattern of alpha helical structure. In an indirect ELISA, purified S25-229 showed strong reactivity with mouse anti-PEDV sera. In addition, immunization of mice with 20 μg of purified S25-229 elicited highly potent serum IgG titers. Finally, mouse antisera against S25-229 showed immune reactivity with native PEDV S protein in an immunofluorescence assay. These results suggest that purified S25-229 may have potential to be used as a subunit vaccine against PEDV variant strains. PMID:27260969

  8. Characterization of Shiga Toxin Subtypes and Virulence Genes in Porcine Shiga Toxin-Producing Escherichia coli.

    PubMed

    Baranzoni, Gian Marco; Fratamico, Pina M; Gangiredla, Jayanthi; Patel, Isha; Bagi, Lori K; Delannoy, Sabine; Fach, Patrick; Boccia, Federica; Anastasio, Aniello; Pepe, Tiziana

    2016-01-01

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx 2e (81%) was the most common Stx variant, followed by stx 1a (14%), stx 2d (3%), and stx 1c (1%). The STEC serogroups that carried stx 2d were O15:H27, O159:H16 and O159:H-. Similar to stx 2a and stx 2c, the stx 2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections. PMID:27148249

  9. Characterization of Shiga toxin subtypes and virulence genes in porcine Shiga toxin-producing Escherichia coli

    DOE PAGESBeta

    Baranzoni, Gian Marco; Fratamico, Pina M.; Gangiredla, Jayanthi; Patel, Isha; Bagi, Lori K.; Delannoy, Sabine; Fach, Patrick; Boccia, Federica; Anastasio, Aniello; Pepe, Tiziana

    2016-04-21

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using amore » Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. Furthermore, the present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.« less

  10. Characterization of Shiga Toxin Subtypes and Virulence Genes in Porcine Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Baranzoni, Gian Marco; Fratamico, Pina M.; Gangiredla, Jayanthi; Patel, Isha; Bagi, Lori K.; Delannoy, Sabine; Fach, Patrick; Boccia, Federica; Anastasio, Aniello; Pepe, Tiziana

    2016-01-01

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections. PMID:27148249

  11. Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus.

    PubMed Central

    Krempl, C; Schultze, B; Laude, H; Herrler, G

    1997-01-01

    Enteropathogenic transmissible gastroenteritis virus (TGEV), a porcine coronavirus, is able to agglutinate erythrocytes because of sialic acid binding activity. Competitive inhibitors that may mask the sialic acid binding activity can be inactivated by sialidase treatment of virions. Here, we show that TGEV virions with efficient hemagglutinating activity were also obtained when cells were treated with sialidase prior to infection. This method was used to analyze TGEV mutants for hemagglutinating activity. Recently, mutants with strongly reduced enteropathogenicity that have point mutations or a deletion of four amino acids within residues 145 to 155 of the S protein have been described. Here, we show that in addition to their reduced pathogenicity, these mutants also have lost hemagglutinating activity. These results connect sialic acid binding activity with the enteropathogenicity of TGEV. PMID:9060696

  12. Carbohydrate-binding specificities of potential probiotic Lactobacillus strains in porcine jejunal (IPEC-J2) cells and porcine mucin.

    PubMed

    Valeriano, Valerie Diane; Bagon, Bernadette B; Balolong, Marilen P; Kang, Dae-Kyung

    2016-07-01

    Bacterial lectins are carbohydrate-binding adhesins that recognize glycoreceptors in the gut mucus and epithelium of hosts. In this study, the contribution of lectin-like activities to adhesion of Lactobacillus mucosae LM1 and Lactobacillus johnsonii PF01, which were isolated from swine intestine, were compared to those of the commercial probiotic Lactobacillus rhamnosus GG. Both LM1 and PF01 strains have been reported to have good adhesion ability to crude intestinal mucus of pigs. To confirm this, we quantified their adhesion to porcine gastric mucin and intestinal porcine enterocytes isolated from the jejunum of piglets (IPEC-J2). In addition, we examined their carbohydrate-binding specificities by suspending bacterial cells in carbohydrate solutions prior to adhesion assays. We found that the selected carbohydrates affected the adherences of LM1 to IPEC-J2 cells and of LGG to mucin. In addition, compared to adhesion to IPEC-J2 cells, adhesion to mucin by both LM1 and LGG was characterized by enhanced specific recognition of glycoreceptor components such as galactose, mannose, and N-acetylglucosamine. Hydrophobic interactions might make a greater contribution to adhesion of PF01. A similar adhesin profile between a probiotic and a pathogen, suggest a correlation between shared pathogen-probiotic glycoreceptor recognition and the ability to exclude enteropathogens such as Escherichia coli K88 and Salmonella Typhimurium KCCM 40253. These findings extend our understanding of the mechanisms of the intestinal adhesion and pathogen-inhibition abilities of probiotic Lactobacillus strains. PMID:27350617

  13. [Enteropathogens associated with diarrheal disease in infants (< 5 years old) in a population sample in Greater Metropolitan Criciúma, Santa Catarina State, Brazil].

    PubMed

    Schnack, Felice Jaqueline; Fontana Ld, Lidiani de Medeiros; Barbosa, Paulo Roberto; Silva, Loraine Storch Meyer da; Baillargeon, Clair Maria Martinello; Barichello, Tatiana; Póvoa, Marinete Marins; Cavasini, Carlos Eug nio; Machado, Ricardo Luiz Dantas

    2003-01-01

    Enteropathogens were investigated in 94 children with diarrhea and 45 age-matched controls, 0 to 5 years old, attending an outpatient unit in Criciúma, Santa Catarina State, Brazil. Cryptosporidium (85.1%) topped the list of parasite isolates, followed by Entamoeba histolytica (56.4%) and Giardia lamblia (4.3%). Four samples contained enteropathogenic Escherichia coli (4.3%). Samonella and Shiguella were not detected. Only one sample contained rotavirus (1.1%). PMID:12973585

  14. In Vitro Evaluation of Swine-Derived Lactobacillus reuteri: Probiotic Properties and Effects on Intestinal Porcine Epithelial Cells Challenged with Enterotoxigenic Escherichia coli K88.

    PubMed

    Wang, Zhilin; Wang, Li; Chen, Zhuang; Ma, Xianyong; Yang, Xuefen; Zhang, Jian; Jiang, Zongyong

    2016-06-28

    Probiotics are considered as the best effective alternatives to antibiotics. The aim of this study was to characterize the probiotic potential of lactobacilli for use in swine farming by using in vitro evaluation methods. A total of 106 lactic acid bacterial isolates, originating from porcine feces, were first screened for the capacity to survive stresses considered important for putative probiotic strains. Sixteen isolates showed notable acid and bile resistance, antibacterial activity, and adherence to intestinal porcine epithelial cells (IPEC-1). One isolate, LR1, identified as Lactobacillus reuteri, was selected for extensive study of its probiotic and functional properties in IPEC-1 cell models. L. reuteri LR1 exhibited good adhesion to IPEC-1 cells and could inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to IPEC-1 cells. L. reuteri LR1 could also modulate transcript and protein expression of cytokines involved in inflammation in IPEC-1 cells; the Lactobacillus strain inhibited the ETEC-induced expression of proinflammatory transcripts (IL-6 and TNF-α) and protein (IL-6), and increased the level of anti-inflammatory cytokine (IL-10). Measurement of the permeation of FD-4 showed that L. reuteri LR1 could maintain barrier integrity in monolayer IPEC-1 cells exposed to ETEC. Immunolocalization experiments showed L. reuteri LR1 could also prevent ETEC-induced tight junction ZO-1 disruption. Together, these results indicate that L. reuteri LR1 exhibits desirable probiotic properties and could be a potential probiotic for use in swine production. PMID:26907754

  15. Roles of Hcp family proteins in the pathogenesis of the porcine extraintestinal pathogenic Escherichia coli type VI secretion system.

    PubMed

    Peng, Ying; Wang, Xiangru; Shou, Jin; Zong, Bingbing; Zhang, Yanyan; Tan, Jia; Chen, Jing; Hu, Linlin; Zhu, Yongwei; Chen, Huanchun; Tan, Chen

    2016-01-01

    Hcp (hemolysin-coregulated protein) is considered a vital component of the functional T6SS (Type VI Secretion System), which is a newly discovered secretion system. Our laboratory has previously sequenced the whole genome of porcine extraintestinal pathogenic E. coli (ExPEC) strain PCN033, and identified an integrated T6SS encoding three different hcp family genes. In this study, we first identified a functional T6SS in porcine ExPEC strain PCN033, and demonstrated that the Hcp family proteins were involved in bacterial competition and the interactions with other cells. Interestingly, the three Hcp proteins had different functions. Hcp2 functioned predominantly in bacterial competition; all three proteins were involved in the colonization of mice; and Hcp1 and Hcp3 were predominantly contributed to bacterial-eukaryotic cell interactions. We showed an active T6SS in porcine ExPEC strain PCN033, and the Hcp family proteins had different functions in their interaction with other bacteria or host cells. PMID:27229766

  16. Roles of Hcp family proteins in the pathogenesis of the porcine extraintestinal pathogenic Escherichia coli type VI secretion system

    PubMed Central

    Peng, Ying; Wang, Xiangru; Shou, Jin; Zong, Bingbing; Zhang, Yanyan; Tan, Jia; Chen, Jing; Hu, Linlin; Zhu, Yongwei; Chen, Huanchun; Tan, Chen

    2016-01-01

    Hcp (hemolysin-coregulated protein) is considered a vital component of the functional T6SS (Type VI Secretion System), which is a newly discovered secretion system. Our laboratory has previously sequenced the whole genome of porcine extraintestinal pathogenic E. coli (ExPEC) strain PCN033, and identified an integrated T6SS encoding three different hcp family genes. In this study, we first identified a functional T6SS in porcine ExPEC strain PCN033, and demonstrated that the Hcp family proteins were involved in bacterial competition and the interactions with other cells. Interestingly, the three Hcp proteins had different functions. Hcp2 functioned predominantly in bacterial competition; all three proteins were involved in the colonization of mice; and Hcp1 and Hcp3 were predominantly contributed to bacterial-eukaryotic cell interactions. We showed an active T6SS in porcine ExPEC strain PCN033, and the Hcp family proteins had different functions in their interaction with other bacteria or host cells. PMID:27229766

  17. Incidence of bacterial enteropathogens in foods from Mexico.

    PubMed Central

    Wood, L V; Ferguson, L E; Hogan, P; Thurman, D; Morgan, D R; DuPont, H L; Ericsson, C D

    1983-01-01

    We examined food consumption patterns of U.S. students temporarily living in Guadalajara, Mexico. Consumption of foods prepared in Mexican homes was associated with an increased risk of acquisition of diarrhea. Foods from commercial sources and private Mexican homes in Guadalajara were subsequently examined for contamination with coliforms, fecal coliforms, and bacterial enteropathogens. For comparison, selected restaurant foods were obtained in Houston, Tex. Food obtained from Mexican homes showed generally higher counts of coliforms and fecal coliforms than those obtained from commercial sources in Mexico and Houston. The foods in Mexico, both from homes and commercial sources, commonly contained Escherichia coli and occasionally enterotoxigenic E. coli. Foods in Houston were not contaminated with E. coli or enterotoxigenic E. coli. Salmonella (17 isolates), Shigella (4 isolates), and Aeromonas hydrophila (1 isolate) were found only in the foods obtained from Mexican homes. Enterotoxigenic non-E. coli Enterobacteriaceae was recovered with approximately equal frequency from all food sources. PMID:6354085

  18. The comparative activity of pefloxacin, enoxacin, ciprofloxacin and 13 other antimicrobial agents against enteropathogenic microorganisms.

    PubMed

    Vanhoof, R; Hubrechts, J M; Roebben, E; Nyssen, H J; Nulens, E; Leger, J; De Schepper, N

    1986-01-01

    In this study, we compared the activity of pefloxacin, enoxacin and ciprofloxacin against 269 enteropathogenic strains (Campylobacter jejuni, enteropathogenic Escherichia coli, Salmonella typhi, Shigella spp., Vibrio cholerae and Yersinia enterocolitica) with that of rosoxacin, flumequin, nifuroxazide, erythromycin, chloramphenicol, ampicillin, cefotaxime, tetracycline, amikacin, netilmicin, sulfamethoxazole, trimethoprim and co-trimoxazole. Pefloxacin, enoxacin and ciprofloxacin were always among the most active compounds. Furthermore, resistant strains or strains with elevated MIC values were not found. The MIC90 value for these three compounds was less than or equal to 0.25 mg/l, except for C. jejuni where it was 0.3 mg/l and 1.4 mg/l for pefloxacin and enoxacin, respectively. PMID:3546145

  19. Influence of dietary ingredients on in vitro inflammatory response of intestinal porcine epithelial cells challenged by an enterotoxigenic Escherichia coli (K88).

    PubMed

    Hermes, Rafael G; Manzanilla, Edgar G; Martín-Orúe, Susana M; Pérez, José F; Klasing, Kirk C

    2011-12-01

    Enterotoxigenic Escherichia coli (ETEC) K88 is the main bacterial cause of diarrhea in piglets around weaning and the adhesion of ETEC to the intestinal mucosa is a prerequisite step for its colonization. In this study, the adhesion of a fimbriated ETEC and a non-fimbriated E. coli (NFEC) to the intestinal cells and the activation of the innate immune system were evaluated using a porcine intestinal epithelial cell line (IPEC-J2). The impact of several feedstuffs (wheat bran (WB); casein glycomacropeptide (CGMP); mannan-oligosaccharides (MOS); locust bean extract (LB) and Aspergillus oryzae fermentation extract (AO)) on ETEC attachment and the inflammatory response were also studied. The gene expression of TLR-4; TLR-5; IL-1β; IL-8; IL-10 and TNF-α were quantified using Cyclophilin-A, as a reference gene, and related to a non-challenged treatment. The fimbriated strain was markedly better than the non-fimbriated strain at adherence to intestinal cells and inducing an inflammatory response. All the feedstuffs studied were able to reduce the adhesion of ETEC, with the greatest decrease with CGMP or MOS at highest concentration. Regarding the inflammatory response, the highest dose of WB promoted the lowest relative expression of cytokines and chemokines. All tested feedstuffs were able to reduce the adhesion of ETEC to IPEC-J2 and interfere on the innate inflammatory response; however WB should be further studied according to the beneficial results on the intestinal inflammatory process evidenced in this study. PMID:21944732

  20. Production of Escherichia coli-based virus-like particle vaccine against porcine circovirus type 2 challenge in piglets: Structure characterization and protective efficacy validation.

    PubMed

    Xi, Xiangfeng; Mo, Xiaobing; Xiao, Yan; Yin, Bo; Lv, Chaochao; Wang, Yuzhou; Sun, Zhe; Yang, Qingyuan; Yao, Yali; Xuan, Yajie; Li, Xiangdong; Yuan, Y Adam; Tian, Kegong

    2016-04-10

    We report the strategies leading to the large-production of soluble non-tag full-length porcine circovirus type 2 (PCV2) Cap protein in Escherichia coli. Under neutral pH condition, the purified recombinant Cap protein derived from E. coli expression self-assembles into homogenous round virus-like particle at the similar size of that of the intact PCV2 virus, which is further characterized by Cryo-EM single particle structure determined at 4.5Å. The engineered PCV2 rCap VLP was tested as a subunit vaccine for the protective efficacy against PCV2 challenge on 3-week old piglets. Similar to commercial available PCV2 vaccine, the Cap VLP-immunized piglets developed specific antibody-mediated response and were protected from the virulent SH PCV2 strain challenge. Hence, the production of E. coli based PCV2Cap-VLP could be applied as a cost-friendly and effective subunit vaccine to control PCV2 spreading in developing countries. PMID:26907669

  1. Effect of bovine colostrum, cheese whey, and spray-dried porcine plasma on the in vitro growth of probiotic bacteria and Escherichia coli.

    PubMed

    Champagne, Claude P; Raymond, Yves; Pouliot, Yves; Gauthier, Sylvie F; Lessard, Martin

    2014-05-01

    The aim of this study is to evaluate the effects of defatted colostrum (Col), defatted decaseinated colostrum whey, cheese whey, and spray-dried porcine plasma (SDPP) as supplements of a growth medium (de Man - Rogosa - Sharpe (MRS) broth) on the multiplication of lactic acid bacteria, probiotic bacteria, and potentially pathogenic Escherichia coli. Using automated spectrophotometry (in vitro system), we evaluated the effect of the 4 supplements on maximum growth rate (μ(max)), lag time (LagT), and biomass (OD(max)) of 12 lactic acid bacteria and probiotic bacteria and of an E. coli culture. Enrichment of MRS broth with a Col concentration of 10 g/L increased the μ(max) of 5 of the 12 strains by up to 55%. Negative effects of Col or SDPP on growth rates were also observed with 3 probiotic strains; in one instance μ(max) was reduced by 40%. The most effective inhibitor of E. coli growth was SDPP, and this effect was not linked to its lysozyme content. The positive effect of enrichment with the dairy-based ingredient might be linked to enrichment in sugars and increased buffering power of the medium. These in vitro data suggest that both Col and SDPP could be considered as supplements to animal feeds to improve intestinal health because of their potential to promote growth of probiotic bacteria and to inhibit growth of pathogenic bacteria such as E. coli. PMID:24773334

  2. Induction of pro-inflammatory gene expression by Escherichia coli and mycotoxin zearalenone contamination and protection by a Lactobacillus mixture in porcine IPEC-1 cells.

    PubMed

    Taranu, Ionelia; Marin, Daniela Eliza; Pistol, Gina Cecilia; Motiu, Monica; Pelinescu, Diana

    2015-04-01

    This work investigated the effect of Escherichia coli K88 and zearalenone contamination on pro-inflammatory gene expression (Toll like receptors, cytokines) and signalling molecules and the protective activity of a mixture of Lactobacilli sp. (Lactobacillus plantarum, Lactobacillus acidofilus and Lactobacillus paracasei) in porcine intestinal epithelial cells as part of the local immune system. IPEC-1 cell monolayer was exposed for 1 h to the individual or combined action of E. coli, zearalenone and lactobacilli mixture. Our results showed that TLRs (1-10) and cytokine (IL-1,-6,-8,-10, TNF-α, IFN-γ) genes expressed early (after 1 h of culture) in IPEC-1 cells. E. coli alone increased the TLRs mRNA expression, especially TLR4 and the inflammatory cytokines while ZEA alone showed either no effect or a marginally effect on TLRs, cytokines, and signalling genes when compared to untreated cells. The combined actions of the two contaminants lead to a synergistically up-regulation of key cytokines (IFN-γ, IL-10 and TNF-α) and TLRs (-2,-3,-4,-6, and -10). The live lactobacilli mixture was able to attenuate the pathogen and mycotoxin-induced response by downregulated the majority of inflammatory related genes suggesting that this mixture has an immunomodulatory potential and may be used to lower the inflammatory response. PMID:25640651

  3. Lactobacillus plantarum CS24.2 prevents Escherichia coli adhesion to HT-29 cells and also down-regulates enteropathogen-induced tumor necrosis factor-α and interleukin-8 expression.

    PubMed

    Dhanani, Akhilesh S; Bagchi, Tamishraha

    2013-04-01

    The aim of the present study was to evaluate the potential of Lactobacillus plantarum CS24.2 to antagonize Escherichia coli adhesion and modulate expression of the responses by HT-29 cells of inflammatory molecules to E. coli adhesion. Experiments were performed under different adhesion conditions and findings compared with the responses of Lactobacillus rhamnosus GG. Tests of competitive adhesion, adhesion inhibition and displacement assays were performed for lactobacilli (L. rhamnosus GG and L. plantarum CS24.2) and E. coli O26:H11 to HT-29 cells. Both the lactobacilli significantly reduced E. coli adhesion to HT-29 cells (P < 0.05). The ability of lactobacilli to modulate tumor necrosis factor-α and interleukin-8 expression was analyzed in HT-29 cells stimulated with E. coli using qRT-PCR. L. plantarum CS24.2 significantly down regulated expression of both the genes induced by E. coli in HT-29 cells at 6 hr as well as 24 hr, which was more significant than the corresponding findings for L. rhamnosus GG. The present findings suggest that L. plantarum CS24.2 inhibits pathogen adhesion to a similar extent as does the established probiotic strain L. rhamnosus GG. It may also attenuate tumor necrosis factor-α and interleukin-8 expression in HT-29 cells stimulated with E. coli. PMID:23586634

  4. Enteropathogenic bacteria in the La Paz River of Bolivia.

    PubMed

    Ohno, A; Marui, A; Castro, E S; Reyes, A A; Elio-Calvo, D; Kasitani, H; Ishii, Y; Yamaguchi, K

    1997-10-01

    Diarrheal diseases often result from ingestion of contaminated water or food. The population of La Paz, Bolivia is directly or indirectly exposed to the sewage-contaminated La Paz River. We conducted a bacteriologic survey of the La Paz River to quantify the level of bacterial contamination, with particular reference to enteropathogens. A total bacterial count exceeding 10(6) colony-forming units (CFU)/ml, including lactose fermenting and nonfermenting, gram-negative bacilli of approximately 10(5) CFU/ml, respectively, were detected in river water samples collected near two densely populated areas. A total bacterial count of 10(5) CFU/ml was also detected at the most downstream area of the river near a sparsely populated area. At four sampling locations, several enteropathogens were detected, including five enterotoxigenic Escherichia coli (ETEC) (serotype O6, O15, and O159), two enteropathogenic E. coli (EPEC) (serotype O44), two enteroinvasive E. coli (EIEC) (serotype O29), and three Salmonella O4 group isolates. The heat-labile enterotoxin gene and the invasive toxin gene were detected in all ETEC and EIEC isolates by polymerase chain reaction analysis. Nine isolates of E. coli were found by the agar dilution method to be susceptible to ampicillin, kanamycin, nalidixic acid, tetracycline, and chloramphenicol, and ampicillin resistance was found in only two isolates of EIEC 7-4 (serotype O29) and EPEC 7-5 (serotype O44). Ampicillin resistance was coded on plasmids and transferred conjugatively to E. coli chi1037 at a frequency of 10(-5) CFU/donor by the broth mating method. Strains of Aeromonas caviae, which can cause diarrheal disease in infants, were detected in vegetables grown in fields irrigated by water from the La Paz River. The survival of nine isolates of E. coli in filtered river water was compared with that of laboratory strains (E. coli chi1037, W3110, and ATCC29577). The survival time of seven isolates, excluding two ampicillin-resistant isolates

  5. Genetic Fusions of Heat-Labile Toxoid (LT) and Heat-Stable Toxin b (STb) of Porcine Enterotoxigenic Escherichia coli Elicit Protective Anti-LT and Anti-STb Antibodies ▿

    PubMed Central

    Zhang, Weiping; Francis, David H.

    2010-01-01

    Enterotoxigenic Escherichia coli (ETEC)-associated diarrhea causes a substantial economic loss to swine producers worldwide. The majority of ETEC strains causing porcine diarrhea, especially postweaning diarrhea (PWD), produce heat-labile toxin (LT) and heat-stable toxin b (STb). LT is commonly used in vaccine development, but STb has not been included because of its poor immunogenicity. As a virulence factor in porcine diarrhea, STb needs to be included as an antigen for development of broad-spectrum vaccines. In this study, we used an LT toxoid (LTR192G [hereafter, LT192]) derived from porcine ETEC to carry a mature STb peptide for LT192-STb fusions to enhance STb immunogenicity for potential vaccine application. Anti-LT and anti-STb antibodies were detected in immunized rabbits and pigs. In addition, when challenged with an STb-positive ETEC strain, all 10 suckling piglets borne by immunized gilts remained healthy, whereas 7 out 9 piglets borne by unimmunized gilts developed moderate diarrhea. This study indicates that the LT192-STb fusion enhanced anti-STb immunogenicity and suggests the LT192-STb fusion antigen can be used in future vaccine development against porcine ETEC diarrhea. PMID:20505006

  6. Porcine gonadogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Five images submitted for teaching purposes related to porcine gonadogenesis (2), porcine fetal testicular development (2), and porcine fetal ovarian development. Key words include: Egg cell nests, Embryo, GATA4, Genital ridge, Gonad, Leydig cell, Mesonephros, MIS, Ovary, P450c17, Porcine, Sertoli ...

  7. Preliminary Characterization of the Transcriptional Response of the Porcine Intestinal Cell Line IPEC-J2 to Enterotoxigenic Escherichia coli, Escherichia coli, and E. coli Lipopolysaccharide

    PubMed Central

    Geens, Marisa M.; Niewold, Theo A.

    2010-01-01

    IPEC-J2, a promising in vitro model system, is not well characterized especially on the transcriptional level, in contrast to human counterparts. The aim of this study was to characterize the gene expression in IPEC-J2 cells when coincubated with enterotoxigenic Escherichia coli (ETEC), nonpathogenic E. coli, and E. coli endotoxin. Apical infection of polarized IPEC-J2 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TEER). Microarray analysis showed up-regulation of interleukins when IPEC-J2 were cocultured with E. coli strains this has so far never been measured in this cell line. Highest IL8 expression was found with the ETEC strain possessing the F4 fimbrium, suggesting IPEC-J2 cells to be F4 receptor positive, confirmed in a brush border membrane adhesion assay. It is concluded that the innate immune responses to pathogens and LPS makes the IPEC-J2 cell line a suitable model for research on intestinal host pathogen interaction. PMID:21318186

  8. The novel porcine Lactobacillus sobrius strain protects intestinal cells from enterotoxigenic Escherichia coli K88 infection and prevents membrane barrier damage.

    PubMed

    Roselli, Marianna; Finamore, Alberto; Britti, Maria Serena; Konstantinov, Sergey R; Smidt, Hauke; de Vos, Willem M; Mengheri, Elena

    2007-12-01

    Lactobacilli have a potential to overcome intestinal disorders; however, the exact mode of action is still largely unknown. In this study, we have used the intestinal porcine intestinal IPEC-1 epithelial cells as a model to investigate a possible protective activity of a new Lactobacillus species, the L. sobrius DSM 16698(T), against intestinal injury induced by enterotoxigenic Escherichia coli (ETEC) K88 infection and the underlying mechanisms. Treatment of infected cells with L. sobrius strongly reduced the pathogen adhesion. L. sobrius was also able to prevent the ETEC-induced membrane damage by inhibiting delocalization of zonula occludens (ZO)-1, reduction of occludin amount, rearrangement of F-actin, and dephosphorylation of occludin caused by ETEC. RT-PCR and ELISA experiments showed that L. sobrius counteracted the ETEC-induced increase of IL-8 and upregulated the IL-10 expression. The involvement of IL-8 in the deleterious effects of ETEC was proven by neutralization of IL-8 with a specific antibody. A crucial role of IL-10 was indicated by blockage of IL-10 production with neutralizing anti-IL-10 antibody that fully abrogated the L. sobrius protection. L. sobrius was also able to inhibit the internalization of ETEC, which was likely favored by the leaking barrier. The protective effects were not found with L. amylovorus DSM 20531(T) treatment, a strain derived from cattle waste but phylogenetically closely related to L. sobrius. Together, the data indicate that L. sobrius exerts protection against the harmful effects of ETEC by different mechanisms, including pathogen adhesion inhibition and maintenance of membrane barrier integrity through IL-10 regulation. PMID:18029488

  9. Screening the ability of natural feed ingredients to interfere with the adherence of enterotoxigenic Escherichia coli (ETEC) K88 to the porcine intestinal mucus.

    PubMed

    González-Ortiz, Gemma; Pérez, José Francisco; Hermes, Rafael Gustavo; Molist, Francesc; Jiménez-Díaz, Rufino; Martín-Orúe, Susana María

    2014-02-01

    The inhibition of the attachment of bacteria to the intestine by receptor analogues could be a novel approach to prevent enterotoxigenic Escherichia coli (ETEC) K88-induced diarrhoea in piglets. The objective of the present study was to screen the ability of different feed ingredients (FI) to bind to ETEC K88 (adhesion test, AT) and to block its attachment to the porcine intestinal mucus (blocking test, BT) using in vitro microtitration-based models. In the AT, wheat bran (WB), casein glycomacropeptide (CGMP) and exopolysaccharides exhibited the highest adhesion to ETEC K88 (P< 0·001). In the BT, WB, CGMP and locust bean (LB) reduced the number of ETEC K88 attached to the intestinal mucus (P< 0·001). For WB and LB, fractionation based on their carbohydrate components was subsequently carried out, and each fraction was evaluated individually. None of the WB fractions reduced the adhesion of ETEC K88 to the mucus as did the original extract, suggesting that a protein or glycoprotein could be involved in the recognition process. With regard to the LB fractions, the water-extractable material reduced the adhesion of ETEC K88 (P< 0·001) to the mucus similar to the original extract (P< 0·001), indicating, in this case, that galactomannans or phenolic compounds could be responsible for the recognition process. In conclusion, among the FI screened, the soluble extracts obtained from WB, LB and CGMP exhibited the highest anti-adhesive properties against ETEC K88 in the BT. These results suggest that they may be good candidates to be included in diets of weaned piglets for the prevention of ETEC K88-induced diarrhoea. PMID:24047890

  10. Prediction of Intra-Species Protein-Protein Interactions in Enteropathogens Facilitating Systems Biology Study

    PubMed Central

    Barman, Ranjan Kumar; Jana, Tanmoy; Das, Santasabuj; Saha, Sudipto

    2015-01-01

    Protein-protein interactions in Escherichia coli (E. coli) has been studied extensively using high throughput methods such as tandem affinity purification followed by mass spectrometry and yeast two-hybrid method. This can in turn be used to understand the mechanisms of bacterial cellular processes. However, experimental characterization of such huge amount of interactions data is not available for other important enteropathogens. Here, we propose a support vector machine (SVM)-based prediction model using the known PPIs data of E. coli that can be used to predict PPIs in other enteropathogens, such as Vibrio cholerae, Salmonella Typhi, Shigella flexneri and Yersinia entrocolitica. Different features such as domain-domain association (DDA), network topology, and sequence information were used in developing the SVM model. The proposed model using DDA, degree and amino acid composition features has achieved an accuracy of 82% and 62% on 5-fold cross validation and blind E. coli datasets, respectively. The predicted interactions were validated by Gene Ontology (GO) semantic similarity measure and String PPIs database (experimental PPIs only). Finally, we have developed a user-friendly webserver named EnPPIpred to predict intra-species PPIs in enteropathogens, which will be of great help for the experimental biologists. The webserver EnPPIpred is freely available at http://bicresources.jcbose.ac.in/ssaha4/EnPPIpred/. PMID:26717407

  11. Bacterial enteropathogens and factors associated with seasonal episodes of gastroenteritis in Nsukka, Nigeria.

    PubMed

    Nzeako, Basil; Okafor, Nduka

    2002-01-01

    Each year, between April and October, many children of school age and some young adults in Nsukka, Nigeria suffer from gastroenteritis. The period covers the rainy season in this part of Africa, when manured farmland occasionally is flooded. In view of the number of people suffering diarrhoea and occasionally low-grade fever, it became necessary to investigate the nature of the bacterial agents responsible. Between April and October (1996-1998), 500 loose or watery stools were collected from patients, the ages of which ranged from one month to 31 years. Stools that contained parasites were excluded from the study. Samples were cultured on 5% blood agar and 1% egg-yolk agar (both containing 10 microg/mL ampicillin), MacConkey agar, Shigella Salmonella agar and in alkaline peptone water. Bacterial growths were identified using standard bacteriological procedures. Drinking water and some fruit and vegetables prevalent during this period of the year also were cultured. Of the 500 stool samples tested, 138 (27.6%) grew a range of organisms including Aeromonas hydrophila (65 [13%]), Salmonella spp. (55 [11%]), Shigella spp. (9 [1.8%]) and enteropathogenic Escherichia coli (9 [1.8%]). Drinking water and some vegetables grew Pseudomonas aeruginosa and Enterococcus faecalis, respectively. The highest isolation rate occurred during June and July, corresponding to the period of greatest flooding of arable land. Although no enteropathogens were isolated from the fruit and vegetables examined, they contained E. faecalis--an organism found in faeces. Our findings failed to explain why 72% of the samples grew no bacterial enteropathogens. PMID:12113407

  12. Risk of diarrhoea from shallow groundwater contaminated with enteropathogens in the Kathmandu Valley, Nepal.

    PubMed

    Shrestha, Sadhana; Haramoto, Eiji; Malla, Rabin; Nishida, Kei

    2015-03-01

    Shallow groundwater is the main water source among many alternatives in the Kathmandu Valley, Nepal, which has a rapidly growing population and intermittent piped water supply. Although human pathogens are detected in groundwater, its health effects are unclear. We estimated risk of diarrhoea from shallow groundwater use using quantitative microbial risk assessment. Escherichia coli, Giardia cyst and Cryptosporidium oocyst levels were analysed in dug and tube wells samples. E. coli concentrations were converted to those of enteropathogenic E. coli (EPEC). Risks from EPEC in dug wells and from Cryptosporidium and Giardia in both dug and tube wells were higher than the acceptable limit (<10⁻⁴ infections/person-year) for both drinking and bathing exposures. Risk from protozoan enteropathogens increased the total risk 10,000 times, indicating that ignoring protozoans could lead to serious risk underestimation. Bathing exposure considerably increased risk, indicating that it is an important pathway. Point-of-use (POU) water treatment decreased the risk six-fold and decreased risk overestimation. Because removal efficiency of POU water treatment has the largest impact on total risk, increasing the coverage and efficiency of POU water treatment could be a practical risk management strategy in the Kathmandu Valley and similar settings. PMID:25719484

  13. Simultaneous Detection of Enteropathogenic E. coli and Shiga Toxin-Producing E. coli by Polymerase Chain Reaction

    NASA Astrophysics Data System (ADS)

    Alhaj, N.; Mariana, N. S.; Raha, A. R.; Ishak, Z.

    A PCR for detection of two categories of diarrheagenic Escherichia coli was developed. This method proved to be specific and rapid in detecting virulence genes from enteropathogenic E. coli (EPEC) (eae and bfp) and Shiga toxin-producing E. coli (STEC) (stx1, stx2 and eae) from seventy isolates of various sources. Present results confirm that it is possible and feasible to perform a simultaneous amplification of the virulence genes from two categories of diarrheagenic E. coli (STEC, EPEC) and that this technique becoming a novel diagnostic tool for future water food-borne outbreaks studies.

  14. Evaluation of a multiplex PCR assay for simultaneous detection of bacterial and viral enteropathogens in stool samples of paediatric patients.

    PubMed

    Onori, Manuela; Coltella, Luana; Mancinelli, Livia; Argentieri, Marta; Menichella, Donato; Villani, Alberto; Grandin, Annalisa; Valentini, Diletta; Raponi, Massimiliano; Russo, Cristina

    2014-06-01

    We evaluated a multiplex PCR assay, the Seeplex Diarrhoea ACE detection, that simultaneously detects 15 enteric pathogens, including Salmonella spp., Shigella spp., Vibrio spp., toxin B producer Clostridium difficile, Campylobacter spp., Clostridium perfringens, Yersinia enterocolitica, Aeromonas spp., Escherichia coli O157:H7, verocytotoxin-producing Escherichia coli, adenovirus, Group A rotavirus, norovirus GI and GII, and astrovirus. We compared this assay with clinical methods routinely used in our laboratory, for detecting enteropathogens in stool samples collected from 245 paediatric patients with suspected infectious gastroenteritis. We recovered 61 bacterial pathogens and 121 enteric viruses with our laboratory assays, while we detected 78 bacteria and 167 viruses with the molecular assay. We calculated specificity and sensitivity for both methods after analysis of discordant results and demonstrated greater sensitivity for multiplex PCR than for our routine methods, with the exception of Salmonella spp. and toxigenic C. difficile detection. The multiplex PCR assay proved to be a reliable tool to directly detect the most common enteropathogens in stool samples but with some limitations. PMID:24656922

  15. Porcine E. coli: Virulence-Associated Genes, Resistance Genes and Adhesion and Probiotic Activity Tested by a New Screening Method

    PubMed Central

    Schierack, Peter; Rödiger, Stefan; Kuhl, Christoph; Hiemann, Rico; Roggenbuck, Dirk; Li, Ganwu; Weinreich, Jörg; Berger, Enrico; Nolan, Lisa K.; Nicholson, Bryon; Römer, Antje; Frömmel, Ulrike; Wieler, Lothar H.; Schröder, Christian

    2013-01-01

    We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars. PMID:23658605

  16. Membrane cholesterol plays an important role in enteropathogen adhesion and the activation of innate immunity via flagellin-TLR5 signaling.

    PubMed

    Zhou, Mingxu; Duan, Qiangde; Li, Yinchau; Yang, Yang; Hardwidge, Philip R; Zhu, Guoqiang

    2015-08-01

    Lipid rafts are cholesterol- and sphingolipid-rich ordered microdomains distributed in the plasma membrane that participates in mammalian signal transduction pathways. To determine the role of lipid rafts in mediating interactions between enteropathogens and intestinal epithelial cells, membrane cholesterol was depleted from Caco-2 and IPEC-J2 cells using methyl-β-cyclodextrin. Cholesterol depletion significantly reduced Escherichia coli and Salmonella enteritidis adhesion and invasion into intestinal epithelial cells. Complementation with exogenous cholesterol restored bacterial adhesion to basal levels. We also evaluated the role of lipid rafts in the activation of Toll-like receptor 5 signaling by bacterial flagellin. Depleting membrane cholesterol reduced the ability of purified recombinant E. coli flagellin to activate TLR5 signaling in intestinal cells. These data suggest that both membrane cholesterol and lipid rafts play important roles in enteropathogen adhesion and contribute to the activation of innate immunity via flagellin-TLR5 signaling. PMID:25935453

  17. ENTEROPATHOGENS DETECTED IN A DAYCARE CENTER, SOUTHEASTERN BRAZIL: BACTERIA, VIRUS, AND PARASITE RESEARCH

    PubMed Central

    Castro, Edna Donizetti Rossi; Germini, Marcela Cristina Braga Yassaka; Mascarenhas, Joana D'Arc Pereira; Gabbay, Yvone Benchimol; de Lima, Ian Carlos Gomes; Lobo, Patrícia dos Santos; Fraga, Valéria Daltibari; Conceição, Luciana Moran; Machado, Ricardo Luiz Dantas; Rossit, Andréa Regina Baptista

    2015-01-01

    Introduction: The objective of this study was to determine the prevalence and etiological profile of enteropathogens in children from a daycare center. Methods: From October 2010 to February 2011 stool samples from 100 children enrolled in a government daycare center in the municipality of São José do Rio Preto, in the state of São Paulo, were collected and analyzed. Results: A total of 246 bacteria were isolated in 99% of the fecal samples; 129 were in the diarrheal group and 117 in the non-diarrheal group. Seventy-three strains of Escherichia coli were isolated, 19 of Enterobacter, one of Alcaligenes and one of Proteus. There were 14 cases of mixed colonization with Enterobacter and E. coli. Norovirus and Astrovirus were detected in children with clinical signs suggestive of diarrhea. These viruses were detected exclusively among children residing in urban areas. All fecal samples were negative for the presence of the rotavirus species A and C. The presence of Giardia lamblia, Entamoeba coli, Endolimax nana and hookworm was observed. A significant association was found between food consumption outside home and daycare center and the presence of intestinal parasites. Conclusions: For children of this daycare center, intestinal infection due to pathogens does not seem to have contributed to the occurrence of diarrhea or other intestinal symptoms. The observed differences may be due to the wide diversity of geographical, social and economic characteristics and the climate of Brazil, all of which have been reported as critical factors in the modulation of the frequency of different enteropathogens. PMID:25651323

  18. Genetic Fusions of Heat-Labile (LT) and Heat-Stable (ST) Toxoids of Porcine Enterotoxigenic Escherichia coli Elicit Neutralizing Anti-LT and Anti-STa antibodies ▿

    PubMed Central

    Zhang, Weiping; Zhang, Chengxian; Francis, David H.; Fang, Ying; Knudsen, David; Nataro, James P.; Robertson, Donald C.

    2010-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and farm animals. E. coli fimbriae, or colonization factor antigens (CFAs), and enterotoxins, including heat-labile enterotoxins (LT) and heat-stable enterotoxins (ST), are the key virulence factors in ETEC diarrhea. Unlike fimbriae or LT, STa has not often been included as an antigen in development of vaccines against ETEC diarrhea because of its poor immunogenicity. STa becomes immunogenic only after being coupled with a strongly immunogenic carrier protein. However, native or shorter STa antigens either had to retain toxic activity in order to become antigenic or elicited anti-STa antibodies that were not sufficiently protective. In this study, we genetically mutated the porcine LT (pLT) gene for a pLT192(R→G) toxoid and the porcine STa (pSTa) gene for three full-length pSTa toxoids [STa11(N→K), STa12(P→F), and STa13(A→Q)] and used the full-length pLT192 as an adjuvant to carry the pSTa toxoid for pLT192:pSTa-toxoid fusion antigens. Rabbits immunized with pLT192:pSTa12 or pLT192:pSTa13 fusion protein developed high titers of anti-LT and anti-STa antibodies. Furthermore, rabbit antiserum and antifecal antibodies were able to neutralize purified cholera toxin (CT) and STa toxin. In addition, preliminary data suggested that suckling piglets born by a sow immunized with the pLT192:pSTa13 fusion antigen were protected when challenged with an STa-positive ETEC strain. This study demonstrated that pSTa toxoids are antigenic when fused with a pLT toxoid and that the elicited anti-LT and anti-STa antibodies were protective. This fusion strategy could provide instructive information to develop effective toxoid vaccines against ETEC-associated diarrhea in animals and humans. PMID:19858307

  19. Inhibition of enteropathogens adhesion to human enterocyte-like HT-29 cells by a dairy strain of Propionibacterium acidipropionici.

    PubMed

    Zárate, G; Palacios, J M; Villena, J; Zúñiga-Hansen, M E

    2016-06-01

    Adhesion to the host intestinal mucosa is considered relevant for orally delivered probiotics as it prolongs their persistence in the gut and their health promoting effects. Classical propionibacteria are microorganisms of interest due to their role as dairy starters as well as for their functions as probiotics. Propionibacterium acidipropionici Q4, is a dairy strain isolated from a Swiss-type cheese made in Argentina that displays probiotic potential. In the present work we assessed the ability of this strain to adhere to the human enterocyte-like HT-29 cell line and to counteract the adhesion of two common human enteropathogens, such as Escherichia coli C3 and Salmonella Enteritidis 90/390. The results were compared with those obtained with the well-known probiotic Lactobacillus rhamnosus GG. P. acidipropionici Q4 showed a high adhesion capacity, even higher than the reference strain L. rhamnosus GG (42.3±4.4% and 36.2±2.3%, respectively), whereas adhesion of enteropathogens was significantly lower (25.2±2.2% for E. coli and 21.0±3.4% for S. Enteritidis). Propionibacteria as well as lactobacilli were able to inhibit by exclusion and competition the adherence of E. coli C3 and S. Enteritidis 90/390 whereas only L. rhamnosus GG displaced S. Enteritidis from HT-29 intestinal cells. Inhibition of pathogens by propionibacteria was not exerted by antimicrobials or coaggregation but was mainly due to exclusion by cell surface components, such as proteins and carbohydrates. The relevance of cell surface proteins (CSP) for preventing pathogens infection was confirmed by their concentration dependent effect observed for both pathogens: 100 µg/ml of CSP inhibited E. coli attachment almost as untreated propionibacteria, whereas it partially inhibited the attachment of S. Enteritidis. Results suggest that P. acidipropionici Q4 could be considered for the development of propionibacteria containing functional foods helpful in counteracting enteropathogen infection. PMID

  20. Both enzymatic and non-enzymatic properties of heat-labile enterotoxin are responsible for LT-enhanced adherence of enterotoxigenic Escherichia coli to porcine IPEC-J2 cells.

    PubMed

    Fekete, Peter Z; Mateo, Kristina S; Zhang, Weiping; Moxley, Rodney A; Kaushik, Radhey S; Francis, David H

    2013-06-28

    Previous studies in piglets indicate that heat labile enterotoxin (LT) expression enhances intestinal colonization by K88 adhesin-producing enterotoxigenic Escherichia coli (ETEC) as wild-type ETEC adhered to intestinal epithelium in substantially greater numbers than did non-toxigenic constructs. Enzymatic activity of the toxin was also shown to contribute to the adhesion of ETEC and non-ETEC bacteria to epithelial cells in culture. To further characterize the contribution of LT to host cell adhesion, a nontoxigenic, K88-producing E. coli was transformed with either the gene encoding for LT holotoxin, a catalytically-attenuated form of the toxin [LT(R192G)], or LTB subunits, and resultant changes in bacterial adherence to IPEC-J2 porcine intestinal epithelial cells were measured. Strains expressing LT holotoxin or mutants were able to adhere in significantly higher numbers to IPEC-J2 cells than was an isogenic, toxin-negative construct. LT+ strains were also able to significantly block binding of a wild-type LT+ ETEC strain to IPEC-J2 cells. Adherence of isogenic strains to IPEC-J2 cells was unaltered by cycloheximide treatment, suggesting that LT enhances ETEC adherence to IPEC-J2 cells independent of host cell protein synthesis. However, pretreating IPEC-J2 cells with LT promoted adherence of negatively charged latex beads (a surrogate for bacteria which carry a negative change), which adherence was inhibited by cycloheximide, suggesting LT may induce a change in epithelial cell membrane potential. Overall, these data suggest that LT may enhance ETEC adherence by promoting an association between LTB and epithelial cells, and by altering the surface charge of the host plasma membrane to promote non-specific adherence. PMID:23517763

  1. High Detection Rates of Enteropathogens in Asymptomatic Children Attending Day Care

    PubMed Central

    Enserink, Remko; Scholts, Rianne; Bruijning-Verhagen, Patricia; Duizer, Erwin; Vennema, Harry; de Boer, Richard; Kortbeek, Titia; Roelfsema, Jeroen; Smit, Henriette; Kooistra-Smid, Mirjam; van Pelt, Wilfrid

    2014-01-01

    Background Gastroenteritis morbidity is high among children under the age of four, especially amongst those who attend day care. Objective To determine the prevalence of a range of enteropathogens in the intestinal flora of children attending day care and to relate their occurrence with characteristics of the sampled child and the sampling season. Methods We performed three years of enteropathogen surveillance in a network of 29 child day care centers in the Netherlands. The centers were instructed to take one fecal sample from ten randomly chosen children each month, regardless of gastrointestinal symptoms at time of sampling. All samples were analyzed for the molecular detection of 16 enteropathogenic bacteria, parasites and viruses by real-time multiplex PCR. Results Enteropathogens were detected in 78.0% of the 5197 fecal samples. Of the total, 95.4% of samples were obtained from children who had no gastroenteritis symptoms at time of sampling. Bacterial enteropathogens were detected most often (most prevalent EPEC, 19.9%), followed by parasitic enteropathogens (most prevalent: D. fragilis, 22.1%) and viral enteropathogens (most prevalent: norovirus, 9.5%). 4.6% of samples related to children that experienced symptoms of gastroenteritis at time of sampling. Only rotavirus and norovirus were significantly associated with gastroenteritis among day care attendees. Conclusions Our study indicates that asymptomatic infections with enteropathogens in day care attendees are not a rare event and that gastroenteritis caused by infections with these enteropathogens is only one expression of their presence. PMID:24586825

  2. Effect of plasmid pTENT2 on severity of porcine post-weaning diarrhoea induced by an O149 enterotoxigenic Escherichia coli.

    PubMed

    Goswami, Priti S; Gyles, Carlton L; Friendship, Robert M; Poppe, Cornelis; Kozak, Gosia K; Boerlin, Patrick

    2008-10-15

    A particularly virulent O149:H10 enterotoxigenic Escherichia coli clone harbours a newly characterized plasmid pTENT2 carrying the tetracycline-resistance tetA and the virulence genes estA, paa, and sepA that were not present in less virulent clones. The objectives of this study were to assess whether the additional genes on pTENT2 played a role in the increased severity of post-weaning diarrhoea and if they provided any potential advantage for the emergence of the highly virulent clone. Groups of pigs were dosed orally with isogenic pTENT2-positive and pTENT2-negative ETEC strains, and the clinical and pathological changes were compared between the groups. Two additional groups were given the pTENT2-positive strains and maintained on feed with or without chlortetracycline to assess the effect of subtherapeutic levels of tetracycline on the short-term persistence of the ETEC O149:H10 clone. The severity of diarrhoea within the first few hours post-inoculation was significantly increased (p=0.0408) in animals receiving pTENT2-positive strains as compared to animals receiving pTENT2-negative strains. There were no consistent or significant histopathological differences between any of the groups and no significant difference in the persistence of ETEC between groups. PMID:18502055

  3. Quinolone-Resistant Escherichia coli O127a:K63 Serotype with an Extended-Spectrum-Beta-Lactamase Phenotype from a Food Poisoning Outbreak in China

    PubMed Central

    Hao, Rongzhang; Qiu, Shaofu; Yang, Guang; Su, Wenli; Song, Lixue; Zhang, Jia; Chen, Jiaxu; Jia, Leili; Wang, Ligui

    2012-01-01

    We report an atypical enteropathogenic Escherichia coli O127a:K63 strain with resistance to quinolones and extended-spectrum cephalosporins isolated from a 2010 food poisoning outbreak involving 112 adults in China. Two resistance genes [blaCTX-M-15, aac(6′)-Ib-c] and five mutations (two in gyrA, two in parC, one in parE) coexisted in this enteropathogenic E. coli strain. PMID:22553233

  4. Prevalence and antimicrobial resistance of porcine O157 and non-O157 Shiga toxin-producing Escherichia coli from India.

    PubMed

    Rajkhowa, Swaraj; Sarma, Dilip Kumar

    2014-08-01

    The aims of this study were to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains in pigs as a possible STEC reservoir in India as well as to characterize the STEC strains and to determine the antimicrobial resistance pattern of the strains. A total of 782 E. coli isolates from clinically healthy (n = 473) and diarrhoeic piglets (309) belonging to major pig-producing states of India were screened by the polymerase chain reaction (PCR) assay for the presence of virulence genes characteristic for STEC, that is, Shiga toxin-producing gene(s) (stx1, stx2), intimin (eae), enterohemolysin (hlyA) and STEC autoagglutinating adhesin (Saa). Overall STEC were detected in 113 (14.4%) piglets, and the prevalence of E. coli O157 and non-O157 STEC were 4 (0.5%) and 109 (13.9%), respectively. None of the O157 STEC isolates carried gene encoding for H7 antigen (fliCh7). The various combinations of virulence genes present in the strains studied were stx1 in 4.6%, stx1 in combination with stx2 gene in 5.1%, stx1 in combination with stx2 and ehxA in 0.6%, stx1 in combination with stx2 and eae in 0.2% and stx2 alone in 3.7%. All STEC isolates were found negative for STEC autoagglutinating adhesin (Saa). The number of STEC isolates which showed resistance to antimicrobials such as ampicillin, tetracycline, streptomycin, lincomycin, nalidixic acid, sulfadiazine, penicillin, gentamicin, kanamycin and ceftriaxone were 100, 99, 98, 97, 95, 94, 92, 88, 85 and 85, respectively. Ninety-seven isolates showed resistance to more than 2 antimicrobials, and 8 resistance groups (R1 to R8) were observed. This study demonstrates that pigs in India harbour both O157 and non-O157 STEC, and this may pose serious public health problems in future. PMID:24743858

  5. Diarrheagenic Escherichia coli

    PubMed Central

    Nataro, James P.; Kaper, James B.

    1998-01-01

    Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler’s diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (enteropathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens. PMID:9457432

  6. Effects of the Probiotic Enterococcus faecium and Pathogenic Escherichia coli Strains in a Pig and Human Epithelial Intestinal Cell Model

    PubMed Central

    Lodemann, Ulrike; Strahlendorf, Julia; Schierack, Peter; Klingspor, Shanti; Aschenbach, Jörg R.

    2015-01-01

    The aim of this study has been to elucidate the effect of the probiotic Enterococcus faecium NCIMB 10415 on epithelial integrity in intestinal epithelial cells and whether pre- and coincubation with this strain can reproducibly prevent damage induced by enterotoxigenic (ETEC) and enteropathogenic Escherichia coli (EPEC). Porcine (IPEC-J2) and human (Caco-2) intestinal epithelial cells were incubated with bacterial strains and epithelial integrity was assessed by measuring transepithelial electrical resistance (TEER) and mannitol flux rates. E. faecium alone increased TEER of Caco-2 cells without affecting mannitol fluxes whereas the E. coli strains decreased TEER and concomitantly increased mannitol flux rates in both cell lines. Preincubation with E. faecium had no effect on the TEER decrease induced by E. coli in preliminary experiments. However, in a second set of experiments using a slightly different protocol, E. faecium ameliorated the TEER decrease induced by ETEC at 4 h in IPEC-J2 and at 2, 4, and 6 h in Caco-2 cells. We conclude that E. faecium positively affected epithelial integrity in monoinfected Caco-2 cells and could ameliorate the damage on TEER induced by an ETEC strain. Reproducibility of the results is, however, limited when experiments are performed with living bacteria over longer periods. PMID:25883829

  7. Frequency of virulence genes of Escherichia coli among newborn piglets from an intensive pig farm in Argentina.

    PubMed

    Alustiza, Fabrisio E; Picco, Natalia Y; Bellingeri, Romina V; Terzolo, Horacio R; Vivas, Adriana B

    2012-01-01

    The enterotoxigenic and porcine enteropathogenic Escherichia coli (EtEc and PEPEc) strains are agents associated with swine neonatal diarrhea, causing economic losses in swine production. The main goal of this study was to identify virulence genes of EtEc, verotoxigenic (VtEc) and PEPEc in intestinal strains responsible for swine diseases, by molecular typing using Pcr in newborn piglets from an intensive farm system. Two hundred and sixty seven rectal swabbings from 7-15 days- old landrace x large White crossbred piglets were taken, and 123 randomly selected samples, biochemically compatible with E. coli, were tested for E. coli virulence genes by Pcr. A frequency (%) compatible with: 68 EtEc, 24 VtEc, and 8 EPEc were found. of all E. coli strains studied, 19.51 % carried at least one virulence gene. These data showed conclusively that, in spite of the application of strict sanitary measures in the intensive farm, genes encoding virulence factors of intestinal pathogens compatible with EtEc are still detected; therefore these strains will probably keep circulating among animals. PMID:23267620

  8. Enteropathogens associated with diarrhea among military personnel during Operation Bright Star 96, in Alexandria, Egypt.

    PubMed

    Oyofo, B A; Peruski, L F; Ismail, T F; el-Etr, S H; Churilla, A M; Wasfy, M O; Petruccelli, B P; Gabriel, M E

    1997-06-01

    This study investigated the microbial causes of diarrheal disease among U.S. troops deployed near Alexandria, Egypt, during October 1995. Bacterial causes associated with 19 cases of diarrhea included: enterotoxigenic Escherichia coli (ETEC), 42% (21% heat-stable, 11% heat-labile, and 11% heat-stable/ heat-labile producers); enteropathogenic E. coli (5.3%); and enteroadherent E. coli (42%). Four cases of diarrhea were associated with enteroaggregative E. coli based on probe analysis for enteroaggregative heat-stable enterotoxin 1. Protozoan causes included; Entamoeba histolytica (11%), E. hartmanni (5%), E. nana (5%), Blastocystis hominis (5%), Chilomastix mesnili (11%), Dientamoeba fragilis (5%), Entamoeba coli (5%), and Cryptosporidium (5%). Shigella, Aeromonas, Plesiomonas, Vibrio, Campylobacter, and Salmonella were not detected. Of the eight ETEC cases, one was colonization factor antigen (CFA)/I only, one was both CFA/I and CFA/III, three were CFA/II, two were CFA/IV, and two were CFA-negative. Antibiograms of the ETEC and enteroadherent E. coli strains showed that all isolates were susceptible to norfloxacin, ciprofloxacin, and nalidixic acid but resistant to ampicillin, tetracycline, chloramphenicol, and sulfamethoxazole. PMID:9183160

  9. Evolution of atypical enteropathogenic E. coli by repeated acquisition of LEE pathogenicity island variants.

    PubMed

    Ingle, Danielle J; Tauschek, Marija; Edwards, David J; Hocking, Dianna M; Pickard, Derek J; Azzopardi, Kristy I; Amarasena, Thakshila; Bennett-Wood, Vicki; Pearson, Jaclyn S; Tamboura, Boubou; Antonio, Martin; Ochieng, John B; Oundo, Joseph; Mandomando, Inácio; Qureshi, Shahida; Ramamurthy, Thandavarayan; Hossain, Anowar; Kotloff, Karen L; Nataro, James P; Dougan, Gordon; Levine, Myron M; Robins-Browne, Roy M; Holt, Kathryn E

    2016-01-01

    Atypical enteropathogenic Escherichia coli (aEPEC) is an umbrella term given to E. coli that possess a type III secretion system encoded in the locus of enterocyte effacement (LEE), but lack the virulence factors (stx, bfpA) that characterize enterohaemorrhagic E. coli and typical EPEC, respectively. The burden of disease caused by aEPEC has recently increased in industrialized and developing nations, yet the population structure and virulence profile of this emerging pathogen are poorly understood. Here, we generated whole-genome sequences of 185 aEPEC isolates collected during the Global Enteric Multicenter Study from seven study sites in Asia and Africa, and compared them with publicly available E. coli genomes. Phylogenomic analysis revealed ten distinct widely distributed aEPEC clones. Analysis of genetic variation in the LEE pathogenicity island identified 30 distinct LEE subtypes divided into three major lineages. Each LEE lineage demonstrated a preferred chromosomal insertion site and different complements of non-LEE encoded effector genes, indicating distinct patterns of evolution of these lineages. This study provides the first detailed genomic framework for aEPEC in the context of the EPEC pathotype and will facilitate further studies into the epidemiology and pathogenicity of EPEC by enabling the detection and tracking of specific clones and LEE variants. PMID:27571974

  10. Effect of bovine lactoferricin on enteropathogenic Yersinia adhesion and invasion in HEp-2 cells.

    PubMed

    Di Biase, Assunta Maria; Tinari, Antonella; Pietrantoni, Agostina; Antonini, Giovanni; Valenti, Piera; Conte, Maria Pia; Superti, Fabiana

    2004-05-01

    Bovine lactoferricin, a pepsin-generated antimicrobial peptide from bovine lactoferrin active against a wide range of bacteria, was tested for its ability to influence the adhesion and invasion of Yersinia enterocolitica and Yersinia pseudotuberculosis in HEp-2 cells. The addition of non-cytotoxic and non-bactericidal concentrations of lactoferricin to cell monolayers before infection, under different bacterial growth experimental conditions, was ineffective or resulted in about a 10-fold increase in bacterial adhesion, whereas, in bacteria grown in conditions allowing maximal inv gene expression, a 10-fold inhibition of cell invasion by lactoferricin was observed. To confirm that the anti-invasive activity of lactoferricin was exerted against invasin-mediated bacterial entry, experiments were also performed utilizing Escherichia coli strain HB101 (pRI203), harbouring the inv gene from Y. pseudotuberculosis, which allows penetration of mammalian cells. Under these experimental conditions, lactoferricin was able to inhibit bacterial entry into epithelial cells, demonstrating that this peptide acts on inv-mediated Yersinia species invasion. As the inv gene product is the most important virulence factor in enteropathogenic Yersinia, being responsible for bacterial adherence and penetration within epithelial cells of the intestinal lumen and for the subsequent colonization of regional lymph nodes, these data provide additional information on the protective role of lactoferricin against bacterial infection. PMID:15096550

  11. Enteropathogenic E. coli-induced barrier function alteration is not a consequence of host cell apoptosis

    PubMed Central

    Viswanathan, V. K.; Weflen, Andrew; Koutsouris, Athanasia; Roxas, Jennifer L.; Hecht, Gail

    2012-01-01

    Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen that perturbs intestinal epithelial function. Many of the alterations in the host cells are mediated by effector molecules that are secreted directly into epithelial cells by the EPEC type III secretion system. The secreted effector molecule EspF plays a key role in redistributing tight junction proteins and altering epithelial barrier function. EspF has also been shown to localize to mitochondria and trigger membrane depolarization and eventual host cell death. The relationship, if any, between EspF-induced host cell death and epithelial barrier disruption is presently not known. Site-directed mutation of leucine 16 (L16E) of EspF impairs both mitochondrial localization and consequent host cell death. Although the mutation lies within a region critical for type III secretion, EspF(L16E) is secreted efficiently from EPEC. Despite its inability to promote cell death, EspF(L16E) was not impaired for tight junction alteration or barrier disruption. Consistent with this, the pan-caspase inhibitor Q-VD-OPH, despite reducing EPEC-induced host cell death, had no effect on infection-mediated barrier function alteration. Thus EPEC alters the epithelial barrier independent of its ability to induce host cell death. PMID:18356531

  12. Fast and Sensitive Detection of Enteropathogenic Yersinia by Immunoassays

    PubMed Central

    Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe

    2014-01-01

    Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 103 CFU/ml to 8.8 × 104 CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 105 CFU/ml to 106 CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. PMID:25355759

  13. Fast and sensitive detection of enteropathogenic Yersinia by immunoassays.

    PubMed

    Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe; Simon, Stéphanie

    2015-01-01

    Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 10(3) CFU/ml to 8.8 × 10(4) CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 10(5) CFU/ml to 10(6) CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. PMID:25355759

  14. Antigenic Relationships among Porcine Epidemic Diarrhea Virus and Transmissible Gastroenteritis Virus Strains

    PubMed Central

    Lin, Chun-Ming; Gao, Xiang; Oka, Tomoichiro; Vlasova, Anastasia N; Esseili, Malak A.

    2015-01-01

    ABSTRACT Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are economically important swine enteropathogenic coronaviruses. These two viruses belong to two distinct species of the Alphacoronavirus genus within Coronaviridae and induce similar clinical signs and pathological lesions in newborn piglets, but they are presumed to be antigenically distinct. In the present study, two-way antigenic cross-reactivity examinations between the prototype PEDV CV777 strain, three distinct U.S. PEDV strains (the original highly virulent PC22A, S indel Iowa106, and S 197del PC177), and two representative U.S. TGEV strains (Miller and Purdue) were conducted by cell culture immunofluorescent (CCIF) and viral neutralization (VN) assays. None of the pig TGEV antisera neutralized PEDV and vice versa. One-way cross-reactions were observed by CCIF between TGEV Miller hyperimmune pig antisera and all PEDV strains. Enzyme-linked immunosorbent assays, immunoblotting using monoclonal antibodies and Escherichia coli-expressed recombinant PEDV and TGEV nucleocapsid (N) proteins, and sequence analysis suggested at least one epitope on the N-terminal region of PEDV/TGEV N protein that contributed to this cross-reactivity. Biologically, PEDV strain CV777 induced greater cell fusion in Vero cells than did U.S. PEDV strains. Consistent with the reported genetic differences, the results of CCIF and VN assays also revealed higher antigenic variation between PEDV CV777 and U.S. strains. IMPORTANCE Evidence of antigenic cross-reactivity between porcine enteric coronaviruses, PEDV and TGEV, in CCIF assays supports the idea that these two species are evolutionarily related, but they are distinct species defined by VN assays. Identification of PEDV- or TGEV-specific antigenic regions allows the development of more specific immunoassays for each virus. Antigenic and biologic variations between the prototype and current PEDV strains could explain, at least partially, the

  15. Inheritance of porcine receptors for enterotoxigenic Escherichia coli with fimbriae F4ad and their relation to other F4 receptors.

    PubMed

    Rampoldi, A; Bertschinger, H U; Bürgi, E; Dolf, G; Sidler, X; Bratus, A; Vögeli, P; Neuenschwander, S

    2014-06-01

    Enteric Escherichia coli infections are a highly relevant cause of disease and death in young pigs. Breeding genetically resistant pigs is an economical and sustainable method of prevention. Resistant pigs are protected against colonization of the intestine through the absence of receptors for the bacterial fimbriae, which mediate adhesion to the intestinal surface. The present work aimed at elucidation of the mode of inheritance of the F4ad receptor which according to former investigations appeared quite confusing. Intestines of 489 pigs of an experimental herd were examined by a microscopic adhesion test modified in such a manner that four small intestinal sites instead of one were tested for adhesion of the fimbrial variant F4ad. Segregation analysis revealed that the mixed inheritance model explained our data best. The heritability of the F4ad phenotype was estimated to be 0.7±0.1. There are no relations to the strong receptors for variants F4ab and F4ac. Targeted matings allowed the discrimination between two F4ad receptors, that is, a fully adhesive receptor (F4adRFA) expressed on all enterocytes and at all small intestinal sites, and a partially adhesive receptor (F4adRPA) variably expressed at different sites and often leading to partial bacterial adhesion. In pigs with both F4ad receptors, the F4adRPA receptor is masked by the F4adRFA. The hypothesis that F4adRFA must be encoded by at least two complementary or epistatic dominant genes is supported by the Hardy-Weinberg equilibrium statistics. The F4adRPA receptor is inherited as a monogenetic dominant trait. A comparable partially adhesive receptor for variant F4ab (F4abRPA) was also observed but the limited data did not allow a prediction of the mode of inheritance. Pigs were therefore classified into one of eight receptor phenotypes: A1 (F4abRFA/F4acR+/F4adRFA); A2 (F4abRFA/F4acR+/F4adRPA); B (F4abRFA/F4acR+/F4adR-); C1 (F4abRPA/F4acR-/F4adRFA); C2 (F4abRPA/F4acR-/F4adRPA); D1 (F4abR-/F4acR-/F4ad

  16. Evaluation of a Single Procedure Allowing the Isolation of Enteropathogenic Yersinia along with Other Bacterial Enteropathogens from Human Stools

    PubMed Central

    Savin, Cyril; Leclercq, Alexandre; Carniel, Elisabeth

    2012-01-01

    Enteropathogenic Yersinia are among the most frequent agents of human diarrhea in temperate and cold countries. However, the incidence of yersiniosis is largely underestimated because of the peculiar growth characteristics of pathogenic Yersinia, which make their isolation from poly-contaminated samples difficult. The use of specific procedures for Yersinia isolation is required, but is expensive and time consuming, and therefore is not systematically performed in clinical pathology laboratories. A means to circumvent this problem would be to use a single procedure for the isolation of all bacterial enteropathogens. Since the Statens Serum Institut enteric medium (SSI) has been reported to allow the growth at 37°C of most Gram-negative bacteria, including Yersinia, our study aimed at evaluating its performances for Yersinia isolation, as compared to the commonly used Yersinia-specific semi-selective Cefsulodin-Irgasan-Novobiocin medium (CIN) incubated at 28°C. Our results show that Yersinia pseudotuberculosis growth was strongly inhibited on SSI at 37°C, and therefore that this medium is not suitable for the isolation of this species. All Yersinia enterocolitica strains tested grew on SSI, while some non-pathogenic Yersinia species were inhibited. The morphology of Y. enterocolitica colonies on SSI allowed their differentiation from various other Gram-negative bacteria commonly isolated from stool samples. However, in artificially contaminated human stools, the recovery of Y. enterocolitica colonies on SSI at 37°C was difficult and was 3 logs less sensitive than on CIN at 28°C. Therefore, despite its limitations, the use of a specific procedure (CIN incubated at 28°C) is still required for an efficient isolation of enteropathogenic Yersinia from stools. PMID:22911756

  17. Multiplex PCR for Diagnosis of Enteric Infections Associated with Diarrheagenic Escherichia coli

    PubMed Central

    Vidal, Roberto; Vidal, Maricel; Lagos, Rossana; Levine, Myron; Prado, Valeria

    2004-01-01

    A multiplex PCR for detection of three categories of diarrheagenic Escherichia coli was developed. With this method, enterohemorrhagic E. coli, enteropathogenic E. coli, and enterotoxigenic E. coli were identified in fecal samples from patients with hemorrhagic colitis, watery diarrhea, or hemolytic-uremic syndrome and from food-borne outbreaks. PMID:15071051

  18. Single Multiplex Polymerase Chain Reaction To Detect Diverse Loci Associated with Diarrheagenic Escherichia coli

    PubMed Central

    López-Saucedo, Catalina; Cerna, Jorge F.; Villegas-Sepulveda, Nicolas; Thompson, Rocío; Velazquez, F. Raul; Torres, Javier; Tarr, Phillip I.

    2003-01-01

    We developed and tested a single multiplex polymerase chain reaction (PCR) that detects enterotoxigenic, enteropathogenic, enteroinvasive, and Shiga-toxin–producing Escherichia coli. This PCR is specific, sensitive, and rapid in detecting target isolates in stool and food. Because of its simplicity, economy, and efficiency, this protocol warrants further evaluation in large, prospective studies of polymicrobial substances. PMID:12533296

  19. Optimization of Quantitative PCR Methods for Enteropathogen Detection

    PubMed Central

    Liu, Jie; Gratz, Jean; Amour, Caroline; Nshama, Rosemary; Walongo, Thomas; Maro, Athanasia; Mduma, Esto; Platts-Mills, James; Boisen, Nadia; Nataro, James; Haverstick, Doris M.; Kabir, Furqan; Lertsethtakarn, Paphavee; Silapong, Sasikorn; Jeamwattanalert, Pimmada; Bodhidatta, Ladaporn; Mason, Carl; Begum, Sharmin; Haque, Rashidul; Praharaj, Ira; Kang, Gagandeep; Houpt, Eric R.

    2016-01-01

    Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen’s extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease. PMID:27336160

  20. Porcine astrovirus viremia and high genetic variability in pigs on large holdings in Croatia.

    PubMed

    Brnić, Dragan; Prpić, Jelena; Keros, Tomislav; Roić, Besi; Starešina, Vilim; Jemeršić, Lorena

    2013-03-01

    Astroviruses are emerging viral agents, primarily enteropathogenic in mammals, but recently have been acknowledged to have extra-intestinal implications in humans and mink. Porcine astrovirus is thought to be widely distributed and highly prevalent among pigs, nevertheless its clinical significance remains doubtful as it can be detected in diarrheic as well as in healthy pigs. Recent reports imply the immense genetic variability among porcine astrovirus strains with five distinct lineages being characterized so far. Herein, we report porcine astrovirus circulation in the blood of healthy pigs in different age categories bred on two large industrial holdings in Croatia, with viral RNA seroprevalence of 3.89%. These are the first extra-intestinal findings of astrovirus in pigs, indicating a more complex pathogenesis than previously thought. Partial polymerase sequences of serum-derived strains provisionally clustered into porcine astrovirus lineages 2 and 4, sharing high genetic identity with previously described porcine astrovirus strains. The results were supported by detecting porcine astrovirus strains in composite fecal samples, regardless of pig category or holding tested. Phylogenetic analysis of derived strains suggested the presence of porcine astrovirus lineages previously detected in pig sera with an additional highly genetically divergent lineage 5, reported for the first time in Europe. Moreover, the existence of possible sub lineages should not be excluded. The results obtained in the present study, contribute to knowledge of porcine astrovirus pathogenesis; even though it's possible clinical significance remains unclear. High fecal prevalence accompanied with vast genetic diversity on a relatively confined area, underscores the importance of pigs as porcine astrovirus reservoirs with eventual recombination events as a possible outcome. PMID:23313832

  1. Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.

    PubMed Central

    Bernet, M F; Brassart, D; Neeser, J R; Servin, A L

    1993-01-01

    Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains. Images PMID:8285709

  2. Enteropathogenic E. coli: breaking the intestinal tight junction barrier

    PubMed Central

    Singh, Anand Prakash; Aijaz, Saima

    2016-01-01

    Enteropathogenic E. coli (EPEC) causes acute intestinal infections in infants in the developing world. Infection typically spreads through contaminated food and water and leads to severe, watery diarrhea. EPEC attaches to the intestinal epithelial cells and directly injects virulence factors which modulate multiple signaling pathways leading to host cell dysfunction. However, the molecular mechanisms that regulate the onset of diarrhea are poorly defined. A major target of EPEC is the host cell tight junction complex which acts as a barrier and regulates the passage of water and solutes through the paracellular space. In this review, we focus on the EPEC effectors that target the epithelial barrier, alter its functions and contribute to leakage through the tight junctions. PMID:27239268

  3. Response of early-weaned pigs to an enterotoxigenic Escherichia coli (K88) challenge when fed diets containing spray-dried porcine plasma or pea protein isolate plus egg yolk antibody, zinc oxide, fumaric acid, or antibiotic.

    PubMed

    Owusu-Asiedu, A; Nyachoti, C M; Marquardt, R R

    2003-07-01

    The effect of feeding diets containing either spray-dried porcine plasma (SDPP) or pea protein-isolate (PPI) supplemented with either egg yolk antibodies (EYA) from hens immunized with enterotoxigenic Escherichia coli (ETEC) (K88 and F18) antigens, ZnO, fumaric acid (FA), or carbadox (AB) on pig performance, incidence of scours, and gut morphology was studied in a 14-d experiment. Ninety 10-d-old weaned pigs were assigned to six dietary treatments in a completely randomized design to give five pens per treatment with three pigs per pen. The diets were SDPP without EYA (SDPP - EYA), PPI without EYA (PPI - EYA), PPI with EYA (PPI + EYA), PPI with ZnO (PPI + ZnO), PPI with FA (PPI + FA), or PPI with AB (PPI + AB). Diets were formulated to similar nutrient levels, with AB, EYA, FA, and ZnO at 0.25, 0.5, 2.0, and 0.4% of the diet, respectively. Pigs were weighed and bled on d 0, 7, and 14 to determine plasma urea N (PUN). Pigs were orally challenged with a 6-mL dose of 10(10) cfu/mL ETEC (K88) on d 7. On d 14, three pigs per treatment were killed to obtain sections of the small intestine for histological measurements. Weekly feed intake, BW changes, and gain:feed were determined. Incidence of scours and scour scores were monitored and fecal swabs were taken before and after ETEC challenge for PCR test to detect ETEC (K88). Feeding SDPP or supplementing PPI-based diets with EYA, ZnO, FA, or AB did not affect (P > 0.05) ADG, ADFI (as-fed basis), or gain:feed throughout the study. However, pigs fed PPI - EYA tended to have lower (P = 0.08) ADFI during wk 2 (137.9 g/d) and lower (P < 0.10) ADG from d 0 to 14 (100.1 g/d) than those fed the SDPP - EYA (156.6 g/d), PPI + EYA (151.2 g/d), PPI + ZnO (158.9 g/ d), PPI + FA (155.4 g/d), and PPI + AB (152.6 g/d) diets. Although scours was evident in all pigs 8 h after the ETEC challenge, it lasted only 3 to 5 d in pigs fed SDPP or PPI supplemented with EYA, ZnO, FA, or AB. Pigs fed PPI - EYA continued to have severe diarrhea

  4. Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis.

    PubMed

    Scholz, Roland; Imami, Koshi; Scott, Nichollas E; Trimble, William S; Foster, Leonard J; Finlay, B Brett

    2015-07-01

    Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation

  5. Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli.

    PubMed

    Gujral, Tarunmeet; Kumar, Anoop; Priyamvada, Shubha; Saksena, Seema; Gill, Ravinder K; Hodges, Kim; Alrefai, Waddah A; Hecht, Gail A; Dudeja, Pradeep K

    2015-12-15

    Enteropathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl(-)/HCO3(-) exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl(-)/HCO3(-) exchange activity was measured as DIDS-sensitive (125)I(-) uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 μM) increased apical Cl(-)/HCO3(-) exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis. PMID:26447204

  6. Characteristics of child daycare centres associated with clustering of major enteropathogens.

    PubMed

    Pijnacker, R; Mughini-Gras, L; Vennema, H; Enserink, R; VAN DEN Wijngaard, C C; Kortbeek, T; VAN Pelt, W

    2016-09-01

    Insights into transmission dynamics of enteropathogens in children attending daycare are limited. Here we aimed at identifying daycare centre (DCC) characteristics associated with time-clustered occurrence of enteropathogens in DCC-attending children. For this purpose, we used the KIzSS network, which comprises 43 DCCs that participated in infectious disease surveillance in The Netherlands during February 2010-February 2013. Space-time scan statistics were used to identify clusters of rotavirus, norovirus, astrovirus, Giardia lamblia and Cryptosporidium spp. in a two-dimensional DCC characteristic space constructed using canonical correlation analysis. Logistic regression models were then used to further identify DCC characteristics associated with increased or decreased odds for clustering of enteropathogens. Factors associated with increased odds for enteropathogen clustering in DCCs were having indoor/outdoor paddling pools or sandpits, owning animals, high numbers of attending children, and reporting outbreaks to local health authorities. Factors associated with decreased odds for enteropathogen clustering in DCCs were cleaning child potties in designated waste disposal stations, cleaning vomit with chlorine-based products, daily cleaning of toys, extra cleaning of toys during a suspected outbreak, and excluding children with gastroenteritis. These factors provide targets for reducing the burden of gastrointestinal morbidity associated with time-clustered occurrence of major enteropathogens in DCC attendees. PMID:27483376

  7. Homologous recombination plays minor role in excision of unit-length viral genomes from head-to-tail direct tandem repeats of porcine circovirus during DNA replication in Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously, we demonstrated that a theta-replicating bacterial plasmid containing 1.75 copies of genomic porcine circovirus (PCV) DNA in head-to-tail tandem (HTT) [a partial copy of PCV type 1 (PCV1), a complete copy of PCV type 2 (PCV2) and two origins of DNA replication (Ori)] yielded three differ...

  8. The immune adherence receptor CR1-like existed on porcine erythrocytes membrane

    PubMed Central

    Yin, Wei; Cui, Jiaoyan; Jiang, Junbing; Zhao, Junxing; Fan, Kuohai; Sun, Na; Wang, Zhiwei; Sun, Yaogui; Ma, Haili; Li, Hongquan

    2015-01-01

    In the present study, we obtain a mouse anti-porcine complement receptor type 1 (CR1)-like monoclonal antibody (McAb) and use this McAb to verify the existence of CR1-like protein on porcine erythrocytes. Our results confirm that CR1-like protein is localized on the surface of porcine erythrocytes. Mouse immunoglobulin G inhibited the binding of serum-opsonized green fluorescent protein-expressing Escherichia coli to porcine erythrocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicates that CR1-like McAb reacts with biochemically-purified porcine erythrocyte membrane fractions, with a clear band at 135 kDa to 140 kDa. We postulate that the 135 kDa to 140 kDa membrane protein is the equivalent of the porcine erythrocyte CR1-like protein. PMID:26268676

  9. Single Multiplex PCR Assay To Identify Simultaneously the Six Categories of Diarrheagenic Escherichia coli Associated with Enteric Infections

    PubMed Central

    Vidal, Maricel; Kruger, Eileen; Durán, Claudia; Lagos, Rosanna; Levine, Myron; Prado, Valeria; Toro, Cecilia; Vidal, Roberto

    2005-01-01

    We designed a multiplex PCR for the detection of all categories of diarrheagenic Escherichia coli. This method proved to be specific and rapid in detecting virulence genes from Shiga toxin-producing (stx1, stx2, and eae), enteropathogenic (eae and bfp), enterotoxigenic (stII and lt), enteroinvasive (virF and ipaH), enteroaggregative (aafII), and diffuse adherent (daaE) Escherichia coli in stool samples. PMID:16208019

  10. [Bacteriological studies of travellar's diarrhoea. 5) Analysis of enteropathogenic bacteria at Osaka Airport Quarantine Station from January 1992 through September 3rd, 1994].

    PubMed

    Ueda, Y; Suzuki, N; Mori, H; Miyagi, K; Noda, K; Hirose, H; Takegaki, Y; Hashimoto, S; Oosumi, Y; Miyata, Y; Taguchi, M; Ishibashi, M; Honda, T

    1996-01-01

    During the last 2 years and 8 months before the closure of Osaka Airport Quarantine Station (from Jan. 1992 to Sep. 3, 1994), a total of 7,421,909 overseas travellers were quarantined. 15,919 reported themselves of suffering from diarrhoea. Bacteriological examination of a total of 6,031 individuals' stools were performed. 1) Various enteropathogenic bacteria were isolated from 31.2% of the stools examined. Isolated species were as follows: Plesiomonas shigelloides, 1,127 cases (59.9%); Vibrio parahaemolyticus, 293 cases (15.6%); Salmonella spp., 262 cases (13.9%); Shigella spp., 235 cases (12.5%); Aeromonas sobria, 93 cases (4.9%); V. cholerae non-O1, 69 cases (3.7%). 2) The enteropathogenic bacteria were isolated through out the year without any seasonal variation. 3) The major regions where the travellers were infected with the pathogens are as follows: V. cholerae non-O1 (NAG Vibrio) and enteropathogenic Escherichia coli, South-East and South-West Asia; Vibrio other than NAG, South-East and East Asia; Shigella, widely distributed but especially in India; P. shigelloides and Salmonella, widely distributed. 4) 2 strains of toxigenic (cholera toxin-producing) V. cholerae O139 were isolated from patients who had visited Indonesia and Thailand, respectively. 5) In 320 cases (17%), plural enteropathogenic bacteria were isolated from single patients, suggesting a high frequency of the mixed infections. 6) Among Shigella strains, S. sonnei were isolated the most, followed by S. flexneri (24.7%), S. boydii (8.8%) and S. dysenteriae (2.9%). 7) Among Salmonella serovers, Salmonella Enteritidis was isolated the most frequently (39 cases, 14.1%). 8) 218 (91.6%) of 238 Shigella strains and 103 (37.6%) of 276 Salmonella strains were resistant to one or more drugs tested (SM.CP.TC.KM.ABPC.NA.OFLX). 9) All of the 22 V. cholerae O1 strains were Ogawa, E1 Tor. Among them, 19 were toxigenic strains and 3 were non-toxigenic. 10) O4:K8 was the most frequently isolated serover of V

  11. Campylobacters: the most common bacterial enteropathogens in the Nordic countries.

    PubMed

    Rautelin, H; Hänninen, M L

    2000-10-01

    Campylobacters have been known as important human pathogens since the late 1970s. Campylobacter jejuni and coli are the most common bacterial enteropathogens in the developed countries. During the past years an increasing incidence of campylobacteriosis has been reported in many developed countries. C. jejuni is the most common Campylobacter species while C. coli accounts for about 5-10% of the cases. Although the genome of C. jejuni NCTC 11168 strain was sequenced recently, the exact pathogenetic mechanisms are still not known. Furthermore, there are no reliable animal models available. The epidemiology of this common infection is not well understood; however, eating and handling poultry, contaminated drinking water, and contact with pet animals have been recognized as important risk factors. Most of the cases are sporadic although large water-borne outbreaks have also been reported. Discriminatory typing methods are helpful in tracing the sources and transmission routes. In addition to traditional serotyping, genotyping methods, such as pulsed-field gel electrophoresis, have been developed. As Campylobacter infections probably precede Guillan-Barré syndrome in many cases, a great interest has lately been focused on the possible triggering mechanisms underlying this phenomenon. PMID:11087163

  12. Novel porcine repetitive elements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An analysis of 220 fully sequenced porcine BACs generated by the Comparative Vertebrate Sequencing Initiative (http://www.nisc.nih.gov/) revealed 27 distinct, novel porcine repetitive elements ranging in length from 55 to 1059 nucleotides. This set of fully sequenced BACs covers approximately 1% of...

  13. Prevalence of enteropathogens in stools of rural Maasai children under five years of age in the Maasailand region of the Kenyan Rift Valley.

    PubMed

    Joyce, T; McGuigan, K G; Elmore-Meegan, M; Conroy, R M

    1996-01-01

    Stool samples were collected during August 1994 from seventy rural Maasai children under the age of five years who were living in the Maasailand region of the Kenyan Rift Valley. Microbiological analysis was carried out on these samples to identify which intestinal pathogens were present among the infant population of the Maasai. Of the samples studied 54% were pathogen positive. The most common pathogen isolated was Giardia lamblia which was detected in 31% of the samples. Other pathogens that were detected include: Entamoeba histolytica (23%), Enteropathogenic Escherichia coli (13%), Strongyloides stercoralis (4%), Blastocystis hominis (3%) and Cryptosporidium sp (3%). Although all samples were screened for Campylobacter and rotavirus, neither pathogen was detected. Water samples were taken from all the water sources in the study area and analysed microbiologically. Results showed that all the sources were contaminated with the faecal E. coli whose populations ranged from 14 CFU/100 ml to greater than 1800 CFU/100ml. PMID:8625866

  14. Flies and water as reservoirs for bacterial enteropathogens in urban and rural areas in and around Lahore, Pakistan.

    PubMed Central

    Khalil, K.; Lindblom, G. B.; Mazhar, K.; Kaijser, B.

    1994-01-01

    The study was conducted to isolate and characterize campylobacter, enterotoxigenic Escherichia coli-labile toxin (ETEC-LT), shigella and salmonella in flies and water. The material for the study, flies (n = 300) and water samples (n = 148), was collected from different localities in and around Lahore, Pakistan. Cultivation of the samples was performed on conventional standard media. Membrane filtration technique was used for water prior to culture. Determination of ETEC-LT was done by GM1 ELISA. Results of our study showed that flies and water were reservoirs for all the four pathogens, campylobacter, ETEC-LT, shigella and salmonella. Flies from the village were carrying fewer enteropathogens, while water from the village was found to be more contaminated as compared to the city. Campylobacter and ETEC-LT were the most frequently isolated pathogens in both flies and water. Thus the incidence of diarrhoeal disease in children of developing countries may be decreased by providing plenty of safe drinking water, improving excreta disposal, toilet facilities and giving education in personal hygiene. PMID:7995353

  15. The enteropathogenic E. coli effector EspB facilitates microvillus effacing and antiphagocytosis by inhibiting myosin function.

    PubMed

    Iizumi, Yosuke; Sagara, Hiroshi; Kabe, Yasuaki; Azuma, Motoki; Kume, Kanako; Ogawa, Michinaga; Nagai, Takeshi; Gillespie, Peter G; Sasakawa, Chihiro; Handa, Hiroshi

    2007-12-13

    Enteropathogenic Escherichia coli (EPEC) destroys intestinal microvilli and suppresses phagocytosis by injecting effectors into infected cells through a type III secretion system (TTSS). EspB, a component of the TTSS, is also injected into the cytoplasm of host cells. However, the physiological functions of EspB within the host cell cytoplasm remain unclear. We show that EspB binds to myosins, which are a superfamily of proteins that interact with actin filaments and mediate essential cellular processes, including microvillus formation and phagocytosis. EspB inhibits the interaction of myosins with actin, and an EspB mutant that lacks the myosin-binding region maintained its TTSS function but could not induce microvillus effacing or suppress phagocytosis. Moreover, the myosin-binding region of EspB is essential for Citrobacter rodentium, an EPEC-related murine pathogen, to efficiently infect mice. These results suggest that EspB inhibits myosin functions and thereby facilitates efficient infection by EPEC. PMID:18078690

  16. Commensal effect of pectate lyases secreted from Dickeya dadantii on the proliferation of Escherichia coli O157:H7 on lettuce leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The outbreaks of enterohemorrhagic Escherichia coli O157:H7 from leafy greens are serious food-safety concerns at the present period. Several phytopathogens have been suggested to help persistence and proliferation of the human enteropathogens in phyllosphere. In this work, influence of virulence ...

  17. Insights into Evolution of Escherichia coli O157:H7 from Complete Genome Sequence of Closely Related O55:H7 Precursor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enteropathogenic Escherichia coli (EPEC) is a leading cause of infant mortality and morbidity in developing countries. In spite of this, only two EPEC genomes have been fully sequenced: the typical, model EPEC strain E2348/69 (O127:H6), and the contemporary, atypical EPEC strain CB9615 (O55:H7, Ger...

  18. Immunohistochemistry of porcine skin.

    PubMed

    Wollina, U; Berger, U; Mahrle, G

    1991-01-01

    The present paper reports immunohistological findings in porcine skin, which were obtained by use of mono- and polyclonal antihuman antibodies and either alkaline phosphatase anti-alkaline phosphatase (APAAP) or peroxidase (POX) technique. Epidermal staining was observed with antibodies to keratins (K 8.12, RSKE 60), filaggrin, and calmodulin (ACAM). Staining of connective tissue and vessels was achieved using antibodies to vimentin (V9(1)), collagen type IV, and fibronectin. In general, these antibodies gave a staining pattern similar to that of normal human skin. The similarities of immunoreactivity to poly- and monoclonal antihuman antibodies in porcine and human skin render porcine skin a reliable model in biomedical research. PMID:1710864

  19. Case-Control Study of Enteropathogens Associated with Childhood Diarrhea in Dhaka, Bangladesh

    PubMed Central

    Albert, M. John; Faruque, A. S. G.; Faruque, S. M.; Sack, R. B.; Mahalanabis, D.

    1999-01-01

    The International Centre for Diarrhoeal Disease Research, Bangladesh, is a major center for research into diarrheal diseases. The center treats more than 100,000 patients a year. To obtain useful information representative of all patients, a surveillance system in which a 4% systematic sample of all patients is studied in detail, including etiological agents of diarrhea, was installed in October 1979. The first paper on etiology for the surveillance patients was published in 1982, which identified a potential enteric pathogen in 66% of patients. In subsequent years, several new agents of diarrhea have been identified. To assess the importance of a broader spectrum of diarrheal agents including the ones identified relatively recently, we studied 814 children with diarrhea. The children were up to 5 years of age and were part of the surveillance system. They were matched with an equal number of community controls without diarrhea. The study was conducted from February 1993 to June 1994. A potential enteric pathogen was isolated from 74.8% of diarrheal children and 43.9% of control children (P = 0.0001). Even though the first study was not a case-control study, it identified rotavirus, Campylobacter jejuni, enterotoxigenic Escherichia coli, Shigella spp., and Vibrio cholerae O1 as major pathogens. The present study identified these pathogens as being significantly associated with diarrhea. In addition, the study also identified six additional agents, including enteropathogenic E. coli, Aeromonas spp., V. cholerae O139, enterotoxigenic Bacteroides fragilis, Clostridium difficile, and Cryptosporidium parvum, as being significantly associated with diarrhea. Plesiomonas shigelloides, Salmonella spp., diffusely adherent E. coli, enteroaggregative E. coli, Entamoeba histolytica, and Giardia lamblia were not significantly associated with diarrhea. Enteroinvasive E. coli, enterohemorrhagic E. coli, and Cyclospora cayetanensis were not detected in any of the children. The

  20. Molecular Profiling: Catecholamine Modulation of Gene Expression in Enteropathogenic Bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Investigations of the enteric pathogens Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium and Vibrio parahaemolyticus have demonstrated that these bacteria can respond to the presence of catecholamines, including norepinephrine and/or epinephrine, in their environment by modulating g...

  1. Selected enteropathogens and clinical course in children hospitalized with severe acute gastroenteritis in Barbados

    PubMed Central

    Kumar, Alok; Browne, Chantelle; Scotland, Shauna; Krishnamurthy, Kandamaran; Nielsen, Anders L

    2014-01-01

    Objectives The primary aim of this study was to determine the prevalence of selected bacterial and viral enteropathogens in children hospitalized with acute gastroenteritis and the secondary aim was to characterize the clinical course and the outcome. Methodology A retrospective audit of children (<15 years) admitted with acute gastroenteritis during January 2008 to October 2010. Stool samples were analyzed for bacterial pathogens and for the Rotavirus. Demographics, clinical presentations, hospital course and outcome were extracted from the admission records. Results There were 571 children hospitalized with acute gastroenteritis, which accounted for 11% of all medical hospitalization in children. Overall, 42.9% of these children were ≤12 months in age. Stool test result was documented in 46.6% of children hospitalized with gastroenteritis and an enteropathogen was isolated in 36.8% of cases with documented stool test result. Non-typhoidal Salmonella species was the most commonly isolated enteropathogen accounting for 21.1% of all the documented cases. Rotavirus was identified as an etiological agent in 9.0%. Of the 56 children who had non-typhoidal salmonella gastroenteritis, 54(96.4%) were younger than 5 years. The median duration of hospitalization was 2 days (Range 1 day to 9 days). There were no deaths. Conclusion Non-typhoidal salmonella was the most common enteropathogen isolated and this was followed by the Rotavirus. PMID:25780359

  2. The cytological observation of immune adherence of porcine erythrocyte.

    PubMed

    Sun, Yao-Gui; Yin, Wei; Fan, Xin-Feng; Fan, Kuo-Hai; Jiang, Jun-Bing; Li, Hong-Quan

    2012-10-01

    The immune adherence (IA) between the porcine erythrocytes and the opsonized Escherichia coli carried green fluorescent protein gene (GFP-E.coli) were detected by the fluorescence microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) with an attempt to verify the existence of IA between the porcine erythrocytes and complemented-opsonized microbes. Under fluorescence microscopy, GFP-E.coli opsonized by fresh rabbit serum complement adhered to the erythrocytes and could not be detached by PBS washing, and no IA was observed between the erythrocytes and nonopsonized GFP-E.coli after co-incubation. SEM and TEM also revealed the existence of IA between the serum complement-opsonized GFP-E.coli membrane and the erythrocyte membrane. The partial complement receptor type 1 (CR1)-like gene from porcine was generated by RT-PCR and rapid amplification of cDNA 3' end (3' RACE) (157bp and 578bp), both of which have high similarity with published mammal's CR1 gene. The sequences were spliced based on homology comparison and submitted to GenBank (GenBank Accession No. JX033989). These results indicated that the porcine erythrocytes were able to bind to the opsonized microorganisms. Furthermore, the sequencing results confirmed that the CR1-like gene exists in porcine. PMID:23150925

  3. Spectrum of enteropathogens detected by the FilmArray GI Panel in a multicentre study of community-acquired gastroenteritis.

    PubMed

    Spina, A; Kerr, K G; Cormican, M; Barbut, F; Eigentler, A; Zerva, L; Tassios, P; Popescu, G A; Rafila, A; Eerola, E; Batista, J; Maass, M; Aschbacher, R; Olsen, K E P; Allerberger, F

    2015-08-01

    The European, multicentre, quarterly point-prevalence study of community-acquired diarrhoea (EUCODI) analysed stool samples received at ten participating clinical microbiology laboratories (Austria, Finland, France, Germany, Greece, Ireland, Italy, Portugal, Romania, and the UK) in 2014. On four specified days, each local laboratory submitted samples from ≤20 consecutive patients to the Austrian Study Centre for further testing with the FilmArray GI Panel (BioFire Diagnostics, Salt Lake City, UT, USA). Of the 709 samples from as many patients received, 325 (45.8%) tested negative, 268 (37.8%) yielded only one organism, and 116 (16.4%) yielded multiple organisms. Positivity rates ranged from 41% (30 of 73 samples) in France to 74% (59 of 80 samples) in Romania. With the exception of Entamoeba histolytica and Vibrio cholerae, all of the 22 targeted pathogens were detected at least once. Enteropathogenic Escherichia coli, Campylobacter species, toxigenic Clostridium difficile, enteroaggregative E. coli, norovirus and enterotoxigenic E. coli were the six most commonly detected pathogens. When tested according to local protocols, seven of 128 positive samples (5.5%) yielded multiple organisms. Overall, the FilmArray GI Panel detected at least one organism in 54.2% (384/709) of the samples, as compared with 18.1% (128/709) when testing was performed with conventional techniques locally. This underlines the considerable potential of multiplex PCR to improve routine stool diagnostics in community-acquired diarrhoea. Classic culture methods directed at the isolation of specific pathogens are increasingly becoming second-line tools, being deployed when rapid molecular tests give positive results. This optimizes the yield from stool examinations and dramatically improves the timeliness of diagnosis. PMID:25908431

  4. The enteropathogenic E. coli effector EspH promotes actin pedestal formation and elongation via WASP-interacting protein (WIP)

    PubMed Central

    Wong, Alexander R. C.; Raymond, Benoit; Collins, James W.; Crepin, Valerie F.; Frankel, Gad

    2016-01-01

    Summary Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are diarrheagenic pathogens that colonize the gut mucosa via attaching-and-effacing lesion formation. EPEC and EHEC utilize a type III secretion system (T3SS) to translocate effector proteins that subvert host cell signalling to sustain colonization and multiplication. EspH, a T3SS effector that modulates actin dynamics, was implicated in the elongation of the EHEC actin pedestals. In this study we found that EspH is necessary for both efficient pedestal formation and pedestal elongation during EPEC infection. We report that EspH induces actin polymerization at the bacterial attachment sites independently of the Tir tyrosine residues Y474 and Y454, which are implicated in binding Nck and IRSp53/ITRKS respectively. Moreover, EspH promotes recruitment of neural Wiskott–Aldrich syndrome protein (N-WASP) and the Arp2/3 complex to the bacterial attachment site, in a mechanism involving the C-terminus of Tir and the WH1 domain of N-WASP. Dominant negative of WASP-interacting protein (WIP), which binds the N-WASP WH1 domain, diminished EspH-mediated actin polymerization. This study implicates WIP in EPEC-mediated actin polymerization and pedestal elongation and represents the first instance whereby N-WASP is efficiently recruited to the EPEC attachment sites independently of the Tir:Nck and Tir:IRTKS/IRSp53 pathways. Our study reveals the intricacies of Tir and EspH-mediated actin signalling pathways that comprise of distinct, convergent and synergistic signalling cascades. PMID:22372637

  5. Detection and Characterization of Shiga Toxin Producing Escherichia coli, Salmonella spp., and Yersinia Strains from Human, Animal, and Food Samples in San Luis, Argentina.

    PubMed

    Favier, Gabriela Isabel; Lucero Estrada, Cecilia; Cortiñas, Teresa Inés; Escudero, María Esther

    2014-01-01

    Shiga toxin producing Escherichia coli (STEC), Salmonella spp., and Yersinia species was investigated in humans, animals, and foods in San Luis, Argentina. A total of 453 samples were analyzed by culture and PCR. The antimicrobial susceptibility of all the strains was studied, the genomic relationships among isolates of the same species were determined by PFGE, and the potencial virulence of Y. enterocolitica strains was analyzed. Yersinia species showed higher prevalence (9/453, 2.0%, 95% CI, 0.7-3.3%) than STEC (4/453, 0.9%, 95% CI, 0-1.8%) and Salmonella spp. (3/453, 0.7%, 95% CI, 0-1.5%). Y. enterocolitica and Y. intermedia were isolated from chicken carcasses (6/80, 7.5%, 95% CI, 1.5-13.5%) and porcine skin and bones (3/10, 30%, 95% CI, 0-65%). One STEC strain was recovered from human feces (1/70, 1.4%, 95% CI, 0-4.2%) and STEC stx1/stx2 genes were detected in bovine stools (3/129, 2.3%, 95% CI, 0-5.0%). S. Typhimurium was isolated from human feces (1/70, 1.4%, 95% CI, 0-4.2%) while one S. Newport and two S. Gaminara strains were recovered from one wild boar (1/3, 33%, 95% CI, 0-99%). The knowledge of prevalence and characteristics of these enteropathogens in our region would allow public health services to take adequate preventive measures. PMID:25177351

  6. Detection and Characterization of Shiga Toxin Producing Escherichia coli, Salmonella spp., and Yersinia Strains from Human, Animal, and Food Samples in San Luis, Argentina

    PubMed Central

    Favier, Gabriela Isabel; Lucero Estrada, Cecilia; Cortiñas, Teresa Inés; Escudero, María Esther

    2014-01-01

    Shiga toxin producing Escherichia coli (STEC), Salmonella spp., and Yersinia species was investigated in humans, animals, and foods in San Luis, Argentina. A total of 453 samples were analyzed by culture and PCR. The antimicrobial susceptibility of all the strains was studied, the genomic relationships among isolates of the same species were determined by PFGE, and the potencial virulence of Y. enterocolitica strains was analyzed. Yersinia species showed higher prevalence (9/453, 2.0%, 95% CI, 0.7–3.3%) than STEC (4/453, 0.9%, 95% CI, 0–1.8%) and Salmonella spp. (3/453, 0.7%, 95% CI, 0–1.5%). Y. enterocolitica and Y. intermedia were isolated from chicken carcasses (6/80, 7.5%, 95% CI, 1.5–13.5%) and porcine skin and bones (3/10, 30%, 95% CI, 0–65%). One STEC strain was recovered from human feces (1/70, 1.4%, 95% CI, 0–4.2%) and STEC stx1/stx2 genes were detected in bovine stools (3/129, 2.3%, 95% CI, 0–5.0%). S. Typhimurium was isolated from human feces (1/70, 1.4%, 95% CI, 0–4.2%) while one S. Newport and two S. Gaminara strains were recovered from one wild boar (1/3, 33%, 95% CI, 0–99%). The knowledge of prevalence and characteristics of these enteropathogens in our region would allow public health services to take adequate preventive measures. PMID:25177351

  7. Clinical Implications of Enteroadherent Escherichia coli

    PubMed Central

    Arenas-Hernández, Margarita M.P.; Martínez-Laguna, Ygnacio; Torres, Alfredo G.

    2012-01-01

    Pathogenic Escherichia coli that colonize the small intestine primarily cause gastrointestinal illness in infants and travelers. The main categories of pathogenic E. coli that colonize the epithelial lining of the small intestine are enterotoxigenic E. coli enteropathogenic E. coli and enteroaggregative E. coli. These organisms accomplish their pathogenic process by a complex, coordinated multistage strategy, including non-intimate adherence mediated by various adhesins. These so called “enteroadherent E. coli ” categories subsequently produced toxins or effector proteins that are either secreted to the milieu or injected to the host cell. Finally, destruction of the intestinal microvilli results from the intimate adherence or the toxic effect exerted over the epithelia, resulting in water secretion and diarrhea. In this review, we summarize the current state of knowledge regarding these enteroadherent E. coli strains and the present clinical understanding of how these organisms colonize the human intestine and cause disease. PMID:22798032

  8. Clinical implications of enteroadherent Escherichia coli.

    PubMed

    Arenas-Hernández, Margarita M P; Martínez-Laguna, Ygnacio; Torres, Alfredo G

    2012-10-01

    Pathogenic Escherichia coli that colonize the small intestine primarily cause gastrointestinal illness in infants and travelers. The main categories of pathogenic E. coli that colonize the epithelial lining of the small intestine are enterotoxigenic E. coli, enteropathogenic E. coli, and enteroaggregative E. coli. These organisms accomplish their pathogenic process by a complex, coordinated multistage strategy, including nonintimate adherence mediated by various adhesins. These so called "enteroadherent E. coli" categories subsequently produce toxins or effector proteins that are either secreted to the milieu or injected to the host cell. Finally, destruction of the intestinal microvilli results from the intimate adherence or the toxic effect exerted over the epithelia, resulting in water secretion and diarrhea. In this review, we summarize the current state of knowledge regarding these enteroadherent E. coli strains and the present clinical understanding of how these organisms colonize the human intestine and cause disease. PMID:22798032

  9. Cold Shock Proteins: A Minireview with Special Emphasis on Csp-family of Enteropathogenic Yersinia.

    PubMed

    Keto-Timonen, Riikka; Hietala, Nina; Palonen, Eveliina; Hakakorpi, Anna; Lindström, Miia; Korkeala, Hannu

    2016-01-01

    Bacteria have evolved a number of mechanisms for coping with stress and adapting to changing environmental conditions. Many bacteria produce small cold shock proteins (Csp) as a response to rapid temperature downshift (cold shock). During cold shock, the cell membrane fluidity and enzyme activity decrease, and the efficiency of transcription and translation is reduced due to stabilization of nucleic acid secondary structures. Moreover, protein folding is inefficient and ribosome function is hampered. Csps are thought to counteract these harmful effects by serving as nucleic acid chaperons that may prevent the formation of secondary structures in mRNA at low temperature and thus facilitate the initiation of translation. However, some Csps are non-cold inducible and they are reported to be involved in various cellular processes to promote normal growth and stress adaptation responses. Csps have been shown to contribute to osmotic, oxidative, starvation, pH and ethanol stress tolerance as well as to host cell invasion. Therefore, Csps seem to have a wider role in stress tolerance of bacteria than previously assumed. Yersinia enterocolitica and Yersinia pseudotuberculosis are enteropathogens that can spread through foodstuffs and cause an enteric infection called yersiniosis. Enteropathogenic Yersinia are psychrotrophs that are able to grow at temperatures close to 0°C and thus they set great challenges for the modern food industry. To be able to efficiently control psychrotrophic Yersinia during food production and storage, it is essential to understand the functions and roles of Csps in stress response of enteropathogenic Yersinia. PMID:27499753

  10. Prevalence and genetic diversity of enteropathogenic Yersinia spp. in pigs at farms and slaughter in Lithuania.

    PubMed

    Novoslavskij, Aleksandr; Šernienė, Loreta; Malakauskas, Alvydas; Laukkanen-Ninios, Riikka; Korkeala, Hannu; Malakauskas, Mindaugas

    2013-04-01

    The prevalence of enteropathogenic Yersinia spp. in pigs at farms and slaughter in relation to potential farming risk factors in Lithuania was examined. Pig faeces and carcase swab samples from 11 farms were studied at slaughterhouses. Nine of the 11 farms were visited again 3-5 months later, and pooled feacal samples and environmental samples were collected. Pathogenic Yersinia enterocolitica was found in 64% and Yersinia pseudotuberculosis in 45% of the sampled pig farms. All obtained isolates belonged to bioserotypes 4/O:3 and 2/O:3, respectively. Low biosecurity level was associated with a high prevalence of Y. enterocolitica on farms. Characterization with PFGE of 64 Y. enterocolitica and 27 Y. pseudotuberculosis isolates revealed seven and two different genotypes, respectively. Dominant enteropathogenic Yersinia spp. genotypes were obtained in both pig feacal and carcase samples. The high contamination of pig carcases (25%) with enteropathogenic Yersinia spp. may be an important factor contributing to the high incidence of human yersiniosis in Lithuania. PMID:23102547

  11. Cold Shock Proteins: A Minireview with Special Emphasis on Csp-family of Enteropathogenic Yersinia

    PubMed Central

    Keto-Timonen, Riikka; Hietala, Nina; Palonen, Eveliina; Hakakorpi, Anna; Lindström, Miia; Korkeala, Hannu

    2016-01-01

    Bacteria have evolved a number of mechanisms for coping with stress and adapting to changing environmental conditions. Many bacteria produce small cold shock proteins (Csp) as a response to rapid temperature downshift (cold shock). During cold shock, the cell membrane fluidity and enzyme activity decrease, and the efficiency of transcription and translation is reduced due to stabilization of nucleic acid secondary structures. Moreover, protein folding is inefficient and ribosome function is hampered. Csps are thought to counteract these harmful effects by serving as nucleic acid chaperons that may prevent the formation of secondary structures in mRNA at low temperature and thus facilitate the initiation of translation. However, some Csps are non-cold inducible and they are reported to be involved in various cellular processes to promote normal growth and stress adaptation responses. Csps have been shown to contribute to osmotic, oxidative, starvation, pH and ethanol stress tolerance as well as to host cell invasion. Therefore, Csps seem to have a wider role in stress tolerance of bacteria than previously assumed. Yersinia enterocolitica and Yersinia pseudotuberculosis are enteropathogens that can spread through foodstuffs and cause an enteric infection called yersiniosis. Enteropathogenic Yersinia are psychrotrophs that are able to grow at temperatures close to 0°C and thus they set great challenges for the modern food industry. To be able to efficiently control psychrotrophic Yersinia during food production and storage, it is essential to understand the functions and roles of Csps in stress response of enteropathogenic Yersinia. PMID:27499753

  12. Methods of Analysis of Enteropathogen Infection in the MAL-ED Cohort Study

    PubMed Central

    Platts-Mills, James A.; McCormick, Benjamin J. J.; Kosek, Margaret; Pan, William K.; Checkley, William; Houpt, Eric R.

    2014-01-01

    Studies of diarrheal etiology in low- and middle-income countries have typically focused on children presenting with severe symptoms to health centers and thus are best equipped to describe the pathogens capable of leading to severe diarrheal disease. The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study was designed to evaluate, via intensive community surveillance, the hypothesis that repeated exposure to enteropathogens has a detrimental effect on growth, vaccine response, and cognitive development, which are the primary outcome measures for this study. In the setting of multiple outcomes of interest, a longitudinal cohort design was chosen. Because many or even the majority of enteric infections are asymptomatic, the collection of asymptomatic surveillance stools was a critical element. However, capturing diarrheal stools additionally allowed for the determination of the principle causes of diarrhea at the community level as well as for a comparison between those enteropathogens associated with diarrhea and those that are associated with poor growth, diminished vaccine response, and impaired cognitive development. Here, we discuss the analytical methods proposed for the MAL-ED study to determine the principal causes of diarrhea at the community level and describe the complex interplay between recurrent exposure to enteropathogens and these critical long-term outcomes. PMID:25305292

  13. Seasonal Prevalence of Enteropathogenic Vibrio and Their Phages in the Riverine Estuarine Ecosystem of South Bengal

    PubMed Central

    Mookerjee, Subham; Batabyal, Prasenjit; Sarkar, Madhumanti Halder; Palit, Anup

    2015-01-01

    Diarrheal disease remains an unsolved problem in developing countries. The emergence of new etiological agents (non-cholera vibrios) is a major cause of concern for health planners. We attempted to unveil the seasonal dynamics of entero-pathogenic Vibrios in Gangetic riverine-estuarine ecosystem. 120 surface water samples were collected for a period of one year from 3 sampling sites on the Hooghly river. Five enteropathogenic Vibrio species, V. cholerae (35%), V. parahaemolyticus (22.5%), V. mimicus (19.1%), V. alginolyticus (15.8%) and V. vulnificus (11.6%), were present in the water samples. The vibriophages, V. vulnificus ɸ (17.5%), V. alginolyticus ɸ (17.5%), V. parahaemolyticus ɸ (10%), V. cholerae non-O1/O139 ɸ (26.6%) and V. mimicus ɸ (9.1%), were also detected in these samples. The highest number of Vibrios were noted in the monsoon (20–34°C), and to a lesser extent, in the summer (24–36°C) seasons. Samples positive for phages for any of the identified Vibrio species were mostly devoid of that particular bacterial organism and vice versa. The detection of toxin genes and resistance to β-lactam antibiotics in some environmental enteropathogenic Vibrio species in the aquatic niches is a significant outcome. This finding is instrumental in the south Bengal diarrhoeal incidence. PMID:26340543

  14. Prevalence of Diarrheagenic Escherichia coli in Finns with or without Diarrhea during a Round-the-World Trip

    PubMed Central

    Keskimäki, Markku; Mattila, Leena; Peltola, Heikki; Siitonen, Anja

    2000-01-01

    The incidence of diarrhea and the prevalence of bacterial enteropathogens, viruses, and parasites in feces of subjects with and without diarrhea were evaluated in 204 Finns traveling round the world (from Finland to China, Malaysia, Australia, Fiji, Chile, and Brazil and back to Finland). Special emphasis was placed on the finding of diarrheagenic Escherichia coli (enterotoxigenic, enteropathogenic, Shiga toxin-producing, and enteroaggregative strains) by PCR from growth on primary culture plates. From the PCR-positive samples, corresponding strains were isolated, confirmed as E. coli, and O serotyped. Of all the subjects, 37% experienced a total of 90 episodes of diarrhea. No adenoviruses or rotaviruses were detected, and findings of parasites were insignificant. In contrast, enteropathogenic bacteria were present in 62% of the 65 diarrheal and in 33% of the 127 nondiarrheal samples (P < 0.001); diarrheagenic E. coli strains were found in 35 and 26% of these, respectively (not statistically significant). As a single pathogen, E. coli was found in 20 and 24% of samples (not significant). Of all diarrheagenic E. coli strains, enteropathogenic strains were the most commonly found independently of the clinical picture of the subjects, whereas Salmonella enterica as a single pathogen was the most common non-E. coli organism found in diarrheal samples. Multiple bacterial pathogens were found 10 times more commonly in diarrheal than in nondiarrheal samples (20 versus 2%; P < 0.001). PMID:11101575

  15. Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunoprophylaxis.

    PubMed

    Jung, Kwonil; Saif, Linda J

    2015-05-01

    Porcine epidemic diarrhea virus (PEDV), a member of the genera Alphacoronavirus in the family Coronaviridae, causes acute diarrhea/vomiting, dehydration and high mortality in seronegative neonatal piglets. For the last three decades, PEDV infection has resulted in significant economic losses in the European and Asian pig industries, but in 2013-2014 the disease was also reported in the US, Canada and Mexico. The PED epidemic in the US, from April 2013 to the present, has led to the loss of more than 10% of the US pig population. The disappearance and re-emergence of epidemic PED indicates that the virus is able to escape from current vaccination protocols, biosecurity and control systems. Endemic PED is a significant problem, which is exacerbated by the emergence (or potential importation) of multiple PEDV variants. Epidemic PEDV strains spread rapidly and cause a high number of pig deaths. These strains are highly enteropathogenic and acutely infect villous epithelial cells of the entire small and large intestines although the jejunum and ileum are the primary sites. PEDV infections cause acute, severe atrophic enteritis accompanied by viremia that leads to profound diarrhea and vomiting, followed by extensive dehydration, which is the major cause of death in nursing piglets. A comprehensive understanding of the pathogenic characteristics of epidemic or endemic PEDV strains is needed to prevent and control the disease in affected regions and to develop an effective vaccine. This review focuses on the etiology, epidemiology, disease mechanisms and pathogenesis as well as immunoprophylaxis against PEDV infection. PMID:25841898

  16. Response of early-weaned pigs to an enterotoxigenic Escherichia coli (K88) challenge when fed diets containing spray-dried porcine plasma or pea protein isolate plus egg yolk antibody.

    PubMed

    Owusu-Asiedu, A; Nyachoti, C M; Baidoo, S K; Marquardt, R R; Yang, X

    2003-07-01

    Enterotoxigenic E. coli (ETEC) infection and resulting scours is a major problem for young pigs, especially when purified plant proteins are fed rather than spray-dried porcine plasma (SDPP). The effect of supplementing a pea protein isolate (PPI)-based diet with egg yolk antibodies (EYA) from laying hens immunized with ETEC K88 antigen on piglet performance, incidence of scours, and gut histology was studied in a 14-d trial. Ninety-six 10-d-old weaned pigs were assigned to five dietary treatments in a completely randomized design to give six replicate pens per treatment. The treatments were PPI without EYA (PPI-EYA), PPI with EYA (PPI+EYA), SDPP without EYA (SDPP-EYA), SDPP with EYA (SDPP+EYA), or a combination of PPI and SDPP (PPI+SDPP). Diets were formulated to similar nutrient levels and provided for ad libitum intake. Blood from all pigs was taken on d 0, 7, and 14 for determining plasma urea N (PUN). On d 7, pigs were orally challenged with 6 mL of 10(10) cfu/ mL ETEC K88. Piglets were weighed on d 7 and 14. On d 7, 8, and 14, four pigs per treatment were sacrificed to study the histology of the small intestine. Weekly feed intake, BW changes, and gain:feed were determined. Fecal swabs from 10 pigs per treatment were taken for a PCR test to detect K88 E. coli. Feed efficiency over the 14-d period was not affected (P > 0.78) by dietary treatment. Mean ADFI on an as-fed basis was lower (P < 0.002) in piglets fed PPI-EYA (64.3 g/d) compared with PPI+EYA (94.8 g/d) or SDPP (102 g/d) during wk 1. Piglets fed PPI-EYA tend to have a lower (P < 0.026) overall ADG (84 g/d) than those fed PPI+EYA (123 g/d) or SDPP (127 g/d) (P < 0.006)-based diets. Although scours was evident in all groups of pigs 6 h after the challenge, most of the piglets fed EYA- or SDPP-containing diets recovered 10 to 72 h postchallenge, whereas those fed PPI-EYA continued to have severe diarrhea, resulting in 33% mortality. The PCR results showed that a greater (P < 0.01) percentage of piglets

  17. Real-Time Sensing of Enteropathogenic E. coli-Induced Effects on Epithelial Host Cell Height, Cell-Substrate Interactions, and Endocytic Processes by Infrared Surface Plasmon Spectroscopy

    PubMed Central

    Zlotkin-Rivkin, Efrat; Rund, David; Melamed-Book, Naomi; Zahavi, Eitan Erez; Perlson, Eran; Mercone, Silvana; Golosovsky, Michael; Davidov, Dan; Aroeti, Benjamin

    2013-01-01

    Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell's height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity. PMID:24194932

  18. Porcine prion protein amyloid

    PubMed Central

    Hammarström, Per; Nyström, Sofie

    2015-01-01

    ABSTRACT Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions. PMID:26218890

  19. Antibacterial activity of Lactobacillus spp. isolated from the feces of healthy infants against enteropathogenic bacteria.

    PubMed

    Davoodabadi, Abolfazl; Soltan Dallal, Mohammad Mehdi; Rahimi Foroushani, Abbas; Douraghi, Masoumeh; Sharifi Yazdi, Mohammad Kazem; Amin Harati, Farzaneh

    2015-08-01

    Lactobacilli are normal microflora of the gastrointestinal (GI) tract and are a heterogeneous group of lactic acid bacteria (LAB). Lactobacillus strains with Probiotic activity may have health Benefits for human. This study investigates the probiotic potential of Lactobacillus strains obtained from the feces of healthy infants and also explores antibacterial activity of Lactobacillus strains with probiotic potential against enteropathogenic bacteria. Fecal samples were collected from 95 healthy infants younger than 18 months. Two hundred and ninety Lactobacillus strains were isolated and assessed for probiotic potential properties including ability to survive in gastrointestinal conditions (pH 2.0, 0.3% oxgall), adherence to HT-29 cells and antibiotic resistance. Six strains including Lactobacillus fermentum (4 strains), Lactobacillus paracasei and Lactobacillus plantarum showed good probiotic potential and inhibited the growth of enteropathogenic bacteria including ETEC H10407, Shigella flexneri ATCC 12022, Shigella sonnei ATCC 9290, Salmonella enteritidis H7 and Yersinia enterocolitica ATCC 23715. These Lactobacillus strains with probiotic potential may be useful for prevention or treatment of diarrhea, but further in vitro and in vivo studies on these strains are still required. PMID:25930687

  20. Denaturing gradient gel electrophoresis as a tool to determine batch similarity of probiotic cultures of porcine cecal bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A reconstituted porcine-derived continuous-flow (RPCF) culture of gastrointestinal bacteria effective against enterotoxigenic Escherichia coli in weanling pigs has been maintained for several years and used as a parent culture from which to derive additional RPCF cultures. The integrity and health ...

  1. Enteroadherent Escherichia coli as a cause of diarrhea among children in Mexico.

    PubMed Central

    Mathewson, J J; Oberhelman, R A; Dupont, H L; Javier de la Cabada, F; Garibay, E V

    1987-01-01

    Enteropathogenic Escherichia coli (EPEC) often exhibits localized adherence or diffuse adherence to HEp-2 cells. We recently provided evidence that HEp-2 cell-adherent or enteroadherent E. coli (EAEC) not belonging to EPEC serogroups was the cause of diarrhea among U.S. travelers to Mexico. In the present study, we looked for EAEC and EPEC in stool specimens from 154 children with acute diarrhea and 137 well children seen at several outpatient clinics in Guadalajara, Mexico. EAEC showing localized adherence (EAEC-L) was isolated from 13.0% of the patients and 0.7% of the controls (P less than 0.0001). EAEC showing diffuse adherence (EAEC-D) was recovered from 20.8% of the patients and 7.3% of the controls (P less than 0.001). EPEC was isolated from 4.5 and 6.7% of the patients and controls, respectively. Among all enteropathogens, only enterotoxigenic E. coli occurred as commonly (21.4%) as EAEC-D and EAEC-L did in children with diarrhea. Of the EAEC-L strains isolated from children with diarrhea, 20% belonged to recognized EPEC serogroups, and 3.1% of EAEC-D strains belonged to recognized EPEC serogroups. This study suggests that EAEC may be an important pediatric enteropathogen in Mexican children with diarrhea and further supports the observation that adherence to HEp-2 cells may be a marker of virulence independent of EPEC serogroup among E. coli strains. PMID:3312288

  2. Activity of essential oils from Brazilian medicinal plants on Escherichia coli.

    PubMed

    Duarte, Marta Cristina Teixeira; Leme, Ewerton Eduardo; Delarmelina, Camila; Soares, Andressa Almeida; Figueira, Glyn Mara; Sartoratto, Adilson

    2007-05-01

    Essential oils obtained from leaves of 29 medicinal plants commonly used in Brazil were screened against 13 different Escherichia coli serotypes. The oils were obtained by water-distillation using a Clevenger-type system and their minimal inhibitory concentration (MIC) were determined by microdilution method. Essential oil from Cymbopogon martinii exhibited a broad inhibition spectrum, presenting strong activity (MIC between 100 and 500 microg/mL) against 10 out of 13 Escherichia coli serotypes: three enterotoxigenic, two enteropathogenic, three enteroinvasive and two shiga-toxin producers. C. winterianus inhibited strongly two enterotoxigenic, one enteropathogenic, one enteroinvasive and one shiga-toxin producer serotypes. Aloysia triphylla also shows good potential to kill Escherichia coli with moderate to strong inhibition. Other essential oils showed antimicrobial properties, however with a more restricted action against the serotypes studied. Chemical analysis of Cymbopogon martinii essential oil performed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC-MS) showed the presence of compounds with known antimicrobial activity, including geraniol, geranyl acetate and trans-cariophyllene, which tested separately, indicated geraniol as antimicrobial active compound. The significant antibacterial activity of Cymbopogon martinii oil suggests that they could serve as a source for compounds with therapeutic potential. PMID:17210236

  3. Development of monoclonal antibodies specifically recognizing the endogenous sterile alpha motif and HD domain 1 protein in porcine cell lines.

    PubMed

    Yang, Shen; Zhou, Yan-Jun; Zhan, Yuan; Yu, Ling-Xue; Jiang, Yi-Feng; Tong, Wu; Tong, Guang-Zhi

    2014-10-01

    The sterile alpha motif and HD domain 1 (SAMHD1) protein has been identified as a novel innate immunity restriction factor that participates in processes crucial to the viral life cycle. In the present study, we describe a procedure to generate monoclonal antibodies (MAbs) against porcine SAMHD1 and investigate its characteristics to analyze the expression of endogenous SAMHD1. The open reading frame of porcine SAMHD1 was cloned into the prokaryotic expression vector pCold-TF DNA to construct a recombinant plasmid pcold-pSAMHD1 and induce expression of recombinant porcine SAMHD1 protein by IPTG in Escherichia coli Rosetta. The purified recombinant porcine SAMHD1 protein was used to prepare MAbs of SAMHD1. After subcloning five times hybridoma cell clones expressing SAMHD1, MAbs were generated. Western blot analysis and indirect immunofluorescence assay showed that the overexpressed porcine SAMHD1 in 293T cells and endogenous SAMHD1 protein in porcine cell lines could be specifically recognized by the MAbs produced in this study. In conclusion, specific MAbs of porcine SAMHD1 are reported, and these MAbs provide a valuable tool for further studies of SAMHD1-mediated signaling in virus-infected cells to elucidate the underlying antiviral mechanism. PMID:25358004

  4. Development of Monoclonal Antibodies Specifically Recognizing the Endogenous Sterile Alpha Motif and HD Domain 1 Protein in Porcine Cell Lines

    PubMed Central

    Yang, Shen; Zhou, Yan-jun; Zhan, Yuan; Yu, Ling-xue; Jiang, Yi-feng; Tong, Wu

    2014-01-01

    The sterile alpha motif and HD domain 1 (SAMHD1) protein has been identified as a novel innate immunity restriction factor that participates in processes crucial to the viral life cycle. In the present study, we describe a procedure to generate monoclonal antibodies (MAbs) against porcine SAMHD1 and investigate its characteristics to analyze the expression of endogenous SAMHD1. The open reading frame of porcine SAMHD1 was cloned into the prokaryotic expression vector pCold-TF DNA to construct a recombinant plasmid pcold-pSAMHD1 and induce expression of recombinant porcine SAMHD1 protein by IPTG in Escherichia coli Rosetta. The purified recombinant porcine SAMHD1 protein was used to prepare MAbs of SAMHD1. After subcloning five times hybridoma cell clones expressing SAMHD1, MAbs were generated. Western blot analysis and indirect immunofluorescence assay showed that the overexpressed porcine SAMHD1 in 293T cells and endogenous SAMHD1 protein in porcine cell lines could be specifically recognized by the MAbs produced in this study. In conclusion, specific MAbs of porcine SAMHD1 are reported, and these MAbs provide a valuable tool for further studies of SAMHD1-mediated signaling in virus-infected cells to elucidate the underlying antiviral mechanism. PMID:25358004

  5. Tight Junction Disruption Induced by Type 3 Secretion System Effectors Injected by Enteropathogenic and Enterohemorrhagic Escherichia coli

    PubMed Central

    Ugalde-Silva, Paul; Gonzalez-Lugo, Octavio; Navarro-Garcia, Fernando

    2016-01-01

    The intestinal epithelium consists of a single cell layer, which is a critical selectively permeable barrier to both absorb nutrients and avoid the entry of potentially harmful entities, including microorganisms. Epithelial cells are held together by the apical junctional complexes, consisting of adherens junctions, and tight junctions (TJs), and by underlying desmosomes. TJs lay in the apical domain of epithelial cells and are mainly composed by transmembrane proteins such as occludin, claudins, JAMs, and tricellulin, that are associated with the cytoplasmic plaque formed by proteins from the MAGUK family, such as ZO-1/2/3, connecting TJ to the actin cytoskeleton, and cingulin and paracingulin connecting TJ to the microtubule network. Extracellular bacteria such as EPEC and EHEC living in the intestinal lumen inject effectors proteins directly from the bacterial cytoplasm to the host cell cytoplasm, where they play a relevant role in the manipulation of the eukaryotic cell functions by modifying or blocking cell signaling pathways. TJ integrity depends on various cell functions such as actin cytoskeleton, microtubule network for vesicular trafficking, membrane integrity, inflammation, and cell survival. EPEC and EHEC effectors target most of these functions. Effectors encoded inside or outside of locus of enterocyte effacement (LEE) disrupt the TJ strands. EPEC and EHEC exploit the TJ dynamics to open this structure, for causing diarrhea. EPEC and EHEC secrete effectors that mimic host proteins to manipulate the signaling pathways, including those related to TJ dynamics. In this review, we focus on the known mechanisms exploited by EPEC and EHEC effectors for causing TJ disruption. PMID:27606286

  6. Pathogenicity Island O-122 in enteropathogenic Escherichia coli strains is associated with diarrhea severity in children from Lima Peru.

    PubMed

    Mercado, Erik H; Piscoche, Cristian; Contreras, Carmen; Durand, David; Riveros, Maribel; Ruiz, Joaquim; Ochoa, Theresa J

    2016-06-01

    EPEC is an attaching and effacing diarrheal pathogen that carries a large pathogenicity island, locus for enterocyte effacement (LEE). Recently, the pathogenicity island PAI O-122 was described among non-LEE effectors and found to be associated with diarrhea among atypical EPEC strains. It is unknown if incomplete PAI O-122 could be associated with diarrhea duration and severity. To identify these virulence determinants we analyzed 379 EPEC strains isolated from Peruvian children. EPEC was diagnosed by PCR(eae+, stx-) and classified as typical(t-EPEC) or atypical(a-EPEC). To characterize PAI O-122 we amplified three modules by PCR: Module 1(pagC), Module 2(senA, nleB and nleE) and Module 3(lifA/efa-1). To characterize the large ORF lifA/efa-1 we amplified the regions known as efa-N, efa-M and efa-C. Clinical information was obtained from the cohort study. A total of 379 EPEC strains were able to analyze PAI O-122 genes, 128 (10.4%) EPEC strains were isolated from 1235 diarrhea episodes and 251(9.2%) from 2734 healthy controls. t-EPEC strains were isolated from 14.8% (19/128) of children with diarrhea and 25/251(10.0%) from healthy controls. The most frequent PAI O-122 genes were nleE(37.7%), senA(34.6%) and nleB(37.5%), with similar prevalence among diarrhea and control samples. However, lifA/efa-1 was more common among diarrhea cases than healthy control cases (30.5% vs. 21.1%, p<0.05). The presence of complete PAI O-122 was associated with diarrhea episodes of higher severity among single pathogen infection (33.3% vs. 1.8%, p<0.05) mainly due to the presence of a complete lifA/efa-1 gene. In summary, the gene lifA/efa-1 is significantly associated with diarrheal episodes of higher severity, suggesting to be an important virulent factor. PMID:27236730

  7. Prevalence and Characterization of Shiga Toxin-Producing and Enteropathogenic Escherichia coli in Shellfish-Harvesting Areas and Their Watersheds

    PubMed Central

    Balière, Charlotte; Rincé, Alain; Blanco, Jorge; Dahbi, Ghizlane; Harel, Josée; Vogeleer, Philippe; Giard, Jean-Christophe; Mariani-Kurkdjian, Patricia; Gourmelon, Michèle

    2015-01-01

    more strains formed a strong biofilm at 18 than at 30°C. Finally, more than 85% of analyzed strains were found to be sensitive to the 16 tested antibiotics. These data suggest the low risk of human infection by STEC if shellfish from these shellfish-harvesting areas were consumed. PMID:26648928

  8. Tight Junction Disruption Induced by Type 3 Secretion System Effectors Injected by Enteropathogenic and Enterohemorrhagic Escherichia coli.

    PubMed

    Ugalde-Silva, Paul; Gonzalez-Lugo, Octavio; Navarro-Garcia, Fernando

    2016-01-01

    The intestinal epithelium consists of a single cell layer, which is a critical selectively permeable barrier to both absorb nutrients and avoid the entry of potentially harmful entities, including microorganisms. Epithelial cells are held together by the apical junctional complexes, consisting of adherens junctions, and tight junctions (TJs), and by underlying desmosomes. TJs lay in the apical domain of epithelial cells and are mainly composed by transmembrane proteins such as occludin, claudins, JAMs, and tricellulin, that are associated with the cytoplasmic plaque formed by proteins from the MAGUK family, such as ZO-1/2/3, connecting TJ to the actin cytoskeleton, and cingulin and paracingulin connecting TJ to the microtubule network. Extracellular bacteria such as EPEC and EHEC living in the intestinal lumen inject effectors proteins directly from the bacterial cytoplasm to the host cell cytoplasm, where they play a relevant role in the manipulation of the eukaryotic cell functions by modifying or blocking cell signaling pathways. TJ integrity depends on various cell functions such as actin cytoskeleton, microtubule network for vesicular trafficking, membrane integrity, inflammation, and cell survival. EPEC and EHEC effectors target most of these functions. Effectors encoded inside or outside of locus of enterocyte effacement (LEE) disrupt the TJ strands. EPEC and EHEC exploit the TJ dynamics to open this structure, for causing diarrhea. EPEC and EHEC secrete effectors that mimic host proteins to manipulate the signaling pathways, including those related to TJ dynamics. In this review, we focus on the known mechanisms exploited by EPEC and EHEC effectors for causing TJ disruption. PMID:27606286

  9. Neonatal Escherichia coli infections in domestic mammals: transmissibility of pathogenic characteristics.

    PubMed

    Smith, H W

    1976-01-01

    Apart from the fact that different serotypes are involved, natural and experimental Escherichia coli infection in domestic mammals closely resembles natural E. coli infection in human beings. Some of the important characteristics of E. coli strains that cause disease in domestic mammals are determined by transmissible plasmids. These include enterotoxin, haemolysin and K88 antigen in piglet enteropathogenic strains and enterotoxin and K99 antigen production in calf and lamb enteropathogenic strains; most strains that cause generalized infections in young domestic mammals, i.e. invasive strains, also produce plasmid-determined colicine V. These are all good reasons for employing young domestic mammals as the animal model for studying certain aspects of E. coli infection in human beings. Exploiting the fact that plasmids can be introduced into bacterial cells by conjugation and can be removed from them by "curing", bacterial strains were created that differed from each other, as far as could be determined, only by the presence or absence of one or more of these plasmid-determined properties. These strains, or cell-free preparations of them, were then given by mouth to piglets, calves, lambs and baby rabbits. The results showed that the K88 antigen, probably on account of its adhesive properties, permitted pig enteropathogenic strains of E. coli to proliferate in the small intestine of piglets; the K99 antigen performed a similar function in calf and lamb enteropathogenic strains. The enterotoxin produced by the proliferating organisms was then chiefly responsible for the subsequent movement of fluid from the body into the small intestine and the consequent diarrhoea. Possession of the Col V plasmid contributed significantly to the virulence of invasive strains of E. coli by enabling them to resist more successfully the defence mechanisms of the host. PMID:791599

  10. Escherichia Coli

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  11. Detection and Characterization of Porcine Endogenous Retrovirus in Porcine Plasma and Porcine Factor VIII

    PubMed Central

    Takefman, Daniel M.; Wong, Susan; Maudru, Thomas; Peden, Keith; Wilson, Carolyn A.

    2001-01-01

    The pig genome contains porcine endogenous retroviruses (PERVs) capable of infecting human cells. Detection of infectious retrovirus in porcine peripheral blood mononuclear cells and endothelial cells suggested to us that pig plasma is likely to contain PERV. Both PERV env sequences and viral reverse transcriptase (RT) activity were detected in all plasma samples isolated from four NIH minipigs. To detect infectious virus from plasma, we performed a culture assay using three cell lines of feline, swine, and human origin that had previously been shown to be permissive for PERV. Infectious virus was successfully cultured from all four NIH minipig plasmas on the swine cell line ST-IOWA. Using RT-PCR with env-specific primers, we could detect expression of PERV class C envelope in the supernatant of ST-IOWA cells that had been exposed to each pig plasma. We next examined a pig plasma derivative, Hyate:C (porcine factor VIII), and found evidence of PERV particles, since all six lots examined were positive for PERV RNA and RT activity. However, infectious virus could not be detected in clinical lots of Hyate:C, suggesting that the manufacturing process might reduce the load of infectious virus to levels below detectable limits of the assay. Detection of infectious virus in porcine plasma confirms and extends the previous findings that certain porcine cells express PERV when manipulated in vitro and clearly demonstrates that there are porcine cells that express infectious PERV constitutively in vivo. PMID:11312325

  12. Both direct and indirect effects account for the pro-inflammatory activity of enteropathogenic mycotoxins on the human intestinal epithelium: Stimulation of interleukin-8 secretion, potentiation of interleukin-1{beta} effect and increase in the transepithelial passage of commensal bacteria

    SciTech Connect

    Maresca, Marc; Yahi, Nouara; Younes-Sakr, Lama; Boyron, Marilyn; Caporiccio, Bertrand; Fantini, Jacques

    2008-04-01

    Mycotoxins are fungal secondary metabolites responsible of food-mediated intoxication in animals and humans. Deoxynivalenol, ochratoxin A and patulin are the best known enteropathogenic mycotoxins able to alter intestinal functions resulting in malnutrition, diarrhea, vomiting and intestinal inflammation in vivo. Although their effects on intestinal barrier and transport activities have been extensively characterized, the mechanisms responsible for their pro-inflammatory effect are still poorly understood. Here we investigated if mycotoxin-induced intestinal inflammation results from a direct and/or indirect pro-inflammatory activity of these mycotoxins on human intestinal epithelial cells, using differentiated Caco-2 cells as model and interleukin 8 (IL-8) as an indicator of intestinal inflammation. Deoxynivalenol was the only mycotoxin able to directly increase IL-8 secretion (10- to 15-fold increase). We also investigated if these mycotoxins could indirectly stimulate IL-8 secretion through: (i) a modulation of the action of pro-inflammatory molecules such as the interleukin-1beta (IL-1{beta}), and/or (ii) an increase in the transepithelial passage of non-invasive commensal Escherichia coli. We found that deoxynivalenol, ochratoxin A and patulin all potentiated the effect of IL-1{beta} on IL-8 secretion (ranging from 35% to 138% increase) and increased the transepithelial passage of commensal bacteria (ranging from 12- to 1544-fold increase). In addition to potentially exacerbate established intestinal inflammation, these mycotoxins may thus participate in the induction of sepsis and intestinal inflammation in vivo. Taken together, our results suggest that the pro-inflammatory activity of enteropathogenic mycotoxins is mediated by both direct and indirect effects.

  13. Concurrent infection of enterocytes with Eimeria scabra and other enteropathogens in swine.

    PubMed

    Koudela, B; Vítovec, J; Stĕrba, J

    1990-02-01

    Bacteria were detected in the enterocytes of the distal jejunum in weaned pigs on Days 7 and 9 post-infection (DPI) with Eimeria scabra in addition to the developmental stages of the coccidia. Short rod-shaped bacteria were identified in approximately 60% of the enterocytes that contained developmental stages of E. scabra. No such bacteria were observed in cells where coccidia were absent. Gamonts of cryptosporidia were also observed within the microvillous zone of the enterocytes in the distal jejunum of weaned pigs on DPI 9 with E. scabra. Cryptosporidia were present only in enterocytes harbouring stages of E. scabra. Chlamydial particles were also found in the cytoplasm of enterocytes 7 DPI with E. scabra. The presence of other enteropathogens exclusively in the enterocytes containing developmental stages of coccidia suggests that the coccidium E. scabra facilitates the invasion and development of bacteria, cryptosporidia and chlamydia in the enterocytes. PMID:2343533

  14. 7 CFR 1230.611 - Porcine animal.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Porcine animal. 1230.611 Section 1230.611 Agriculture... CONSUMER INFORMATION Procedures for the Conduct of Referendum Definitions § 1230.611 Porcine animal. The term Porcine animal means a swine, that is raised: (a) As a feeder pig, that is, a young pig sold...

  15. 7 CFR 1230.611 - Porcine animal.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Porcine animal. 1230.611 Section 1230.611 Agriculture... CONSUMER INFORMATION Procedures for the Conduct of Referendum Definitions § 1230.611 Porcine animal. The term Porcine animal means a swine, that is raised: (a) As a feeder pig, that is, a young pig sold...

  16. 7 CFR 1230.611 - Porcine animal.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 10 2012-01-01 2012-01-01 false Porcine animal. 1230.611 Section 1230.611 Agriculture... CONSUMER INFORMATION Procedures for the Conduct of Referendum Definitions § 1230.611 Porcine animal. The term Porcine animal means a swine, that is raised: (a) As a feeder pig, that is, a young pig sold...

  17. 7 CFR 1230.611 - Porcine animal.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 10 2013-01-01 2013-01-01 false Porcine animal. 1230.611 Section 1230.611 Agriculture... CONSUMER INFORMATION Procedures for the Conduct of Referendum Definitions § 1230.611 Porcine animal. The term Porcine animal means a swine, that is raised: (a) As a feeder pig, that is, a young pig sold...

  18. 7 CFR 1230.611 - Porcine animal.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 10 2014-01-01 2014-01-01 false Porcine animal. 1230.611 Section 1230.611 Agriculture... CONSUMER INFORMATION Procedures for the Conduct of Referendum Definitions § 1230.611 Porcine animal. The term Porcine animal means a swine, that is raised: (a) As a feeder pig, that is, a young pig sold...

  19. Evolutionary Genetics of a New Pathogenic Escherichia Species: Escherichia albertii and Related Shigella boydii Strains

    PubMed Central

    Hyma, Katie E.; Lacher, David W.; Nelson, Adam M.; Bumbaugh, Alyssa C.; Janda, J. Michael; Strockbine, Nancy A.; Young, Vincent B.; Whittam, Thomas S.

    2005-01-01

    A bacterium originally described as Hafnia alvei induces diarrhea in rabbits and causes epithelial damage similar to the attachment and effacement associated with enteropathogenic Escherichia coli. Subsequent studies identified similar H. alvei-like strains that are positive for an intimin gene (eae) probe and, based on DNA relatedness, are classified as a distinct Escherichia species, Escherichia albertii. We determined sequences for multiple housekeeping genes in five E. albertii strains and compared these sequences to those of strains representing the major groups of pathogenic E. coli and Shigella. A comparison of 2,484 codon positions in 14 genes revealed that E. albertii strains differ, on average, at ∼7.4% of the nucleotide sites from pathogenic E. coli strains and at 15.7% from Salmonella enterica serotype Typhimurium. Interestingly, E. albertii strains were found to be closely related to strains of Shigella boydii serotype 13 (Shigella B13), a distant relative of E. coli representing a divergent lineage in the genus Escherichia. Analysis of homologues of intimin (eae) revealed that the central conserved domains are similar in E. albertii and Shigella B13 and distinct from those of eae variants found in pathogenic E. coli. Sequence analysis of the cytolethal distending toxin gene cluster (cdt) also disclosed three allelic groups corresponding to E. albertii, Shigella B13, and a nontypeable isolate serologically related to S. boydii serotype 7. Based on the synonymous substitution rate, the E. albertii-Shigella B13 lineage is estimated to have split from an E. coli-like ancestor ∼28 million years ago and formed a distinct evolutionary branch of enteric pathogens that has radiated into groups with distinct virulence properties. PMID:15629933

  20. Radiation sensitivity of bacteria and virus in porcine xenoskin for dressing agent

    NASA Astrophysics Data System (ADS)

    Jo, Eu-Ri; Jung, Pil-Mun; Choi, Jong-il; Lee, Ju-Woon

    2012-08-01

    In this study, gamma irradiation sensitivities of bacteria and viruses in porcine skin were evaluated to establish the optimum sterilization condition for the dressing material and a xenoskin graft. Escherichia coli and Bacillus subtilis were used as model pathogens and inoculated at 106-107 log CFU/g. As model viruses, porcine parvovirus (PPV), bovine viral diarrhea virus (BVDV), and poliovirus were used and inoculated at 105-106 TCID50/g into porcine skin. The D10 value of E. coli was found to be 0.25±0.1 kGy. B. subtilis endospores produced under stressful environmental conditions showed lower radiation sensitivity as D10 was 3.88±0.3 kGy in porcine skin. The D10 values of PPV, BVDV, and poliovirus were found to be 1.73±0.2, 3.81±0.2, and 6.88±0.3 kGy, respectively. These results can offer the basic information required for inactivating pathogens by gamma irradiation and achieving dressing material and porcine skin grafts.

  1. Structure of the Cyclomodulin Cif from Pathogenic Escherichia coli

    SciTech Connect

    Hsu, Y.; Jubelin, G; Taieb, F; Nougayrède, J; Oswald, E; Stebbins, C

    2008-01-01

    Bacterial pathogens have evolved a sophisticated arsenal of virulence factors to modulate host cell biology. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) use a type III protein secretion system (T3SS) to inject microbial proteins into host cells. The T3SS effector cycle inhibiting factor (Cif) produced by EPEC and EHEC is able to block host eukaryotic cell-cycle progression. We present here a crystal structure of Cif, revealing it to be a divergent member of the superfamily of enzymes including cysteine proteases and acetyltransferases that share a common catalytic triad. Mutation of these conserved active site residues abolishes the ability of Cif to block cell-cycle progression. Finally, we demonstrate that irreversible cysteine protease inhibitors do not abolish the Cif cytopathic effect, suggesting that another enzymatic activity may underlie the biological activity of this virulence factor.

  2. The in vitro and in vivo effects of constitutive light expression on a bioluminescent strain of the mouse enteropathogen Citrobacter rodentium.

    PubMed

    Read, Hannah M; Mills, Grant; Johnson, Sarah; Tsai, Peter; Dalton, James; Barquist, Lars; Print, Cristin G; Patrick, Wayne M; Wiles, Siouxsie

    2016-01-01

    Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive) light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. In this study, we compared the growth of the bioluminescent C. rodentium strain ICC180 with its non-bioluminescent parent (strain ICC169) in a wide variety of environments. To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2,000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggest that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments. PMID:27366640

  3. The in vitro and in vivo effects of constitutive light expression on a bioluminescent strain of the mouse enteropathogen Citrobacter rodentium

    PubMed Central

    Read, Hannah M.; Mills, Grant; Johnson, Sarah; Tsai, Peter; Dalton, James; Barquist, Lars; Print, Cristin G.; Patrick, Wayne M.

    2016-01-01

    Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive) light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. In this study, we compared the growth of the bioluminescent C. rodentium strain ICC180 with its non-bioluminescent parent (strain ICC169) in a wide variety of environments. To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2,000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggest that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments. PMID:27366640

  4. Porcine Dentin Sialophosphoprotein

    PubMed Central

    Yamakoshi, Yasuo; Lu, Yuhe; Hu, Jan C.-C.; Kim, Jung-Wook; Iwata, Takanori; Kobayashi, Kazuyuki; Nagano, Takatoshi; Yamakoshi, Fumiko; Hu, Yuanyuan; Fukae, Makoto; Simmer, James P.

    2008-01-01

    Dentin sialophosphoprotein (DSPP) is critical for proper mineralization of tooth dentin, and mutations in DSPP cause inherited dentin defects. Dentin phosphoprotein (DPP) is the C-terminal cleavage product of DSPP that binds collagen and induces intrafibrillar mineralization. We isolated DPP from individual pigs and determined that its N-terminal and C-terminal domains are glycosylated and that DPP averages 155 phosphates per molecule. Porcine DPP is unstable at low pH and high temperatures, and complexing with collagen improves its stability. Surprisingly, we observed DPP size variations on SDS-PAGE for DPP isolated from individual pigs. These variations are not caused by differences in proteolytic processing or degrees of phosphorylation or glycosylation, but rather to allelic variations in Dspp. Characterization of the DPP coding region identified 4 allelic variants. Among the 4 alleles, 27 sequence variations were identified, including 16 length polymorphisms ranging from 3 to 63 nucleotides. None of the length variations shifted the reading frame, and all localized to the highly redundant region of the DPP code. The 4 alleles encode DPP domains having 551, 575, 589, or 594 amino acids and completely explain the DPP size variations. DPP length variations are polymorphic and are not associated with dentin defects. PMID:18359767

  5. Pathological and bacteriological characterization of neonatal porcine diarrhoea of uncertain aetiology.

    PubMed

    Larsson, Jenny; Aspán, Anna; Lindberg, Ronny; Grandon, Rodrigo; Båverud, Viveca; Fall, Nils; Jacobson, Magdalena

    2015-08-01

    Neonatal porcine diarrhoea of uncertain aetiology has been reported from a number of countries. This study investigated 50 diarrhoeic and 19 healthy piglets from 10 affected Swedish herds. The piglets were blood-sampled for analysis of serum γ-globulin and necropsied, and the intestines were sampled for histopathology and cultured for Escherichia coli, Clostridium perfringens and Clostridium difficile. Escherichia coli isolates (n = 276) were examined by PCR for virulence genes encoding LT, STa, STb, EAST1, VT2e, F4, F5, F6, F18, F41, AIDA-I, intimin, and for the genes aaiC and aggR. Selected isolates were analysed for additional virulence genes by a microarray and subjected to O-typing. Clostridium perfringens isolates (n = 152) were examined by PCR for genes encoding major toxins, enterotoxin and beta2-toxin. There was no difference in serum γ-globulin concentration between diarrhoeic and non-diarrhoeic piglets, and pathological lesions in the intestines were generally mild. Porcine enterotoxigenic Escherichia coli, a common cause of piglet diarrhoea, was only found in two piglets. Further, the virulence gene profiling did not suggest involvement of other diarrhoeogenic pathotypes of Escherichia coli. Growth of Clostridium perfringens did not differ between diarrhoeic and non-diarrhoeic piglets. All isolates were type A, all were negative for enterotoxin, and 151 of 152 isolates were beta2-toxin positive. In pigs ≥ 2  days old, moderate to profuse growth of Clostridium difficile was more common in the controls. In conclusion, it was not possible to relate Escherichia coli, Clostridium perfringens type A and C or Clostridium difficile to neonatal porcine diarrhoea in any of the investigated herds. PMID:26272503

  6. Porcine Reproductive and Respiratory Syndrome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Porcine reproductive and respiratory syndrome (PRRS) first appeared in the late 1980s, though serologic evidence indicates that it had been circulating in swine for some time prior to being recognized. PRRS has since become a highly significant infectious disease affecting swine production worldwid...

  7. Induction of Haemophilus influenzae type b capsular antibody in neonatal rabbits by gastrointestinal colonization with cross-reacting Escherichia coli.

    PubMed

    Myerowitz, R L; Handzel, Z T; Scheerson, R; Robbins, J B

    1973-02-01

    In two separate experiments, newborn rabbits were fed a live suspension of either of two Escherichia coli strains which possess a "K" antigen cross-reactive with the capsular polysaccharide of Haemophilus influenzae type b. Both feedings were harmless and resulted in fecal excretion of the fed E. coli in most animals as well as active immunization of fed animals toward H. influenzae type b. Feeding non-enteropathogenic, cross-reacting E coli to newborns may be a method for inducing active immunity toward H. influenzae type b diseases by accelerating the acquisition of "natural" immunity. PMID:4572607

  8. Epidemiologic studies of Escherichia coli diarrheal infections in a low socioeconomic level peri-urban community in Santiago, Chile.

    PubMed

    Levine, M M; Ferreccio, C; Prado, V; Cayazzo, M; Abrego, P; Martinez, J; Maggi, L; Baldini, M M; Martin, W; Maneval, D

    1993-11-15

    The incidence of diarrhea due to six categories of diarrheogenic Escherichia coli was determined in two pediatric cohorts in a low socioeconomic level community in Santiago, Chile, with access to chlorinated water. An age cross-sectional cohort of 340 children aged birth to 47 months was assembled. A newborn cohort was assembled by enrolling 10-12 newborns monthly for 12 months. Episodes of diarrhea were detected by twice weekly household visits. E. coli from stool cultures of cases and matched controls were hybridized with DNA probes specific for enterotoxigenic, enteroinvasive, enteropathogenic, enterohemorrhagic, enteroaggregative, and diffuse adherence E. coli. Overall, the incidence of diarrhea was low (2.1 episodes/infant/year). Nevertheless, a putative E. coli enteropathogen was found in a large proportion of diarrheal episodes, particularly during the summer. In both cohorts, enterotoxigenic E. coli were important pathogens. Enteropathogenic E. coli were incriminated during the first year of life in the newborn cohort, where they were found significantly more often in cases (p = 0.021) than in controls; beyond this age, isolation rates were similar. In contrast, the relative risk of isolation of diffuse adherence E. coli increased with age in the age cross-sectional cohort, where, overall, the difference in rate of isolation between cases and controls was significant (p = 0.0024). Enteroinvasive and enterohemorrhagic E. coli were isolated infrequently. Enteroaggregative E. coli were encountered equally in cases and controls. Facile transmission of E. coli enteropathogens is occurring in this community despite the availability of potable water. PMID:8237973

  9. Bacterial enteropathogens of neonates admitted to an urban diarrhoeal hospital in Bangladesh.

    PubMed

    Khan, A M; Hossain, M S; Khan, A I; Chisti, M J; Chowdhury, F; Faruque, A S G; Salam, M A

    2009-04-01

    Data on the aetiology of diarrhoea in neonates are scarce, especially from developing countries including Bangladesh. A retrospective review of the electronic database of the Microbiology Laboratory of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), was carried out to examine enteropathogens associated with diarrhoea in neonates. Stool specimens of the neonates on admission to the Dhaka Hospital of ICDDR,B were collected and sent to the laboratory for direct plating onto taurocholate tellurite gelatin agar, Salmonella-Shigella agar and MacConkey's agar. Stool specimens of 2511 neonates of either sex were examined. Bacterial pathogens were recovered from the stools of 699 (27.8%) of these neonates--a single bacterial pathogen from 670 neonates and more than one pathogen from 29 neonates. Vibrio cholerae, Shigella, Salmonella, Aeromonas spp. and Plesiomonas shigelloides were isolated from 294, 108, 52, 222 and 19, respectively, of the neonates. The year-wise isolation of these pathogens varied between 4.9-23.4%, 2.7-5.4%, 0-4.7%, 0-19.4% and 0-1.6%, respectively, of the neonates. The results of the study indicate that infection by V. cholerae, Shigella spp., Salmonella spp., Aeromonas and P. shigelloides is common in neonatal diarrhoea in Bangladesh. PMID:18840632

  10. Lactobacillus brevis Strains from Fermented Aloe vera Survive Gastroduodenal Environment and Suppress Common Food Borne Enteropathogens

    PubMed Central

    Kim, Young-Wook; Jeong, Young-Ju; Kim, Ah-Young; Son, Hyun-Hee; Lee, Jong-Am; Jung, Cheong-Hwan; Kim, Chae-Hyun; Kim, Jaeman

    2014-01-01

    Five novel Lactobacillus brevis strains were isolated from naturally fermented Aloe vera leaf flesh. Each strain was identified by Random Amplified Polymorphic DNA (RAPD) analysis and 16S rRNA sequence comparison. These strains were highly tolerant to acid, surviving in pH2.5 for up to 4 hours, and resistant to 5% bile salts at 37°C for 18 hours. Due to its tolerance to acid and bile salts, one strain passed through the gastric barrier and colonised the intestine after oral administration. All five strains inhibited the growth of many harmful enteropathogens without restraining most of normal commensals in the gut and hence named POAL (Probiotics Originating from Aloe Leaf) strains. Additionally, each strain exhibited discriminative resistance to a wide range of antibiotics. The L. brevis POAL strains, moreover, expressed high levels of the glutamate decarboxylase (GAD) gene which produces a beneficial neurotransmitter, γ-aminobutyric acid (GABA). These characteristics in all suggest that the novel L. brevis strains should be considered as potential food additives and resources for pharmaceutical research. PMID:24598940

  11. Enteropathogens associated with acute diarrhea in children from households with high socioeconomic level in uruguay.

    PubMed

    Varela, Gustavo; Batthyány, Lara; Bianco, María Noel; Pérez, Walter; Pardo, Lorena; Algorta, Gabriela; Robino, Luciana; Suárez, Ramón; Navarro, Armando; Pírez, María Catalina; Schelotto, Felipe

    2015-01-01

    Infectious diarrhea, a common disease of children, deserves permanent monitoring in all social groups. To know the etiology and clinical manifestations of acute diarrhea in children up to 5 years of age from high socioeconomic level households, we conducted a descriptive, microbiological, and clinical study. Stools from 59 children with acute community-acquired diarrhea were examined, and their parents were interviewed concerning symptoms and signs. Rotavirus, adenovirus, and norovirus were detected by commercially available qualitative immunochromatographic lateral flow rapid tests. Salmonella, Campylobacter, Yersinia, and Shigella were investigated by standard bacteriological methods and diarrheagenic E. coli by PCR assays. We identified a potential enteric pathogen in 30 children. The most frequent causes of diarrhea were enteropathogenic E. coli (EPEC), viruses, Campylobacter, Salmonella, and Shiga-toxin-producing E. coli (STEC). Only 2 patients showed mixed infections. Our data suggest that children with viral or Campylobacter diarrhea were taken to the hospital earlier than those infected with EPEC. One child infected with STEC O26 developed "complete" HUS. The microbiological results highlight the importance of zoonotic bacteria such as atypical EPEC, Campylobacter, STEC, and Salmonella as pathogens associated with acute diarrhea in these children. The findings also reinforce our previous communications about the regional importance of non-O157 STEC strains in severe infant food-borne diseases. PMID:25861274

  12. Inactivation of enteropathogenic E. coli by solar disinfection (SODIS) under simulated sunlight conditions

    NASA Astrophysics Data System (ADS)

    Ubomba-Jaswa, E.; Boyle, M. A. R.; McGuigan, K. G.

    2008-02-01

    Solar Disinfection (SODIS) is a low cost water treatment method currently used in communities that do not have year round access to safe water. However, there is still reluctance in widespread adoption of this treatment method due to a number of limitations. An important limitation is the lack of SODIS inactivation studies on some waterborne pathogens in the developing world. SODIS inactivation of enteropathogenic E. coli (EPEC), a major cause of infantile diarrhoea is reported for the first time under simulated sunlight conditions and following a natural temperature profile. EPEC was exposed to simulated sunlight (885Wm-2) for periods up to a cumulative time of 4 hours. Inactivation was determined by a log reduction in growth of the organisms. The temperature (°C) of the water was taken at every time point. After 4 hours exposure EPEC was completely inactivated (7 log reduction) by SODIS. Imposing a realistic water temperature profile (min-max) concomitant with irradiation produces a greater kill of EPEC. Maintaining simulated sunlight experiments at a high fixed temperature may result in over --estimation of inactivation. Following a natural water temperature profile will result in more reliable inactivation comparable with those that might be obtained under natural sunlight conditions.

  13. Enteropathogens Associated with Acute Diarrhea in Children from Households with High Socioeconomic Level in Uruguay

    PubMed Central

    Batthyány, Lara; Bianco, María Noel; Pérez, Walter; Pardo, Lorena; Algorta, Gabriela; Robino, Luciana; Suárez, Ramón; Navarro, Armando; Pírez, María Catalina; Schelotto, Felipe

    2015-01-01

    Infectious diarrhea, a common disease of children, deserves permanent monitoring in all social groups. To know the etiology and clinical manifestations of acute diarrhea in children up to 5 years of age from high socioeconomic level households, we conducted a descriptive, microbiological, and clinical study. Stools from 59 children with acute community-acquired diarrhea were examined, and their parents were interviewed concerning symptoms and signs. Rotavirus, adenovirus, and norovirus were detected by commercially available qualitative immunochromatographic lateral flow rapid tests. Salmonella, Campylobacter, Yersinia, and Shigella were investigated by standard bacteriological methods and diarrheagenic E. coli by PCR assays. We identified a potential enteric pathogen in 30 children. The most frequent causes of diarrhea were enteropathogenic E. coli (EPEC), viruses, Campylobacter, Salmonella, and Shiga-toxin-producing E. coli (STEC). Only 2 patients showed mixed infections. Our data suggest that children with viral or Campylobacter diarrhea were taken to the hospital earlier than those infected with EPEC. One child infected with STEC O26 developed “complete” HUS. The microbiological results highlight the importance of zoonotic bacteria such as atypical EPEC, Campylobacter, STEC, and Salmonella as pathogens associated with acute diarrhea in these children. The findings also reinforce our previous communications about the regional importance of non-O157 STEC strains in severe infant food-borne diseases. PMID:25861274

  14. Prevalence of enteropathogenic bacteria in treated effluents and receiving water bodies and their potential health risks.

    PubMed

    Teklehaimanot, Giorgis Z; Genthe, B; Kamika, I; Momba, M N B

    2015-06-15

    The failure of wastewater treatment plants to produce effluents of a high microbiological quality is a matter of great concern in terms of water resource pollution. A more serious concern is that this water source is used by communities in developing countries for multiple purposes, which include drinking, recreation and agriculture. The current study investigated the prevalence and potential health risks of enteropathogenic bacteria (Salmonella typhimurium, Shigella dysenteriae and Vibrio cholerae) in the treated effluents of three selected South African Wastewater Treatment Works as well as their receiving water bodies. Culture-based and polymerase chain reaction techniques were used to detect and identify the pathogenic bacteria. The conventional methods revealed that of the 272 water samples collected, 236 samples (86.8%) tested presumptively positive for Salmonella spp., 220 samples (80.9%) for Shigella spp. and 253 samples (93.0%) for V. cholerae. Molecular test results indicated that out of the randomly selected presumptive positive samples (145), zero to 60% of samples were positive for S. typhimurium and S. dysenteriae and 20% to 60% for V. cholerae. For the health risk assessment, the daily combined risk of S. typhimurium, S. dysenteriae and V. cholerae infection was above the lowest acceptable risk limit of 10(-4) as estimated by the World Health Organization for drinking water. This study showed that the target treated wastewater effluents and their receiving water bodies could pose a potential health risk to the surrounding communities. PMID:25777950

  15. Lactobacillus brevis strains from fermented aloe vera survive gastroduodenal environment and suppress common food borne enteropathogens.

    PubMed

    Kim, Young-Wook; Jeong, Young-Ju; Kim, Ah-Young; Son, Hyun-Hee; Lee, Jong-Am; Jung, Cheong-Hwan; Kim, Chae-Hyun; Kim, Jaeman

    2014-01-01

    Five novel Lactobacillus brevis strains were isolated from naturally fermented Aloe vera leaf flesh. Each strain was identified by Random Amplified Polymorphic DNA (RAPD) analysis and 16S rRNA sequence comparison. These strains were highly tolerant to acid, surviving in pH2.5 for up to 4 hours, and resistant to 5% bile salts at 37°C for 18 hours. Due to its tolerance to acid and bile salts, one strain passed through the gastric barrier and colonised the intestine after oral administration. All five strains inhibited the growth of many harmful enteropathogens without restraining most of normal commensals in the gut and hence named POAL (Probiotics Originating from Aloe Leaf) strains. Additionally, each strain exhibited discriminative resistance to a wide range of antibiotics. The L. brevis POAL strains, moreover, expressed high levels of the glutamate decarboxylase (GAD) gene which produces a beneficial neurotransmitter, γ-aminobutyric acid (GABA). These characteristics in all suggest that the novel L. brevis strains should be considered as potential food additives and resources for pharmaceutical research. PMID:24598940

  16. The prevalence of swine enteropathogens in Brazilian grower and finish herds

    PubMed Central

    Viott, A.M.; Lage, A.P.; Cruz, E.C.C.; Guedes, R.M.C.

    2013-01-01

    Diarrhoea among growing and finishing pigs is an important problem in many herds. The prevalence of L. intracellularis, B. pilosicoli, B. hyodysenteriae, Salmonella spp., enterotoxigenic E. coli, Trichuris suis and the occurrence of mixed infection were investigated. Fecal samples for forty-six herds with diarrhea or a history of diarrhea were randomly collected in Minas Gerais state, Brazil. The enteric pathogens were detected by culture (E. coli and Salmonella sp.), PCR (L. intracellularis and Brachyspira spp.) and eggs counts (T. suis). The overall herd prevalence of L. intracellularis, Salmonella enterica serotype Typhimurium and enterotoxigenic E. coli were 19.56%, 6.52%, 10.86% respectively. Mixed infection was diagnosed in 30.43% of herds, and L. intracellularis and Salmonella enterica serotype Typhimurium are main pathogens association (10.87%). B. pilosicoli was diagnosed only in two herds, always associated with mixed infections. B. hyodysenteriae and T. suis were not demonstrated in any sample. These pathogens have been reported world-wide but studies regarding epidemiology in Brazil are few. This study contributes to establish of prevention programs for the control enteropathogens in grower finish herds in Brazil. PMID:24159297

  17. Extracellular signal-regulated kinase (ERK) activation is required for porcine epidemic diarrhea virus replication.

    PubMed

    Kim, Youngnam; Lee, Changhee

    2015-10-01

    Porcine epidemic diarrhea virus (PEDV) is a highly enteropathogenic coronavirus of swine that causes acute enteritis with high mortality in nursery piglets. To date, the cellular factors involved in PEDV replication have not been well defined. The extracellular signal-regulated kinase (ERK) that serves as a critical component of cellular signal transduction pathways to modulate a variety of cellular functions has been shown to regulate several viral infections. In the present study, we found that PEDV activates ERK1/2 early in infection independently of viral replication. The PEDV-induced ERK1/2 activation resulted in the phosphorylation of its downstream substrate Elk-1 in infected cells. Treatment with ERK inhibitors or ERK1/2 knockdown significantly suppressed viral progeny production. Inhibition of ERK activation also diminished viral protein expression and genomic and subgenomic RNA transcription. These findings indicate that the ERK signaling pathway plays an important role in the PEDV life cycle and beneficially contributes to viral infection. PMID:26115165

  18. Mechanical characterization of porcine corneas.

    PubMed

    Boschetti, F; Triacca, V; Spinelli, L; Pandolfi, A

    2012-03-01

    An experimental program has been carried out in order to investigate the mechanical behavior of porcine corneas. We report the results of inflation tests on the whole cornea and uniaxial tests on excised corneal strips, performed on 51 fresh porcine eyes. Uniaxial tests have been performed on specimens cut from previously inflated corneas. The cornea behavior is characterized by means of elastic stiffness, measured on both average pressure-apex displacement and average uniaxial stress-strain curves; and by means of transversal contraction coefficient, peak stress, and failure stress measured on uniaxial stress-strain curves. Uniaxial tests performed on excised strips allowed to measure the anisotropy in the corneal stiffness and to compare the stiffness of the cornea with the one of the sclera. Viscous properties of the cornea have been obtained through uniaxial relaxation curves on excised corneal strips. The relevant geometrical parameters have been measured and, with the aid of the elastic thin shell theory, a stress-strain curve has been derived from the average inflation test data and compared with similar data available in the literature. The experimental system has been developed in view of future applications to the mechanical testing of both porcine and human corneas. PMID:22482683

  19. Amino acid residues in the Ler protein critical for derepression of the LEE5 promoter in enteropathogenic E. coli.

    PubMed

    Choi, Su-Mi; Jeong, Jae-Ho; Choy, Hyon E; Shin, Minsang

    2016-08-01

    Enteropathogenic E. coli causes attaching and effacing (A/E) intestinal lesions. The genes involved in the formation of A/E lesions are encoded within a chromosomal island comprising of five major operons, LEE1-5. The global regulator H-NS represses the expression of these operons. Ler, a H-NS homologue, counteracts the H-NS-mediated repression. Using a novel genetic approach, we identified the amino acid residues in Ler that are involved in the interaction with H-NS: I20 and L23 in the C-terminal portion of α-helix 3, and I42 in the following unstructured linker region. PMID:27480636

  20. Expression and purification of soluble porcine cystatin 11 in Pichia pastoris.

    PubMed

    Fan, Kuohai; Jiang, Junbing; Wang, Zhirui; Fan, Ruicheng; Yin, Wei; Sun, Yaogui; Li, Hongquan

    2014-11-01

    Cystatin 11 (CST11) belongs to the cystatin type 2 family of cysteine protease inhibitors and exhibits antimicrobial activity in vitro. In this study, we describe the expression and purification of recombinant porcine CST11 in the Pichia pastoris system. We then assess its antimicrobial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis by liquid growth inhibition assay. Kinetic studies indicate that the recombinant porcine CST11 has high potency against E. coli and S. aureus. Scanning electronic microscope analysis showed that CST11 might be targeting the bacterial membrane and, thus, could potentially be developed as a therapeutic agent for inhibiting microbe infection without the risk of antibiotic resistance. PMID:25161037

  1. 7 CFR 1230.18 - Porcine animal.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Porcine animal. 1230.18 Section 1230.18 Agriculture... animal. Porcine animal means a swine, that is raised as (a) a feeder pig, that is, a young pig sold to another person to be finished for slaughtering over a period of more than 1 month; (b) for...

  2. 7 CFR 1230.18 - Porcine animal.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Porcine animal. 1230.18 Section 1230.18 Agriculture... animal. Porcine animal means a swine, that is raised as (a) a feeder pig, that is, a young pig sold to another person to be finished for slaughtering over a period of more than 1 month; (b) for...

  3. 7 CFR 1230.18 - Porcine animal.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 10 2013-01-01 2013-01-01 false Porcine animal. 1230.18 Section 1230.18 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... animal. Porcine animal means a swine, that is raised as (a) a feeder pig, that is, a young pig sold...

  4. 7 CFR 1230.18 - Porcine animal.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 10 2012-01-01 2012-01-01 false Porcine animal. 1230.18 Section 1230.18 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... animal. Porcine animal means a swine, that is raised as (a) a feeder pig, that is, a young pig sold...

  5. 7 CFR 1230.18 - Porcine animal.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 10 2014-01-01 2014-01-01 false Porcine animal. 1230.18 Section 1230.18 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... animal. Porcine animal means a swine, that is raised as (a) a feeder pig, that is, a young pig sold...

  6. [Bacteriological study of traveller's diarrhoea. 4) Isolation of enteropathogenic bacteria from patients with traveller's diarrhoea at Osaka Airport Quarantine Station during 1984-1991].

    PubMed

    Yoshida, A; Noda, K; Omura, K; Miyagi, K; Mori, H; Suzuki, N; Takai, S; Matsumoto, Y; Hayashi, K; Miyata, Y

    1992-10-01

    During the last 8 years (1984 to 1991), 16,639,233 overseas travellers were quarantined at Osaka Airport Quarantine Station and 38,326 travellers reported that they were (or had been) suffering from diarrhoea. Bacteriological examination of stools from 12,573 persons revealed the following results. 1) Various enteropathogenic bacteria were isolated from 3,669 cases (29.2%) examined. The predominant species of bacteria isolated were as follows: Salmonella, 1049 cases; Plesiomonas shigelloides, 1030 cases; Vibrio parahaemolyticus, 789 cases; Shigella, 607 cases; enterotoxigenic Escherichia coli, 422 cases; Vibrio cholerae non-O1, 212 cases. 2) There were no apparent seasonal variations in the isolation rate of these pathogens. 3) The suspected regions for infection with these pathogens were as follows: a) Salmonella, Enterotoxigenic E. coli and Plesiomonas, mainly South-East and South-West Asia. b) Shigella, South-West Asia, especially India (59.8%). c) V. parahaemolyticus and V. fluvialis, mainly South-East and East Asia. d) V. cholerae non-O1, V. mimicus, almost restricted to Asia, mainly South-East Asia. 4) 22 strains of V. cholerae O1 were isolated and 19 were Ogawa, E1 Tor. Of these strains, 13 were cholera toxin-producing strains and 9 were non-toxigenic strains. 5) Several pathogens (mixed infection) were isolated simultaneously from 670 cases. 6) The 1247 Salmonella strains were identified into 98 serovars. 7) Of 624 Shigella strains isolated, 57.9% were S. sonnei, 29.2% were S. flexneri, 8.6% were S. boydii, 4.3% were S. dysenteriae. 8) The most predominant serovar of V. parahaemolyticus was O4:K8. Of 1,247 strains isolated, 9.8% were not producing thermostable direct hemolysin (TDH). 9) 570 (91.3%) of 624 Shigella strains and 409 (32.8%) of 1,247 Salmonella strains isolated were resistant to any one of the drugs tested (SM. CP. TC. KM. ABPC. NA. OFLX). The resistance rate and the number of multiple drug-resistance strains increased year by year. 10

  7. Molecular characterization of diarrheagenic Escherichia coli from Libya.

    PubMed

    Ali, Mostafa Mohamed M; Mohamed, Zienat Kamel; Klena, John D; Ahmed, Salwa Fouad; Moussa, Tarek A A; Ghenghesh, Khalifa Sifaw

    2012-05-01

    Diarrheagenic Escherichia coli (DEC) are important enteric pathogens that cause a wide variety of gastrointestinal diseases, particularly in children. Escherichia coli isolates cultured from 243 diarrheal stool samples obtained from Libyan children and 50 water samples were screened by polymerase chain reaction (PCR) for genes characteristic of enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), and enteroinvasive E. coli (EIEC). The DEC were detected in 21 (8.6%) children with diarrhea; 10 (4.1%) cases were identified as EAEC, 3 (1.2%) as EPEC, and 8 (3.3%) were ETEC; EHEC, and EIEC were not detected. All DEC were grouped phylogenetically by PCR with the majority (> 70%) identified as phylogenetic groups A and B1. The EAEC isolates were also tested for eight genes associated with virulence using PCR. Multi-virulence (≥ 3 virulence factors) was found in 50% of EAEC isolates. Isolated EAEC possessed different virulence traits and belonged to different phylogenetic groups indicating their heterogeneity. PMID:22556089

  8. Molecular Characterization of Diarrheagenic Escherichia coli from Libya

    PubMed Central

    Ali, Mostafa Mohamed M.; Mohamed, Zienat Kamel; Klena, John D.; Ahmed, Salwa Fouad; Moussa, Tarek A. A.; Ghenghesh, Khalifa Sifaw

    2012-01-01

    Diarrheagenic Escherichia coli (DEC) are important enteric pathogens that cause a wide variety of gastrointestinal diseases, particularly in children. Escherichia coli isolates cultured from 243 diarrheal stool samples obtained from Libyan children and 50 water samples were screened by polymerase chain reaction (PCR) for genes characteristic of enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), and enteroinvasive E. coli (EIEC). The DEC were detected in 21 (8.6%) children with diarrhea; 10 (4.1%) cases were identified as EAEC, 3 (1.2%) as EPEC, and 8 (3.3%) were ETEC; EHEC, and EIEC were not detected. All DEC were grouped phylogenetically by PCR with the majority (> 70%) identified as phylogenetic groups A and B1. The EAEC isolates were also tested for eight genes associated with virulence using PCR. Multi-virulence (≥ 3 virulence factors) was found in 50% of EAEC isolates. Isolated EAEC possessed different virulence traits and belonged to different phylogenetic groups indicating their heterogeneity. PMID:22556089

  9. Chronic diarrhea among adults in Kigali, Rwanda: association with bacterial enteropathogens, rectocolonic inflammation, and human immunodeficiency virus infection.

    PubMed

    Clerinx, J; Bogaerts, J; Taelman, H; Habyarimana, J B; Nyirabareja, A; Ngendahayo, P; Van de Perre, P

    1995-11-01

    One hundred patients with chronic diarrhea were seen in the Department of Internal Medicine at the Centre Hospitalier de Kigali, Rwanda; stool and/or rectal swab culture was performed for these patients, and they underwent rectoscopy and serological testing for human immunodeficiency virus type 1 (HIV-1). Enteropathogenic bacteria were isolated from 39 (39%) of the patients: Shigella species (22 of 100 patients tested), non-typhi Salmonella (11/100), Aeromonas species (5/60), and Campylobacter species (4/60). Rectocolitis was seen in 70 (70%) of the patients. HIV-1 antibodies were detected in 82 (94%) of 87 patients tested. Cytomegalovirus was not found in rectal biopsy specimens from 29 patients. Entamoeba histolytica was detected in two of 31 rectal smears. Idiopathic ulcerative colitis was diagnosed for two HIV-1-seropositive patients. One or more AIDS-defining diseases were found in 32 (32%) of the patients, and 72 (72%) fulfilled the World Health Organization's clinical case definition criteria for AIDS. Chronic diarrhea, as seen in a hospital setting in a region highly endemic for HIV-1 infection, is strongly associated with HIV-1 infection, with rectocolonic inflammation, and with infection due to enteropathogenic bacteria. PMID:8589155

  10. Production of Shiga-like toxin among Escherichia coli strains and other bacteria isolated from diarrhea in São Paulo, Brazil.

    PubMed Central

    Giraldi, R; Guth, B E; Trabulsi, L R

    1990-01-01

    An elevated level of Shiga-like toxin I (SLT-I) production was found in 1 of 466 Escherichia coli strains studied. Among the 34 sonic lysates obtained from classical enteropathogenic E. coli, 5 produced SLT-I. The Aeromonas, Citrobacter, Edwardsiella, Enterobacter, Klebsiella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Yersinia, and Vibrio strains also studied were not SLT producers, except for a Shigella dysenteriae type 1 strain. Although SLT-I-producing E. coli strains were isolated from diarrhea, they seem to be an uncommon cause of disease in children less than 1 year old in our community. PMID:2199511

  11. Prevalence and characteristics of intimin-producing Escherichia coli strains isolated from healthy chickens in Korea.

    PubMed

    Oh, J-Y; Kang, M-S; An, B-K; Shin, E-G; Kim, M-J; Kim, Y-J; Kwon, Y-K

    2012-10-01

    Virulent Escherichia coli strains have commonly been associated with diarrheal illness in humans and animals. Typical enteropathogenic Escherichia coli (EPEC) with intimin gene (eaeA) and E. coli adherence factor plasmid, or atypical EPEC with only eaeA have been implicated in human cases. In the present study, we investigated the prevalence of virulence-associated genes including eaeA in the E. coli strains isolated from cloacal specimens of 184 chicken flocks in 7 provinces in Korea between 2009 and 2010. When 7 virulence genes (VT1, VT2, LT, and ST for enterotoxigenic E. coli; eaeA and bfpA for enteropathogenic E. coli; and aggR for enteroaggregative E. coli) were screened by multiplex PCR, a total of 30 E. coli strains carrying only the eaeA gene were detected from 184 flocks that were identified as atypical enteropathogenic Escherichia coli (aEPEC). The aEPEC strains were analyzed by eae subtyping, phylogenetic grouping PCR, and serotyping. Twelve (40%) of 30 aEPEC strains possessed an eae-β subtype, followed by θ (30%), ε (16.7%), and β1 (13.3%). Eight (26.7%) of 30 aEPEC strains were designated into the phylogenetic group A. Two (6.7%) and 3 (10%) aEPEC strains were classified into the phylogenetic group B2 and D, respectively. A total of 15 (50%) aEPEC strains were serotyped to groups O24, O25, O26, O71, O80, O103, and O157, and the remaining strains were nontypeable. In analyzing the genetic diversity among the 30 aEPEC isolates by the pulsed-field gel electrophoresis method with XbaI-digestion, the pulsed-field gel electrophoresis profiling produced 20 different patterns, but isolates within the same group did not show clear geographic or breed relationships. Our data indicate that healthy chickens may constitute an important natural reservoir of aEPEC strains, and suggest that transmission to humans could not be excluded. PMID:22991525

  12. Escherichia coli-induced epithelial hyporesponsiveness to secretagogues is associated with altered CFTR localization.

    PubMed

    Ohland, Christina L; DeVinney, Rebekah; MacNaughton, Wallace K

    2012-04-01

    Both pathogenic and commensal strains of Escherichia coli colonize the human intestinal tract. Pathogenic strains differ only in the expression of virulence factors, many of which comprise a type III secretion system (TTSS). Little is known regarding the effect of E. coli on the intestinal epithelial response to the secretagogues that drive ion secretion, despite its importance in causing clinically significant diarrhoea. Using Ussing chambers to measure electrogenic ion transport of T84 intestinal epithelial cell monolayers, we found that all strains of E. coli tested (pathogenic, commensal, probiotic and lab strain) significantly reduced cAMP-dependent ion secretion after 4-8 h exposure. Enteropathogenic E. coli mutants lacking a functional TTSS caused similar hyposecretion while not causing significant apoptosis (as shown by caspase-3 cleavage) or necrosis (lactate dehydrogenase release), as did the commensal strain F18, indicating that epithelial cell death was not the cause of hyposecretion. Enteropathogenic E. coli and the TTSS mutant significantly reduced cell surface expression of the apical anion channel, cystic fibrosis transmembrane conductance regulator, which is likely the mechanism behind the pathogen-induced hyposecretion. However, F18 did not cause cystic fibrosis transmembrane conductance regulator mislocalization and the commensal-induced mechanism remains unclear. PMID:22212348

  13. A comparison of Luminex xTAG® Gastrointestinal Pathogen Panel (xTAG GPP) and routine tests for the detection of enteropathogens circulating in Southern China.

    PubMed

    Deng, Jiankai; Luo, Xin; Wang, Ruilian; Jiang, Lingxiao; Ding, Xixia; Hao, Wei; Peng, Yongzheng; Jiang, Changhong; Yu, Nan; Che, Xiaoyan

    2015-11-01

    To investigate whether Luminex xTAG® Gastrointestinal Pathogen Panel (xTAG GPP) is applicable for the diagnosis of diarrhea and surveillance of enteropathogens circulating in Southern China, 290 stool samples were tested for 15 kinds of enteropathogens using xTAG GPP and compared to the results from the routine tests, including culture; immunochromatography; real-time PCR; microscopy; and a third method, gene sequencing. One hundred fifty-nine samples were positive, yielding a total of 181 enteropathogens (69 bacteria and 112 viruses), with rotavirus being most prevalent (39.0%, 62/159). The overall sensitivity and specificity of xTAG GPP were 96.3% (93.3-98.2%) and 99.8% (99.6-99.9%), respectively, with a combination of the methods as the gold standard. The coinfection rates detected by the routine tests and xTAG GPP were 10.0% (25 double and 4 triple infections) and 12.1% (29 double, 4 triple and 2 quadruple infections), respectively. xTAG GPP is a powerful tool for the identification of multiple enteropathogens. PMID:26318973

  14. A porcine model of osteosarcoma

    PubMed Central

    Saalfrank, A; Janssen, K-P; Ravon, M; Flisikowski, K; Eser, S; Steiger, K; Flisikowska, T; Müller-Fliedner, P; Schulze, É; Brönner, C; Gnann, A; Kappe, E; Böhm, B; Schade, B; Certa, U; Saur, D; Esposito, I; Kind, A; Schnieke, A

    2016-01-01

    We previously produced pigs with a latent oncogenic TP53 mutation. Humans with TP53 germline mutations are predisposed to a wide spectrum of early-onset cancers, predominantly breast, brain, adrenal gland cancer, soft tissue sarcomas and osteosarcomas. Loss of p53 function has been observed in >50% of human cancers. Here we demonstrate that porcine mesenchymal stem cells (MSCs) convert to a transformed phenotype after activation of latent oncogenic TP53R167H and KRASG12D, and overexpression of MYC promotes tumorigenesis. The process mimics key molecular aspects of human sarcomagenesis. Transformed porcine MSCs exhibit genomic instability, with complex karyotypes, and develop into sarcomas on transplantation into immune-deficient mice. In pigs, heterozygous knockout of TP53 was sufficient for spontaneous osteosarcoma development in older animals, whereas homozygous TP53 knockout resulted in multiple large osteosarcomas in 7–8-month-old animals. This is the first report that engineered mutation of an endogenous tumour-suppressor gene leads to invasive cancer in pigs. Unlike in Trp53 mutant mice, osteosarcoma developed in the long bones and skull, closely recapitulating the human disease. These animals thus promise a model for juvenile osteosarcoma, a relatively uncommon but devastating disease. PMID:26974205

  15. Virulence Gene Regulation in Escherichia coli.

    PubMed

    Mellies, Jay L; Barron, Alex M S

    2006-01-01

    Escherichia colicauses three types of illnesses in humans: diarrhea, urinary tract infections, and meningitis in newborns. The acquisition of virulence-associated genes and the ability to properly regulate these, often horizontally transferred, loci distinguishes pathogens from the normally harmless commensal E. coli found within the human intestine. This review addresses our current understanding of virulence gene regulation in several important diarrhea-causing pathotypes, including enteropathogenic, enterohemorrhagic,enterotoxigenic, and enteroaggregativeE. coli-EPEC, EHEC, ETEC and EAEC, respectively. The intensely studied regulatory circuitry controlling virulence of uropathogenicE. coli, or UPEC, is also reviewed, as is that of MNEC, a common cause of meningitis in neonates. Specific topics covered include the regulation of initial attachment events necessary for infection, environmental cues affecting virulence gene expression, control of attaching and effacing lesionformation, and control of effector molecule expression and secretion via the type III secretion systems by EPEC and EHEC. How phage control virulence and the expression of the Stx toxins of EHEC, phase variation, quorum sensing, and posttranscriptional regulation of virulence determinants are also addressed. A number of important virulence regulators are described, including the AraC-like molecules PerA of EPEC, CfaR and Rns of ETEC, and AggR of EAEC;the Ler protein of EPEC and EHEC;RfaH of UPEC;and the H-NS molecule that acts to silence gene expression. The regulatory circuitry controlling virulence of these greatly varied E. colipathotypes is complex, but common themes offerinsight into the signals and regulators necessary forE. coli disease progression. PMID:26443571

  16. (PCG) Protein Crystal Growth Porcine Elastase

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Porcine Elastase. This enzyme is associated with the degradation of lung tissue in people suffering from emphysema. It is useful in studying causes of this disease. Principal Investigator on STS-26 was Charles Bugg.

  17. Sirtuin Inhibition Adversely Affects Porcine Oocyte Meiosis

    PubMed Central

    Zhang, Liang; Ma, Rujun; Hu, Jin; Ding, Xiaolin; Xu, Yinxue

    2015-01-01

    Sirtuins have been implicated in diverse biological processes, including oxidative stress, energy metabolism, cell migration, and aging. Here, we employed Sirtuin inhibitors, nicotinamide (NAM) and Sirtinol, to investigate their effects on porcine oocyte maturation respectively. The rate of polar body extrusion in porcine oocytes decreased after treatment with NAM and Sirtinol, accompanied with the failure of cumulus cell expansion. We further found that NAM and Sirtinol significantly disrupted oocyte polarity, and inhibited the formation of actin cap and cortical granule-free domain (CGFD). Moreover, the abnormal spindles and misaligned chromosomes were readily detected during porcine oocyte maturation after treatment with NAM and Sirtinol. Together, these results suggest that Sirtuins are involved in cortical polarity and spindle organization in porcine oocytes. PMID:26176547

  18. Porcine Head Response to Blast

    PubMed Central

    Shridharani, Jay K.; Wood, Garrett W.; Panzer, Matthew B.; Capehart, Bruce P.; Nyein, Michelle K.; Radovitzky, Raul A.; Bass, Cameron R. ‘Dale’

    2012-01-01

    Recent studies have shown an increase in the frequency of traumatic brain injuries related to blast exposure. However, the mechanisms that cause blast neurotrauma are unknown. Blast neurotrauma research using computational models has been one method to elucidate that response of the brain in blast, and to identify possible mechanical correlates of injury. However, model validation against experimental data is required to ensure that the model output is representative of in vivo biomechanical response. This study exposes porcine subjects to primary blast overpressures generated using a compressed-gas shock tube. Shock tube blasts were directed to the unprotected head of each animal while the lungs and thorax were protected using ballistic protective vests similar to those employed in theater. The test conditions ranged from 110 to 740 kPa peak incident overpressure with scaled durations from 1.3 to 6.9 ms and correspond approximately with a 50% injury risk for brain bleeding and apnea in a ferret model scaled to porcine exposure. Instrumentation was placed on the porcine head to measure bulk acceleration, pressure at the surface of the head, and pressure inside the cranial cavity. Immediately after the blast, 5 of the 20 animals tested were apneic. Three subjects recovered without intervention within 30 s and the remaining two recovered within 8 min following respiratory assistance and administration of the respiratory stimulant doxapram. Gross examination of the brain revealed no indication of bleeding. Intracranial pressures ranged from 80 to 390 kPa as a result of the blast and were notably lower than the shock tube reflected pressures of 300–2830 kPa, indicating pressure attenuation by the skull up to a factor of 8.4. Peak head accelerations were measured from 385 to 3845 G’s and were well correlated with peak incident overpressure (R2 = 0.90). One SD corridors for the surface pressure, intracranial pressure (ICP), and head acceleration are

  19. HIGH PREVALENCE OF ENTEROINVASIVE ESCHERICHIA COLI ISOLATED IN A REMOTE REGION OF NORTHERN COASTAL ECUADOR

    PubMed Central

    VIEIRA, NADIA; BATES, SARAH J.; SOLBERG, OWEN D.; PONCE, KARINA; HOWSMON, REBECCA; CEVALLOS, WILLIAM; TRUEBA, GABRIEL; RILEY, LEE; EISENBERG, JOSEPH N. S.

    2008-01-01

    Enteroinvasive Escherichia coli (EIEC) causes dysentery; however, it is less widely reported than other etiological agents in studies of diarrhea worldwide. Between August 2003 and July 2005, stool samples were collected in case-control studies in 22 rural communities in northwestern Ecuador. Infection was assessed by PCR specific for LT and STa genes of enterotoxigenic E. coli (ETEC), the bfp gene of enteropathogenic E. coli (EPEC), and the ipaH gene of both enteroinvasive E. coli and Shigellae. The pathogenic E. coli most frequently identified were EIEC (3.2 cases/100 persons) and Shigellae (1.5 cases/100 persons), followed by ETEC (1.3 cases/100 persons), and EPEC (0.9 case/100 persons). EIEC exhibited similar risk-factor relationships with other pathotypes analyzed but different age-specific infection rates. EIEC was the predominant diarrheagenic bacteria isolated in our community-based study, a unique observation compared with other regions of the world. PMID:17360879

  20. Role of virulence factors on host inflammatory response induced by diarrheagenic Escherichia coli pathotypes.

    PubMed

    Sanchez-Villamil, Javier; Navarro-Garcia, Fernando

    2015-01-01

    Pathogens are able to breach the intestinal barrier, and different bacterial species can display different abilities to colonize hosts and induce inflammation. Inflammatory response studies induced by enteropathogens as Escherichia coli are interesting since it has acquired diverse genetic mobile elements, leading to different E. coli pathotypes. Diarrheagenic E. coli secrete toxins, effectors and virulence factors that exploit the host cell functions to facilitate the bacterial colonization. Many bacterial proteins are delivered to the host cell for subverting the inflammatory response. Hereby, we have highlighted the specific processes used by E. coli pathotypes, by that subvert the inflammatory pathways. These mechanisms include an arrangement of pro- and anti-inflammatory responses to favor the appropriate environmental niche for the bacterial survival and growth. PMID:26059623

  1. Antimicrobial activity of selected synbiotics targeted for the elderly against pathogenic Escherichia coli strains.

    PubMed

    Likotrafiti, E; Tuohy, K M; Gibson, G R; Rastall, R A

    2016-01-01

    The aim of the present study was to evaluate the antimicrobial activity of two synbiotic combinations, Lactobacillus fermentum with short-chain fructooligosaccharides (FOS-LF) and Bifidobacterium longum with isomaltooligosaccharides (IMO-BL), against enterohaemorrhagic Escherichia coli O157:H7 and enteropathogenic E. coli O86. Antimicrobial activity was determined (1) by co-culturing the synbiotics and pathogens in batch cultures, and (2) with the three-stage continuous culture system (gut model), inoculated with faecal slurry from an elderly donor. In the co-culture experiments, IMO-BL was significantly inhibitory to both E. coli strains, while FOS-LF was slightly inhibitory or not inhibitory. Factors other than acid production appeared to play a role in the inhibition. In the gut models, both synbiotics effectively inhibited E. coli O157 in the first vessel, but not in vessels 2 and 3. E. coli O86 was not significantly inhibited. PMID:26754553

  2. Cloning and characterization of porcine resistin gene.

    PubMed

    Dai, M H; Xia, T; Chen, X D; Gan, L; Feng, S Q; Qiu, H; Peng, Y; Yang, Z Q

    2006-02-01

    Resistin is a member of resistin-like molecules (RELMs) and a hormone secreted from mature adipocytes in rodents and leukocytes in human. We now report the cloning and characterization of the full-length porcine resistin cDNA and gene. Sequence analysis indicated that the pig resistin cDNA sequence had an open reading frame of 330 bp encoding a 12 kDa protein of 109 amino acids. The deduced amino acid sequence showed 75.2% identity to the human resistin. The porcine resistin gene was composed of four exons and had exactly the same exon structure as the human resistin gene. The tissue distribution of porcine resistin mRNA was assessed by semi-quantitative RT-PCR. Resistin gene expression was the highest in porcine leukocytes and low in adipose tissue. Resistin protein could be detected in porcine serum by western blotting and it circulated in serum as dimers and trimers. We provided the first evidence that resistin was abundantly expressed in porcine leukocytes and had an expression pattern similar to that in human resistin mRNA and protein. This suggests that the pig may be a suitable animal model for studying the function of resistin in human insulin resistance. PMID:16023825

  3. Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, June 2016.

    PubMed

    Xavier, Basil Britto; Lammens, Christine; Ruhal, Rohit; Kumar-Singh, Samir; Butaye, Patrick; Goossens, Herman; Malhotra-Kumar, Surbhi

    2016-07-01

    We identified a novel plasmid-mediated colistin-resistance gene in porcine and bovine colistin-resistant Escherichia coli that did not contain mcr-1. The gene, termed mcr-2, a 1,617 bp phosphoethanolamine transferase harboured on an IncX4 plasmid, has 76.7% nucleotide identity to mcr-1. Prevalence of mcr-2 in porcine colistin-resistant E. coli (11/53) in Belgium was higher than that of mcr-1 (7/53). These data call for an immediate introduction of mcr-2 screening in ongoing molecular epidemiological surveillance of colistin-resistant Gram-negative pathogens. PMID:27416987

  4. SEROLOGICAL CROSS-REACTIONS BETWEEN ESCHERICHIA COLI 0157 AND OTHER SPECIES OF THE GENUS ESCHERICHIA

    EPA Science Inventory

    Escherichia hermannii, a sorbitol-negative species of the genus Escherichia, has been reported to be agglutinated by Escherichia coli 0157 and four sorbitol-negative species of the genus Escherichia: . hermannii (24 isolates), Escherichia fergusonii (12 isolates), Escherichia vul...

  5. Porcine IPEC-J2 Intestinal Epithelial Cells in Microbiological Investigations

    PubMed Central

    Brosnahan, Amanda J.; Brown, David R.

    2011-01-01

    IPEC-J2 cells are porcine intestinal columnar epithelial cells that were isolated from neonatal piglet mid-jejunum. This cell line forms polarized monolayers with high transepithelial electrical resistance when cultured on 0.4 μm pore-size filters. The cell line is unique in that it is derived from small intestinal tissue (compared to the common human colon-derived lines HT-29, T84, and Caco-2) and is not transformed (compared to the porcine small intestinal line, IPI-2I). Porcine intestinal epithelial cells more closely mimic human physiology than analogous rodent cell lines (e.g. IEC-6 or IEC-18), which is important in studies of zoonotic infections; in addition, they provide specificity to study porcine-derived infections. IPEC-J2 cells are increasingly being used in microbiological studies to examine the interactions of various animal and human pathogens, including Salmonella enterica and pathogenic Escherichia coli, with intestinal epithelial cells. The IPEC-J2 cell line has also been employed in some probiotic studies, in which the cells have been used as an initial screening tool for adhesiveness and anti-inflammatory properties of the potential probiotic microorganisms. The validity of these studies is not clear as follow-up studies to assess the efficacy of the probiotics in vivo have not been published to date. The aims of this review are to provide a comprehensive overview of the microbiological studies that have been conducted with IPEC-J2 cells and a reference guide of key cellular and immune markers that have been identified in this cell line that may prove to be useful in future studies. PMID:22074860

  6. Isolation and characterization of a Korean porcine epidemic diarrhea virus strain KNU-141112.

    PubMed

    Lee, Sunhee; Kim, Youngnam; Lee, Changhee

    2015-10-01

    Severe outbreaks of porcine epidemic diarrhea virus (PEDV) have re-emerged in Korea and rapidly swept across the country, causing tremendous economic losses to producers and customers. Despite the availability of PEDV vaccines in the domestic market, the disease continues to plague the Korean pork industry, raising issues regarding their protective efficacy and new vaccine development. Therefore, PEDV isolation in cell culture is urgently needed to develop efficacious vaccines and diagnostic assays and to conduct further studies on the virus biology. In the present study, one Korean PEDV strain, KOR/KNU-141112/2014, was successfully isolated and serially propagated in Vero cells for over 30 passages. The in vitro and in vivo characteristics of the Korean PEDV isolate were investigated. Virus production in cell culture was confirmed by cytopathology, immunofluorescence, and real-time RT-PCR. The infectious virus titers of the viruses during the first 30 passages ranged from 10(5.1) to 10(8.2) TCID50 per ml. The inactivated KNU-141112 virus was found to mediate potent neutralizing antibody responses in immunized guinea pigs. Animal studies showed that KNU-141112 virus causes severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis, indicating that strain KNU-141112 is highly enteropathogenic in the natural host. In addition, the entire genomes or complete S genes of KNU-141112 viruses at selected cell culture passages were sequenced to assess the genetic stability and relatedness. Our genomic analyses indicated that the Korean isolate KNU-141112 is genetically stable during the first 30 passages in cell culture and is grouped within subgroup G2b together with the recent re-emergent Korean strains. PMID:26196056

  7. Construction of an infectious genomic clone of porcine parvovirus: effect of the 5'-end on DNA replication.

    PubMed

    Casal, J I; Diaz-Aroca, E; Ranz, A I; Manclus, J J

    1990-08-01

    The linear single-stranded DNA genome of the porcine parvovirus, an autonomous parvovirus, was cloned in duplex form into the bacterial plasmid pUC18 using a simple and reliable method. These clones were stable during propagation in Escherichia coli JM109. The recombinant clones of porcine parvovirus were infectious when transfected into monolayers of swine testes cells as identified by the development of cytopathic effect, indirect immunofluorescence with specific antiserum, and hemagglutination assays. DNA isolated from progeny virus arising from transfected infectious clones was found to be indistinguishable from wild-type DNA by restriction enzyme analysis. Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogeneous, cloned DNA. The presence of the turn of the 5'-end loop seems to be necessary to get stable infectious clones. PMID:2371779

  8. Porcine Epidemic Diarrhea Virus 3C-Like Protease Regulates Its Interferon Antagonism by Cleaving NEMO

    PubMed Central

    Wang, Dang; Fang, Liurong; Shi, Yanling; Zhang, Huan; Gao, Li; Peng, Guiqing; Chen, Huanchun; Li, Kui

    2015-01-01

    ABSTRACT Porcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus causing lethal watery diarrhea in piglets. Since 2010, a PEDV variant has spread rapidly in China, and it emerged in the United States in 2013, posing significant economic and public health concerns. The ability to circumvent the interferon (IFN) antiviral response, as suggested for PEDV, promotes viral survival and regulates pathogenesis of PEDV infections, but the underlying mechanisms remain obscure. Here, we show that PEDV-encoded 3C-like protease, nsp5, is an IFN antagonist that proteolytically cleaves the nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), an essential adaptor bridging interferon-regulatory factor and NF-κB activation. NEMO is cleaved at glutamine 231 (Q231) by PEDV, and this cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of PEDV nsp5 abrogated NEMO cleavage and the inhibition of IFN induction. Structural analysis suggests that several key residues outside the catalytic sites of PEDV nsp5 probably impact NEMO cleavage by modulating potential interactions of nsp5 with their substrates. These data show that PEDV nsp5 disrupts type I IFN signaling by cleaving NEMO. Previously, we and others demonstrated that NEMO is also cleaved by 3C or 3C-like proteinases of picornavirus and artertivirus. Thus, NEMO probably represents a prime target for 3C or 3C-like proteinases of different viruses. IMPORTANCE The continued emergence and reemergence of porcine epidemic diarrhea virus (PEDV) underscore the importance of studying how this virus manipulates the immune responses of its hosts. During coevolution with its hosts, PEDV has acquired mechanisms to subvert host innate immune responses for its survival advantage. At least two proteins encoded by PEDV have been identified as interferon (IFN

  9. Lentiviral vector gene transfer to porcine airways.

    PubMed

    Sinn, Patrick L; Cooney, Ashley L; Oakland, Mayumi; Dylla, Douglas E; Wallen, Tanner J; Pezzulo, Alejandro A; Chang, Eugene H; McCray, Paul B

    2012-01-01

    In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1-based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF).Molecular Therapy - Nucleic Acids (2012) 1, e56; doi:10.1038/mtna.2012.47; published online 27 November 2012. PMID:23187455

  10. Pet, an Autotransporter Enterotoxin from Enteroaggregative Escherichia coli

    PubMed Central

    Eslava, Carlos; Navarro-García, Fernando; Czeczulin, John R.; Henderson, Ian R.; Cravioto, Alejandro; Nataro, James P.

    1998-01-01

    Enteroaggregative Escherichia coli (EAEC) is an emerging cause of diarrheal illness. Clinical data suggest that diarrhea caused by EAEC is predominantly secretory in nature, but the responsible enterotoxin has not been described. Work from our laboratories has implicated a ca. 108-kDa protein as a heat-labile enterotoxin and cytotoxin, as evidenced by rises in short-circuit current and falls in tissue resistance in rat jejunal tissue mounted in an Ussing chamber. Here we report the genetic cloning, sequencing, and characterization of this high-molecular-weight heat-labile toxin. The toxin (designated the plasmid-encoded toxin [Pet]) is encoded on the 65-MDa adherence-related plasmid of EAEC strain 042. Nucleotide sequence analysis suggests that the toxin is a member of the autotransporter class of proteins, characterized by the presence of a conserved C-terminal domain which forms a β-barrel pore in the bacterial outer membrane and through which the mature protein is transported. The Pet toxin is highly homologous to the EspP protease of enterohemorrhagic E. coli and to EspC of enteropathogenic E. coli, an as yet cryptic protein. In addition to its potential role in EAEC infection, Pet represents the first enterotoxin within the autotransporter class of secreted proteins. We hypothesize that other closely related members of this class may also produce enterotoxic effects. PMID:9632580

  11. Comparative Genomic Indexing Reveals the Phylogenomics of Escherichia coli Pathogens

    PubMed Central

    Anjum, Muna F.; Lucchini, Sacha; Thompson, Arthur; Hinton, Jay C. D.; Woodward, Martin J.

    2003-01-01

    The Escherichia coli O26 serogroup includes important food-borne pathogens associated with human and animal diarrheal disease. Current typing methods have revealed great genetic heterogeneity within the O26 group; the data are often inconsistent and focus only on verotoxin (VT)-positive O26 isolates. To improve current understanding of diversity within this serogroup, the genomic relatedness of VT-positive and -negative O26 strains was assessed by comparative genomic indexing. Our results clearly demonstrate that irrespective of virulence characteristics and pathotype designation, the O26 strains show greater genomic similarity to each other than to any other strain included in this study. Our data suggest that enteropathogenic and VT-expressing E. coli O26 strains represent the same clonal lineage and that VT-expressing E. coli O26 strains have gained additional virulence characteristics. Using this approach, we established the core genes which are central to the E. coli species and identified regions of variation from the E. coli K-12 chromosomal backbone. PMID:12874348

  12. Identification of Diarrheagenic Escherichia coli Strains from Avian Organic Fertilizers

    PubMed Central

    Puño-Sarmiento, Juan; Gazal, Luis Eduardo; Medeiros, Leonardo P.; Nishio, Erick K.; Kobayashi, Renata K. T.; Nakazato, Gerson

    2014-01-01

    The Brazilian poultry industry generates large amounts of organic waste, such as chicken litter, which is often used in agriculture. Among the bacteria present in organic fertilizer are members of the Enterobacteriaceae family. The objective of this study was to detect the presence of diarrheagenic Escherichia coli (DEC) strains in avian organic fertilizer, and assess the potential damage they can cause in humans due to antimicrobial resistance. The presence of DEC pathotypes and phylogenetic groups were detected by multiplex-PCR. Phenotypic assays, such as tests for adhesion, cytotoxicity activity, biofilm formation and especially antimicrobial susceptibility, were performed. Fifteen DEC strains from 64 E. coli were isolated. Among these, four strains were classified as enteropathogenic (EPEC; 6.2%), three strains as Shiga toxin-producing (STEC; 4.7%), 10 strains as enteroaggregative (EAEC; 12.5%), but two of these harbored the eaeA gene too. The low number of isolated strains was most likely due to the composting process, which reduces the number of microorganisms. These strains were able to adhere to HEp-2 and HeLa cells and produce Shiga-toxins and biofilms; in addition, some of the strains showed antimicrobial resistance, which indicates a risk of the transfer of resistance genes to human E. coli. These results showed that DEC strains isolated from avian organic fertilizers can cause human infections. PMID:25170683

  13. Restriction of Porcine Endogenous Retrovirus by Porcine APOBEC3 Cytidine Deaminases ▿

    PubMed Central

    Dörrschuck, Eva; Fischer, Nicole; Bravo, Ignacio G.; Hanschmann, Kay-Martin; Kuiper, Heidi; Spötter, Andreas; Möller, Ronny; Cichutek, Klaus; Münk, Carsten; Tönjes, Ralf R.

    2011-01-01

    Xenotransplantation of porcine cells, tissues, and organs shows promise to surmount the shortage of human donor materials. Among the barriers to pig-to-human xenotransplantation are porcine endogenous retroviruses (PERV) since functional representatives of the two polytropic classes, PERV-A and PERV-B, are able to infect human embryonic kidney cells in vitro, suggesting that a xenozoonosis in vivo could occur. To assess the capacity of human and porcine cells to counteract PERV infections, we analyzed human and porcine APOBEC3 (A3) proteins. This multigene family of cytidine deaminases contributes to the cellular intrinsic immunity and act as potent inhibitors of retroviruses and retrotransposons. Our data show that the porcine A3 gene locus on chromosome 5 consists of the two single-domain genes A3Z2 and A3Z3. The evolutionary relationships of the A3Z3 genes reflect the evolutionary history of mammals. The two A3 genes encode at least four different mRNAs: A3Z2, A3Z3, A3Z2-Z3, and A3Z2-Z3 splice variant A (SVA). Porcine and human A3s have been tested toward their antiretroviral activity against PERV and murine leukemia virus (MuLV) using novel single-round reporter viruses. The porcine A3Z2, A3Z3 and A3Z2-Z3 were packaged into PERV particles and inhibited PERV replication in a dose-dependent manner. The antiretroviral effect correlated with editing by the porcine A3s with a trinucleotide preference for 5′ TGC for A3Z2 and A3Z2-Z3 and 5′ CAC for A3Z3. These results strongly imply that human and porcine A3s could inhibit PERV replication in vivo, thereby reducing the risk of infection of human cells by PERV in the context of pig-to-human xenotransplantation. PMID:21307203

  14. Rifaximin Does Not Induce Toxin Production or Phage-Mediated Lysis of Shiga Toxin-Producing Escherichia coli▿

    PubMed Central

    Ochoa, Theresa J.; Chen, Jane; Walker, Christopher M.; Gonzales, Elsa; Cleary, Thomas G.

    2007-01-01

    Diarrhea in children is often caused by enteropathogen infections that might benefit from early empirical antibiotic therapy. However, when the definition of the pathogen requires sophisticated laboratory studies, the etiology of enteritis is not known early in illness. Empirical therapy may be dangerous if the child is infected with a Shiga toxin-producing Escherichia coli (STEC) strain because antimicrobials may increase Shiga toxin (Stx) release, resulting in increased risk of microangiopathic hemolytic anemia with acute renal failure (hemolytic-uremic syndrome [HUS]) and death. There is a need for antimicrobials that would be effective against multiple bacterial enteropathogens yet not induce Stx release or increase the risk of HUS. Rifaximin has been evaluated in adults for treatment of bacterial enteritis and has a good record for safety and efficacy, but it has not been evaluated extensively in children with gastroenteritis. We therefore evaluated rifaximin's potential for phage induction, drug-induced bacteriolysis, and toxin release in 57 STEC strains (26 O157 and 31 non-O157 strains). Growth in ciprofloxacin, a known Stx phage inducer, caused bacteriolysis and release of toxin in 25/26 (96%) O157 strains and 15/31 (48%) non-O157 strains. In contrast, rifaximin did not induce phage replication or lysis in any strain. Toxin release in the presence of rifaximin was not different from release in the absence of antibiotic. Rifaximin, unlike many antibiotics used to treat pediatric gastroenteritis, does not induce phage-mediated bacteriolysis and Stx release. PMID:17526759

  15. Prevalence of Escherichia coli O157:H7 in Children with Bloody Diarrhea Referring to Abuzar Teaching Hospital, Ahvaz, Iran

    PubMed Central

    Khosravi, Azar Dokht; Sheikh, Ahmad Farajzadeh; Ahmadzadeh, Ali; Shamsizadeh, Ahmad

    2016-01-01

    Introduction Escherichia coli O157: H7 are recognized as important aetiological agents of diarrhea in children, particularly in developed countries. Aim The aim of the study was to determine the rates of detection of E. coli O157: H7strains among children in Ahvaz, Iran. Materials and Methods From June 2010 to December 2010, 137 diarrheal stool samples of children were collected. E.coli was identified by standard microbiological techniques. O157 or O157:H7 subtypes discerned by serological tests. Results Of the 137 E. coli isolates, enteropathogens were found in 53 (38.7%) of the patients as follow: Shigella spp. (75.5%), EPEC (enteropathogenic E. coli) (16.9%), Campylobacter spp. (3.8%) and Salmonella spp. (3.8%). None of the isolated E. coli was O157:H7 serotype. Conclusion This shows that non-O157:H7 E. coli are the major cause of paediatric infections in this region of Iran. PMID:26894066

  16. Faecal excretion of rotavirus and other enteropathogens in newborns of the high and low socio-economic stratum in Santiago, Chile.

    PubMed Central

    Spencer, E.; Araya, M.; Sandino, A. M.; Pacheco, I.; Brunser, O.

    1988-01-01

    Faecal excretion of enteropathogens was studied in newborns in their first week of life. Rotavirus was investigated in 225 neonates, of whom 107 belonged to the low socio-economic stratum (SES) and 118 to the high SES. Half of each group were delivered by caesarean section. Rotavirus was detected in 10 infants (4.4%). Eight of them had been in the same ward and excreted the same viral electrophoretype. Enteropathogenic bacteria were isolated from 8 out of 57 (14.0%) newborns. Positive cultures were equally distributed by SES and route of delivery. Giardia lamblia was the only parasite detected, in one infant (2.6%) of the high SES. None of the children developed symptoms. Faecal excretion of enteropathogens ended spontaneously within a week in all cases. It is suggested that the lack of symptomatology and the spontaneous termination of the faecal excretion are related to immaturity of the small intestinal mucosa, that does not allow the completion of the steps that must take place during a successful infectious event. Images Fig. 1 Fig. 2 Fig. 3 PMID:2846331

  17. Bayesian Estimation of the True Prevalence and of the Diagnostic Test Sensitivity and Specificity of Enteropathogenic Yersinia in Finnish Pig Serum Samples

    PubMed Central

    Vilar, M. J.; Ranta, J.; Virtanen, S.; Korkeala, H.

    2015-01-01

    Bayesian analysis was used to estimate the pig's and herd's true prevalence of enteropathogenic Yersinia in serum samples collected from Finnish pig farms. The sensitivity and specificity of the diagnostic test were also estimated for the commercially available ELISA which is used for antibody detection against enteropathogenic Yersinia. The Bayesian analysis was performed in two steps; the first step estimated the prior true prevalence of enteropathogenic Yersinia with data obtained from a systematic review of the literature. In the second step, data of the apparent prevalence (cross-sectional study data), prior true prevalence (first step), and estimated sensitivity and specificity of the diagnostic methods were used for building the Bayesian model. The true prevalence of Yersinia in slaughter-age pigs was 67.5% (95% PI 63.2–70.9). The true prevalence of Yersinia in sows was 74.0% (95% PI 57.3–82.4). The estimates of sensitivity and specificity values of the ELISA were 79.5% and 96.9%. PMID:26539540

  18. Quantitative proteomics and bioinformatic analysis provide new insight into the dynamic response of porcine intestine to Salmonella Typhimurium

    PubMed Central

    Collado-Romero, Melania; Aguilar, Carmen; Arce, Cristina; Lucena, Concepción; Codrea, Marius C.; Morera, Luis; Bendixen, Emoke; Moreno, Ángela; Garrido, Juan J.

    2015-01-01

    The enteropathogen Salmonella Typhimurium (S. Typhimurium) is the most commonly non-typhoideal serotype isolated in pig worldwide. Currently, one of the main sources of human infection is by consumption of pork meat. Therefore, prevention and control of salmonellosis in pigs is crucial for minimizing risks to public health. The aim of the present study was to use isobaric tags for relative and absolute quantification (iTRAQ) to explore differences in the response to Salmonella in two segment of the porcine gut (ileum and colon) along a time course of 1, 2, and 6 days post infection (dpi) with S. Typhimurium. A total of 298 proteins were identified in the infected ileum samples of which, 112 displayed significant expression differences due to Salmonella infection. In colon, 184 proteins were detected in the infected samples of which 46 resulted differentially expressed with respect to the controls. The higher number of changes in protein expression was quantified in ileum at 2 dpi. Further biological interpretation of proteomics data using bioinformatics tools demonstrated that the expression changes in colon were found in proteins involved in cell death and survival, tissue morphology or molecular transport at the early stages and tissue regeneration at 6 dpi. In ileum, however, changes in protein expression were mainly related to immunological and infection diseases, inflammatory response or connective tissue disorders at 1 and 2 dpi. iTRAQ has proved to be a proteomic robust approach allowing us to identify ileum as the earliest response focus upon S. Typhimurium in the porcine gut. In addition, new functions involved in the response to bacteria such as eIF2 signaling, free radical scavengers or antimicrobial peptides (AMP) expression have been identified. Finally, the impairment at of the enterohepatic circulation of bile acids and lipid metabolism by means the under regulation of FABP6 protein and FXR/RXR and LXR/RXR signaling pathway in ileum has been

  19. Frequency of enteropathogen detection in suckling and weaned pigs with diarrhea in Japan.

    PubMed

    Katsuda, Ken; Kohmoto, Mariko; Kawashima, Kenji; Tsunemitsu, Hiroshi

    2006-07-01

    Fecal samples from suckling (n=153) and weaned (n=116) piglets with diarrhea in Japan were examined for shedding of viral, bacterial, and parasitic pathogens using culture, microscopic, and polymerase chain reaction methods. In suckling piglets, diarrhea was attributed to infection with a single etiologic agent in 60.8% of cases and with combinations of agents in 22.2%. In weaned piglets, diarrhea was attributed to a single etiologic agent in 43.1% and to combinations of agents in 47.4% of cases. Rotavirus was the most prevalent agent in suckling (67.3%) and weaned (65.5%) piglets. The detection of other pathogens was associated with age of the animals examined. Coccidia were predominantly isolated from suckling piglets, whereas Escherichia coli was found predominantly in weaned piglets. Although a relationship was not observed between detection rate of rotavirus and age of piglets, a single group of rotavirus was detected in 87.5% of suckling piglets whereas multiple groups were detected in 51.6% of weaned piglets. The results of this study confirm that diarrhea in piglets can, to a variable degree, be causally associated with multiple agents. Additionally, these results suggest reasons why this syndrome can be difficult to control. PMID:16921873

  20. Occurrence, virulence genes and antibiotic resistance of enteropathogenic Escherichia coli (EPEC) from twelve bovine farms in the north-east of Ireland.

    PubMed

    Bolton, D J; Ennis, C; McDowell, D

    2014-03-01

    Cattle faecal samples (n = 480) were collected from a cluster of 12 farms, and PCR screened for the presence of the intimin gene (eae). Positive samples were cultured, and colonies were examined for the presence of eae and verocytotoxin (vtx) genes. Colonies which were positive for the intimin gene and negative for the verocytotoxin genes were further screened using PCR for a range of virulence factors including bfpA, espA, espB, tir ehxA, toxB, etpD, katP, saa, iha, lpfAO157/OI-141 and lpfAO157/OI-154. Of the 480 faecal samples, 5.8% (28/480) were PCR positive, and one isolate was obtained from each. All 28 isolates obtained were bfpA negative and therefore atypical EPEC (aEPEC). The serotypes detected included O2:H27, O8:H36, O15:H2, O49:H+, O84:H28, O105:H7 and O132:H34 but half of the isolates could not be serogrouped using currently available antisera. Twenty-two (79%) of the isolates carried the tir gene but only 25% were espB positive, and all other virulence genes tested for were scarce or absent. Several isolates showed intermediate resistance to ciprofloxacin, kanamycin, nalidixic acid, minocycline and tetracycline; full resistance to nalidixic acid or tetracycline with one isolate (O-:H8) displaying resistance to aminoglycosides (kanamycin and streptomycin), quinolones (nalidixic acid) and sulphonamides. This study provides further evidence that cattle are a potential source of aEPEC and add to the very limited data currently available on virulence genes and antibiotic resistance in this pathogenic E. coli group in animals. PMID:23782867

  1. Clonal analysis and virulent traits of pathogenic extraintestinal Escherichia coli isolates from swine in China

    PubMed Central

    2012-01-01

    Background Extraintestinal pathogenic Escherichia coli (ExPEC) can cause a variety of infections outside the gastrointestinal tract in humans and animals. Infections due to swine ExPECs have been occurring with increasing frequency in China. These ExPECs may now be considered a new food-borne pathogen that causes cross-infections between humans and pigs. Knowledge of the clonal structure and virulence genes is needed as a framework to improve the understanding of phylogenetic traits of porcine ExPECs. Results Multilocus sequence typing (MLST) data showed that the isolates investigated in this study could be placed into four main clonal complexes, designated as CC10, CC1687, CC88 and CC58. Strains within CC10 were classified as phylogroup A, and these accounted for most of our porcine ExPEC isolates. Isolates in the CC1687 clonal complex, formed by new sequence types (STs), was classified as phylogroup D, with CC88 isolates considered as B2 and CC58 isolates as B1. Porcine ExPECs in these four clonal complexes demonstrated significantly different virulence gene patterns. A few porcine ExPECs were indentified in phylogroup B2, the phylogroup in which human ExPECs mainly exist. However some STs in the four clonal groups of porcine ExPECs were reported to cause extraintestinal infections in human, based on data in the MLST database. Conclusion Porcine ExPECs have different virulence gene patterns for different clonal complexes. However, these strains are mostly fell in phylogenentic phylogroup A, B1 and D, which is different from human ExPECs that concentrate in phylogroup B2. Our findings provide a better understanding relating to the clonal structure of ExPECs in diseased pigs and indicate a need to re-evaluate their contribution to human ExPEC diseases. PMID:22909380

  2. Hygienic aspects of porcine gullets.

    PubMed

    Bijker, P G; Mossel, D A; van Logtestijn, J G

    1985-01-01

    In an attempt to elaborate good manufacturing practices, including the collection, processing and storage of porcine gullets, their bacterial condition immediately after collection (100 samples), as well as that of deep frozen gullets just before incorporation into meat products (40 samples), was assessed. Fresh gullets were found to be contaminated to a high degree: poured plate colony count at 30°C (PPCC) approximately 10(6) to 10(7) and Enterobacteriaceae approximately 10(3) to 10(4) cfu g(-1). Deep frozen gullets showed even higher counts: PPCC approximately 10(7) to 10(8) and Enterobacteriaceae approximately 10(4) to 10(5) cfu g(-1). Hygiene during collection was visually assessed in six abattoirs and found to be satisfactory in two, moderate in three and poor in one. The effects of processing, by cleaning or removal of the mucus membrane, on bacterial condition, pH, colour and odour were assessed before and during storage at 4°C and 20°C. Both cleaning and removal of the mucus membrane resulted in up to approximately a tenfold reduction of colony counts. After 7 days' storage at 4°C these were significantly lower than those of unprocessed gullets (P < 0·01). Processed gullets stored at 4°C were no longer fit for consumption after 4 days' storage. It being impossible to achieve a marked improvement in the bacteriological condition of gullets, the incorporation of these products into sausages should be discouraged and their use in petfoods only allowed under reasonable conditions of hygiene and chilling. PMID:22055164

  3. Immunomodulatory and antimicrobial efficacy of Lactobacilli against enteropathogenic infection of Salmonella typhi: In-vitro and in-vivo study.

    PubMed

    Mazaya, Basem; Hamzawy, Mohamed A; Khalil, Mahmoud A F; Tawkol, Wael M; Sabit, Hussein

    2015-12-01

    Salmonellosis-induced diarrhea, is one of the commonest cause of childhood mortality in developing countries. Using of probiotics is viewed as a promising means for reducing the pathogenic loads of bacterial infection. The current study aimed to evaluate the potential antimicrobial and immunomodulatory efficacy of isolated lactobacillus strains against the enteropathogenic effect of S. Typhi. Different Lactobacillus strains were isolated from 13 dairy products. Their antimicrobial activities were tested against different bacterial strains. Six groups of CD1 mice were treated for 8 days as follows: group (1) untreated control; group (2) was challenged with single inoculation S. typhi, and groups (3) and (4) were treated with Lactobacillus plantarum (LA5) or Lactobacillus paracsi (LA7) for 7 days, respectively. Groups (5) and (6) were challenged with S. typhi, and then treated with either LA5 or LA 7 for 7 days, respectively. Isolated Lactobacillus showed antimicrobial activity against wide range of bacterial strains. Salmonellosis showed high widal titer, induced significant disturbance of TNF and IL-1β, while sever changes of the histological patterns of the intestinal villi and hepatocytes have been illustrated. LA5 or LA7 succeeded to eradicate typhoid infection, restore the values of inflammatory cytokines to typical levels of control group, and improve histological pictures of intestinal and hepatic tissues. It can be concluded that lactobacilli are promising candidate in protection and eradication against bacterial infection induced by S. Typhi due to its antimicrobial, anti-inflammatory, and immunomodulatory activities. PMID:26303120

  4. The enteropathogenic E. coli effector EspF targets and disrupts the nucleolus by a process regulated by mitochondrial dysfunction.

    PubMed

    Dean, Paul; Scott, Jon A; Knox, Andrew A; Quitard, Sabine; Watkins, Nicholas J; Kenny, Brendan

    2010-01-01

    The nucleolus is a multifunctional structure within the nucleus of eukaryotic cells and is the primary site of ribosome biogenesis. Almost all viruses target and disrupt the nucleolus--a feature exclusive to this pathogen group. Here, using a combination of bio-imaging, genetic and biochemical analyses, we demonstrate that the enteropathogenic E. coli (EPEC) effector protein EspF specifically targets the nucleolus and disrupts a subset of nucleolar factors. Driven by a defined N-terminal nucleolar targeting domain, EspF causes the complete loss from the nucleolus of nucleolin, the most abundant nucleolar protein. We also show that other bacterial species disrupt the nucleolus, dependent on their ability to deliver effector proteins into the host cell. Moreover, we uncover a novel regulatory mechanism whereby nucleolar targeting by EspF is strictly controlled by EPEC's manipulation of host mitochondria. Collectively, this work reveals that the nucleolus may be a common feature of bacterial pathogenesis and demonstrates that a bacterial pathogen has evolved a highly sophisticated mechanism to enable spatio-temporal control over its virulence proteins. PMID:20585567

  5. Detection of Bacillus cereus with enteropathogenic potential by multiplex real-time PCR based on SYBR Green I.

    PubMed

    Wehrle, Esther; Didier, Andrea; Moravek, Maximilian; Dietrich, Richard; Märtlbauer, Erwin

    2010-06-01

    In order to meet the growing demand for fast and reliable detection of potentially toxinogenic Bacillus cereus, we developed a multiplex real-time PCR assay based on SYBR Green I with subsequent melting curve analysis. We designed and selected primers specific for genes of toxins responsible for diarrhoea (nheA, hblD and cytK1) and emesis (ces). A panel of 337 Bacillus strains was applied to the novel method on Light Cycler 2.0 with average melting temperature (T(m)) values of 73.85 degrees C (nheA), 87.01 degrees C (hblD), 78.66 degrees C (ces) and 82.19 degrees C (cytK1). An adapted version of the assay was also successfully run on Light Cycler 480 using one third (113 strains) of the total test panel. Verification of PCR results by conventional PCR as well as immunoassays and cytotoxicity tests gave an overall excellent correlation. Distinct melting peaks were only observed in B. cereus and B. cereus group strains but not in other Bacilli and Gram-positive or Gram-negative bacteria. Artificial contamination of three different food matrices with distinct bacterial counts revealed a detection limit of 10(1) CFU/g B. cereus cells after overnight enrichment. Thus, the novel multiplex real-time PCR turned out to be a reliable method for identification of B. cereus with enteropathogenic potential. PMID:19944752

  6. Preponderance of toxigenic Escherichia coli in stool pathogens correlates with toxin detection in accessible drinking-water sources.

    PubMed

    Igbokwe, H; Bhattacharyya, S; Gradus, S; Khubbar, M; Griswold, D; Navidad, J; Igwilo, C; Masson-Meyers, D; Azenabor, A A

    2015-02-01

    Since early detection of pathogens and their virulence factors contribute to intervention and control strategies, we assessed the enteropathogens in diarrhoea disease and investigated the link between toxigenic strains of Escherichia coli from stool and drinking-water sources; and determined the expression of toxin genes by antibiotic-resistant E. coli in Lagos, Nigeria. This was compared with isolates from diarrhoeal stool and water from Wisconsin, USA. The new Luminex xTAG GPP (Gastroplex) technique and conventional real-time PCR were used to profile enteric pathogens and E. coli toxin gene isolates, respectively. Results showed the pathogen profile of stool and indicated a relationship between E. coli toxin genes in water and stool from Lagos which was absent in Wisconsin isolates. The Gastroplex technique was efficient for multiple enteric pathogens and toxin gene detection. The co-existence of antibiotic resistance with enteroinvasive E. coli toxin genes suggests an additional prognostic burden on patients. PMID:24787554

  7. Volunteer Challenge With Enterotoxigenic Escherichia coli That Express Intestinal Colonization Factor Fimbriae CS17 and CS19

    PubMed Central

    McKenzie, Robin; Porter, Chad K.; Cantrell, Joyce A.; DeNearing, Barbara; O’Dowd, Aisling; Grahek, Shannon L.; Sincock, Stephanie A.; Woods, Colleen; Sebeny, Peter; Sack, David A.; Tribble, David R.; Bourgeois, A. Louis

    2011-01-01

    Human challenges with enterotoxigenic Escherichia coli (ETEC) have broadened our understanding of this important enteropathogen. We report findings from the first challenge studies using ETEC-expressing colonization factor fimbria CS17 and CS19. LSN03-016011/A (LT, CS17) elicited a dose-dependent effect, with the upper dose (6 × 109 organisms) causing diarrhea in 88% of recipients. WS0115A (LTSTp, CS19) also showed a dose response, with a 44% diarrhea rate at 9 × 109 organisms. Both strains elicited homologous antifimbrial and anti-LT antibody seroconversion. These studies establish the relative pathogenicity of ETEC expressing newer class 5 fimbriae and suggest suitability of the LT|CS17-ETEC challenge model for interventional trials. PMID:21628659

  8. Virulence Genes in Expanded-Spectrum-Cephalosporin-Resistant and -Susceptible Escherichia coli Isolates from Treated and Untreated Chickens.

    PubMed

    Baron, S; Delannoy, S; Bougeard, S; Larvor, E; Jouy, E; Balan, O; Fach, P; Kempf, I

    2016-03-01

    This study investigated antimicrobial resistance, screened for the presence of virulence genes involved in intestinal infections, and determined phylogenetic groups of Escherichia coli isolates from untreated poultry and poultry treated with ceftiofur, an expanded-spectrum cephalosporin. Results show that none of the 76 isolates appeared to be Shiga toxin-producing E. coli or enteropathogenic E. coli. All isolates were negative for the major virulence factors/toxins tested (ehxA, cdt, heat-stable enterotoxin [ST], and heat-labile enterotoxin [LT]). The few virulence genes harbored in isolates generally did not correlate with isolate antimicrobial resistance or treatment status. However, some of the virulence genes were significantly associated with certain phylogenetic groups. PMID:26666927

  9. Volunteer challenge with enterotoxigenic Escherichia coli that express intestinal colonization factor fimbriae CS17 and CS19.

    PubMed

    McKenzie, Robin; Porter, Chad K; Cantrell, Joyce A; Denearing, Barbara; O'Dowd, Aisling; Grahek, Shannon L; Sincock, Stephanie A; Woods, Colleen; Sebeny, Peter; Sack, David A; Tribble, David R; Bourgeois, A Louis; Savarino, Stephen J

    2011-07-01

    Human challenges with enterotoxigenic Escherichia coli (ETEC) have broadened our understanding of this important enteropathogen. We report findings from the first challenge studies using ETEC-expressing colonization factor fimbria CS17 and CS19. LSN03-016011/A (LT, CS17) elicited a dose-dependent effect, with the upper dose (6 × 10(9) organisms) causing diarrhea in 88% of recipients. WS0115A (LTSTp, CS19) also showed a dose response, with a 44% diarrhea rate at 9 × 10(9) organisms. Both strains elicited homologous antifimbrial and anti-LT antibody seroconversion. These studies establish the relative pathogenicity of ETEC expressing newer class 5 fimbriae and suggest suitability of the LT|CS17-ETEC challenge model for interventional trials. PMID:21628659

  10. Genetic Mapping of the Antigenic Determinants of Two Polysaccharide K Antigens, K10 and K54, in Escherichia coli

    PubMed Central

    Ørskov, Ida; Nyman, Kate

    1974-01-01

    The genes controlling synthesis of the Escherichia coli acidic polysaccharide capsular antigens K10 and K54 were transferred by conjugation to E. coli strains of other serotypes. The genes concerned with these K antigen determinants showed genetic linkage with the serA locus. We propose to name the K antigen-controlling gene kpsA. The genetic determinants of the two K antigens could also be transferred to enteropathogenic serotypes, even though such strains have never been found in nature with special acidic polysaccharide K antigens. A noncapsulated derivative, K−, of the K10 strain can transfer the genetic determinant of the K antigen, demonstrating the probable existence of another chromosomal locus involved in the production of such acidic polysaccharide K antigens. PMID:4138850

  11. Porcine cancer models for translational oncology

    PubMed Central

    Sieren, Jessica C.; Quelle, Dawn; Meyerholz, David K.; Rogers, Christopher S.

    2014-01-01

    Large-animal cancer models are needed to advance the development of innovative and clinically applicable tumor diagnostic, therapeutic, and monitoring technologies. We developed a genetically modified porcine model of cancer based on a TP53 mutation, and established its utility for tracking tumorigenesis in vivo through non-invasive clinical imaging approaches. PMID:27308376

  12. The Porcine Immunology and Nutrition Resource Database

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diverse genomics-based databases have been developed to facilitate research with human and rodent models. Current porcine gene databases, however, lack the nutritional and immunological orientation and robust annotation to design effective molecular tools to study relevant pig models. To address t...

  13. Splicing variants of porcine synphilin-1.

    PubMed

    Larsen, Knud; Madsen, Lone Bruhn; Farajzadeh, Leila; Bendixen, Christian

    2015-09-01

    Parkinson's disease (PD), idiopathic and familial, is characterized by degradation of dopaminergic neurons and the presence of Lewy bodies (LB) in the substantia nigra. LBs contain aggregated proteins of which α-synuclein is the major component. The protein synphilin-1 interacts and colocalizes with α-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinson's disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human (90%) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation. PMID:26101749

  14. Virulence factors of lactose-negative Escherichia coli strains isolated from children with diarrhea in Somalia.

    PubMed Central

    Nicoletti, M; Superti, F; Conti, C; Calconi, A; Zagaglia, C

    1988-01-01

    Lactose-negative Escherichia coli strains were isolated at high frequency from children with diarrhea in Somalia during a 2-year study on diarrheal diseases. Sixty-four of these strains, considered to be a representative sample, were characterized for virulence factors, plasmid profiles, and antibiotic resistance. Of these strains, 5 were recognized as enteroinvasive E. coli (they were serotyped as O135:K-:H-), 6 belonged to classical enteropathogenic E. coli serotypes, 9 were able to adhere to tissue culture cells (of these, 4 showed a pattern of localized adherence and 1 was an enteropathogenic strain), 18 were both adherent and hemolytic, and 8 were simply hemolytic. None hybridized with 32P-labeled heat-labile or heat-stable (a and b) enterotoxin gene probes or produced moderate or high-level cytotoxic effects on HeLa cells. Of the 64 strains examined, 24 produced mannose-resistant hemagglutination with human, chicken, and monkey erythrocytes. One of these was serotyped as O4:K-:H8, and a rabbit O antiserum raised against this strain allowed us to establish that 23 strains had the same O antigen. The 23 O4 strains were hemolytic and were not enterotoxic for rabbit ileal loops, and intact bacteria were able to destroy tissue culture cell monolayers very rapidly. The uniformity of the antibiotic resistance pattern and of the plasmid DNA content, together with the fact that they were isolated in different years and in different children, suggests that the O4 strains must be epidemiologically relevant in Somalia. A possible diarrheagenic role for the adherent-hemolytic E. coli strains is also discussed. Images PMID:3281977

  15. Production of monoclonal antibodies to porcine interleukin-18 and their use for immunoaffinity purification of recombinant porcine interleukin-18.

    PubMed

    Muneta, Y; Shimoji, Y; Yokomizo, Y; Mori, Y

    2000-03-01

    We have recently reported the cloning and expression of porcine interleukin-18 (IL-18). In this study, we describe the production of anti-porcine IL-18 monoclonal antibodies (mAb) and their use in the purification of a large amount of recombinant porcine IL-18 by immunoaffinity column chromatography. Five monoclonal antibodies (2-2-B, 2-5-B, 2-13-C, 3-1-C and 5-3-B) were established and characterized. Three (2-2-B, 3-1-C and 5-3-B) of them were of IgG1 subclass, and the other two were IgMs. Epitope analysis of the three IgG1 mAbs showed that they recognized the same epitope. All five mAbs demonstrated reactivity with baculovirus generated porcine IL-18 by immunoblot analysis. Biologically active porcine IL-18 was obtained by immunoaffinity chromatography using anti-porcine IL-18 mAb at more than 85% purity from culture supernatants of Trichoplusia ni (Tn5) derived cells infected with recombinant baculovirus containing the coding sequence of porcine mature IL-18. These results suggest that the anti-porcine IL-18 mAbs established in this study are useful for one-step purification of porcine mature IL-18 as well as the detection of porcine IL-18 by immunoblotting. PMID:10699583

  16. One-step spray-dried polyelectrolyte microparticles enhance the antigen cross-presentation capacity of porcine dendritic cells.

    PubMed

    Devriendt, Bert; Baert, Kim; Dierendonck, Marijke; Favoreel, Herman; De Koker, Stefaan; Remon, Jean Paul; De Geest, Bruno G; Cox, Eric

    2013-06-01

    Vaccination is regarded as the most efficient and cost-effective way to prevent infectious diseases. Vaccine design nowadays focuses on the implementation of safer recombinant subunit vaccines. However, these recombinant subunit antigens are often poor immunogens and several strategies are currently under investigation to enhance their immunogenicity. The encapsulation of antigens in biodegradable microparticulate delivery systems seems a promising strategy to boost their immunogenicity. Here, we evaluate the capacity of polyelectrolyte complex microparticles (PECMs), fabricated by single step spray-drying, to deliver antigens to porcine dendritic cells and how these particles affect the functional maturation of dendritic cells (DCs). As clinically relevant model antigen F4 fimbriae, a bacterial adhesin purified from a porcine-specific enterotoxigenic Escherichia coli strain was chosen. The resulting antigen-loaded PECMs are efficiently internalised by porcine monocyte-derived DCs. F4 fimbriae-loaded PECMs (F4-PECMs) enhanced CD40 and CD25 surface expression by DCs and this phenotypical maturation correlated with an increased secretion of IL-6 and IL-1β. More importantly, F4-PECMs enhance both the T cell stimulatory and antigen presentation capacity of DCs. Moreover, PECMs efficiently promoted the CD8(+) T cell stimulatory capacity of dendritic cells, indicating an enhanced ability to cross-present the encapsulated antigens. These results could accelerate the development of veterinary and human subunit vaccines based on polyelectrolyte complex microparticles to induce protective immunity against a variety of extra- and intracellular pathogens. PMID:23207327

  17. Pathogenicity and molecular characterization of emerging porcine reproductive and respiratory syndrome virus in Vietnam in 2007.

    PubMed

    Metwally, S; Mohamed, F; Faaberg, K; Burrage, T; Prarat, M; Moran, K; Bracht, A; Mayr, G; Berninger, M; Koster, L; To, T L; Nguyen, V L; Reising, M; Landgraf, J; Cox, L; Lubroth, J; Carrillo, C

    2010-10-01

    In 2007, Vietnam experienced swine disease outbreaks causing clinical signs similar to the 'porcine high fever disease' that occurred in China during 2006. Analysis of diagnostic samples from the disease outbreaks in Vietnam identified porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV-2). Additionally, Escherichia coli and Streptococcus equi subspecies zooepidemicus were cultured from lung and spleen, and Streptococcus suis from one spleen sample. Genetic characterization of the Vietnamese PRRSV isolates revealed that this virus belongs to the North American genotype (type 2) with a high nucleotide identity to the recently reported Chinese strains. Amino acid sequence in the nsp2 region revealed 95.7-99.4% identity to Chinese strain HUN4, 68-69% identity to strain VR-2332 and 58-59% identity to strain MN184. A partial deletion in the nsp2 gene was detected; however, this deletion did not appear to enhance the virus pathogenicity in the inoculated pigs. Animal inoculation studies were conducted to determine the pathogenicity of PRRSV and to identify other possible agents present in the original specimens. Pigs inoculated with PRRSV alone and their contacts showed persistent fever, and two of five pigs developed cough, neurological signs and swollen joints. Necropsy examination showed mild to moderate bronchopneumonia, enlarged lymph nodes, fibrinous pericarditis and polyarthritis. PRRSV was re-isolated from blood and tissues of the inoculated and contact pigs. Pigs inoculated with lung and spleen tissue homogenates from sick pigs from Vietnam developed high fever, septicaemia, and died acutely within 72 h, while their contact pigs showed no clinical signs throughout the experiment. Streptococcus equi subspecies zooepidemicus was cultured, and PRRSV was re-isolated only from the inoculated pigs. Results suggest that the cause of the swine deaths in Vietnam is a multifactorial syndrome with PRRSV as a major factor. PMID

  18. Stability of Reference Gene Expression After Porcine Sapelovirus Infection in Porcine Intestinal Epithelial Cells.

    PubMed

    Huang, Yong; Chen, Yabing; Sun, Huan; Lan, Daoliang

    2016-01-01

    Intestinal epithelial cells, which serve as the first physical barrier to protect intestinal tract from external antigens, have an important role in the local innate immunity. Screening of reference genes that have stable expression levels after viral infection in porcine intestinal epithelial cells is critical for ensuring the reliability of the expression analysis on anti-infection genes in porcine intestinal epithelial cells. In this study, nine common reference genes in pigs, including ACTB, B2M, GAPDH, HMBS, SDHA, HPRT1, TBP, YWHAZ, and RPL32, were chosen as the candidate reference genes. Porcine sapelovirus (PSV) was used as a model virus to infect porcine intestinal epithelial cell line (IPEC-J2). The expression stability of the nine genes was assessed by the geNorm, NormFinder, and BestKeeper software. Moreover, RefFinder program was used to evaluate the analytical results of above three softwares, and a relative expression experiment of selected target gene was used to verify the analysis results. The comprehensive results indicated that the gene combination of TBP and RPL32 has the most stable expression, which could be considered as an appropriate reference gene for research on gene expression after PSV infection in IPEC-J2cells. The results provided essential data for expression analysis of anti-infection genes in porcine intestinal epithelial cells. PMID:27092424

  19. Coincidental detection of genomes of porcine parvoviruses and porcine circovirus type 2 infecting pigs in Japan

    PubMed Central

    SAEKHOW, Prayuth; KISHIZUKA, Shingo; SANO, Natsuha; MITSUI, Hiroko; AKASAKI, Hajime; MAWATARI, Takahiro; IKEDA, Hidetoshi

    2015-01-01

    The infection status of 15 viruses in 120 pigs aged about 6 months was investigated based on tonsil specimens collected from a slaughterhouse. Only 5 species of porcine parvoviruses and porcine circovirus type 2 (PCV2) were detected at high frequencies; 67% for porcine parvovirus (PPV) (PPV-Kr or -NADL2 as the new abbreviation), 58% for PPV2 (CnP-PARV4), 39% for PPV3 (P-PARV4), 33% for PPV4 (PPV4), 55% for PBo-likeV (PBoV7) and 80% for PCV2. A phylogenetic analysis of PPV3 suggested that Japanese PPV3s showed a slight variation, and possibly, there were farms harboring homogeneous or heterogeneous PPV3s. Statistical analyses indicated that the detection of PCV2 was significantly coincidental with each detection of PPV, PPV2 and PPV3, and PPV and PPV4 were also coincidentally detected. The concurrent infection with PCV2 and porcine parvoviruses in the subclinically infected pigs may resemble the infection status of pigs with the clinical manifestations of porcine circovirus associated disease which occurs in 3–5 months old pigs and is thought to be primarily caused by the PCV2 infection. PMID:26166811

  20. Coincidental detection of genomes of porcine parvoviruses and porcine circovirus type 2 infecting pigs in Japan.

    PubMed

    Saekhow, Prayuth; Kishizuka, Shingo; Sano, Natsuha; Mitsui, Hiroko; Akasaki, Hajime; Mawatari, Takahiro; Ikeda, Hidetoshi

    2016-01-01

    The infection status of 15 viruses in 120 pigs aged about 6 months was investigated based on tonsil specimens collected from a slaughterhouse. Only 5 species of porcine parvoviruses and porcine circovirus type 2 (PCV2) were detected at high frequencies; 67% for porcine parvovirus (PPV) (PPV-Kr or -NADL2 as the new abbreviation), 58% for PPV2 (CnP-PARV4), 39% for PPV3 (P-PARV4), 33% for PPV4 (PPV4), 55% for PBo-likeV (PBoV7) and 80% for PCV2. A phylogenetic analysis of PPV3 suggested that Japanese PPV3s showed a slight variation, and possibly, there were farms harboring homogeneous or heterogeneous PPV3s. Statistical analyses indicated that the detection of PCV2 was significantly coincidental with each detection of PPV, PPV2 and PPV3, and PPV and PPV4 were also coincidentally detected. The concurrent infection with PCV2 and porcine parvoviruses in the subclinically infected pigs may resemble the infection status of pigs with the clinical manifestations of porcine circovirus associated disease which occurs in 3-5 months old pigs and is thought to be primarily caused by the PCV2 infection. PMID:26166811

  1. Tissue Distribution of Porcine FTO and Its Effect on Porcine Intramuscular Preadipocytes Proliferation and Differentiation

    PubMed Central

    Chen, Xiaoling; Zhou, Bo; Luo, Yanliu; Huang, Zhiqing; Jia, Gang; Liu, Guangmang; Zhao, Hua

    2016-01-01

    The fat mass and obesity associated (FTO) gene plays an important role in adipogenesis. However, its function during porcine intramuscular preadipocyte proliferation and differentiation remains poorly understood. In this study, we prepared the antiserum against porcine FTO (pFTO), which was used to determine its subcellular localization and tissue distribution. Our data indicated that pFTO was localized predominantly in the nucleus. Real-time quantitative PCR and western blot analysis showed that pFTO was highly expressed in the lung and subcutaneous adipose tissue. Overexpression of pFTO in porcine intramuscular preadipocytes significantly promoted cell proliferation and lipid deposition. Furthermore, overexpression of pFTO in differentiating porcine intramuscular preadipocytes also significantly increased the mRNA levels of adipocyte differentiation transcription factors peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), lipoprotein lipase (LPL) and fatty acid synthase (FAS). Our findings provide the first functional evidence to reveal a role of pFTO in porcine intramuscular preadipocyte proliferation and differentiation. PMID:26964098

  2. Porcine Parvovirus: Natural and Experimental Infections of the Porcine Fetus and Prevalence In Mature Swine 1

    PubMed Central

    Redman, D. R.; Bohl, E. H.; Ferguson, L. C.

    1974-01-01

    Antibodies against porcine parvovirus were detected in 17 of 116 prenursing pig sera. Antibodies against transmissible gastroenteritis or ECPO-6 (an enterovirus) were not detected in prenursing sera of the pigs tested. Seventy-seven percent of 129 serum samples from 23 Ohio farms and 82% of 96 samples from slaughter plants in Ohio were serologically positive for porcine parvovirus. Mummies or other abnormalities were not observed in newly born pigs exposed to porcine parvovirus by the transuterine route 101 days after gestation. Indirect evidence suggested that the virus had not spread to other fet uses following exposure after 101 days at least not in a sufficient amount of time to stimulate detectable antibody. Direct intrafetal exposure to porcine parvovirus (i.m. injection, transutero) after 62 days of gestation resulted in dealth and mummification of the two fetuses, and apparently in the subsequent spread of the virus, as five of nine live pigs born were serologically positive for porcine parvovirus and these five pigs had not been injected with the virus. Immunoglobulin G was detected in all newborn pigs irregardless of known antigenic stimulation or the presence of specific antibody. In general, the presence of immunoglobulin M or immunoglobulin A in fetal serum was correlated with a history of antigenic stimulation or the presence of detectable antibody. PMID:4426705

  3. Serological Prevalence of Enteropathogenic Yersinia spp. in Pigs and Wild Boars from Different Production Systems in the Moravian Region, Czech Republic.

    PubMed

    Lorencova, Alena; Babak, Vladimir; Lamka, Jiri

    2016-05-01

    Human yersiniosis caused by pathogenic Yersinia spp. is one of the most common reported zoonoses in the European Union and pigs are considered as the major reservoir of these bacteria. Serological testing represents a suitable method to obtain information about the prevalence of enteropathogenic Yersinia spp. in food animals. The prevalence of antibodies against enteropathogenic Yersinia spp. was studied in 319 slaughtered pigs and 135 wild boars from different production systems in the Moravian region (Czech Republic) using a commercially available ELISA test (an apparent prevalence). The seroprevalence was significantly associated with the type of breeding system, with the lowest seroprevalence being observed in household-raised pigs (13/29, 44.8%). No significant difference between the prevalence of anti-Yersinia antibodies in conventional (146/180, 81.1%) and organic pigs (92/110, 83.6%) was found. Antibodies were found in 65.9% (89/135) of wild boars without a significant difference between adult (23/41, 56.1%) and young (66/94, 70.2%) animals. Seropositivity was significantly higher in domestic (251/319, 78.7% in total) compared to feral pigs. A Bayesian approach taking into account the sensitivity and specificity of the ELISA test was used to estimate the true prevalence of anti-Yersinia antibodies in pigs and wild boars. According to our results, domestic pigs and wild boars proved to be an important reservoir of enteropathogenic Yersinia in the Czech Republic. Attention should be paid to good hygienic practice during slaughtering and handling of meat to prevent meat contamination and subsequently human infection. PMID:26982547

  4. Porcine Sialoadhesin: A Newly Identified Xenogeneic Innate Immune Receptor

    PubMed Central

    Brock, Linda G.; Delputte, Peter L.; Waldman, Joshua P.; Nauwynck, Hans J.; Rees, Michael A.

    2012-01-01

    Extracorporeal porcine liver perfusion is being developed as a bridge to liver allotransplantation for patients with fulminant hepatic failure. This strategy is limited by porcine Kupffer cell destruction of human erythrocytes, mediated by lectin binding of a sialic acid motif in the absence of antibody and complement. Sialoadhesin, a macrophage restricted lectin that binds sialic acid, was originally described as a sheep erythrocyte binding receptor. Given similarities between sialoadhesin and the unidentified macrophage lectin in our model, we hypothesized porcine sialoadhesin contributed to recognition of human erythrocytes. Two additional types of macrophages were identified to bind human erythrocytes - spleen and alveolar. Expression of sialoadhesin was confirmed by immunofluorescence in porcine tissues and by flow cytometry on primary macrophages. A stable transgenic cell line expressing porcine sialoadhesin (pSn CHO) bound human erythrocytes, while a sialoadhesin mutant cell line did not. Porcine macrophage and pSn CHO recognition of human erythrocytes was inhibited approximately 90% by an anti-porcine sialoadhesin monoclonal antibody and by human erythrocyte glycoproteins. Furthermore, this binding was substantially reduced by sialidase treatment of erythrocytes. These data support the hypothesis that porcine sialoadhesin is a xenogeneic receptor that mediates porcine macrophage binding of human erythrocytes in a sialic acid-dependent manner. PMID:22958948

  5. Porcine hokovirus in wild boar in Portugal.

    PubMed

    Miranda, Carla; Coelho, Catarina; Vieira-Pinto, Madalena; Thompson, Gertrude

    2016-04-01

    Porcine hokovirus (PHoV), also referred to as porcine parvovirus 4 (P-PARV4), a recently discovered parvovirus of swine that is closely related to human parvovirus 4/5 (H-PARV4/5), was first described in Hong Kong. To evaluate the occurrence of P-PARV4 in Portuguese wild boars in the hunting season of 2011/2012, liver and serum samples were tested. P-PARV4 was detected in 24 % of the wild boars analyzed. Phylogenetic analysis showed a close relationship between the P-PARV4 isolates and other P-PARV4 reference strains. This virus appears to be emerging, with yet unknown implications for public health. PMID:26711454

  6. Lawsonia intracellularis and Porcine Circovirus type-2 infection in Estonia.

    PubMed

    Järveots, T; Saar, T; Põdersoo, D; Rüütel-Boudinot, S; Sütt, S; Tummeleht, L; Suuroja, T; Lindjärv, R

    2016-01-01

    The present study describes the reasons of post-weaning distress in Estonian pig herds. Here we examined the natural cases of Lawsonia intracellularis and porcine circovirus 2 (PCV2) infection and co-infections. The presence of L. intracellularis in swine herds were tested by PCR and by histopathological methods, whereas PCV2 was detected by real-time-PCR and immunohistochemical stainings. Seven of the 11 investigated herds with signs of post-weaning wasting were infected with L. intracellularis and all 11 herds with PCV2. From the analysed samples 22.2% were infected with L. intracellularis and 25% with PCV2. The results of microbiological studies suggested that the piglets suffered from enteritis and pneumonia. Escherichia coli and Pasteurella multocida often aggravated the process of illness. The frequency of L. intracellularis was high in pigs 7-12 weeks old (18.5-42.7%) and PCV2 infection was too high in pigs 7-12 weeks old (24.8-32.7%). E. coli was often a co-factor with L. intracellularis and PCV2. The primary reasons of post weaning wasting were PCV2 and E. coli, later aggravated by L. intracellularis and other pathogens. Our results indicated that different pathogens have an important role in developing post-weaning wasting. Proliferative intestinal inflammation caused by L. intracellularis is mainly characterised by its localization and morphological findings. The main gross lesions were the enlargement of mesenteric lymph nodes and thickening of the wall of ileum. In post-weaning multi-systemic wasting syndrome there are characteristic histological lesions in lymphoid tissues. They consist of a variable degree of lymphocyte depletion, together with histiocytic and/or multinucleate giant cell infiltration. This basic lymphoid lesions is observable in almost all tissues of a single severely affected animal, including lymph nodes, Peyer's patches and spleen. Sporadically, multifocal coagulative necrosis may be observed. PMID:27487502

  7. Expression of colonization factor CS5 of enterotoxigenic Escherichia coli (ETEC) is enhanced in vivo and by the bile component Na glycocholate hydrate.

    PubMed

    Nicklasson, Matilda; Sjöling, Åsa; von Mentzer, Astrid; Qadri, Firdausi; Svennerholm, Ann-Mari

    2012-01-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of acute watery diarrhoea in developing countries. Colonization factors (CFs) on the bacterial surface mediate adhesion to the small intestinal epithelium. Two of the most common CFs worldwide are coli surface antigens 5 and 6 (CS5, CS6). In this study we investigated the expression of CS5 and CS6 in vivo, and the effects of bile and sodium bicarbonate, present in the human gut, on the expression of CS5. Five CS5+CS6 ETEC isolates from adult Bangladeshi patients with acute diarrhoea were studied. The level of transcription from the CS5 operon was approximately 100-fold higher than from the CS6 operon in ETEC bacteria recovered directly from diarrhoeal stool without sub-culturing (in vivo). The glyco-conjugated primary bile salt sodium glycocholate hydrate (NaGCH) induced phenotypic expression of CS5 in a dose-dependent manner and caused a 100-fold up-regulation of CS5 mRNA levels; this is the first description of NaGCH as an enteropathogenic virulence inducer. The relative transcription levels from the CS5 and CS6 operons in the presence of bile or NaGCH in vitro were similar to those in vivo. Another bile salt, sodium deoxycholate (NaDC), previously reported to induce enteropathogenic virulence, also induced expression of CS5, whereas sodium bicarbonate did not. PMID:22563407

  8. Cloning of Porcine Pituitary Tumor Transforming Gene 1 and Its Expression in Porcine Oocytes and Embryos.

    PubMed

    Xie, Bingkun; Qin, Zhaoxian; Liu, Shuai; Nong, Suqun; Ma, Qingyan; Chen, Baojian; Liu, Mingjun; Pan, Tianbiao; Liao, D Joshua

    2016-01-01

    The maternal-to-embryonic transition (MET) is a complex process that occurs during early mammalian embryogenesis and is characterized by activation of the zygotic genome, initiation of embryonic transcription, and replacement of maternal mRNA with embryonic mRNA. The objective of this study was to reveal the temporal expression and localization patterns of PTTG1 during early porcine embryonic development and to establish a relationship between PTTG1 and the MET. To achieve this goal, reverse transcription-polymerase chain reaction (RT-PCR) was performed to clone porcine PTTG1. Subsequently, germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, zygotes, 2-, 4-, and 8-cell-stage embryos, morulas, and blastocysts were produced in vitro and their gene expression was analyzed. The results revealed that the coding sequence of porcine PTTG1 is 609-bp in length and that it encodes a 202-aa polypeptide. Using qRT-PCR, PTTG1 mRNA expression was observed to be maintained at high levels in GV- and MII-stage oocytes. The transcript levels in oocytes were also significantly higher than those in embryos from the zygote to blastocyst stages. Immunohistochemical analyses revealed that porcine PTTG1 was primarily localized to the cytoplasm and partially localized to the nucleus. Furthermore, the PTTG1 protein levels in MII-stage oocytes and zygotes were significantly higher than those in embryos from the 2-cell to blastocyst stage. After fertilization, the level of this protein began to decrease gradually until the blastocyst stage. The results of our study suggest that porcine PTTG1 is a new candidate maternal effect gene (MEG) that may participate in the processes of oocyte maturation and zygotic genome activation during porcine embryogenesis. PMID:27058238

  9. Cloning of Porcine Pituitary Tumor Transforming Gene 1 and Its Expression in Porcine Oocytes and Embryos

    PubMed Central

    Liu, Shuai; Nong, Suqun; Ma, Qingyan; Chen, Baojian; Liu, Mingjun; Pan, Tianbiao; Liao, D. Joshua

    2016-01-01

    The maternal-to-embryonic transition (MET) is a complex process that occurs during early mammalian embryogenesis and is characterized by activation of the zygotic genome, initiation of embryonic transcription, and replacement of maternal mRNA with embryonic mRNA. The objective of this study was to reveal the temporal expression and localization patterns of PTTG1 during early porcine embryonic development and to establish a relationship between PTTG1 and the MET. To achieve this goal, reverse transcription-polymerase chain reaction (RT-PCR) was performed to clone porcine PTTG1. Subsequently, germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, zygotes, 2-, 4-, and 8-cell-stage embryos, morulas, and blastocysts were produced in vitro and their gene expression was analyzed. The results revealed that the coding sequence of porcine PTTG1 is 609-bp in length and that it encodes a 202-aa polypeptide. Using qRT-PCR, PTTG1 mRNA expression was observed to be maintained at high levels in GV- and MII-stage oocytes. The transcript levels in oocytes were also significantly higher than those in embryos from the zygote to blastocyst stages. Immunohistochemical analyses revealed that porcine PTTG1 was primarily localized to the cytoplasm and partially localized to the nucleus. Furthermore, the PTTG1 protein levels in MII-stage oocytes and zygotes were significantly higher than those in embryos from the 2-cell to blastocyst stage. After fertilization, the level of this protein began to decrease gradually until the blastocyst stage. The results of our study suggest that porcine PTTG1 is a new candidate maternal effect gene (MEG) that may participate in the processes of oocyte maturation and zygotic genome activation during porcine embryogenesis. PMID:27058238

  10. Tissue Sampling Guides for Porcine Biomedical Models.

    PubMed

    Albl, Barbara; Haesner, Serena; Braun-Reichhart, Christina; Streckel, Elisabeth; Renner, Simone; Seeliger, Frank; Wolf, Eckhard; Wanke, Rüdiger; Blutke, Andreas

    2016-04-01

    This article provides guidelines for organ and tissue sampling adapted to porcine animal models in translational medical research. Detailed protocols for the determination of sampling locations and numbers as well as recommendations on the orientation, size, and trimming direction of samples from ∼50 different porcine organs and tissues are provided in the Supplementary Material. The proposed sampling protocols include the generation of samples suitable for subsequent qualitative and quantitative analyses, including cryohistology, paraffin, and plastic histology; immunohistochemistry;in situhybridization; electron microscopy; and quantitative stereology as well as molecular analyses of DNA, RNA, proteins, metabolites, and electrolytes. With regard to the planned extent of sampling efforts, time, and personnel expenses, and dependent upon the scheduled analyses, different protocols are provided. These protocols are adjusted for (I) routine screenings, as used in general toxicity studies or in analyses of gene expression patterns or histopathological organ alterations, (II) advanced analyses of single organs/tissues, and (III) large-scale sampling procedures to be applied in biobank projects. Providing a robust reference for studies of porcine models, the described protocols will ensure the efficiency of sampling, the systematic recovery of high-quality samples representing the entire organ or tissue as well as the intra-/interstudy comparability and reproducibility of results. PMID:26883152

  11. Emerging Escherichia Pathogen

    PubMed Central

    Permpalung, Nitipong; Sentochnik, Deborah E.

    2013-01-01

    Escherichia hermannii was first identified as a new species in 1982. It has rarely been reported as a human pathogen. We report the first case of E. hermannii as the sole pathogen in a catheter-related bloodstream infection. PMID:23740732

  12. Genetic recombination. [Escherichia coli

    SciTech Connect

    Stahl, F.W.

    1987-02-01

    The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

  13. Pathogenic Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli, a member of the Enterobacteriaceae family, is a part of the normal flora of the intestinal tract of humans and a variety of animals. E. coli strains are classified on the basis of antigenic differences in two surface components (serotyping), the somatic antigen (O) of the lipopoly...

  14. PATHOGENIC ESCHERICHIA COLI

    EPA Science Inventory

    Escherichia coli is a bacterial species which inhabits the gastrointestinal tract of man and warm-blooded animals. Because of the ubiquity of this bacterium in the intestinal flora, it serves as an important indicator organism of fecal contamination. E. coli, aside from serving a...

  15. Development of colloidal gold-based immunochromatographic assay for rapid detection of Mycoplasma suis in porcine plasma.

    PubMed

    Meng, Kai; Sun, Wenjing; Zhao, Peng; Zhang, Limei; Cai, Dongjie; Cheng, Ziqiang; Guo, Huijun; Liu, Jianzhu; Yang, Dubao; Wang, Shujing; Chai, Tongjie

    2014-05-15

    A one-step immunochromatographic assay using gold nanoparticles coated with polyclonal antibody (pAb) against Mycoplasma suis (M. suis) was developed in this study for the detection of M. suis in porcine plasma. The colloidal gold was prepared by the reduction of gold salt with sodium citrate coupled with pAb against M. suis. The pAb was produced by immunizing the BALB/c mice with recombinant MSG1 (rMSG1) protein from M. suis expressed in Escherichia coli. The optimal concentrations of the capture antibody and the coating antibody were 12 μg/ml and 1.5 mg/ml, respectively, and that of the blocking buffer was 1% bovine serum albumin. The lower detection limit of the immunochromatographic assay test was 100 ng/ml with visual detection under optimal conditions of analysis. Classical swine fever virus, porcine reproductive and respiratory syndrome virus, swine pneumonia mycoplasma, swine toxoplasma, and porcine parvovirus were used to evaluate the specificity of the immunochromatographic strips. No cross-reaction of the antibodies with other related swine pathogens was observed. This qualitative test based on the visual evaluation of the results did not require any equipment. The assay time for M. suis detection was less than 10 min, suitable for rapid detection at the grassroots level. The one-step colloidal gold immunochromatographic strips that we developed had high specificity and sensitivity. Therefore, this method would be feasible, convenient, rapid, and effective for detecting M. suis in porcine plasma. PMID:24434494

  16. Production of a subunit vaccine candidate against porcine post-weaning diarrhea in high-biomass transplastomic tobacco.

    PubMed

    Kolotilin, Igor; Kaldis, Angelo; Devriendt, Bert; Joensuu, Jussi; Cox, Eric; Menassa, Rima

    2012-01-01

    Post-weaning diarrhea (PWD) in piglets is a major problem in piggeries worldwide and results in severe economic losses. Infection with Enterotoxigenic Escherichia coli (ETEC) is the key culprit for the PWD disease. F4 fimbriae of ETEC are highly stable proteinaceous polymers, mainly composed of the major structural subunit FaeG, with a capacity to evoke mucosal immune responses, thus demonstrating a potential to act as an oral vaccine against ETEC-induced porcine PWD. In this study we used a transplastomic approach in tobacco to produce a recombinant variant of the FaeG protein, rFaeG(ntd/dsc), engineered for expression as a stable monomer by N-terminal deletion and donor strand-complementation (ntd/dsc). The generated transplastomic tobacco plants accumulated up to 2.0 g rFaeG(ntd/dsc) per 1 kg fresh leaf tissue (more than 1% of dry leaf tissue) and showed normal phenotype indistinguishable from wild type untransformed plants. We determined that chloroplast-produced rFaeG(ntd/dsc) protein retained the key properties of an oral vaccine, i.e. binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. Additionally, the plant biomass matrix was shown to delay degradation of the chloroplast-produced rFaeG(ntd/dsc) in gastrointestinal conditions, demonstrating a potential to function as a shelter-vehicle for vaccine delivery. These results suggest that transplastomic plants expressing the rFaeG(ntd/dsc) protein could be used for production and, possibly, delivery of an oral vaccine against porcine F4+ ETEC infections. Our findings therefore present a feasible approach for developing an oral vaccination strategy against porcine PWD. PMID:22879967

  17. The Role of Protegrins and Other Elastase-Activated Polypeptides in the Bactericidal Properties of Porcine Inflammatory Fluids

    PubMed Central

    Shi, Jishu; Ganz, Tomas

    1998-01-01

    The mammalian host response to infection includes the production and secretion of antimicrobial peptides from phagocytes and epithelial cells. Protegrins, a group of broadly microbicidal peptides isolated originally from porcine neutrophil lysates, were found to be stored as inactive proforms in porcine neutrophil granules but could be activated extracellularly by neutrophil elastase. We assessed the biological role of protegrins and other elastase-activated polypeptides in the microbicidal activity of neutrophil secretions and inflammatory fluids. When stimulated with phorbol myristate acetate (PMA), neutrophils generated stable microbicidal activity against both Escherichia coli and Listeria monocytogenes under normal-salt conditions and in the presence of 0 to 10% serum. The generation of these antimicrobial substances was dependent on neutrophil elastase, since it was inhibited by 1 mM N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone when it was present during activation, but not when this inhibitor was added afterwards. However, elastase-dependent activation of proprotegrins to protegrins in PMA-stimulated neutrophils was not inhibited by the presence of 1 to 2% serum. Porcine neutrophils also released antibacterial activity during phagocytosis of latex beads, and this too was dependent in large part on elastase-activated polypeptides, including protegrins. Moreover, protegrins were found at bactericidal concentrations in cell-free abscess fluid from naturally infected pigs. Taken together, these studies show that protegrins and other elastase-activated polypeptides are important stable antibacterial factors in porcine neutrophil secretions. The potential host defense role of elastase as an activating enzyme for the precursors of microbicidal peptides must be taken into account when therapeutic inhibitors of neutrophil elastase are evaluated for clinical use as anti-inflammatory agents. PMID:9673240

  18. In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination.

    PubMed

    Kwon, Hyung-Jun; Ryu, Young Bae; Kim, Young-Min; Song, Naaleum; Kim, Cha Young; Rho, Mun-Chual; Jeong, Jae-Ho; Cho, Kyoung-Oh; Lee, Woo Song; Park, Su-Jin

    2013-08-01

    Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2-5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1-5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection. PMID:23746631

  19. Exploring the genetic basis for porcine circovirus pathogenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Porcine circoviruses are members of the Circovirus genus within the Circoviridae family. Association of porcine circovirus type 2 (PCV2) with post-weaning multisystemic wasting syndrome (PMWS) was first reported in western Canada in 1996. Shortly thereafter the disease was recognized in Europe. Sub...

  20. Outbreak investigation of porcine epidemic diarrhea in swine in Ontario.

    PubMed

    Pasma, Tim; Furness, Mary Catherine; Alves, David; Aubry, Pascale

    2016-01-01

    Porcine epidemic diarrhea virus was first diagnosed in Ontario in January of 2014. An outbreak investigation was conducted and it was hypothesized that feed containing spray-dried porcine plasma contaminated with the virus was a risk factor in the introduction and spread of the disease in Ontario. PMID:26740705

  1. Porcine bocaviruses: genetic analysis and prevalence in Chinese swine population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Among members of the Bocavirus genus, that contain three open reading frames (ORFs), of the Parvovirinae subfamily, porcine bocaviruses (PoBoVs) exhibit the most genetic diversity. Based on the ORF2-encoded VP1 classification, the six reported porcine bocaviruses were grouped into four species: PoBo...

  2. Stability of and Attachment to Lettuce by a Culturable Porcine Sapovirus Surrogate for Human Caliciviruses

    PubMed Central

    Zhang, Zhenwen

    2012-01-01

    Human noroviruses (HuNoVs) are the leading cause of food-borne illness, accounting for 58% of U.S. cases. Because HuNoVs are unculturable, surrogates are needed to investigate transmission routes and evaluate disinfection methods. However, the current surrogates, feline calicivirus (FCV) and murine NoV (MNV), are less tolerant than HuNoVs to acid and chlorine, respectively. Porcine sapovirus (SaV) is the only culturable enteropathogenic calicivirus. In this study, the resistance of SaV to physicochemical treatments was compared to that of HuNoVs (by reverse transcription-PCR), FCV, and MNV (by infectivity assays). Sapovirus and HuNoV (viral RNA) showed similar resistances to heat (56°C) and to different concentrations of chlorine. However, SaV was more resistant than HuNoVs to ethanol treatment (60% and 70%). Like HuNoVs, SaV was stable at pH 3.0 to 8.0, with a <1.0 log10 50% tissue culture infective dose (TCID50) reduction at pH 3.0 compared to the value for pH 4.0 to 8.0. SaV and MNV showed similar resistances, and both were more resistant than FCV to heat inactivation (56°C). FCV was more resistant than MNV and SaV to ethanol, and all three viruses showed similar resistances to treatment with low concentrations of chlorine for 1 min. Those results indicate that SaV is a promising surrogate for HuNoVs. Next, we used SaV as a surrogate to examine virus attachment to lettuce at different pHs. Sapovirus attached to lettuce leaves significantly at its capsid isoelectric point (pH 5.0), and the attached viral particles remained infectious on lettuce after 1 week of storage at 4°C. The culturable SaV is a good surrogate for studying HuNoV contamination and transmission in leafy greens and potential disinfectants. PMID:22447610

  3. Reactomes of porcine alveolar macrophages infected with porcine reproductive and respiratory syndrome virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Porcine reproductive and respiratory syndrome (PRRS) has devastated pig industries worldwide for many years. It is caused by a small RNA virus (PRRSV), which targets almost exclusively pig monocytes or macrophages. In the present study, five SAGE (serial analysis of gene expression) libraries derive...

  4. Production of porcine TNFα by ADAM17-mediated cleavage negatively regulates porcine reproductive and respiratory syndrome virus infection.

    PubMed

    Li, Ren; Guo, Longjun; Gu, Weihong; Luo, Xiaolei; Zhang, Jian; Xu, Yunfei; Tian, Zhijun; Feng, Li; Wang, Yue

    2016-06-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes a series of inflammatory reactions in sites of infection, companied by the upregulation of key inflammatory factor TNFα. TNFα, which serves as a "master regulator" of inflammatory cytokine production, is mainly produced by macrophages at the early infection stage. Here, we showed that porcine alveolar macrophages produced a great amount of soluble TNFα upon PRRSV infection. Furthermore, we found that TNFα had great anti-PRRSV effect. Next, by using inhibitor and genetic modification methods, we addressed that porcine TNFα production was mediated by ADAM17. Lastly, we proved that the (78)Arg-Ser-Ser motif of porcine TNFα contained the essential information for efficient cleavage. Taken together, our findings provide the direct evidence that ADAM17 cleaves porcine TNFα, which represents a new view for identifying potential therapeutic targets in anti-PRRSV therapy. PMID:26724939

  5. Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells.

    PubMed

    Peraino, Jaclyn Stromp; Schenk, Marian; Li, Guoying; Zhang, Huiping; Farkash, Evan A; Sachs, David H; Huang, Christene A; Duran-Struuck, Raimon; Wang, Zhirui

    2013-12-15

    Regulatory T cells (Tregs) have been widely recognized as crucial players in controlling immune responses. Because their major role is to ensure that the immune system is not over reactive, Tregs have been the focus of multiple research studies including those investigating transplantation tolerance, autoimmunity and cancer treatment. On their surface Tregs constitutively express CD25, a high affinity receptor for the cytokine interleukin-2 (IL-2). The reagents constructed in this study were generated by genetically linking porcine IL-2 to the truncated diphtheria toxin (DT390). This reagent functions by first binding to the cell surface via the porcine IL-2/porcine CD25 interaction then the DT390 domain facilitates internalization followed by inhibition of protein synthesis resulting in cell death. Four versions of the porcine IL-2 fusion toxin were designed in an interest to find the most effective isoform: 1) monovalent glycosylated porcine IL-2 fusion toxin (Gly); 2) monovalent non-N-glycosylated porcine IL-2 fusion toxin (NonGly); 3) bivalent glycosylated porcine IL-2 fusion toxin (Bi-Gly); 4) bivalent non-N-glycosylated porcine IL-2 fusion toxin (Bi-NonGly). Using a porcine CD25(+) B cell lymphoma cell line (LCL13271) in vitro analysis of the fusion toxins' ability to inhibit protein synthesis demonstrated that the Bi-NonGly fusion toxin is the most efficient reagent. These in vitro results are consistent with binding affinity as the Bi-NonGly fusion toxin binds strongest to CD25 on the same LCL13271 cells. The Bi-Gly fusion toxin significantly prolonged the survival (p=0.028) of tumor-bearing NOD/SCID IL-2 receptor γ(-/-) (NSG) mice injected with LCL13271 cells compared with untreated controls. This recombinant protein has great potential to function as a useful tool for in vivo depletion of porcine CD25(+) cells for studying immune regulation. PMID:24055128

  6. Development of a Diphtheria Toxin-Based Recombinant Porcine IL-2 Fusion Toxin for Depleting Porcine CD25+ Cells

    PubMed Central

    Peraino, Jaclyn Stromp; Schenk, Marian; Li, Guoying; Zhang, Huiping; Farkash, Evan A.; Sachs, David H.; Huang, Christene A.; Duran-Struuck, Raimon; Wang, Zhirui

    2013-01-01

    Regulatory T cells (Tregs) have been widely recognized as crucial players in controlling immune responses. Because their major role is to ensure that the immune system is not over reactive, Tregs have been the focus of multiple research studies including those investigating transplantation tolerance, autoimmunity and cancer treatment. On their surface Tregs constitutively express CD25, a high affinity receptor for the cytokine interleukin-2 (IL-2). The reagents constructed in this study were generated by genetically linking porcine IL-2 to the truncated diphtheria toxin (DT390). This reagent functions by first binding to the cell surface via the porcine IL-2/porcine CD25 interaction then the DT390 domain facilitates internalization followed by inhibition of protein synthesis resulting in cell death. Four versions of the porcine IL-2 fusion toxin were designed in an interest to find the most effective isoform: 1) monovalent glycosylated porcine IL-2 fusion toxin (Gly); 2) monovalent non-N-glycosylated porcine IL-2 fusion toxin (NonGly); 3) bivalent glycosylated porcine IL-2 fusion toxin (Bi-Gly); 4) bivalent non-N-glycosylated porcine IL-2 fusion toxin (Bi-NonGly). Using a porcine CD25+ B cell lymphoma cell line (LCL13271) in vitro analysis of the fusion toxins’ ability to inhibit protein synthesis demonstrated that the Bi-NonGly fusion toxin is the most efficient reagent. These in vitro results are consistent with binding affinity as the Bi-NonGly fusion toxin binds strongest to CD25 on the same LCL13271 cells. The Bi-Gly fusion toxin significantly prolonged the survival (p=0.028) of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with LCL13271 cells compared with untreated controls. This recombinant protein has great potential to function as a useful tool for in vivo depletion of porcine CD25+ cells for studying immune regulation. PMID:24055128

  7. Escherichia vulneris: isolation and treatment.

    PubMed

    Dye, K R; Dall, L; Yuhas, J; Brockert, J L

    1984-12-01

    We have described two cases of Escherichia vulneris wound infections resistant to ampicillin given orally, but susceptible to higher blood levels obtainable by parenchymal administration. PMID:6505777

  8. [Bacteriological studies of traveller's diarrhoea (6). Analysis of enteropathogenic bacteria at Kansai Airport Quarantine Station from September 4th, 1994 through December 1996].

    PubMed

    Ueda, Y; Suzuki, N; Furukawa, T; Takegaki, Y; Takahashi, N; Miyagi, K; Noda, K; Hirose, H; Hashimoto, S; Miyamoto, H; Yano, S; Miyata, Y; Taguchi, M; Ishibashi, M; Honda, T

    1999-02-01

    During the period of investigation from Sept. 4, 1994 to Dec, 1996, a total of 11,446,534 overseas travellers were quarantined at Kansai Airport Quarantine Station, and 22,187 voluntarily reported of episodes suffering from diarrhoea. Bacteriological examination of the stools a total of 9,299 individuals' was performed, and the following results were obtained. 1) Various enteropathogenic bacteria were isolated from 33.3% of the stools examined. Bacterial species isolated were as follows: Plesiomonas shigelloides, 2,066 cases (66.7%); Vibrio parahaemolyticus, 358 cases (11.6%); Aeromonas sobria, 360 cases (11.6%); Shigella spp., 291 cases (9.4%); Salmonella spp., 183 cases (5.9%); A. hydrophila, 126 cases (4.1%); and V. cholerae non-O1, 121 cases (3.9%). However, ETEC was not done with an object of test. 2) In 502 cases (16.2%), plural enteropathogenic bacteria were isolated from single patient, suggesting high frequency of a mixed infections. 3) From Feb. to Mar. 1995, thirteen cases cholera were found from patients who had travelled to Bali, Indonesia. Cases with enteropathogenic bacteria other than V. cholerae O1 were found without any seasonal variation. 4) The major regions where the travellers were infected with the pathogens are as follows: Vibrio spp., were from only Asia; Shigella, widely distributed but especially in India and Indonesia; P. shigelloides and Salmonella, widely distributed. 5) Among the Shigella strains, S. sonnei were isolated the most, followed by S. flexneri, S. boydii and S. dysenteriae. A strain of S. boydii provisional serovar E 16553 was isolated from a patient infected in India. 6) Among the Salmonella serovars, Salmonella Enteritidis was isolated the most frequently (49 cases, 25.7%). 7) 265 (89.2%) of 297 Shigella strains, 52 (27.2%) of 19] Salmonella strains, and 19 (95.0%) of 20 V. cholerae O1 were resistant to one or more drugs tested (SM. CP. TC. KM. ABPC. NA. OFLX). 8) All of the 20 V. cholerae O1 strains were Ogawa, E1 Tor

  9. Molecular characterization and expression of porcine Siglec-5.

    PubMed

    Escalona, Z; Álvarez, B; Uenishi, H; Toki, D; Yuste, M; Revilla, C; Gómez del Moral, M; Alonso, F; Ezquerra, A; Domínguez, J

    2014-05-01

    In this study we describe the characterization of the porcine orthologue of Siglec-5. A cDNa clone was obtained from a porcine cDNa library derived from swine small intestine which encodes a 555 a-a type 1 transmembrane protein with sequence homology to human Siglec-5. This protein consists of four Ig-like domains, a transmembrane region, and a cytoplasmic tail with two tyrosine-based signalling motifs. When expressed as a recombinant protein fused to the Fc region of human IgG1, porcine Siglec-5 was able to bind porcine red blood cells in a sialic acid-dependent manner. Monoclonal antibodies (mAb) were developed against porcine Siglec-5 and used to analyse its expression in bone marrow and blood cells, and lymphoid tissues. Porcine Siglec-5 expression was mainly restricted to myelomonocytic cells and their precursors, being detected also, although at low levels, on plasmacytoid dendritic cells and B lymphocytes. In lymphoid tissues, ellipsoids of the spleen and subcapsular and medullar sinuses of lymph nodes were positive for Siglec-5. These mAbs were able to precipitate, from granulocyte lysates, a protein of approximately 85 kDa under non-reducing conditions, indicating that porcine Siglec-5 is expressed as a monomer in the plasma membrane. PMID:24382335

  10. A novel culture system for adult porcine intestinal crypts.

    PubMed

    Khalil, Hassan A; Lei, Nan Ye; Brinkley, Garrett; Scott, Andrew; Wang, Jiafang; Kar, Upendra K; Jabaji, Ziyad B; Lewis, Michael; Martín, Martín G; Dunn, James C Y; Stelzner, Matthias G

    2016-07-01

    Porcine models are useful for investigating therapeutic approaches to short bowel syndrome and potentially to intestinal stem cell (ISC) transplantation. Whereas techniques for the culture and genetic manipulation of ISCs from mice and humans are well established, similar methods for porcine stem cells have not been reported. Jejunal crypts were isolated from murine, human, and juvenile and adult porcine small intestine, suspended in Matrigel, and co-cultured with syngeneic intestinal subepithelial myofibroblasts (ISEMFs) or cultured without feeder cells in various culture media. Media containing epidermal growth factor, noggin, and R-spondin 1 (ENR medium) were supplemented with various combinations of Wnt3a- or ISEMF-conditioned medium (CM) and with glycogen synthase kinase 3 inhibitor (GSK3i), and their effects were studied on cultured crypts. Cell lineage differentiation was assessed by immunohistochemistry and quantitative polymerase chain reaction. Cultured porcine cells were serially passaged and transduced with a lentiviral vector. Whereas ENR medium supported murine enteroid growth, it did not sustain porcine crypts beyond 5 days. Supplementation of Wnt3a-CM and GSK3i resulted in the formation of complex porcine enteroids with budding extensions. These enteroids contained a mixture of stem and differentiated cells and were successfully passaged in the presence of GSK3i. Crypts grown in media supplemented with porcine ISEMF-CM formed spheroids that were less well differentiated than enteroids. Enteroids and spheroids were transfected with a lentivirus with high efficiency. Thus, our method maintains juvenile and adult porcine crypt cells long-term in culture. Porcine enteroids and spheroids can be successfully passaged and transduced by using lentiviral vectors. PMID:26928041

  11. Amino-Acid Sequence of Porcine Pepsin

    PubMed Central

    Tang, J.; Sepulveda, P.; Marciniszyn, J.; Chen, K. C. S.; Huang, W-Y.; Tao, N.; Liu, D.; Lanier, J. P.

    1973-01-01

    As the culmination of several years of experiments, we propose a complete amino-acid sequence for porcine pepsin, an enzyme containing 327 amino-acid residues in a single polypeptide chain. In the sequence determination, the enzyme was treated with cyanogen bromide. Five resulting fragments were purified. The amino-acid sequence of four of the fragments accounted for 290 residues. Because the structure of a 37-residue carboxyl-terminal fragment was already known, it was not studied. The alignment of these fragments was determined from the sequence of methionyl-peptides we had previously reported. We also discovered the locations of activesite aspartyl residues, as well as the pairing of the three disulfide bridges. A minor component of commercial crystalline pepsin was found to contain two extra amino-acid residues, Ala-Leu-, at the amino-terminus of the molecule. This minor component was apparently derived from a different site of cleavage during the activation of porcine pepsinogen. PMID:4587252

  12. Tissue Remodelling following Resection of Porcine Liver

    PubMed Central

    Nygård, Ingvild Engdal; Mortensen, Kim Erlend; Hedegaard, Jakob; Conley, Lene Nagstrup; Bendixen, Christian; Sveinbjørnsson, Baldur; Revhaug, Arthur

    2015-01-01

    Aim. To study genes regulating the extracellular matrix (ECM) and investigate the tissue remodelling following liver resection in porcine. Methods. Four pigs with 60% partial hepatectomy- (PHx-) induced liver regeneration were studied over six weeks. Four pigs underwent sham surgery and another four pigs were used as controls of the normal liver growth. Liver biopsies were taken upon laparotomy, after three and six weeks. Gene expression profiles were obtained using porcine-specific oligonucleotide microarrays. Immunohistochemical staining was performed and a proliferative index was assessed. Results. More differentially expressed genes were associated with the regulation of ECM in the resection group compared to the sham and control groups. Secreted protein acidic and rich in cysteine (SPARC) and collagen 1, alpha 2 (COL1A2) were both upregulated in the early phase of liver regeneration, validated by immunopositive cells during the remodelling phase of liver regeneration. A broadened connective tissue was demonstrated by Masson's Trichrome staining, and an immunohistochemical staining against pan-Cytokeratin (pan-CK) demonstrated a distinct pattern of migrating cells, followed by proliferating cell nuclear antigen (PCNA) positive nuclei. Conclusions. The present study demonstrates both a distinct pattern of PCNA positive nuclei and a deposition of ECM proteins in the remodelling phase of liver regeneration. PMID:26240819

  13. Phylogeny and evolution of porcine parvovirus.

    PubMed

    Ren, Xiaofeng; Tao, Ye; Cui, Jin; Suo, Siqingaowa; Cong, Yingying; Tijssen, Peter

    2013-12-26

    Porcine parvovirus (PPV), a member of the genus Parvovirus, family Parvoviridae, is a significant causative agent in porcine reproductive failure, causing serious economic losses in the swine industry. Previous phylogenetic studies based on the NS1 or VP2 genes indicated that current PPV strains diverged 30 years ago and that VP2 was under neutral or positive selection. Our analysis of NS1, VP2 and complete ORFs indicated that the most recent common ancestor of PPV strains existed about 250 years ago and that the 127-nt repeat in the 3'NTR was present in viruses of some subclades that evolved about 80 years ago. Nucleotide substitution rates of NS1 and VP2 genes were 3.03 × 10(-5) and 1.07 × 10(-4), respectively. Both the NS1 and VP2 proteins were under purifying selection and recombination did not contribute to the genetic diversity of PPV. As expected, surface amino acids are hydrophilic and make up the majority of mutations in the VP2 protein; residues in VP2 interfaces were substituted gradually, often in conjunction with complementary substitutions in the neighboring VP2. PMID:24050995

  14. Molecular epidemiology and evolution of porcine parvoviruses.

    PubMed

    Streck, André Felipe; Canal, Cláudio Wageck; Truyen, Uwe

    2015-12-01

    Porcine parvovirus (PPV), recently named Ungulate protoparvovirus 1, is considered to be one of the most important causes of reproductive failure in swine. Fetal death, mummification, stillbirths and delayed return to estrus are predominant clinical signs commonly associated with PPV infection in a herd. It has recently been shown that certain parvoviruses exhibit a nucleotide substitution rate close to that commonly determined for RNA viruses. However, the PPV vaccines broadly used in the last 30 years have most likely reduced the genetic diversity of the virus and led to the predominance of strains with a capsid profile distinct from that of the original vaccine-based strains. Furthermore, a number of novel porcine parvovirus species with yet-unknown veterinary relevance and characteristics have been described during the last decade. In this review, an overview of PPV molecular evolution is presented, highlighting characteristics of the various genetic elements, their evolutionary rate and the discovery of new capsid profiles driven by the currently used vaccines. PMID:26453771

  15. Interactions of porcine circovirus 2 with its hosts.

    PubMed

    Ren, Linzhu; Chen, Xinrong; Ouyang, Hongsheng

    2016-08-01

    Porcine circovirus 2 (PCV2) can cause porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD), which are widely presented in swine-producing countries. Since the discovery of this virus, considerable efforts have been devoted to understanding this pathogen and its interactions with its host. Here, we review the current state of knowledge on interactions between host cell factors and PCV2 with respect to viral proliferation, virus-induced cell apoptosis and autophagy, and host antiviral defenses during PCV2 infection. We also review mouse model systems for PCV2 infection. PMID:27016220

  16. Pathogenic Escherichia coli.

    PubMed

    Kaper, James B; Nataro, James P; Mobley, Harry L

    2004-02-01

    Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes. PMID:15040260

  17. Porcine circovirus type 2 detection in in vitro produced porcine blastocysts after virus sperm exposure.

    PubMed

    Galeati, Giovanna; Zannoni, Augusta; Spinaci, Marcella; Bucci, Diego; Ostanello, Fabio; Panarese, Serena; Tamanini, Carlo; Sarli, Giuseppe

    2016-04-01

    This study was aimed at assessing the capability of semen experimentally infected with porcine circovirus type 2 (PCV2) to produce porcine blastocysts PCR positive for PCV2. Embryos were obtained from in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes or by parthenogenesis. Sperm suspension was exposed to PCV2b and utilized for IVF. PCV2 spiked semen did not reveal any reduction in sperm viability or motility but its ability to produce infected blastocysts was irrelevant as only one out of 15 blastocysts obtained by IVF were PCV2b; however two blastocysts were PCV2a positive. Furthermore, the presence of PCV2 was demonstrated also in embryos obtained by parthenogenesis (one out of 17 was PCV2b and one PCV2a positive). Even if PCV2 firmly attaches to the surface of spermatozoa, experimentally spiked sperm were not effective in infecting oocytes during IVF and in producing PCR positive embryos. The infected blastocysts we obtained derived most probably from infected oocytes recovered at the abattoir. PMID:26434667

  18. Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells

    SciTech Connect

    Cong, Yingying; Li, Xiaoxue; Bai, Yunyun; Lv, Xiaonan; Herrler, Georg; Enjuanes, Luis; Zhou, Xingdong; Qu, Bo; Meng, Fandan; Cong, Chengcheng; Ren, Xiaofeng; Li, Guangxing

    2015-04-15

    Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells. - Highlights: • PEDV infection of polarized intestinal epithelial cells (IECs) was characterized. • Porcine aminpeptidase N (pAPN) facilitated PEDV infection in IECs. • PEDV entry into and release from polarized cell via its apical membrane. • PEDV infection may proceed by lateral spread of virus in IECs.

  19. Molecular epidemiology of Escherichia coli diarrhea in children in Hong Kong.

    PubMed Central

    Biswas, R; Nelson, E A; Lewindon, P J; Lyon, D J; Sullivan, P B; Echeverria, P

    1996-01-01

    This pediatric hospital-based study of 388 diarrhea cases and 306 controls analyzed predominant E. coli colonies from primary culture (253 cases and 177 controls) with eight DNA probes for enteropathogenic, enterotoxigenic, enteroaggregative, and diffusely adherent E. coli. Only enteropathogenic E. coli adherence factor was identified significantly more frequently in cases (10) than in controls (0). PMID:8940481

  20. Genome-wide inactivation of porcine endogenous retroviruses (PERVs).

    PubMed

    Yang, Luhan; Güell, Marc; Niu, Dong; George, Haydy; Lesha, Emal; Grishin, Dennis; Aach, John; Shrock, Ellen; Xu, Weihong; Poci, Jürgen; Cortazio, Rebeca; Wilkinson, Robert A; Fishman, Jay A; Church, George

    2015-11-27

    The shortage of organs for transplantation is a major barrier to the treatment of organ failure. Although porcine organs are considered promising, their use has been checked by concerns about the transmission of porcine endogenous retroviruses (PERVs) to humans. Here we describe the eradication of all PERVs in a porcine kidney epithelial cell line (PK15). We first determined the PK15 PERV copy number to be 62. Using CRISPR-Cas9, we disrupted all copies of the PERV pol gene and demonstrated a >1000-fold reduction in PERV transmission to human cells, using our engineered cells. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that PERVs can be inactivated for clinical application of porcine-to-human xenotransplantation. PMID:26456528

  1. Escherichia coli Serogroups Isolated from Streams in Pennsylvania, 1965 to 19721

    PubMed Central

    Glantz, Paul J.

    1973-01-01

    Of 3,200 cultures of Escherichia coli isolated from streams in Pennsylvania over a 7-year period, 82.46% or 2,639 were O serogrouped. The largest number of cultures (33.4%) belonged to O groups 1 to 26, and the second highest number (16.8%) belonged to O groups 60 to 88. The individual E. coli O groups most frequently isolated were ADO3, 18ac, 2a, 3, 7, 73, 139, and OX13. Practically every known standard E. coli O group was found in the streams. It was not possible to identify the K and H antigen of every E. coli isolate. Serotypes of E. coli O2a:K1:H6, O26:K60:H11, O55:K59:H27, O86:K62:H2, 112ab:K68:H2, 125ab:K70:H21, 128ab:K67:H2, and O138:K81:H14 known to be pathogenic for humans and animals were identified. Cultures having the same K antigen but a different H antigen for enteropathogenic E. coli O groups 6, 18ab, 18ac, 111ab, 126, 127a, 139, 141, and 147 were also isolated. PMID:4586930

  2. A PCR-ELISA for detecting Shiga toxin-producing Escherichia coli.

    PubMed

    Ge, Beilei; Zhao, Shaohua; Hall, Robert; Meng, Jianghong

    2002-03-01

    A sensitive and specific PCR-ELISA was developed to detect Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in food. The assay was based on the incorporation of digoxigenin-labeled dUTP and a biotin-labeled primer specific for Shiga toxin genes during PCR amplification. The labeled PCR products were bound to streptavidin-coated wells of a microtiter plate and detected by an ELISA. The specificity of the PCR was determined using 39 bacterial strains, including STEC, enteropathogenic E. coli, E. coli K12, and Salmonella. All of the STEC strains were positive, and non-STEC organisms were negative. The ELISA detecting system was able to increase the sensitivity of the PCR assay by up to 100-fold, compared with a conventional gel electrophoresis. The detection limit of the PCR-ELISA was 0.1-10 CFU dependent upon STEC serotypes, and genotypes of Shiga toxins. With the aid of a simple DNA extraction system, PrepMan, the PCR-ELISA was able to detect ca. 10(5) CFU of STEC per gram of ground beef without any culture enrichment. The entire procedure took about 6 h. Because of its microtiter plate format, PCR-ELISA is particularly suitable for large-scale screening and compatible with future automation. PMID:11909738

  3. A Survey for Escherichia coli Virulence Factors in Asymptomatic Free-Ranging Parrots.

    PubMed

    Becker Saidenberg, André; Robaldo Guedes, Neiva Maria; Fernandes Seixas, Gláucia Helena; da Costa Allgayer, Mariangela; Pacífico de Assis, Erica; Fabio Silveira, Luis; Anne Melville, Priscilla; Benites, Nilson Roberti

    2012-01-01

    Parrots in captivity are frequently affected by Escherichia coli (E. coli) infections. The objective of this study was to collect information on the carrier state for E. coli pathotypes in asymptomatic free-ranging parrots. Cloacal swabs were collected from nestlings of Hyacinth, Lear's macaws and Blue-fronted Amazon parrots and tested by polymerase chain reaction (PCR) for virulence factors commonly found in enteropathogenic, avian pathogenic, and uropathogenic E. coli strains. In total, 44 samples were cultured and E. coli isolates were yielded, from which DNA was extracted and processed by PCR. Genes commonly found in APEC isolates from Blue-fronted Amazon parrots and Hyacinth macaws were expressed in 14 of these 44 samples. One atypical EPEC isolate was obtained from a sample from Lear's macaw. The most commonly found gene was the increased serum survival (iss) gene. This is the first report, that describes such pathotypes in asymptomatic free-living parrots. The findings of this study suggest the presence of a stable host/parasite relationship at the time of the sampling brings a new understanding to the role that E. coli plays in captive and wild parrots. Such information can be used to improve husbandry protocols as well as help conservation efforts of free-living populations. PMID:23738135

  4. The rabbit as a new reservoir host of enterohemorrhagic Escherichia coli.

    PubMed

    García, Alexis; Fox, James G

    2003-12-01

    We investigated the prevalence of enterohemorrhagic Escherichia coli (EHEC) in rabbits acquired from two commercial vendors and a local petting zoo. Fecal samples from 34 Dutch Belted (DB) and 15 New Zealand White (NZW) rabbits were cultured; and isolates were biotyped, serotyped, tested by polymerase chain reaction (PCR), and genotyped by repetitive-element sequence-based PCR (Rep-PCR). Seven (25%) of 28 DB rabbits acquired from one commercial source were positive for EHEC, including O153:H- and O153:H7. One (9%) of 11 NZW rabbits from the same source was positive for eae-, stx1+ O153 strains. In contrast, six DB rabbits from another commercial source and four rabbits from a petting zoo were negative for EHEC. Rep-PCR demonstrated that the O153 EHEC and O145 enteropathogenic E. coli were two distinct clones. Our study indicates that rabbits are a new reservoir host of EHEC that may pose a zoonotic risk for humans. PMID:14720401

  5. The Rabbit as a New Reservoir Host of Enterohemorrhagic Escherichia coli

    PubMed Central

    Fox, James G.

    2003-01-01

    We investigated the prevalence of enterohemorrhagic Escherichia coli (EHEC) in rabbits acquired from two commercial vendors and a local petting zoo. Fecal samples from 34 Dutch Belted (DB) and 15 New Zealand White (NZW) rabbits were cultured; and isolates were biotyped, serotyped, tested by polymerase chain reaction (PCR), and genotyped by repetitive-element sequence–based PCR (Rep-PCR). Seven (25%) of 28 DB rabbits acquired from one commercial source were positive for EHEC, including O153:H- and O153:H7. One (9%) of 11 NZW rabbits from the same source was positive for eae-, stx1+ O153 strains. In contrast, six DB rabbits from another commercial source and four rabbits from a petting zoo were negative for EHEC. Rep-PCR demonstrated that the O153 EHEC and O145 enteropathogenic E. coli were two distinct clones. Our study indicates that rabbits are a new reservoir host of EHEC that may pose a zoonotic risk for humans. PMID:14720401

  6. tir- and stx-Positive Escherichia coli in Stream Waters in a Metropolitan Area

    PubMed Central

    Higgins, James A.; Belt, Kenneth T.; Karns, Jeffrey S.; Russell-Anelli, Jonathan; Shelton, Daniel R.

    2005-01-01

    Diarrheagenic Escherichia coli, which may include the enteropathogenic E. coli and the enterohemorrhagic E. coli, are a significant cause of diarrheal disease among infants and children in both developing and developed areas. Disease outbreaks related to freshwater exposure have been documented, but the presence of these organisms in the urban aquatic environment is not well characterized. From April 2002 through April 2004 we conducted weekly surveys of streams in the metropolitan Baltimore, Md., area for the prevalence of potentially pathogenic E. coli by using PCR assays targeting the tir and stx1 and stx2 genes. Coliforms testing positive for the presence of the tir gene were cultured from 653 of 1,218 samples (53%), with a greater prevalence associated with urban, polluted streams than in suburban and forested watershed streams. Polluted urban streams were also more likely to test positive for the presence of one of the stx genes. Sequence analysis of the tir amplicon, as well as the entire tir gene from three isolates, indicated that the pathogenic E. coli present in the stream waters has a high degree of sequence homology with the E. coli O157:H7 serotype. Our data indicate that pathogenic E. coli are continually deposited into a variety of stream habitats and suggest that this organism may be a permanent member of the gastrointestinal microflora of humans and animals in the metropolitan Baltimore area. PMID:15870341

  7. Potential role of Escherichia coli DNA mismatch repair proteins in colon cancer.

    PubMed

    Khan, Shahanavaj

    2015-12-01

    The epithelium of gastrointestinal tract organizes many innate defense systems against microbial intruders such as integrity of epithelial, rapid eviction of infected cells, quick turnover of epithelial cell, intrinsic immune responses and autophagy. However, Enteropathogenic Escherichia coli (EPEC) are equipped with well developed infectious tricks that evade the host defense systems and utilize the gastrointestinal epithelium as a multiplicative foothold. During multiplication on and within the epithelium, EPEC secrete various toxins that can weaken, usurp, and use many host cellular systems. However, the possible mechanisms of pathogenesis are still poorly elusive. Recent study reveals the existence of EPEC in colorectal cancer patients and their potential role in depletion of DNA mismatch repair (MMR) proteins of host cell in colonic cell lines. The EPEC colonised intracellularly in colon mucosa of colorectal carcinoma whereas extracellular strain was detected in mucosa of normal colon cells. Interestingly, alteration in MutS, MutL complexes and MUTYH of mammalian cells may be involved in development of CRC. These data propose that MMR of E. coli may be potential therapeutic targets and early detection biomarkers for CRC. This article reviews the potential role of E. coli MutS, MutL and MutY protein in CRC aetiology. PMID:26014615

  8. Microarray Evaluation of Antimicrobial Resistance and Virulence of Escherichia coli Isolates from Portuguese Poultry.

    PubMed

    Mendonça, Nuno; Figueiredo, Rui; Mendes, Catarina; Card, Roderick M; Anjum, Muna F; da Silva, Gabriela Jorge

    2016-01-01

    The presence of antimicrobial resistance and virulence factors of 174 Escherichia coli strains isolated from healthy Portuguese Gallus gallus was evaluated. Resistance profiles were determined against 33 antimicrobials by microbroth dilution. Resistance was prevalent for tetracycline (70%) and ampicillin (63%). Extended-spectrum beta-lactamase (ESBL) phenotype was observed in 18% of the isolates. Multidrug resistance was found in 56% of isolates. A subset of 74 isolates were screened by DNA microarrays for the carriage of 88 antibiotic resistance genes and 62 virulence genes. Overall, 37 different resistance genes were detected. The most common were tet(A) (72%), blaTEM (68%), and sul1 (47%), while 21% isolates harbored an ESBL gene (blaCTX-M group 1, group 2, or group 9). Of these, 96% carried the increased serum survival (iss) virulence gene, while 89% presented the enterobactin siderophore receptor protein (iroN), 70% the temperature-sensitive hemagglutinin (tsh), and 68% the long polar fimbriae (lpfA) virulence genes associated with extraintestinal pathogenic E. coli. In conclusion, prevalence of antibiotic resistant E. coli from the microbiota of Portuguese chickens was high, including to extended spectrum cephalosporins. The majority of isolates seems to have the potential to trigger extraintestinal human infection due to the presence of some virulence genes. However, the absence of genes specific for enteropathogenic E. coli reduces the risk for human intestinal infection. PMID:27025519

  9. Enterohaemorrhagic Escherichia coli inhibits recycling endosome function and trafficking of surface receptors

    PubMed Central

    Clements, Abigail; Stoneham, Charlotte A; Furniss, R Christopher D; Frankel, Gad

    2014-01-01

    Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC) manipulate many cell processes by injecting effector proteins from the bacteria into the host cell via a Type III secretion system. In this paper we report that the effector protein EspG disrupts recycling endosome function. In particular, we found that following transferrin binding and endocytosis EspG reduces recycling of the transferrin receptor (TfR), the prototypical recycling protein, from an intracellular location to the cell surface, resulting in an accumulation of TfR within the cell. The surface levels of three receptors [TfR, epidermal growth factor receptor (EGFR) and β1 integrin] were tested and found to be reduced dependent on EspG translocation. Furthermore, disruption of recycling endosome function and the reduced surface presentation of receptors was dependent on the previously reported RabGAP activity and ARF binding ability of EspG. This paper therefore supports the previous hypothesis that EspG acts as an enzyme scaffold perturbing cell signalling events, in this case altering recycling endosome function and cell surface receptor levels during infection. PMID:24898821

  10. A Survey for Escherichia coli Virulence Factors in Asymptomatic Free-Ranging Parrots

    PubMed Central

    Becker Saidenberg, André; Robaldo Guedes, Neiva Maria; Fernandes Seixas, Gláucia Helena; da Costa Allgayer, Mariangela; Pacífico de Assis, Erica; Fabio Silveira, Luis; Anne Melville, Priscilla; Benites, Nilson Roberti

    2012-01-01

    Parrots in captivity are frequently affected by Escherichia coli (E. coli) infections. The objective of this study was to collect information on the carrier state for E. coli pathotypes in asymptomatic free-ranging parrots. Cloacal swabs were collected from nestlings of Hyacinth, Lear's macaws and Blue-fronted Amazon parrots and tested by polymerase chain reaction (PCR) for virulence factors commonly found in enteropathogenic, avian pathogenic, and uropathogenic E. coli strains. In total, 44 samples were cultured and E. coli isolates were yielded, from which DNA was extracted and processed by PCR. Genes commonly found in APEC isolates from Blue-fronted Amazon parrots and Hyacinth macaws were expressed in 14 of these 44 samples. One atypical EPEC isolate was obtained from a sample from Lear's macaw. The most commonly found gene was the increased serum survival (iss) gene. This is the first report, that describes such pathotypes in asymptomatic free-living parrots. The findings of this study suggest the presence of a stable host/parasite relationship at the time of the sampling brings a new understanding to the role that E. coli plays in captive and wild parrots. Such information can be used to improve husbandry protocols as well as help conservation efforts of free-living populations. PMID:23738135

  11. Comparative Genomics Provides Insight into the Diversity of the Attaching and Effacing Escherichia coli Virulence Plasmids

    PubMed Central

    Hazen, Tracy H.; Kaper, James B.; Nataro, James P.

    2015-01-01

    Attaching and effacing Escherichia coli (AEEC) strains are a genomically diverse group of diarrheagenic E. coli strains that are characterized by the presence of the locus of enterocyte effacement (LEE) genomic island, which encodes a type III secretion system that is essential to virulence. AEEC strains can be further classified as either enterohemorrhagic E. coli (EHEC), typical enteropathogenic E. coli (EPEC), or atypical EPEC, depending on the presence or absence of the Shiga toxin genes or bundle-forming pilus (BFP) genes. Recent AEEC genomic studies have focused on the diversity of the core genome, and less is known regarding the genetic diversity and relatedness of AEEC plasmids. Comparative genomic analyses in this study demonstrated genetic similarity among AEEC plasmid genes involved in plasmid replication conjugative transfer and maintenance, while the remainder of the plasmids had sequence variability. Investigation of the EPEC adherence factor (EAF) plasmids, which carry the BFP genes, demonstrated significant plasmid diversity even among isolates within the same phylogenomic lineage, suggesting that these EAF-like plasmids have undergone genetic modifications or have been lost and acquired multiple times. Global transcriptional analyses of the EPEC prototype isolate E2348/69 and two EAF plasmid mutants of this isolate demonstrated that the plasmid genes influence the expression of a number of chromosomal genes in addition to the LEE. This suggests that the genetic diversity of the EAF plasmids could contribute to differences in the global virulence regulons of EPEC isolates. PMID:26238712

  12. Seasonal distribution and prevalence of diarrheagenic Escherichia coli in different aquatic environments in Taiwan.

    PubMed

    Huang, Wen-Chien; Hsu, Bing-Mu; Kao, Po-Min; Tao, Chi-Wei; Ho, Ying-Ning; Kuo, Chun-Wei; Huang, Yu-Li

    2016-02-01

    Diarrheagenic Escherichia coli (DEC) are the most common agents of diarrhea. Waterborne DEC could pose a potential health risk to human through agricultural, household, recreational, and industrial use. There are few published reports on the detection of DEC and its seasonal distribution in aquatic environments. The presence of DEC in different types of aquatic environments was investigated in this study. Water samples were collected from major rivers, water reservoirs, and recreational hot springs throughout Taiwan. Moreover, an intensive water sampling plan was carried out along Puzih River. The detection of DEC target genes was used to determine the presence of enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), and Shiga toxin-producing E. coli (STEC). Among the 383 water samples analyzed, DEC was found in 122 (31.8%) samples. The detection rate varied by genotype, raging from 3.6% for STEC to 17.2% for EPEC. The DEC detection rate was higher from river waters than reservoirs and hot springs. In addition, DEC was detected at a higher rate in spring and summer. The presence of EPEC was significantly associated with total coliform levels among hot spring samples. Moreover, the presence of ETEC in river water samples was associated with heterotrophic plate counts. Water with EPEC differed significantly in pH from Puzih River samples. These results suggest that seasonal characteristics may affect the presence of DEC in different aquatic environments, and water quality indicators may be indicative of the presence of DEC. PMID:26454073

  13. Evolutionary origin of pathogenic determinants in enterotoxigenic Escherichia coli and Vibrio cholerae O1.

    PubMed Central

    Yamamoto, T; Gojobori, T; Yokota, T

    1987-01-01

    Three families of the evolutionarily related pathogenic determinants in enterotoxigenic Escherichia coli and Vibrio cholerae O1, a family of cholera enterotoxin (CT) and heat-labile enterotoxin (LT) including CT, LTh, and LTp, a family of heat-stable enterotoxin I (STI) including STIa and STIb, and a family of K88 enteroadhesion fimbriae including K88ab, K88ac, and K88ad were analyzed for synonymous (silent) nucleotide substitutions by using the gene nucleotide sequences of earlier reports and the LTp gene nucleotide sequence presented in this paper. The data suggested that the divergences between LT and CT and between STIa and STIb occurred in the remote past, whereas those between LTh and LTp and between members of the K88 family occurred very recently. We concluded that the LT gene is a foreign gene that has been acquired by E. coli to form an enteropathogen. This provides evolutionary evidence of species-to-species transfer of pathogenic determinants in procaryotes. PMID:3546273

  14. Microarray Evaluation of Antimicrobial Resistance and Virulence of Escherichia coli Isolates from Portuguese Poultry

    PubMed Central

    Mendonça, Nuno; Figueiredo, Rui; Mendes, Catarina; Card, Roderick M.; Anjum, Muna F.; da Silva, Gabriela Jorge

    2016-01-01

    The presence of antimicrobial resistance and virulence factors of 174 Escherichia coli strains isolated from healthy Portuguese Gallus gallus was evaluated. Resistance profiles were determined against 33 antimicrobials by microbroth dilution. Resistance was prevalent for tetracycline (70%) and ampicillin (63%). Extended-spectrum beta-lactamase (ESBL) phenotype was observed in 18% of the isolates. Multidrug resistance was found in 56% of isolates. A subset of 74 isolates were screened by DNA microarrays for the carriage of 88 antibiotic resistance genes and 62 virulence genes. Overall, 37 different resistance genes were detected. The most common were tet(A) (72%), blaTEM (68%), and sul1 (47%), while 21% isolates harbored an ESBL gene (blaCTX-M group 1, group 2, or group 9). Of these, 96% carried the increased serum survival (iss) virulence gene, while 89% presented the enterobactin siderophore receptor protein (iroN), 70% the temperature-sensitive hemagglutinin (tsh), and 68% the long polar fimbriae (lpfA) virulence genes associated with extraintestinal pathogenic E. coli. In conclusion, prevalence of antibiotic resistant E. coli from the microbiota of Portuguese chickens was high, including to extended spectrum cephalosporins. The majority of isolates seems to have the potential to trigger extraintestinal human infection due to the presence of some virulence genes. However, the absence of genes specific for enteropathogenic E. coli reduces the risk for human intestinal infection. PMID:27025519

  15. Virulence genes, antibiotic resistance and integrons in Escherichia coli strains isolated from synanthropic birds from Spain.

    PubMed

    Sacristán, C; Esperón, F; Herrera-León, S; Iglesias, I; Neves, E; Nogal, V; Muñoz, M J; de la Torre, A

    2014-01-01

    The aim of this study was to determine the presence of virulence genes and antibiotic resistance profiles in 164 Escherichia coli strains isolated from birds (feral pigeons, hybrid ducks, house sparrows and spotless starlings) inhabiting urban and rural environments. A total of eight atypical enteropathogenic E. coli strains were identified: one in a house sparrow, four in feral pigeons and three in spotless starlings. Antibiotic resistance was present in 32.9% (54) of E. coli strains. The dominant type of resistance was to tetracycline (21.3%), ampicillin (19.5%) and sulfamethoxazole (18.9%). Five isolates had class 1 integrons containing gene cassettes encoding for dihydrofolate reductase A (dfrA) and aminoglycoside adenyltransferase A (aadA), one in a feral pigeon and four in spotless starlings. To our knowledge, the present study constitutes the first detection of virulence genes from E. coli in spotless starlings and house sparrows, and is also the first identification worldwide of integrons containing antibiotic resistance gene cassettes in E. coli strains from spotless starlings and pigeons. PMID:24689431

  16. FUT1 genetic variants impact protein glycosylation of porcine intestinal mucosa.

    PubMed

    Hesselager, Marianne O; Everest-Dass, Arun V; Thaysen-Andersen, Morten; Bendixen, Emøke; Packer, Nicolle H

    2016-06-01

    A massive use of antibiotics in industrial pig production is a major cause of the rapidly rising bacterial resistance to antibiotics. An enhanced understanding of infectious diseases and of host-microbe interactions has the potential to explore alternative ways to improve pig health and reduce the need for antibiotics. Host-microbe interactions depend on host-expressed glycans and microbe-carrying lectins. In this study, a G > A (nucleotide 307) missense mutation in the porcine α1,2fucosyltransferase 1 gene (FUT1), which has been reported to prevent infections by the common porcine enteric pathogen F18 fimbriated Escherichia coli, provided a unique opportunity to study glycan structures potentially involved in intestinal infections. N- and O-Linked glycans of the intestinal mucosa proteins were characterized in detail using LC-MS/MS. Relative abundances of all glycans were determined and compared between four heterozygous pigs (FUT1-307(A/G)) and four age-matched homozygous pigs from the same 2 litters carrying the missense FUT1 gene constellation (FUT1-307(A/A)). None of the characterized 48 N-linked glycans was found to be regulated by the FUT1 missense mutation, while 11 of the O-linked glycans showed significantly altered abundances between the two genotypes. The overall abundance of H-antigen carrying structures was decreased fivefold, while H-antigen precursors and sialylated structures were relatively more abundant in pigs with the FUT1 missense mutation. These results provide insight into the role of FUT1 on intestinal glycosylation, improve our understanding of how variation in FUT1 can modulate host-microbe interactions, and suggest that the FUT1 genetic variant may help to improve pig gut health. PMID:26858341

  17. Sequence and regulation of the porcine FSHR gene promoter.

    PubMed

    Wu, Wangjun; Han, Jing; Cao, Rui; Zhang, Jinbi; Li, Bojiang; Liu, Zequn; Liu, Kaiqing; Li, Qifa; Pan, Zengxiang; Chen, Jie; Liu, Honglin

    2015-03-01

    Follicle-stimulating hormone (FSH) plays a crucial role in animal reproduction and exerts its physiological functions by interacting with the FSH receptor (FSHR). The FSHR is exclusively expressed in granulose cells in the ovary and its expression level is closely related to granulose cell differentiation and follicle maturation. In mammal, most of the follicles undergo atresia, while follicle atresia is mainly caused by granulosa cell apoptosis. However, knowledge on the transcriptional regulatory mechanisms of the porcine FSHR gene in granulosa cell is still limited. In this study, approximately 2.1kb of the proximal promoter sequence of the porcine FSHR gene were obtained by genome walking, and the regulatory elements and transcription factors in the porcine FSHR promoter sequence were predicted. Furthermore, the core promoter region (-1195/-598) of the porcine FSHR gene was identified using a luciferase assay. Subsequently, the relationship between expression levels of the porcine FSHR gene and histone H3K9 acetylation levels around the core promoter region (-787/-572) in vivo and invitro were analyzed. Our results showed that an increased FSHR gene expression level was accompanied with an increase in histone H3K9 acetylation levels, suggesting that histone H3K9 acetylation could regulate the expression of the porcine FSHR gene. PMID:25599592

  18. [Research Advances in the Porcine Deltacoronavirus].

    PubMed

    Fang, Puxian; Fang, Liurong; Dong, Nan; Xiao, Shaobo

    2016-03-01

    The deltacoronavirus is a new member of the subfamily Coronaviridae of the family Coronaviridae. Deltacoronaviruses can infect birds and mammals. Deltacoronaviruses were detected in early 2007 in Asian leopard cats and Chinese ferret badgers. In 2014, porcine deltacoronavirus (PDCoV) infection spread rapidly in the USA. Moreover, cell culture-adapted PDCoV has been obtained from infected piglets. Animal experiments have confirmed that the isolated PDCoV is highly pathogenic and causes severe diarrhea in piglets. Thus, the PDCoV can be considered to be a good model to study the deltacoronavirus. In this review, we discuss the etiology, epidemiology, pathogenicity, culture, and diagnostic methods of the PDCoV. PMID:27396171

  19. Tiamulin resistance in porcine Brachyspira pilosicoli isolates.

    PubMed

    Pringle, M; Landén, A; Franklin, A

    2006-02-01

    There are few studies on antimicrobial susceptibility of Brachyspira pilosicoli, therefore this study was performed to investigate the situation among isolates from pigs. The tiamulin and tylosin susceptibility was determined by broth dilution for 93 and 86 porcine B. pilosicoli isolates, respectively. The isolates came from clinical samples taken in Swedish pig herds during the years 2002 and 2003. The tylosin minimal inhibitory concentration (MIC) was >16 microg/ml for 50% (n=43) of the isolates tested. A tiamulin MIC >2 microg/ml was obtained for 14% (n=13) of the isolates and these were also tested against doxycycline, salinomycin, valnemulin, lincomycin and aivlosin. For these isolates the susceptibility to salinomycin and doxycycline was high but the MICs for aivlosin varied. The relationship between the 13 tiamulin resistant isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Among the 13 isolates 10 different PFGE patterns were identified. PMID:16253666

  20. Nursing supports neonatal porcine testicular development.

    PubMed

    Rahman, K M; Lovich, J E; Lam, C; Camp, M E; Wiley, A A; Bartol, F F; Bagnell, C A

    2014-07-01

    The lactocrine hypothesis suggests a mechanism whereby milk-borne bioactive factors delivered to nursing offspring affect development of neonatal tissues. The objective of this study was to assess whether nursing affects testicular development in neonatal boars as reflected by: (1) Sertoli cell number and proliferation measured by GATA-4 expression and proliferating cell nuclear antigen immunostaining patterns; (2) Leydig cell development and steroidogenic activity as reflected by insulin-like factor 3 (INSL3), and P450 side chain cleavage (scc) enzyme expression; and (3) expression of estrogen receptor-alpha (ESR1), vascular endothelial growth factor (VEGF) A, and relaxin family peptide receptor (RXFP) 1. At birth, boars were randomly assigned (n = 6-7/group) to nurse ad libitum or to be pan fed porcine milk replacer for 48 h. Testes were collected from boars at birth, before nursing and from nursed and replacer-fed boars at 50 h on postnatal day (PND) 2. Sertoli cell proliferating cell nuclear antigen labeling index increased (P < 0.01) from birth to PND 2 in nursed, but not in replacer-fed boars. Sertoli cell number and testicular GATA-4 protein levels increased (P < 0.01) from PND 0 to PND 2 only in nursed boars. Neither age nor nursing affected testicular INSL3, P450scc, ESR1, or VEGFA levels. However, testicular relaxin family peptide receptor 1 (RXFP1) levels increased (P < 0.01) with age and were greater in replacer-fed boars on PND 2. Results suggest that nursing supports neonatal porcine testicular development and provide additional evidence for the importance of lactocrine signaling in pigs. PMID:24906933

  1. How Active Are Porcine Endogenous Retroviruses (PERVs)?

    PubMed Central

    Denner, Joachim

    2016-01-01

    Porcine endogenous retroviruses (PERVs) represent a risk factor if porcine cells, tissues, or organs were to be transplanted into human recipients to alleviate the shortage of human transplants; a procedure called xenotransplantation. In contrast to human endogenous retroviruses (HERVs), which are mostly defective and not replication-competent, PERVs are released from normal pig cells and are infectious. PERV-A and PERV-B are polytropic viruses infecting cells of several species, among them humans; whereas PERV-C is an ecotropic virus infecting only pig cells. Virus infection was shown in co-culture experiments, but also in vivo, in the pig, leading to de novo integration of proviruses in certain organs. This was shown by measurement of the copy number per cell, finding different numbers in different organs. In addition, recombinations between PERV-A and PERV-C were observed and the recombinant PERV-A/C were found to be integrated in cells of different organs, but not in the germ line of the animals. Here, the evidence for such in vivo activities of PERVs, including expression as mRNA, protein and virus particles, de novo infection and recombination, will be summarised. These activities make screening of pigs for provirus number and PERV expression level difficult, especially when only blood or ear biopsies are available for analysis. Highly sensitive methods to measure the copy number and the expression level will be required when selecting pigs with low copy number and low expression of PERV as well as when inactivating PERVs using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (CRISPR/Cas) technology. PMID:27527207

  2. Electromechanical Reshaping of Ex Vivo Porcine Trachea

    PubMed Central

    Hussain, Syed; Manuel, Cyrus T.; Protsenko, Dmitriy E.; Wong, Brian J. F.

    2015-01-01

    Objectives The trachea is a composite cartilaginous structure particularly prone to various forms of convexities. Electromechanical reshaping (EMR) is an emerging technique used to reshape cartilaginous tissues by applying electric current in tandem with imposed mechanical deformation to achieve shape change. In this study, EMR was used to reshape tracheal cartilage rings to demonstrate the feasibility of this technology as a potentially minimally invasive procedure to alter tracheal structure. Study Design Controlled laboratory study using ex vivo porcine tracheae. Methods The natural concavity of each porcine tracheal ring was reversed around a cork mandrel. Two pairs of electrodes were inserted along the long axis of the tracheal ring and placed 1.5 millimeters from the midline. Current was applied over a range of voltages (3 volts [V], 4V, and 5V) for either 2 or 3 minutes. The degree of EMR-induced reshaping was quantified from photographs using digital techniques. Confocal imaging with fluorescent live and dead assays was conducted to determine viability of the tissue after EMR. Results Specimens that underwent EMR for 2 or 3 minutes at 4V or 5V were observed to have undergone significant (P <.05) reshaping relative to the control. Viability results demonstrated that EMR reshaping occurs at the expense of tissue injury, although the extent of injury is modest relative to conventional techniques. Conclusion EMR reshapes tracheal cartilage rings as a function of voltage and application time. It has potential as a minimally invasive and cost-efficient endoscopic technology to treat pathologic tracheal convexities. Given our findings, consideration of EMR for use in larger ex vivo tracheal segments and animal studies is now plausible. Level of Evidence N/A. PMID:25692713

  3. Porcine circovirus diseases: a review of PMWS.

    PubMed

    Baekbo, P; Kristensen, C S; Larsen, L E

    2012-03-01

    This article is a review on post-weaning multisystemic wasting syndrome (PMWS), the first described disease among the porcine circovirus diseases (PCVD). Post-weaning multisystemic wasting syndrome has, since its appearance in Canada in 1991, been seen in all major pig producing countries. To diagnose PMWS at herd level typical clinical appearance consisting of wasting and increased mortality must be combined with finding at autopsy of diseased pigs, where typical microscopic findings in the lymphatic tissue must be present. Post-weaning multisystemic wasting syndrome significantly increases the mortality and reduces the daily weight gain in weaner pig and/or in finishing pigs. Post-weaning multisystemic wasting syndrome can be transmitted by pig-to-pig contact and some studies point at airborne transmission as a possibility. Studies in Europe have shown several risk factors that either increase or decrease the risk for a pig herd to be affected by PMWS. At the pig level, studies have shown the importance of maternal immunity as protection for subsequent development of PMWS. To control PMWS, good production management and control of other diseases are crucial. Since 2004, commercial vaccines against Porcine Circo Virus type 2 have been coming on the market and many studies have shown great benefits of these to control PMWS. Today, sow vaccines as well as piglet vaccines are available in most countries. An extensive meta-analysis of many of the vaccines has shown a comparable good efficacy of the vaccines in significantly reducing mortality and increasing weight gain of the pigs. PMID:22252114

  4. Porcine Models of Muscular Dystrophy1

    PubMed Central

    Selsby, Joshua T.; Ross, Jason W.; Nonneman, Dan; Hollinger, Katrin

    2015-01-01

    Duchenne muscular dystrophy is a progressive, fatal, X-linked disease caused by a failure to accumulate the cytoskeletal protein dystrophin. This disease has been studied using a variety of animal models including fish, mice, rats, and dogs. While these models have contributed substantially to our mechanistic understanding of the disease and disease progression, limitations inherent to each model have slowed the clinical advancement of therapies, which necessitates the development of novel large-animal models. Several porcine dystrophin-deficient models have been identified, although disease severity may be so severe as to limit their potential contributions to the field. We have recently identified and completed the initial characterization of a natural porcine model of dystrophin insufficiency. Muscles from these animals display characteristic focal necrosis concomitant with decreased abundance and localization of dystrophin-glycoprotein complex components. These pigs recapitulate many of the cardinal features of muscular dystrophy, have elevated serum creatine kinase activity, and preliminarily appear to display altered locomotion. They also suffer from sudden death preceded by EKG abnormalities. Pig dystrophinopathy models could allow refinement of dosing strategies in human-sized animals in preparation for clinical trials. From an animal handling perspective, these pigs can generally be treated normally, with the understanding that acute stress can lead to sudden death. In summary, the ability to create genetically modified pig models and the serendipitous discovery of genetic disease in the swine industry has resulted in the emergence of new animal tools to facilitate the critical objective of improving the quality and length of life for boys afflicted with such a devastating disease. PMID:25991703

  5. Temperature- and medium-dependent secretion of proteins by Shiga toxin-producing Escherichia coli.

    PubMed Central

    Ebel, F; Deibel, C; Kresse, A U; Guzmán, C A; Chakraborty, T

    1996-01-01

    Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal disease in humans and livestock, and these bacteria have recently emerged as a leading cause of renal failure in children. In this study, we have examined medium- and temperature-dependent production of secreted proteins from a STEC O26 serotype strain. Growth of bacteria in Luria broth led to the detection of secreted polypeptides of 104, 55, 54, and 37 kDa (p104, p55, p54, and p37, respectively). When grown in serum-free tissue culture medium, only p104, p37 and two additional polypeptides of 25 and 22 kDa (p25 and p22) were present in supernatant fluids. Production of these polypeptides was growth temperature dependent and induced in cultures grown at 37 degrees C. N-terminal amino acid sequencing revealed that p104 was homologous to the secreted p110 of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a family of secreted proteins in pathogenic bacteria of which the immunoglobulin A protease of Neisseria gonorrhoeae is the prototype. The N-terminal amino acid sequences of p55 and p54 were unique to the STEC strain, while p37 and p25 were found to be highly homologous to the similarly sized EspA and EspB proteins, previously detected in culture supernatants of EPEC. Molecular cloning and sequencing of STEC espB alleles from two different serotypes showed that the encoded polypeptides were about 80% homologous. A monoclonal antibody raised against STEC EspB also cross-reacted with its EPEC analog and allowed us to demonstrate medium- and temperature-dependent production of this important virulence factor in STEC and EPEC strains of differing serotypes. PMID:8890194

  6. Vaccines against Infections Caused by Salmonella, Shigella, and Pathogenic Escherichia coli.

    PubMed

    Guzman, Carlos A; Borsutzky, Stefan; Favre, Didier; Dietrich, Guido

    2004-12-01

    Infectious diseases represent one of the most common causes of death worldwide, with the enteropathogenic bacteria Salmonella and Shigella and pathogenic Escherichia coli being among the most detrimental. Currently, vaccination represents the preferred method of preventing such infections. For stimulating the adaptive immune response, immunizations are frequently based on formulations which include inactivated whole-cell vaccines, live attenuated vaccines, or subunit vaccines. These can be administered via a parenteral or mucosal route, the latter having the advantage that it most closely mimics the actual course of infection. In addition to the type of vaccine and method of application, important consideration needs to be paid to safety, efficacy, and cost, which are often major bottlenecks in the successful implementation of vaccines. In this chapter we take a limited look at the history surrounding vaccinations involving Salmonella, Shigella, and pathogenic E. coli. Salmonella infections, which can lead to typhoid fever, are becoming increasing difficult to treat with antibiotics due to multi-drug-resistant strains. At present, the parenteral Vi-based subunit vaccines and the live attenuated oral vaccine Ty21a have proven to be the vaccines of choice, with high levels of protective efficacy and limited side effects. Shigella infections are responsible for the diarrheal disease shigellosis. Various live and nonliving mucosal and parenteral vaccines have been tested, with the most promising candidates evolving around those that stimulate the production of O-antigen-specific antibodies. Pathogenic Escherichia coli infections can lead to severe diseases due to the bacterium's production of several specific toxins. Vaccines against this bacterium target its toxins, as well as surface-exposed antigens, all of which have been found to be effective as immunogens. PMID:26443352

  7. Antimicrobial resistance and virulence factors in Escherichia coli from swedish dairy calves

    PubMed Central

    2012-01-01

    Background In Sweden, knowledge about the role of enteropathogenic Escherichia coli in neonatal calf diarrhea and the occurrence of antimicrobial resistance in E. coli from young calves is largely unknown. This has therapeutic concern and such knowledge is also required for prudent use of antimicrobials. Methods In a case control study Esherichia coli isolated from faecal samples from dairy calves were phenotyped by biochemical fingerprinting and analyzed for virulence genes by PCR. Antimicrobial susceptibility was tested by determination of minimum inhibitory concentration (MIC). Farm management data were collected and Fisher's exact test and univariable and multivariable logistic regression analysis were performed. Results Of 95 E. coli tested for antimicrobial susceptibility 61% were resistant to one or more substances and 28% were multi-resistant. The virulence gene F5 (K99) was not found in any isolate. In total, 21 out of 40 of the investigated virulence genes were not detected or rarely detected. The virulence genes espP, irp, and fyuA were more common in resistant E. coli than in fully susceptible isolates (P < 0.05). The virulence gene terZ was associated with calf diarrhea (P ≤ 0.01). The participating 85 herds had a median herd size of 80 lactating cows. Herds with calf diarrhea problems were larger (> 55 cows; P < 0.001), had higher calf mortality (P ≤ 0.01) and calf group feeders were more in use (P < 0.05), compared to herds without calf diarrhea problems. There was no association between calf diarrhea and diversity of enteric E. coli. Conclusions Antimicrobial resistance was common in E. coli from pre-weaned dairy calves, occurring particularly in calves from herds experiencing calf diarrhea problems. The results indicate that more factors than use of antimicrobials influence the epidemiology of resistant E. coli. Enteropathogenic E. coli seems to be an uncommon cause of neonatal calf diarrhea in Swedish dairy herds. In practice, calf diarrhea

  8. IscR Regulates Synthesis of Colonization Factor Antigen I Fimbriae in Response to Iron Starvation in Enterotoxigenic Escherichia coli

    PubMed Central

    Haines, Sara; Arnaud-Barbe, Nadège; Poncet, David; Reverchon, Sylvie; Wawrzyniak, Julien; Nasser, William

    2015-01-01

    ABSTRACT Iron availability functions as an environmental cue for enteropathogenic bacteria, signaling arrival within the human host. As enterotoxigenic Escherichia coli (ETEC) is a major cause of human diarrhea, the effect of iron on ETEC virulence factors was evaluated here. ETEC pathogenicity is directly linked to production of fimbrial colonization factors and secretion of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). Efficient colonization of the small intestine further requires at least the flagellin binding adhesin EtpA. Under iron starvation, production of the CFA/I fimbriae was increased in the ETEC H10407 prototype strain. In contrast, LT secretion was inhibited. Furthermore, under iron starvation, gene expression of the cfa (CFA/I) and etp (EtpBAC) operons was induced, whereas transcription of toxin genes was either unchanged or repressed. Transcriptional reporter fusion experiments focusing on the cfa operon further showed that iron starvation stimulated cfaA promoter activity in ETEC, indicating that the impact of iron on CFA/I production was mediated by transcriptional regulation. Evaluation of cfaA promoter activity in heterologous E. coli single mutant knockout strains identified IscR as the regulator responsible for inducing cfa fimbrial gene expression in response to iron starvation, and this was confirmed in an ETEC ΔiscR strain. The global iron response regulator, Fur, was not implicated. IscR binding sites were identified in silico within the cfaA promoter and fixation confirmed by DNase I footprinting, indicating that IscR directly binds the promoter region to induce CFA/I. IMPORTANCE Pathogenic enterobacteria modulate expression of virulence genes in response to iron availability. Although the Fur transcription factor represents the global regulator of iron homeostasis in Escherichia coli, we show that several ETEC virulence factors are modulated by iron, with expression of the major fimbriae under the control of the iron

  9. Porcine cathelicidins efficiently complex and deliver nucleic acids to plasmacytoid dendritic cells and can thereby mediate bacteria-induced IFN-α responses.

    PubMed

    Baumann, Arnaud; Démoulins, Thomas; Python, Sylvie; Summerfield, Artur

    2014-07-01

    Cathelicidins constitute potent antimicrobial peptides characterized by a high cationic charge that enables strong interactions with nucleic acids. In fact, the only human cathelicidin LL-37 triggers rapid sensing of nucleic acids by plasmacytoid dendritic cells (pDC). Among the porcine cathelicidins, phylogenetic analysis of the C-terminal mature peptide showed that porcine myeloid antimicrobial peptide (PMAP)-36 was the most closely related of the 11 porcine cathelicidins to human LL-37. Despite several investigations evaluating potent antimicrobial functions of porcine cathelicidins, nothing is known about their ability to promote pDC activation. We therefore investigated the capacity of the proline-arginine-rich 39-aa peptide, PMAP-23, PMAP-36, and protegrin-1 to complex with bacterial DNA or synthetic RNA molecules and facilitate pDC activation. We demonstrate that these peptides mediate a rapid and efficient uptake of nucleic acids within minutes, followed by robust IFN-α responses. The highest positively charged cathelicidin, PMAP-36, was found to be the most potent peptide tested for this effect. The peptide-DNA complexes were internalized and also found to associate with the cell membranes of pDC. The amphipathic conformation typical of PMAP-36 was not required for IFN-α induction in pDC. We also demonstrate that PMAP-36 can mediate IFN-α induction in pDC stimulated by Escherichia coli, which alone fail to activate pDC. This response was weaker with a scrambled PMAP-36, relating to its lower antimicrobial activity. Collectively, our data suggest that the antimicrobial and nucleic acid-complexing properties of cathelicidins can mediate pDC activation-promoting adaptive immune responses against microbial infections. PMID:24899499

  10. Genome Sequences of the Novel Porcine Parvovirus 3, Identified in Guangxi Province, China.

    PubMed

    Zhong, Hui; Li, Xiangmin; Zhao, Zekai; An, Chunjing; Wan, Peng; Wu, Mengge; Chen, Huanchun; Qian, Ping

    2016-01-01

    Porcine parvovirus 3 is a novel parvovirus that infects pigs. Here, we report two genome sequences of porcine parvovirus 3 strains GX1 and GX2, which are highly prevalent in Guangxi province. It will help in understanding the epidemiology and molecular characteristics of the porcine parvovirus 3. PMID:26941135

  11. Genome Sequences of the Novel Porcine Parvovirus 3, Identified in Guangxi Province, China

    PubMed Central

    Zhong, Hui; Li, Xiangmin; Zhao, Zekai; An, Chunjing; Wan, Peng; Wu, Mengge; Chen, Huanchun

    2016-01-01

    Porcine parvovirus 3 is a novel parvovirus that infects pigs. Here, we report two genome sequences of porcine parvovirus 3 strains GX1 and GX2, which are highly prevalent in Guangxi province. It will help in understanding the epidemiology and molecular characteristics of the porcine parvovirus 3. PMID:26941135

  12. Molecular characterization of porcine SARM1 and its role in regulating TLRs signaling during highly pathogenic porcine reproductive and respiratory syndrome virus infection in vivo.

    PubMed

    Zhou, Xiang; Jiang, Tengfei; Du, Xiaochuan; Zhou, Ping; Jiang, Zhihua; Michal, Jennifer J; Liu, Bang

    2013-01-01

    Toll-like receptors (TLRs) are important pattern-recognition receptors (PRRs) that trigger innate immune response and mediate acquired immunity. Evidence has shown that SARM1 (sterile-α and TIR motif containing protein 1) is one of five TIR domain-containing adaptor proteins involved in TLRs signaling transduction. In the present study, a full-length cDNA sequence was cloned for the porcine SARM1 gene, which contains nine exons. Using the radiation hybrid mapping approach, we assigned the porcine gene to SSC12 q13. Under the normal condition, porcine SARM1 was highly expressed in brain and spleen. Polyinosinic-polycytidylic acid (poly (I:C)) weakly induced the porcine SARM1 expression in the early stimulation. We found that porcine SARM1 protein is localized in mitochondria and attenuates NF-κB activation induced by stimulation and infection. The quantitative real-time PCR (Q-PCR) analysis showed that the expression of porcine SARM1 significantly decreased in several tissues of Tongcheng pigs infected with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Gene-interaction network analysis for porcine SARM1 in porcine alveolar macrophages (PAMs) showed that down-regulation of SARM1 gene in infected Tongcheng pig may modulate TRIF-depend TLRs signaling and regulate the expression of disease-resistant genes and inflammatory genes. Our findings provide evidence that porcine SARM1 may play an important role in immune regulation with PRRSV infection. PMID:22366489

  13. Faecal excretion of entero-pathogens in a Pakistani family returning to the U.K. after a visit to Rawalpindi.

    PubMed

    Harvey, R W; Price, T H

    1980-10-01

    The family consisted of two parents and five children. While the father remained in Cardiff, the mother and all the children visited Rawalpindi, Pakistan, for 6 weeks to stay with relatives. Travel was by flight from Heathrow airport to Pakistan and by a short road journey to Rawalpindi. Mrs M. - the mother - as a guest, did no cooking on the holiday. The house which they were living in had a piped water supply, thought to be treated. There was no flush toilet but a commode was available and was emptied daily. All the children had gastro-enteritis symptoms for 2-3 days after arrival. Ru M. - a daughter - had the most severe illness and was treated by a local doctor. Diarrhoea in the three girls persisted on return to U.K. A faecal swab from Ru M. showed her to be excreting S. typhi (degraded Vi phage type). She was admitted to hospital. Faecal samples from the remaining members of the family were taken and examined for entero-pathogens. The father, Fa. M., who had not left Cardiff, had negative stools and remained free from infection. All other family members were excreting one or more entero-pathogens, including a Campylobacter sp., three types of Sh. flexneri and one type of Sh. boydii. A subsequent faecal sample revealed that one of the male children, A.M., was excreting S. typhi phage type B2. The two typhoid infections were apparently unconnected.The media used for microbiological diagnosis in this incident are discussed and contrasted with those employed for routine Salmonella examination of environmental samples. The advantages of selenite F in clinical diagnosis are noted.Antibiotic therapy was used for both typhoid cases but was not employed for the Shigella infections. The clinical condition of those involved in this incident might well have failed to arouse suspicion and the question arises whether food handlers returning from holiday in tropical and subtropical areas should have bacterological investigations before going back to their employment. PMID

  14. Functional verification of a porcine myostatin propeptide mutant.

    PubMed

    Ma, Dezun; Jiang, Shengwang; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Xiao, Gaojun; Yang, Jinzeng; Cui, Wentao

    2015-10-01

    Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14%, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37%, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61%, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells. PMID:26174475

  15. Isolation, Culture and Identification of Porcine Skeletal Muscle Satellite Cells

    PubMed Central

    Li, Bo-jiang; Li, Ping-hua; Huang, Rui-hua; Sun, Wen-xing; Wang, Han; Li, Qi-fa; Chen, Jie; Wu, Wang-jun; Liu, Hong-lin

    2015-01-01

    The objective of this study was to establish the optimum protocol for the isolation and culture of porcine muscle satellite cells. Mononuclear muscle satellite cells are a kind of adult stem cell, which is located between the basal lamina and sarcolemma of muscle fibers and is the primary source of myogenic precursor cells in postnatal muscle. Muscle satellite cells are a useful model to investigate the mechanisms of muscle growth and development. Although the isolation and culture protocols of muscle satellite cells in some species (e.g. mouse) have been established successfully, the culture system for porcine muscle satellite cells is very limited. In this study, we optimized the isolation procedure of porcine muscle satellite cells and elaborated the isolation and culture process in detail. Furthermore, we characterized the porcine muscle satellite cells using the immunofluorecence. Our study provides a reference for the isolation of porcine muscle satellite cells and will be useful for studying the molecular mechanisms in these cells. PMID:26104526

  16. Clinical comparison of St. Jude and porcine mitral valve prostheses.

    PubMed

    Douglas, P S; Hirshfeld, J W; Edie, R N; Stephenson, L W; Gleason, K; Edmunds, L H

    1988-01-01

    One hundred and six consecutive patients who had mitral valve replacement with either a St. Jude or porcine heterograft prosthesis were prospectively studied. The 2 groups are similar with respect to 67 clinical and operative factors and allow comparison of valve performance as an independent variable. Total follow-up is 3,312 patient-months (mean 36 months, range 2-57 months, 94% complete). There are no statistical differences in symptomatic improvement or mortality by life table analysis. Valve-related complications expressed as percent per patient-year are: reoperation: 1.8 St. Jude and 3.8 porcine; endocarditis: 1.2 and 1.9; regurgitant murmur: 2.3 and 1.9; hemolysis: 1.8 and 0.0; late thromboembolism: 1.8 and 1.0; hemorrhage: 2.9 and 2.9; and valve failure: 0.0 and 1.0. There were no significant differences found. Actuarial survival at 3 years was 78% in St. Jude and 81% in porcine patients. Forty-six percent of patients with St. Jude valves and 55% of patients with porcine valves were alive and free of all complications at latest follow-up. The clinical performance of St. Jude and porcine mitral valves are similar over this period of intermediate follow-up. PMID:3360831

  17. Porcine respiratory disease complex: Interaction of vaccination and porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, and Mycoplasma hyopneumoniae.

    PubMed

    Chae, Chanhee

    2016-06-01

    Porcine respiratory disease is a multifactorial and complex disease caused by a combination of infectious pathogens, environmental stressors, differences in production systems, and various management practices; hence the name porcine respiratory disease complex (PRDC) is used. Porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and Mycoplasma hyopneumoniae are considered to be the most important pathogens that cause PRDC. Although interactions among the three major respiratory pathogens are well documented, it is also necessary to understand the interaction between vaccines and the three major respiratory pathogens. PRRSV and M. hyopneumoniae are well known to potentiate PCV2-associated lesions; however, PRRSV and mycoplasmal vaccines can both enhance PCV2 viraemia regardless of the effects of the actual PRRSV or M. hyopneumoniae infection. On the other hand, M. hyopneumoniae potentiates the severity of pneumonia induced by PRRSV, and vaccination against M. hyopneumoniae alone is also able to decrease PRRSV viraemia and PRRSV-induced lung lesions in dually infected pigs. This review focuses on (1) interactions between PCV2, PRRSV, and M. hyopneumoniae; and (2) interactions between vaccines and the three major respiratory pathogens. PMID:27256017

  18. Enterohemorrhagic Escherichia coli Adhesins.

    PubMed

    McWilliams, Brian D; Torres, Alfredo G

    2014-06-01

    Adhesins are a group of proteins in enterohemorrhagic Escherichia coli (EHEC) that are involved in the attachment or colonization of this pathogen to abiotic (plastic or steel) and biological surfaces, such as those found in bovine and human intestines. This review provides the most up-to-date information on these essential adhesion factors, summarizing important historical discoveries and analyzing the current and future state of this research. In doing so, the proteins intimin and Tir are discussed in depth, especially regarding their role in the development of attaching and effacing lesions and in EHEC virulence. Further, a series of fimbrial proteins (Lpf1, Lpf2, curli, ECP, F9, ELF, Sfp, HCP, and type 1 fimbria) are also described, emphasizing their various contributions to adherence and colonization of different surfaces and their potential use as genetic markers in detection and classification of different EHEC serotypes. This review also discusses the role of several autotransporter proteins (EhaA-D, EspP, Saa and Sab, and Cah), as well as other proteins associated with adherence, such as flagella, EibG, Iha, and OmpA. While these proteins have all been studied to varying degrees, all of the adhesins summarized in this article have been linked to different stages of the EHEC life cycle, making them good targets for the development of more effective diagnostics and therapeutics. PMID:26103974

  19. Recurrent Escherichia coli bacteremia.

    PubMed Central

    Maslow, J N; Mulligan, M E; Arbeit, R D

    1994-01-01

    Escherichia coli is the most common gram-negative organism associated with bacteremia. While recurrent E. coli urinary tract infections are well-described, recurrent E. coli bacteremia appears to be uncommon, with no episodes noted in multiple series of patients with gram-negative bacteremias. We report on 5 patients with recurrent bloodstream infections identified from a series of 163 patients with E. coli bacteremia. For each patient, the isolates from each episode were analyzed by pulsed-field gel electrophoresis (PFGE) and ribotyping and for the presence of E. coli virulence factors. For each of four patients, the index and recurrent episodes of bacteremia represented the same strain as defined by PFGE, and the strains were found to carry one or more virulence factors. The remaining patient, with two episodes of bloodstream infection separated by a 4-year interval, was infected with two isolates that did not carry any virulence factors and that were clonally related by ribotype analysis but differed by PFGE. All five patients had either a local host defense defect (three patients) or impaired systemic defenses (one patient) or both (one patient). Thus, recurrent E. coli bacteremia is likely to represent a multifactorial process that occurs in patients with impaired host defenses who are infected with virulent isolates. Images PMID:7910828

  20. Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells.

    PubMed

    Cong, Yingying; Li, Xiaoxue; Bai, Yunyun; Lv, Xiaonan; Herrler, Georg; Enjuanes, Luis; Zhou, Xingdong; Qu, Bo; Meng, Fandan; Cong, Chengcheng; Ren, Xiaofeng; Li, Guangxing

    2015-04-01

    Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells. PMID:25681796

  1. Preservation and transplantation of porcine testis tissue.

    PubMed

    Zeng, W; Snedaker, A K; Megee, S; Rathi, R; Chen, F; Honaramooz, A; Dobrinski, I

    2009-01-01

    Grafting of immature mammalian testis tissue to mouse hosts can preserve the male germline. To make this approach applicable to a clinical or field situation, it is imperative that the testis tissue and/or spermatozoa harvested from grafted tissue are preserved successfully. The aim of the present study was to evaluate protocols for the preservation of testis tissue in a porcine model. Testis tissue was stored at 4 degrees C for short-term preservation or cryopreserved by slow-freezing, automated slow-freezing or vitrification for long-term storage. Preserved tissue was transplanted ectopically to mouse hosts and recovered xenografts were analysed histologically. In addition, spermatozoa were harvested from xenografts and cryopreserved. Total cell viability and germ cell viability remained high after tissue preservation. Complete spermatogenesis occurred in xenografts preserved by cooling up to 48 h, whereas spermatogenesis progressed to round spermatids in the xenografts that were frozen-thawed before grafting. Approximately 50% of spermatozoa harvested from xenografts remained viable after freezing and thawing. The in vivo developmental potential of cryopreserved tissue was reduced despite high post-thaw viability. Therefore, it is important to evaluate germ cell differentiation in vivo in addition to cell viability in vitro when optimising freezing protocols for testis tissue. PMID:19261226

  2. Genetic and antigenic changes in porcine rubulavirus

    PubMed Central

    Sánchez-Betancourt, José I.; Trujillo, María E.; Mendoza, Susana E.; Reyes-Leyva, Julio; Alonso, Rogelio A.

    2012-01-01

    Blue eye disease, caused by a porcine rubulavirus (PoRV), is an emergent viral swine disease that has been endemic in Mexico since 1980. Atypical outbreaks were detected in 1990 and 2003. Growing and adult pigs presented neurological signs, mild neurological signs were observed in piglets, and severe reproductive problems were observed in adults. Amino acid sequence comparisons and phylogenetic analysis of the hemagglutinin-neuraminidase (HN) protein revealed genetically different lineages. We used cross-neutralization assays, with homologous and heterologous antisera, to determine the antigenic relatedness values for the PoRV isolates. We found antigenic changes among several strains and identified a highly divergent one, making up a new serogroup. It seems that genetically and antigenically different PoRV strains are circulating simultaneously in the swine population in the geographical region studied. The cross neutralization studies suggest that the HN is not the only antigenic determinant participating in the antigenic changes among the different PoRV strains. PMID:22754092

  3. Porcine deltacoronavirus: histological lesions and genetic characterization.

    PubMed

    Wang, Leyi; Hayes, Jeff; Sarver, Craig; Byrum, Beverly; Zhang, Yan

    2016-01-01

    First identified in 2012 in a surveillance study in Hong Kong, porcine deltacoronavirus (PDCoV) is a proposed member of the genus Deltacoronavirus of the family Coronaviridae. In February of 2014, PDCoV was detected in pigs with clinical diarrheal symptoms for the first time in the USA. Since then, it has been detected in more than 20 states in the USA and in other countries, including Canada, South Korea, and mainland China. So far, histological lesions in the intestines of pigs naturally infected with PDCoV under field conditions have not been reported. In this report, we describe the characteristic histological lesions in the small intestine that were associated with PDCoV infection, as evidenced by detection of viral nucleic acid by RT-PCR. In addition, we performed genomic analysis to determine the genetic relationship of all PDCoV strains from the four countries. We found that PDCoV mainly caused histological lesions in the small intestines of naturally infected piglets. Sequence analysis demonstrated that the PDCoV strains of different countries are closely related and shared high nucleotide sequence similarity; however, deletion patterns in the spike and 3' untranslated regions are different among the strains from mainland China, Hong Kong, the USA, and South Korea. Our study highlights the fact that continual surveillance is needed to trace the evolution of this virus. PMID:26475155

  4. Antioxidant capacity of hydrolyzed porcine tissues

    PubMed Central

    Damgaard, Trine D; Otte, Jeanette A H; Meinert, Lene; Jensen, Kirsten; Lametsch, René

    2014-01-01

    The antioxidative capacity of seven different porcine tissue hydrolysates (colon, appendix, rectum, pancreas, heart, liver, and lung) were tested by four different assays, including iron chelation, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) radical scavenging, and inhibition of lipid oxidation. All hydrolyzed tissues displayed antioxidant capacity in all four assays, with colon, liver, and appendix as the three most potent inhibitors of lipid oxidation (47, 29, and 27 mmol/L trolox equivalent antioxidant capacity [TEAC], respectively) and liver, colon, pancreas, and appendix as the four most potent iron chelators (92% ± 1.1, 79.3% ± 3.2, 77.1% ± 1.8, and 77% ± 2.3, respectively). Furthermore, colon and appendix showed good radical scavenging capacities with ABTS scavenging of 86.4% ± 2.1 and 84.4% ± 2.9 and DPPH scavenging of 17.6% ± 0.3 and 17.1% ± 0.2, respectively. Our results provide new knowledge about the antioxidant capacity of a variety of animal by-products, which can be transformed into antioxidant hydrolysates, thereby creating added value. PMID:24936298

  5. Genetic and antigenic changes in porcine rubulavirus.

    PubMed

    Sánchez-Betancourt, José I; Trujillo, María E; Mendoza, Susana E; Reyes-Leyva, Julio; Alonso, Rogelio A

    2012-01-01

    Blue eye disease, caused by a porcine rubulavirus (PoRV), is an emergent viral swine disease that has been endemic in Mexico since 1980. Atypical outbreaks were detected in 1990 and 2003. Growing and adult pigs presented neurological signs, mild neurological signs were observed in piglets, and severe reproductive problems were observed in adults. Amino acid sequence comparisons and phylogenetic analysis of the hemagglutinin-neuraminidase (HN) protein revealed genetically different lineages. We used cross-neutralization assays, with homologous and heterologous antisera, to determine the antigenic relatedness values for the PoRV isolates. We found antigenic changes among several strains and identified a highly divergent one, making up a new serogroup. It seems that genetically and antigenically different PoRV strains are circulating simultaneously in the swine population in the geographical region studied. The cross neutralization studies suggest that the HN is not the only antigenic determinant participating in the antigenic changes among the different PoRV strains. PMID:22754092

  6. Ultrafast laser machining of porcine sclera

    NASA Astrophysics Data System (ADS)

    Góra, W. S.; Carter, R. M.; Dhillon, B.; Hand, D. P.; Shephard, J. D.

    2015-07-01

    The use of ultrafast lasers (pulsed lasers with pulse lengths of a few picoseconds or less) offers the possibility for minimally invasive removal of soft ophthalmic tissue. The potential for using pico- and femtosecond pulses for modification of scleral tissue has been reported elsewhere [1-6] and has resulted in the introduction of new, minimally invasive, procedures into clinical practice [3, 5-10]. Our research is focused on finding optimal parameters for picosecond laser machining of scleral tissue without introducing any unwanted collateral damage to the tissue. Experiments were carried out on hydrated porcine sclera in vitro, which has similar collagen organization, histology and water content (~70%) to human tissue. In this paper we present a 2D finite element ablation model which employs a one-step heating process. It is assumed that the incident laser radiation that is not reflected is absorbed in the tissue according to the Beer-Lambert law and transformed into heat energy. The experimental setup uses an industrial picosecond laser (TRUMPF TruMicro 5x50) with 5.9 ps pulses at 1030 nm, with pulse energies up to 125 μJ and a focused spot diameter of 35 μm. The use of a scan head allows flexibility in designing various scanning patterns. We show that picosecond pulses are capable of modifying scleral tissue without introducing collateral damage. This offers a possible route for minimally invasive sclerostomy. Many scanning patterns including single line ablation, square and circular cavity removal were tested.

  7. Picosecond laser ablation of porcine sclera

    NASA Astrophysics Data System (ADS)

    Góra, Wojciech S.; Harvey, Eleanor M.; Dhillon, Baljean; Parson, Simon H.; Maier, Robert R. J.; Hand, Duncan P.; Shephard, Jonathan D.

    2013-03-01

    Lasers have been shown to be successful in certain medical procedures and they have been identified as potentially making a major contribution to the development of minimally invasive procedures. However, the uptake is not as widespread and there is scope for many other applications where laser devices may offer a significant advantage in comparison to the traditional surgical tools. The purpose of this research is to assess the potential of using a picosecond laser for minimally invasive laser sclerostomy. Experiments were carried out on porcine scleral samples due to the comparable properties to human tissue. Samples were prepared with a 5mm diameter trephine and were stored in lactated Ringer's solution. After laser machining, the samples were fixed in 3% glutaraldehyde, then dried and investigated under SEM. The laser used in the experiments is an industrial picosecond TRUMPF TruMicro laser operating at a wavelength of 1030nm, pulse length of 6ps, repetition rate of 1 kHz and a focused spot diameter of 30μm. The laser beam was scanned across the samples with the use of a galvanometer scan head and various ablation patterns were investigated. Processing parameters (pulse energy, spot and line separation) which allow for the most efficient laser ablation of scleral tissue without introducing any collateral damage were investigated. The potential to create various shapes, such as linear incisions, square cavities and circular cavities was demonstrated.

  8. Porcine parvovirus: DNA sequence and genome organization.

    PubMed

    Ranz, A I; Manclús, J J; Díaz-Aroca, E; Casal, J I

    1989-10-01

    We have determined the nucleotide sequence of an almost full-length clone of porcine parvovirus (PPV). The sequence is 4973 nucleotides (nt) long. The 3' end of virion DNA shows a Y-shaped configuration homologous to rodent parvoviruses. The 5' end of virion DNA shows a repetition of 127 nt at the carboxy terminus of the capsid proteins. The overall organization of the PPV genome is similar to those of other autonomous parvoviruses. There are two large open reading frames (ORFs) that almost entirely cover the genome, both located in the same frame of the complementary strand. The left ORF encodes the non-structural protein NS1 and the right ORF encodes the capsid proteins (VP1, VP2 and VP3). Promoter analysis, location of splicing sites and putative amino acid sequences for the viral proteins show a high homology of PPV with feline panleukopenia virus and canine parvoviruses (FPV and CPV) and rodent parvovirus. Therefore we conclude that PPV is related to the Kilham rat virus (KRV) group of autonomous parvoviruses formed by KRV, minute virus of mice, Lu III, H-1, FPV and CPV. PMID:2794971

  9. Identification and Analysis of the Porcine MicroRNA in Porcine Cytomegalovirus-Infected Macrophages Using Deep Sequencing

    PubMed Central

    Liu, Xiao; Liao, Shan; Xu, Zhiwen; Zhu, Ling; Yang, Fan; Guo, Wanzhu

    2016-01-01

    Porcine cytomegalovirus (PCMV; genus Cytomegalovirus, subfamily Betaherpesvirinae, family Herpesviridae) is an immunosuppressive virus that mainly inhibits the immune function of T lymphocytes and macrophages, which has caused substantial damage in the farming industry. In this study, we obtained the miRNA expression profiles of PCMV-infected porcine macrophages via high-throughput sequencing. The comprehensive analysis of miRNA profiles showed that 239 miRNA database-annotated and 355 novel pig-encoded miRNAs were detected. Of these, 130 miRNAs showed significant differential expression between the PCMV-infected and uninfected porcine macrophages. The 10 differentially expressed pig-encoded miRNAs were further determined by stem-loop reverse-transcription polymerase chain reaction, and the results were consistent with the high-throughput sequencing. Gene Ontology analysis of the target genes of miRNAs in PCMV-infected porcine macrophages showed that the differentially expressed miRNAs are mainly involved in immune and metabolic processes. This is the first report of the miRNA transcriptome in porcine macrophages and an analysis of the miRNA regulatory mechanisms during PCMV infection. Further research into the regulatory mechanisms of miRNAs during immunosuppressive viral infections should contribute to the treatment and prevention of immunosuppressive viruses. PMID:26943793

  10. E. coli Nissle 1917 Affects Salmonella Adhesion to Porcine Intestinal Epithelial Cells

    PubMed Central

    Schierack, Peter; Kleta, Sylvia; Tedin, Karsten; Babila, Julius Tachu; Oswald, Sibylle; Oelschlaeger, Tobias A.; Hiemann, Rico; Paetzold, Susanne; Wieler, Lothar H.

    2011-01-01

    Background The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known. Methodology/Principal Findings In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra- and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN. Conclusions We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion. PMID:21379575

  11. Preparation of a porcine plasma protein composite film and its application.

    PubMed

    Lee, Ji-Hyun; Song, Kyung Bin

    2015-01-01

    To use blood released from slaughtering houses, a porcine plasma protein (PPP)/nanoclay composite film was prepared. The tensile strength and elongation at break values of the PPP composite film with 5% nanoclay were 10.01 MPa and 6.55%, respectively. The PPP composite film containing 1% grapefruit seed extract (GSE) was applied to pork meat, and the populations of inoculated Escherichia coli O157:H7 and Listeria monocytogenes in the pork meat packaged with the PPP composite film decreased by 0.8 and 1.0 log CFU/g, respectively, after 7 days of storage compared to the populations of the control. In addition, thiobarbituric acid values in the pork meat packaged with the PPP composite film were less than those of the control sample during storage. These results suggest that the PPP nanocomposite film containing 1% GSE can be used as a packaging material to maintain the quality of pork meat. PMID:25248798

  12. Molecular cloning, expression and characterization of the porcine β defensin 2 in E. coli.

    PubMed

    Li, Chun-li; Zhao, Yan-cong; Song, Xiao-yan; Huang, Xian-xian; Zhao, Wei-dong

    2013-06-01

    Porcine β defensin 2(pBD2)is a cationic 37-amino acid antimicrobial peptide with disulfide bonds. Synthetic pBD2 had broad antimicrobial activity against pathogenic bacteria, and thus pBD2 could be a good candidate as a bactericidal agent for pigs. This study reported the successful recombinant expression of pBD2 in Escherichia coli and analysis of its antimicrobial activity, its hemolytic activity, salt-tolerance and thermal stability as well. The pBD2 gene, obtained by RT-PCR using the tongue total RNA as a template and cloned into pET30a expression vector, was transformed into E. coli BL21 (DE3) plysS. The recombinant pBD2 was expressed after induction by IPTG and purified by His tag affinity column with 95% purity. The recombinant pBD2 exhibited antimicrobial activity against both Gram-positive S. aureus and Gram-negative E. coli including the multi-resistant E. coli. The minimum inhibitory concentration (MIC) of recombinant pBD2 against tested bacteria was 10 μg/mL, and the recombinant pBD2 could kill 50% E. coli at 14.39 μg/mL and S. aureus at 21.1 μg/mL. In addition, pBD2 showed low hemolytic activity, salt-tolerance and thermal stability, the properties would be important for its application in practice. PMID:22973850

  13. Immunological methods for the detection of porcine lymphotropic herpesviruses (PLHV).

    PubMed

    Plotzki, Elena; Keller, Martina; Ehlers, Bernhard; Denner, Joachim

    2016-07-01

    Porcine lymphotropic herpesviruses (PLHV-1, -2, and -3) are widespread in pigs and closely related to the human pathogenic gammaherpesviruses Epstein-Barr virus (human herpesvirus 4, HHV-4) and Kaposi sarcoma herpesvirus (HHV-8). In minipigs, PLHV-1 causes a porcine post-transplantation lymphoproliferative disorder (PTLD) after experimental transplantations. Porcine PTLD comes with clinical symptoms similar to those of human PTLD, a serious complication of solid organ and allogeneic bone marrow transplantation linked to HHV-4. Since PLHVs may be transmitted from donor pigs to the human recipient of xenotransplants (pig cells, tissues or organs), sensitive and specific methods should be developed to detect and eliminate PLHVs. Here we describe an ELISA and a Western blot assay using recombinant glycoprotein B of PLHV-1. Using both assays, the presence of specific antibodies in different pig breeds as well as in German slaughterhouse workers was analysed. Antibodies were detected in some animals, but not in human subjects. PMID:27036503

  14. Comparison of human and porcine skin for characterization of sunscreens

    NASA Astrophysics Data System (ADS)

    Weigmann, Hans-Jürgen; Schanzer, Sabine; Patzelt, Alexa; Bahaban, Virginie; Durat, Fabienne; Sterry, Wolfram; Lademann, Jürgen

    2009-03-01

    The universal sun protection factor (USPF) characterizing sunscreen efficacy based on spectroscopically determined data, which were obtained using the tape stripping procedure. The USPF takes into account the complete ultraviolet (UV) spectral range in contrast to the classical sun protection factor (SPF). Until now, the USPF determination has been evaluated only in human skin. However, investigating new filters not yet licensed excludes in vivo investigation on human skin but requires the utilization of a suitable skin model. The penetration behavior and the protection efficacy of 10 commercial sunscreens characterized by USPF were investigated, comparing human and porcine skin. The penetration behavior found for typical UV filter substances is nearly identical for both skin types. The comparison of the USPF obtained for human and porcine skin results in a linear relation between both USPF values with a correlation factor R2=0.98. The results demonstrate the possibility for the use of porcine skin to determine the protection efficacy of sunscreens.

  15. Spatial Clustering of Porcine Cysticercosis in Mbulu District, Northern Tanzania

    PubMed Central

    Ngowi, Helena A.; Kassuku, Ayub A.; Carabin, Hélène; Mlangwa, James E. D.; Mlozi, Malongo R. S.; Mbilinyi, Boniface P.; Willingham, Arve L.

    2010-01-01

    Background Porcine cysticercosis is caused by a zoonotic tapeworm, Taenia solium, which causes serious disease syndromes in human. Effective control of the parasite requires knowledge on the burden and pattern of the infections in order to properly direct limited resources. The objective of this study was to establish the spatial distribution of porcine cysticercosis in Mbulu district, northern Tanzania, to guide control strategies. Methodology/Principal Findings This study is a secondary analysis of data collected during the baseline and follow-up periods of a randomized community trial aiming at reducing the incidence rate of porcine cysticercosis through an educational program. At baseline, 784 randomly selected pig-keeping households located in 42 villages in 14 wards were included. Lingual examination of indigenous pigs aged 2–12 (median 8) months, one randomly selected from each household, were conducted. Data from the control group of the randomized trial that included 21 of the 42 villages were used for the incidence study. A total of 295 pig-keeping households were provided with sentinel pigs (one each) and reassessed for cysticercosis incidence once or twice for 2–9 (median 4) months using lingual examination and antigen ELISA. Prevalence of porcine cysticercosis was computed in Epi Info 3.5. The prevalence and incidence of porcine cysticercosis were mapped at household level using ArcView 3.2. K functions were computed in R software to assess general clustering of porcine cysticercosis. Spatial scan statistics were computed in SatScan to identify local clusters of the infection. The overall prevalence of porcine cysticercosis was 7.3% (95% CI: 5.6, 9.4; n = 784). The K functions revealed a significant overall clustering of porcine cysticercosis incidence for all distances between 600 m and 5 km from a randomly chosen case household based on Ag-ELISA. Lingual examination revealed clustering from 650 m to 6 km and between 7.5 and 10 km. The

  16. Porcine survival model to simulate acute upper gastrointestinal bleedings.

    PubMed

    Prosst, Ruediger L; Schurr, Marc O; Schostek, Sebastian; Krautwald, Martina; Gottwald, Thomas

    2016-06-01

    The existing animal models used for the simulation of acute gastrointestinal bleedings are usually non-survival models. We developed and evaluated a new porcine model (domestic pig, German Landrace) in which the animal remains alive and survives the artificial bleeding without any cardiovascular impairment. This consists of a bleeding catheter which is implanted into the stomach, then subcutaneously tunnelled from the abdomen to the neck where it is exteriorized and fixed with sutures. Using the injection of porcine blood, controllable and reproducible acute upper gastrointestinal bleeding can be simulated while maintaining normal gastrointestinal motility and physiology. Depending on the volume of blood applied through the gastric catheter, the bleeding intensity can be varied from traces of blood to a massive haemorrhage. This porcine model could be valuable, e.g. for testing the efficacy of new bleeding diagnostics in large animals before human use. PMID:26306615

  17. MicroRNA expression profiles of porcine skeletal muscle.

    PubMed

    Zhou, B; Liu, H L; Shi, F X; Wang, J Y

    2010-10-01

    MicroRNAs (miRNAs) are endogenous non-coding RNAs of ∼22 nucleotides in length that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. To evaluate the roles of miRNA in porcine skeletal muscle, miRNA expression profiles were investigated using longissimus muscle tissue from pigs at embryonic day 90 (E90) and postpartum day 120 (PD120). First, we used previously known miRNA sequences from humans and mice to perform blast searches against the porcine expressed sequence tag (EST) database; 98 new miRNA candidates were identified according to a range of filtering criteria. These miRNA candidates and 73 known miRNAs (miRBase 13.0) from pigs were chosen for porcine miRNA microarray analysis. A total of 16 newly identified miRNAs and 31 previously known miRNAs were detected in porcine skeletal muscle tissues. During later foetal development at E90, miR-1826, miR-26a, miR-199b and let-7 were highly expressed, whilst miR-1a, miR-133a, miR-26a and miR-1826 showed highest abundance during the fast growing stage at PD120. Using the 47 miRNAs detected by the microarray assay, we performed further investigations using the publicly available porcine mRNA database from NCBI and computed potential target hits using the software rnahybrid. This study identified 16 new miRNA candidates, computed potential target hits for 18 miRNA families and determined the miRNA expression profiles in porcine skeletal muscle tissues at different developmental stages. These results provide a valuable resource for investigators interested in post-transcriptional gene regulation in pigs and related animals. PMID:20331612

  18. Membrane barrier of a porcine hepatocyte bioartificial liver.

    PubMed

    Nyberg, Scott L; Yagi, Toshikazu; Matsushita, Takakazu; Hardin, Joseph; Grande, Joseph P; Gibson, Lawrence E; Platt, Jeffrey L

    2003-03-01

    Pores in the membrane of a bioartificial liver (BAL) allow it to function as a semipermeable barrier between its contents (i.e., liver cells) and components of the recipient's immune system. This study is designed to assess the influence of pore size on immune response to a BAL containing porcine hepatocytes. Sixteen healthy dogs were divided into four groups (four dogs per group) based on pore size of the BAL membrane and level of exposure to porcine hepatocytes. Group 1 dogs were administered porcine hepatocytes by intraperitoneal injection and served as positive controls. Group 2 dogs were exposed to porcine hepatocytes in a large-pore (200-nm) BAL, and group 3 dogs were exposed to porcine hepatocytes in a small-pore (10-nm) BAL. Group 4 dogs were exposed to a no-cell (unloaded) BAL and served as negative controls. Intraperitoneal injection of hepatocytes or 3 hours of BAL hemoperfusion was performed day 0 and 3 weeks later on day 21. Biochemical, humoral, and cellular measures of immune response were collected until day 44. The initiation of BAL hemoperfusion was associated with a rapid decline in CH(50) levels of complement and transient neutropenia and thrombocytopenia during all BAL exposures. Xenoreactive antibody response to BAL was increased by use of membranes with large pores and secondary exposures. Skin testing on day 42 showed a delayed-type hypersensitivity response to porcine hepatocytes that also correlated with level of previous antigen exposure. BAL treatment was associated with both immediate and elicited immunologic responses. The immediate response was transient and not influenced by membrane pore size, whereas elicited responses were influenced by pore size of the BAL during previous exposures. PMID:12619028

  19. Prevalence and Characteristics of eae- and stx-Positive Strains of Escherichia coli from Wild Birds in the Immediate Environment of Tokyo Bay ▿

    PubMed Central

    Kobayashi, Hideki; Kanazaki, Mika; Hata, Eiji; Kubo, Masanori

    2009-01-01

    The prevalence and characteristics of eae- and stx-positive Escherichia coli strains in wild birds in the immediate environment of Tokyo Bay, Japan, was examined using cloacal swab samples taken from 447 birds belonging to 62 species. PCR screening showed that the prevalences of stx- and eae-positive strains of Escherichia coli were 5% (23/447) and 25% (113/447), respectively. Four strains of stx2f-positive E. coli were isolated from two feral pigeons, an oriental turtle dove and a barn swallow. In contrast, 39 eae-positive E. coli strains were isolated, and most of the strains possessed a subtype of intimin that is classified as a minor group of human intimins, such as intimin υ, κ, and μ. Moreover, these strains did not possess any of the other pathogenic genes tested, such as stxs, ehxA, bfp, or irp. Thus, wild birds were considered to be a reservoir of atypical enteropathogenic E. coli. PMID:18997019

  20. Prevalence of emerging porcine parvoviruses and their co-infections with porcine circovirus type 2 in China.

    PubMed

    Sun, Jianhui; Huang, Liping; Wei, Yanwu; Wang, Yiping; Chen, Dongjie; Du, Wenjuan; Wu, Hongli; Liu, Changming

    2015-05-01

    A total of 450 samples from domestic pigs in China were tested for porcine parvoviruses (PPVs) and co-infections with porcine circovirus type 2 (PCV2), and their complete capsid genes were sequenced. The prevalence of PPV1, PPV2, PPV3, PPV4, and PCV2 was 5.56 %, 39.56 %, 45.11 %, 21.56 %, and 47.33 %, respectively, and co-infection with PCV2 occurred in 4 % (PPV1), 22.44 % (PPV2), 24 % (PPV3), and 12 % (PPV4) of the samples. Phylogenetic analysis revealed two main lineages for each virus, and residues that differentiated these viruses were identified. The co-infections of emerging PPVs and PCV2 were prevalent, indicating their cooperative roles in porcine circovirus-associated diseases. PMID:25742931

  1. Genetic relationships among pathogenic Escherichia coli of serogroup O157.

    PubMed Central

    Whittam, T S; Wilson, R A

    1988-01-01

    Escherichia coli strains of serotype O157:H7 are a newly described clonal pathogenic form associated with recent outbreaks of hemorrhagic colitis in humans. Although O157 strains of various H types have long been recognized as enterotoxigenic in animals, little is known about how these pathogenic animal strains are related to those of serotype O157:H7. To determine the genetic relatedness of O157:H7 isolates to animal O157 strains, we examined 194 O157 isolates, representing 12 distinct flagellar antigens (H serotypes), obtained from a variety of animal and human infections. To characterize isolates, we assayed allelic variation at 19 enzyme loci by multilocus enzyme electrophoresis. Genotypic comparisons of isolates revealed extensive variation among 33 distinct clonal genotypes that differed, on average, at 44% of the enzyme loci. K88 fimbriae were expressed in 72% of the isolates and occurred in a diversity of chromosomal genotypic backgrounds. Five major clonal groups were recognized; one group was clearly associated with porcine colibacillosis, and another was associated with human urinary tract infections. The O157:H7 genotype was not closely allied with any of the major groups of clones. The results indicate that O157 E. coli are genetically diverse and strongly suggest that the O157:H7 lineage was not recently derived from other pathogenic strains of the O157 serogroup. PMID:2457555

  2. Inhibition of Endosome-Lysosome System Acidification Enhances Porcine Circovirus 2 Infection of Porcine Epithelial Cells▿

    PubMed Central

    Misinzo, Gerald; Delputte, Peter L.; Nauwynck, Hans J.

    2008-01-01

    Recently, Misinzo et al. (G. Misinzo, P. Meerts, M. Bublot, J. Mast, H. M. Weingartl, and H. J. Nauwynck, J. Gen. Virol. 86:2057-2068, 2005) reported that inhibiting endosome-lysosome system acidification reduced porcine circovirus 2 (PCV2) infection of monocytic 3D4/31 cells. The present study examined the effect of inhibiting endosome-lysosome system acidification in epithelial cells, since epithelial cells support PCV2 infection in vivo and are used in culturing PCV2 in vitro. Ammonium chloride (NH4Cl), chloroquine diphosphate (CQ), and monensin were used to inhibit endosome-lysosome system acidification. NH4Cl, CQ, or monensin increased PCV2 (Stoon-1010) infection by 726% ± 110%, 1,212% ± 34%, and 1,100% ± 179%, respectively, in porcine kidney (PK-15) cells; by 128% ± 7%, 158% ± 3%, and 142% ± 11% in swine kidney cells; by 160% ± 28%, 446% ± 50%, and 162% ± 56% in swine testicle (ST) cells; and by 313% ± 25%, 611% ± 86%, and 352% ± 44% in primary kidney epithelial cells. Similarly, increased PCV2 infection was observed with six other PCV2 strains in PK-15 cells treated with endosome-lysosome system acidification inhibitors. The mechanism behind increased PCV2 infection was further investigated in PK-15 cells using CQ. PCV2 infection of PK-15 cells was increased only when CQ was added early during PCV2 infection. CQ did not affect PCV2 virus-like particle (VLP) attachment to PK-15 cells but increased the disassembly of internalized PCV2 VLPs. In untreated PK-15 cells, internalized PCV2 VLPs localized within the endosome-lysosome system. PCV2 infection of untreated 3D4/31 and PK-15 cells and CQ-treated PK-15 cells was blocked by a serine protease inhibitor [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride] but not by aspartyl protease (pepstatin A), cysteine protease (E-64), and metalloprotease (phosphoramidon) inhibitors. These results suggest that serine protease-mediated PCV2 disassembly is enhanced in porcine epithelial cells but inhibited

  3. Analysis of porcine MHC using microarrays.

    PubMed

    Gao, Yu; Wahlberg, Per; Marthey, Sylvain; Esquerré, Diane; Jaffrézic, Florence; Lecardonnel, Jérome; Hugot, Karine; Rogel-Gaillard, Claire

    2012-07-15

    The major histocompatibility complex (MHC) in Mammals is one of the most gene dense regions of the genome and contains the polymorphic histocompatibility gene families known to be involved in pathogen response and control of auto-immunity. The MHC is a complex genetic system that provides an interesting model system to study genome expression regulation and genetic diversity at the megabase scale. The pig MHC or SLA (Swine Leucocyte Antigen) complex spans 2.4 megabases and 151 loci have been annotated. We will review key results from previous RNA expression studies using microarrays containing probes specific to annotated loci within SLA and in addition present novel data obtained using high-density tiling arrays encompassing the whole SLA complex. We have focused on transcriptome modifications of porcine peripheral blood mononuclear cells stimulated with a mixture of phorbol myristate acetate and ionomycin known to activate B and T cell proliferation. Our results show that numerous loci mapping to the SLA complex are affected by the treatment. A general decreased level of expression for class I and II genes and an up-regulation of genes involved in peptide processing and transport were observed. Tiling array-based experiments contributed to refined gene annotations as presented for one SLA class I gene referred to as SLA-11. In conclusion, high-density tiling arrays can serve as an excellent tool to draw comprehensive transcription maps, and improve genome annotations for the SLA complex. We are currently studying their relevance to characterize SLA genetic diversity in combination with high throughput next generation sequencing. PMID:21561666

  4. Reactomes of Porcine Alveolar Macrophages Infected with Porcine Reproductive and Respiratory Syndrome Virus

    PubMed Central

    Jiang, Zhihua; Zhou, Xiang; Michal, Jennifer J.; Wu, Xiao-Lin; Zhang, Lifan; Zhang, Ming; Ding, Bo; Liu, Bang; Manoranjan, Valipuram S.; Neill, John D.; Harhay, Gregory P.; Kehrli, Marcus E.; Miller, Laura C.

    2013-01-01

    Porcine reproductive and respiratory syndrome (PRRS) has devastated pig industries worldwide for many years. It is caused by a small RNA virus (PRRSV), which targets almost exclusively pig monocytes or macrophages. In the present study, five SAGE (serial analysis of gene expression) libraries derived from 0 hour mock-infected and 6, 12, 16 and 24 hours PRRSV-infected porcine alveolar macrophages (PAMs) produced a total 643,255 sequenced tags with 91,807 unique tags. Differentially expressed (DE) tags were then detected using the Bayesian framework followed by gene/mRNA assignment, arbitrary selection and manual annotation, which determined 699 DE genes for reactome analysis. The DAVID, KEGG and REACTOME databases assigned 573 of the DE genes into six biological systems, 60 functional categories and 504 pathways. The six systems are: cellular processes, genetic information processing, environmental information processing, metabolism, organismal systems and human diseases as defined by KEGG with modification. Self-organizing map (SOM) analysis further grouped these 699 DE genes into ten clusters, reflecting their expression trends along these five time points. Based on the number one functional category in each system, cell growth and death, transcription processes, signal transductions, energy metabolism, immune system and infectious diseases formed the major reactomes of PAMs responding to PRRSV infection. Our investigation also focused on dominant pathways that had at least 20 DE genes identified, multi-pathway genes that were involved in 10 or more pathways and exclusively-expressed genes that were included in one system. Overall, our present study reported a large set of DE genes, compiled a comprehensive coverage of pathways, and revealed system-based reactomes of PAMs infected with PRRSV. We believe that our reactome data provides new insight into molecular mechanisms involved in host genetic complexity of antiviral activities against PRRSV and lays a strong

  5. Prevalence and Antibiogram Profiling of Escherichia coli Pathotypes Isolated from the Kat River and the Fort Beaufort Abstraction Water

    PubMed Central

    Nontongana, Nolonwabo; Sibanda, Timothy; Ngwenya, Elvis; Okoh, Anthony I.

    2014-01-01

    Escherichia coli is a widespread bacterium encompassing a variety of strains, ranging from highly pathogenic strains, causing worldwide outbreaks of severe diseases to avirulent, well characterized safe laboratory strains. This study evaluated the prevalence and antibiogram profiles of E. coli pathotypes isolated from the Kat River and Fort Beaufort abstraction water. A total of 171 out of 278 confirmed E. coli isolates were positive for at least one pathogenic determinant and these included enteropathogenic E. coli (6%), enterotoxigenic E. coli (47%), uropathogenic E. coli (2%), neonatal meningitis E. coli (5%), diffusely adherent E. coli (1%) and enterohaemorrhagic E. coli (1%). Interestingly, enteroinvasive and enteroaggregative E. coli were not detected. The phenotypic antibiogram profiles of the isolates revealed that all were resistant to penicillin G, while 98% and 38% of the pathotypes were resistant to ampicillin and trimethoprim-sulphamethoxazole, respectively. About 8% of the isolates were resistant to streptomycin. More than half of the isolates exhibited multiple antibiotic resistance with 44% being resistant to three antibiotics and 8% resistant to four antibiotics. We conclude that the Kat River is a reservoir of potentially virulent antibiotic resistant E. coli strains that can cause serious health risks to humans who drink raw water from this river, or in the case that consumption of treated drinking water coincides with failed drinking water processes. PMID:25119699

  6. Identification and characterization of microcin S, a new antibacterial peptide produced by probiotic Escherichia coli G3/10.

    PubMed

    Zschüttig, Anke; Zimmermann, Kurt; Blom, Jochen; Goesmann, Alexander; Pöhlmann, Christoph; Gunzer, Florian

    2012-01-01

    Escherichia coli G3/10 is a component of the probiotic drug Symbioflor 2. In an in vitro assay with human intestinal epithelial cells, E. coli G3/10 is capable of suppressing adherence of enteropathogenic E. coli E2348/69. In this study, we demonstrate that a completely novel class II microcin, produced by probiotic E. coli G3/10, is responsible for this behavior. We named this antibacterial peptide microcin S (MccS). Microcin S is coded on a 50.6 kb megaplasmid of E. coli G3/10, which we have completely sequenced and annotated. The microcin S operon is about 4.7 kb in size and is comprised of four genes. Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S. Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay. Moreover, growth of E. coli transformed with a plasmid containing mcsS under control of an araC PBAD activator-promoter is inhibited upon mcsS induction. Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10. PMID:22479389

  7. Genotype comparison of sorbitol-negative Escherichia coli isolates from healthy broiler chickens from different commercial farms.

    PubMed

    Lefebvre, B; Gattuso, M; Moisan, H; Malouin, F; Diarra, M S

    2009-07-01

    Hybridization on arrays was used to assess the presence of virulence-associated genes and to determine the relatedness of 32 non-O157 sorbitol-negative Escherichia coli isolates from healthy broiler chickens. These isolates were from commercial farms that used feed supplemented with different antimicrobial agents (virginiamycin, bacitracin, salinomycin, narasin, nicarbazin, or diclazuril). For each isolate, fluorescent probes were made from genomic DNA and were hybridized on DNA arrays composed of genes associated with general functions, virulence, iron uptake systems, and DNA repair genes (e.g., mut genes). Hybridization on arrays results showed that isolates from the same farm tended to be clustered but actually represented 18 genetically distinct groups of isolates. Results revealed that some isolates showed similarity to human uropathogenic E. coli or avian pathogenic E. coli. Four avian pathogenic E. coli-like isolates were detected. Another isolate possessed the intimin gene (eaeA) and typical genes of the type 3 secretion system associated with enteropathogenic E. coli and enterohemorrhagic E. coli strains. Genes from a second system (secondary type 3 secretion system) homologous to that found in Salmonella Typhimurium were detected in many isolates. Several of the studied isolates also possessed the aerobactin, salmochelin, and yersiniabactin genes involved in iron acquisition in pathogenic bacteria. Our results clearly suggest that commensal E. coli isolates from chickens are reservoirs of virulence-associated genes and may represent colibacillosis and zoonotic risks. PMID:19531720

  8. Prevalence and antibiogram profiling of Escherichia coli pathotypes isolated from the Kat River and the Fort Beaufort abstraction water.

    PubMed

    Nontongana, Nolonwabo; Sibanda, Timothy; Ngwenya, Elvis; Okoh, Anthony I

    2014-08-01

    Escherichia coli is a widespread bacterium encompassing a variety of strains, ranging from highly pathogenic strains, causing worldwide outbreaks of severe diseases to avirulent, well characterized safe laboratory strains. This study evaluated the prevalence and antibiogram profiles of E. coli pathotypes isolated from the Kat River and Fort Beaufort abstraction water. A total of 171 out of 278 confirmed E. coli isolates were positive for at least one pathogenic determinant and these included enteropathogenic E. coli (6%), enterotoxigenic E. coli (47%), uropathogenic E. coli (2%), neonatal meningitis E. coli (5%), diffusely adherent E. coli (1%) and enterohaemorrhagic E. coli (1%). Interestingly, enteroinvasive and enteroaggregative E. coli were not detected. The phenotypic antibiogram profiles of the isolates revealed that all were resistant to penicillin G, while 98% and 38% of the pathotypes were resistant to ampicillin and trimethoprim-sulphamethoxazole, respectively. About 8% of the isolates were resistant to streptomycin. More than half of the isolates exhibited multiple antibiotic resistance with 44% being resistant to three antibiotics and 8% resistant to four antibiotics. We conclude that the Kat River is a reservoir of potentially virulent antibiotic resistant E. coli strains that can cause serious health risks to humans who drink raw water from this river, or in the case that consumption of treated drinking water coincides with failed drinking water processes. PMID:25119699

  9. Application of DNA hybridization techniques in the assessment of diarrheal disease among refugess in Thailand. [Shigella; Escherichia coli; Campylobacter; Cryptosporidium

    SciTech Connect

    Taylor, D.N.; Echeverria, P.; Pitarangsi, C.; Seriwatana, J.; Sethabutr, O.; Bodhidatta, L.; Brown, C.; Herrmann, J.E.; Blacklow, N.R.

    1988-01-01

    The epidemiology and etiology of acute diarrheal disease were determined in a Hmong refugee camp on the Thai-Laotian border from April 11 to May 14, 1985. DNA hybridization techniques were used to detect Shigella species, enteroinvasive Escherichia coli, and enterotoxigenic E. coli. A monoclonal enzyme-linked immunosorbent assay was used to detect rotavirus, and standard microbiology was used to detect other enteropathogens. The age-specific diarrheal disease rates were 47 episodes per month per 1000 children less than five years old and 113 episodes per month per 1000 children less than one year old. Rotavirus, enterotoxigenic E. coli, Campylobacter, and Cryptosporidium were the predominant pathogens in children less than two years old. The DNA probe hybridized with 94% of 31 specimens identified as enterotoxigenic E. coli by the standard assays and with none of the specimens in which the standard assays were negative. The probe for Shigella and enteroinvasive E. coli hybridized in eight of 10 stools that contained Shigella and four of 314 stools from which Shigella and enteroinvasive E. coli were not isolated. The use of DNA probes allows specimens to be collected in remote areas with a minimum amount of equipment and technical expertise so that they can be easily transported to a central laboratory for further processing.

  10. Characterization of Escherichia coli isolated from calf diarrhea in and around Kombolcha, South Wollo, Amhara Region, Ethiopia.

    PubMed

    Gebregiorgis, Ashenafi; Tessema, Tesfaye Sisay

    2016-02-01

    This study was carried out from October 2012 to end of February 2013 in and around Kombolcha, Amhara regional state, Ethiopia, using a total of 201 neonatal calves aged 1 day to 4 months and suffering from diarrhea. The objectives of the study were to isolate Escherichia coli from diarrheic calves, and to determine E. coli biotypes and risk factors associated with its isolation. The fecal samples were collected, transported, and processed following standard microbiological procedures. Seventy-four isolates of E. coli were identified. Yellowish diarrhea, younger age, and low-colostrum feeding were significantly associated with rate of E. coli isolation (P < 0.05). Then the 74 isolates of E. coli were biotyped using fermentation of 9 sugars and grouped into 12 biotypes; the most dominant was biotype III (36.8 %). Finally, by comparing with studies elsewhere, from the 12 isolated biotypes, 3 of them were suggested to be enteropathogenic E. coli (EPEC), entherotoxigenic E. coli (ETEC), and adhesion and effacing E. coli (AEEC) pathogenic strains. The present study showed that E. coli accounted for 37 % of calf diarrhea, with very diverse biotypes. PMID:26563273

  11. Quantitative Mass Spectrometry Identifies Novel Host Binding Partners for Pathogenic Escherichia coli Type III Secretion System Effectors.

    PubMed

    Law, Robyn J; Law, Hong T; Scurll, Joshua M; Scholz, Roland; Santos, Andrew S; Shames, Stephanie R; Deng, Wanyin; Croxen, Matthew A; Li, Yuling; de Hoog, Carmen L; van der Heijden, Joris; Foster, Leonard J; Guttman, Julian A; Finlay, B Brett

    2016-05-01

    Enteropathogenic and enterohemorrhagic Escherichia coli cause enteric diseases resulting in significant morbidity and mortality worldwide. These pathogens remain extracellular and translocate a set of type III secreted effector proteins into host cells to promote bacterial virulence. Effectors manipulate host cell pathways to facilitate infection by interacting with a variety of host targets, yet the binding partners and mechanism of action of many effectors remain elusive. We performed a mass spectrometry screen to identify host targets for a library of effectors. We found five known effector targets and discovered four novel interactions. Interestingly, we identified multiple effectors that interacted with the microtubule associated protein, ensconsin. Using co-immunoprecipitations, we confirmed that NleB1 and EspL interacted with ensconsin in a region that corresponded to its microtubule binding domain. Ensconsin is an essential cofactor of kinesin-1 that is required for intracellular trafficking, and we demonstrated that intracellular trafficking was severely disrupted during wild type EPEC infections but not during infections with ΔnleB1 or ΔespL mutants. Our findings demonstrate the efficacy of quantitative proteomics for identifying effector-host protein interactions and suggest that vesicular trafficking is a crucial cellular process that may be targeted by NleB1 and EspL through their interaction with ensconsin. PMID:27018634

  12. Detection of Pathogenic Escherichia coli and Staphylococcus aureus from Cattle and Pigs Slaughtered in Abattoirs in Vhembe District, South Africa

    PubMed Central

    Tanih, Nicoline F.; Sekwadi, Eunice; Ndip, Roland N.; Bessong, Pascal O.

    2015-01-01

    Pathogenic food-borne bacteria have been associated with severe morbidity and mortality in humans and animals. This study was aimed at determining the prevalence of Staphylococcus aureus, Salmonella spp., and Escherichia coli present in cattle and pigs slaughtered in selected abattoirs in Vhembe District and at determining the susceptibility of the isolates to antibiotics. A total of 176 swab samples (28 cattle and 16 pigs) of the rump, flank, brisket, and neck of the animals were analyzed using standard microbiological methods. E. coli isolates were genotyped to detect pathogenic strains. Of the 176 samples, 104 (67.5%) were positive for E. coli and 50 (32.5%) for S. aureus. There was no statistically significant difference (P > 0.05) in the isolation rate from the different animal parts or abattoirs. Overall, 14/104 (13.46%) of the E. coli isolates were pathogenic strains which included enteropathogenic E. coli (EPEC) (bfpA) 1.9%, enterotoxigenic E. coli (ETEC) (LT) 3.8%, and enteroaggregative E. coli (EAEC) (aaiC) 7.6%. E. coli isolates were resistant (100%) to vancomycin and bacitracin. S. aureus (100%) were resistant to oxacillin and nalidixic acid. The presence of resistant strains of these bacteria in food of animal origin could serve as important vehicles transmitting these bacteria to humans. This finding is of epidemiological significance. PMID:25811040

  13. Identification and Characterization of Microcin S, a New Antibacterial Peptide Produced by Probiotic Escherichia coli G3/10

    PubMed Central

    Zschüttig, Anke; Zimmermann, Kurt; Blom, Jochen; Goesmann, Alexander; Pöhlmann, Christoph; Gunzer, Florian

    2012-01-01

    Escherichia coli G3/10 is a component of the probiotic drug Symbioflor 2. In an in vitro assay with human intestinal epithelial cells, E. coli G3/10 is capable of suppressing adherence of enteropathogenic E. coli E2348/69. In this study, we demonstrate that a completely novel class II microcin, produced by probiotic E. coli G3/10, is responsible for this behavior. We named this antibacterial peptide microcin S (MccS). Microcin S is coded on a 50.6 kb megaplasmid of E. coli G3/10, which we have completely sequenced and annotated. The microcin S operon is about 4.7 kb in size and is comprised of four genes. Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S. Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay. Moreover, growth of E. coli transformed with a plasmid containing mcsS under control of an araC PBAD activator-promoter is inhibited upon mcsS induction. Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10. PMID:22479389

  14. Prevalence and antimicrobial resistance of diarrheagenic Escherichia coli and Shigella species associated with acute diarrhea in Tehran, Iran

    PubMed Central

    Jafari, Fereshteh; Hamidian, Mohammad; Rezadehbashi, Maryam; Doyle, Michael; Salmanzadeh-ahrabi, Siavosh; Derakhshan, Faramarz; Reza Zali, Mohammad

    2009-01-01

    A study was performed to determine the prevalence and antimicrobial resistance of Shigella species and diarrheagenic Escherichia coli isolates cultured from patients with acute diarrhea in Tehran, Iran. Between May 2003 and May 2005, 1120 diarrheal specimens were collected and assayed for bacterial enteropathogens by conventional and molecular methods. Etiological agents were isolated from 564 (50.3%) specimens, and included 305 (54%) E coli, 157 (27.8%) Shigella species, and 102 (18%) from other genera of bacteria. The predominant E coli was Shiga toxin-producing E coli (105 isolates [34.5%]) and the predominant Shigella serotype was Shigella sonnei (88 isolates [56.1%]). A high rate of antibiotic resistance was observed among E coli, with 40 of 53 (75.5%) Shiga toxin-producing E coli isolates resistant to amoxicillin and tetra-cycline, and eight (5.2%) E coli isolates resistant to more than six antibiotics. Most Shigella isolates were resistant to tetracycline (95%) and trimethoprim-sulfamethoxazole (91.7%), with greatest antibiotic resistance observed among S sonnei (53 of 88 [60.2%] isolates). Antibiotic resistance is widespread in diarrheagenic E coli and Shigella in children with acute diarrhea in Tehran, Iran; hence, updated strategies for appropriate use of antimicrobial agents in Iran are needed. PMID:20808457

  15. Isolation of Escherichia coli from piglets in South Korea with diarrhea and characteristics of the virulence genes.

    PubMed

    Kim, Yeong Ju; Kim, Ji Hee; Hur, Jin; Lee, John Hwa

    2010-01-01

    Escherichia coli was isolated from the feces of 122 piglets with diarrhea on 55 farms in Korea. The virulence genes of each isolate were characterized by polymerase chain reaction (PCR). Of the 562 isolates, 191 carried 1 or more of the virulence genes tested for in this study. Of the 191 isolates, 114 (60%) carried 1 or more of the genes for enterotoxigenic E. coli (ETEC) fimbriae F4, F5, F6, F18, and F41 and ETEC toxins LT, STa, and STb, 57 (30%) carried 1 or more of the genes for the Shiga-toxin-producing E. coli (STEC) toxins Stx1, Stx2, and Stx2e, and 21% and 37% carried the gene for enteropathogenic E. coli intimin and for enteroaggregative E. coli toxin, respectively. Collectively, our results indicate that other pathotypes of E. coli as well as ETEC can be strongly associated with diarrhea in piglets. In addition, detection of the genes for Stx1 and Stx2 indicates that pigs are reservoirs of human pathogenic STEC. PMID:20357961

  16. Osteogenic and adipogenic potential of porcine adipose mesenchymal stem cells.

    PubMed

    Qu, Chang-qing; Zhang, Guo-hua; Zhang, Li-jie; Yang, Gong-she

    2007-02-01

    Human, rat, and mouse studies have demonstrated the existence of a population of adipose mesenchymal stem cells (AMSCs) that can undergo multilineage differentiation in vitro. Understanding the clinical potential of AMSCs may require their use in preclinical large-animal models such as pigs. Thus, the objectives of this study were to establish a protocol for the isolation of porcine AMSCs from adipose tissue and to examine their ex vivo differentiation potential to adipocytes and osteoblast. The porcine AMSCs from passage 4 were selected for differentiation analysis. The adipocytes were identified morphologically by staining with Oil Red O, and the adipogenic marker genes were examined by RT-PCR technique. Osteogenic lineage was documented by deposition of calcium stained with Alzarin Red S, visualization of alkaline phosphatase activity, and expression of marker gene. Our result indicates that porcine AMSCs have been successfully isolated and induced differentiation into adipocytes and osteoblasts. This study suggested that porcine AMSCs are also a valuable model system for the study on the mesenchymal lineages for basic research and tissue engineering. PMID:17570023

  17. Porcine Epidemic Diarrhea Virus among Farmed Pigs, Ukraine

    PubMed Central

    Carr, John; Ellis, Richard J.; Steinbach, Falko; Williamson, Susanna

    2015-01-01

    An outbreak of porcine epidemic diarrhea occurred in the summer of 2014 in Ukraine, severely affecting piglets <10 days of age; the mortality rate approached 100%. Full genome sequencing showed the virus to be closely related to strains reported from North America, showing a sequence identity of up to 99.8%. PMID:26584081

  18. Porcine bocavirus: achievements in the past five years.

    PubMed

    Zhou, Feng; Sun, Haoting; Wang, Yuyan

    2014-12-01

    Porcine bocavirus is a recently discovered virus that infects pigs and is classified within the Bocavirus genus (family Parvoviridae, subfamily Parvovirinae). The viral genome constitutes linear single-stranded DNA and has three open reading frames that encode four proteins: NS1, NP1, VP1, and VP2. There have been more than seven genotypes discovered to date. These genotypes have been classified into three groups based on VP1 sequence. Porcine bocavirus is much more prevalent in piglets that are co-infected with other pathogens than in healthy piglets. The virus can be detected using PCR, loop-mediated isothermal amplification, cell cultures, indirect immunofluorescence, and other molecular virology techniques. Porcine bocavirus has been detected in various samples, including stool, serum, lymph nodes, and tonsils. Because this virus was discovered only five years ago, there are still many unanswered questions that require further research. This review summarizes the current state of knowledge and primary research achievements regarding porcine bocavirus. PMID:25514206

  19. Blood gas and hematological changes in experimental peracute porcine pleuropneumonia.

    PubMed Central

    Kiorpes, A L; MacWilliams, P S; Schenkman, D I; Bäckström, L R

    1990-01-01

    The effect of experimental, peracute, porcine pleuropneumonia on arterial blood gases, acid base status, the leukogram, and gross and microscopic lung structure was studied in nine growing pigs (mean weight +/- SD 10.6 +/- 2.0 kg). Pigs were inoculated intranasally with a virulent serotype 5 isolate of Actinobacillus pleuropneumoniae, and all showed signs typical of the disease within four hours. Death occurred in all pigs from 4.5 to 32 hours postinoculation (mean 14 hours). Gross and microscopic changes were typical of porcine pleuropneumonia in all pigs. Changes in the leukogram included a rapid decline in total white cells, segmented neutrophils, lymphocytes, monocytes, and eosinophils. Pigs maintained alveolar ventilation throughout the study as arterial CO2 tension was unchanged; however, arterial O2 tension and pH decreased from (mean +/- SD) 95.2 +/- 5.7 torr and 7.463 +/- 0.018 at baseline to 62.1 +/- 12.3 torr and 7.388 +/- 0.045, respectively, within 90 minutes prior to death. The data showed that in this model of peracute porcine pleuropneumonia, progressive ventilatory failure was not a feature of the disease, and the blood gas values and acid base status were maintained within physiological ranges. The histopathological hematological and physiological findings were consistent with the hypothesis that peracute porcine pleuropneumonia resembles septic shock. Images Fig. 2. Fig. 3. PMID:2106382

  20. Porcine reproductive and respiratory syndrome (PRRS): an immune dysregulatory pandemic

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Porcine reproductive and respiratory disease syndrome (PRRS) is a viral pandemic that especially affects neonates within the "critical window" of immunological development. PRRS was recognized in 1987 and within a few years became pandemic causing an estimated yearly $600,000 economic loss in the US...

  1. DIAGNOSTIC FINDINGS IN PIGS WITH PORCINE MALIGNANT CATARRHAL FEVER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For the first time Ovine Herpesvirus 2 (OvHV-2) was identified in Swiss pigs as the causative agent of Porcine Malignant Catarrhal Fever (MCF). Diseased animals from two farms were observed to show weakness, anorexia, fever up to 41°C, and neurological symptoms, i.e. ataxia, convulsions and hyperest...

  2. Research Advancements in Porcine Derived Mesenchymal Stem Cells.

    PubMed

    Bharti, Dinesh; Shivakumar, Sharath Belame; Subbarao, Raghavendra Baregundi; Rho, Gyu-Jin

    2016-01-01

    In the present era of stem cell biology, various animals such as Mouse, Bovine, Rabbit and Porcine have been tested for the efficiency of their mesenchymal stem cells (MSCs before their actual use for stem cell based application in humans. Among them pigs have many similarities to humans in the form of organ size, physiology and their functioning, therefore they have been considered as a valuable model system for in vitro studies and preclinical assessments. Easy assessability, few ethical issues, successful MSC isolation from different origins like bone marrow, skin, umbilical cord blood, Wharton's jelly, endometrium, amniotic fluid and peripheral blood make porcine a good model for stem cell therapy. Porcine derived MSCs (pMSCs have shown greater in vitro differentiation and transdifferention potential towards mesenchymal lineages and specialized lineages such as cardiomyocytes, neurons, hepatocytes and pancreatic beta cells. Immunomodulatory and low immunogenic profiles as shown by autologous and heterologous MSCs proves them safe and appropriate models for xenotransplantation purposes. Furthermore, tissue engineered stem cell constructs can be of immense importance in relation to various osteochondral defects which are difficult to treat otherwise. Using pMSCs successful treatment of various disorders like Parkinson's disease, cardiac ischemia, hepatic failure, has been reported by many studies. Here, in this review we highlight current research findings in the area of porcine mesenchymal stem cells dealing with their isolation methods, differentiation ability, transplantation applications and their therapeutic potential towards various diseases. PMID:26201864

  3. Porcine circovirus: transcription and rolling-circle DNA replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This review summarizes the molecular studies pertaining to porcine circovirus (PCV) transcription and DNA replication. The genome of PCV is circular, single-stranded DNA and contains 1759-1768 nucleotides. Both the genome-strand (packaged in the virus particle) and the complementary-strand (synthesi...

  4. Age and nursing affect the neonatal porcine uterine transcriptome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The lactocrine hypothesis for maternal programming of neonatal development was proposed to describe a mechanism through which milk-borne bioactive factors, delivered from mother to nursing offspring, could affect development of tissues, including the uterus. Porcine uterine development, initiated be...

  5. Dystrophin deficiency-induced changes in porcine skeletal muscle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel porcine stress syndrome was detected in the U.S. Meat Animal Research Center’s swine research population when two sibling barrows died of apparent stress symptoms (open mouth breathing, vocalization, and refusal to move or stand) after transport at 12 weeks of age. At eight weeks of age, the...

  6. Detection of a Novel Porcine Parvovirus in Chinese Swine Herds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To determine whether the recently reported novel porcine parvovirus type 4 (PPV4) is prevalent in China, a set of PPV4 specific primers were designed and used for the molecular survey of PPV4 among clinical samples. The results indicated a positive detection for PPV4 in Chinese swine herds of 1.84% ...

  7. Antibody to porcine parvovirus in warthog (Phacochoerus aethiopicus).

    PubMed

    Thomson, G R; Peenze, I

    1980-03-01

    Haemagglutination inhibiting antibody to porcine parvovirus was shown to be widespread in all but one of the warthog populations sampled from South Africa and Zimbabwe Rhodesia. In some instances titres as high as greater than or equal to 1/20 000 were detected. PMID:7454234

  8. The first case of porcine epidemic diarrhea in Canada

    PubMed Central

    Ojkic, Davor; Hazlett, Murray; Fairles, Jim; Marom, Anna; Slavic, Durda; Maxie, Grant; Alexandersen, Soren; Pasick, John; Alsop, Janet; Burlatschenko, Sue

    2015-01-01

    In January, 2014, increased mortality was reported in piglets with acute diarrhea on an Ontario farm. Villus atrophy in affected piglets was confined to the small intestine. Samples of colon content were PCR-positive for porcine epidemic diarrhea virus (PEDV). Other laboratory tests did not detect significant pathogens, confirming this was the first case of PED in Canada. PMID:25694663

  9. Methionine deficiency reduces autophagy and accelerates death in intestinal epithelial cells infected with enterotoxigenic Escherichia coli.

    PubMed

    Tang, Yulong; Tan, Bie; Xiong, Xia; Li, Fengna; Ren, Wenkai; Kong, Xiangfeng; Qiu, Wei; Hardwidge, Philip R; Yin, Yulong

    2015-10-01

    Infections by enterotoxigenic Escherichia coli (ETEC) result in large economic losses to the swine industry worldwide. Dietary supplementation with amino acids has been considered as a potential mechanism to improve host defenses against infection. The goal of this study was to determine whether methionine deprivation alters ETEC interactions with porcine intestinal epithelial cells. IPEC-1 cells were cultured in media with or without L-methionine. Methionine deprivation resulted in enhanced ETEC adhesion and increased both the cytotoxicity and apoptotic responses of IPEC-1 cells infected with ETEC. Methionine deprivation inhibited IPEC-1 cell autophagic responses, suggesting that the increased cytotoxicity of ETEC to methionine-deprived IPEC-1 cells might be due to defects in autophagy. PMID:24965529

  10. Mouse intestinal innate immune responses altered by enterotoxigenic Escherichia coli (ETEC) infection.

    PubMed

    Ren, Wenkai; Yin, Jie; Duan, Jielin; Liu, Gang; Zhu, Xiaoping; Chen, Shuai; Li, Tiejun; Wang, Shengping; Tang, Yulong; Hardwidge, Philip R

    2014-11-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of human and porcine morbidity and mortality. The current study was conducted to identify intestinal immunity that is altered in a mouse model of ETEC infection. Innate immune responses and inflammation were analyzed. The activation of signal transduction pathways, including toll like receptor 4 (TLR-4)-nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPK), was analyzed using immunoblotting and PCR array analyses. We found that ETEC infection promoted the expression of pro-inflammatory cytokines through the activation of the NF-κB and MAPK pathways. Meanwhile, ETEC infection affected sIgA transportation and Paneth cell function. These data improve our understanding of how ETEC causes disease in animals. PMID:25267358

  11. Structural and functional annotation of the porcine immunome

    PubMed Central

    2013-01-01

    Background The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems. Results The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated

  12. F4+ enterotoxigenic Escherichia coli (ETEC) adhesion mediated by the major fimbrial subunit FaeG.

    PubMed

    Xia, Pengpeng; Song, Yujie; Zou, Yajie; Yang, Ying; Zhu, Guoqiang

    2015-09-01

    The FaeG subunit is the major constituent of F4(+) fimbriae, associated with glycoprotein and/or glycolipid receptor recognition and majorly contributes to the pathogen attachment to the host cells. To investigate the key factor involved in the fimbrial binding of F4(+) Escherichia coli, both the recombinant E. coli SE5000 strains carrying the fae operon gene clusters that express the different types of fimbriae in vitro, named as rF4ab, rF4ac, and rF4ad, respectively, corresponding to the fimbrial types F4ab, F4ac, and F4ad, and the three isogenic in-frame faeG gene deletion mutants were constructed. The adhesion assays and adhesion inhibition assays showed that ΔfaeG mutants had a significant reduction in the binding to porcine brush border as well as the intestinal epithelial cell lines, while the complemented strain ΔfaeG/pfaeG restored the adhesion function. The recombinant bacterial strains rF4ab, rF4ac, and rF4ad have the same binding property as wild-type F4(+) E. coli strains do and improvement in terms of binding to porcine brush border and the intestinal epithelial cells, and the adherence was blocked by the monoclonal antibody anti-F4 fimbriae. These data demonstrate that the fimbrial binding of F4(+) E. coli is directly mediated by the major FaeG subunit. PMID:25847483

  13. EXTRAINTESTINAL PATHOGENIC ESCHERICHIA COLI (EXPEC)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Extraintestinal pathogenic Escherichia coli (ExPEC) possess virulence traits that allow them to invade, colonize, and induce disease in bodily sites outside of the gastrointestinal tract. Human diseases caused by ExPEC include urinary tract infections, neonatal meningitis, sepsis, pneumonia, surgic...

  14. A Large Chondroitin Sulfate Proteoglycan, Versican, in Porcine Predentin.

    PubMed

    Okahata, Saori; Yamamoto, Ryuji; Yamakoshi, Yasuo; Fukae, Makoto

    2011-01-01

    Proteoglycans and their constituent glycosaminoglycan (GAG) have been proposed to be involved in the inhibition of mineralization in unmineralized tissue, predentin. Among the proteoglycans secreted by odontoblasts, we focused on the large chondroitin sulfate proteoglycan, versican, for its large binding capacity for calcium ions. The aims of this study were the determination of the full-length sequence and splicing variants of the porcine versican, and the detection of versican in the porcine predentin. The complete coding sequence of the porcine versican mRNA was cloned to be 11,775 nucleotides long and encode 3,924 amino acids, and four splicing variants, V0, V1, V2 and V3, were characterized in the isolated porcine cartilage cells. The number of potential GAG attachment sites was 15 in the V0 variant, 13 in the V1 variant, 2 in the V2 variant and 0 in the V3 variant. They were deposited in DDBJ. The V1 variant was determined by RT-PCR in the odontoblasts, dental papilla cells, dental follicle cells, periodontal ligament cells, dental pulp cells, and gingival cells of pigs, although a small amount of the V0 valiant was found in the dental papilla cells. The predentin was prepared from developing porcine permanent incisor tooth germs and its soluble proteins were extracted in order to be partially characterized by protein and proteinase profiles. The versican V1 cleavage products were detected in the predentin extract by Western blotting analysis. These results suggested that the versican splice variant V1 implicates both the control of the mineralization and the activities of the predentin metalloproteinases, because it has 13 GAG chains that bind a large amount of calcium. PMID:22200993

  15. High-level soluble expression of bioactive porcine myostatin propeptide in E. coli.

    PubMed

    Haq, Wing Yeung; Kang, Sang Kee; Lee, Sang Beum; Kang, Hee Chul; Choi, Yun Jaie; Lee, Chin Nyean; Kim, Yong Soo

    2013-10-01

    Myostatin (MSTN) is a potent negative regulator of skeletal muscle mass. The activity of MSTN is suppressed by MSTN propeptide (MSTNPro), the N-terminal part of unprocessed MSTN that is cleaved off during posttranslational MSTN processing. Easy availability of MSTNPro would help to investigate the potential of the protein as an agent to enhance muscle growth in agricultural animal species. Thus, this study was designed to produce bioactive wild-type porcine MSTN propeptide (pMSTNProW) and its mutated form at the BMP-1/TLD proteolytic cleavage site (pMSTNProM) in Escherichia coli. The pMSTNProW and pMSTNProM genes were separately cloned into pMAL-c5X vector downstream of the maltose-binding protein (MBP) gene and were transformed and expressed in soluble forms in E. coli. For each milliliter of cell culture, about 40 μg of soluble MBP-pMSTNProW and MBP-pMSTNProM proteins were purified by amylose resin affinity chromatography. Further purification by anion exchange chromatography of the affinity-purified fractions yielded about 10 μg/mL culture of MBP-pMSTNProW and MBP-pMSTNProM proteins. Factor Xa protease cleaved the fusion partner MBP from MBP-pMSTNPro proteins, and approximately 4.2 μg of pMSTNProW and pMSTNProM proteins were purified per milliliter of culture. MBP-pMSTNProM was resistant to digestion by BMP-1 metalloproteinase, while MBP-pMSTNProW was cleaved into two fragments by BMP-1. Both MBP-pMSTNProW and MBP-pMSTNProM demonstrated their MSTN binding affinities in a pulldown assay. In an in vitro gene reporter assay, both proteins inhibited MSTN bioactivity without a significant difference in their inhibitory capacities, indicating that the cell culture-based gene reporter assay has limitation in detecting the true in vivo biological potencies of mutant forms of MSTNPro proteins at the BMP-1/TLD cleavage site. Current results show that a high-level production of bioactive porcine MSTNpro is possible in E. coli, and it remains to be investigated

  16. Fluorescence in situ hybridization investigation of potentially pathogenic bacteria involved in neonatal porcine diarrhea

    PubMed Central

    2014-01-01

    Background Neonatal diarrhea is a multifactorial condition commonly present on pig farms and leads to economic losses due to increased morbidity and mortality of piglets. Immature immune system and lack of fully established microbiota at birth predispose neonatal piglets to infection with enteric pathogens. The microorganisms that for decades have been associated with enteritis and diarrhea in suckling piglets are: rotavirus A, coronavirus, enterotoxigenic Escherichia coli (ETEC), Clostridium perfringens type C, Cryptosporidium spp., Giardia spp., Cystoisospora suis and Strongyloides ransomi. However, in recent years, the pig industry has experienced an increased number of neonatal diarrhea cases in which the above mentioned pathogens are no longer detected. Potentially pathogenic bacteria have recently received focus in the research on the possible etiology of neonatal diarrhea not caused by common pathogens. The primary aim of this study was to investigate the role of E. coli, Enterococcus spp., C. perfringens and C. difficile in the pathogenesis of neonatal porcine diarrhea with no established casual agents. Fluorescence in situ hybridization with oligonucleotide probes was applied on the fixed intestinal tissue samples from 51 diarrheic and 50 non-diarrheic piglets collected from four Danish farms during outbreaks of neonatal diarrhea not caused by well-known enteric pathogens. Furthermore, an association between the presence of these bacteria and histological lesions was evaluated. Results The prevalence of fluorescence signals specific for E. coli, C. perfringens and C. difficile was similar in both groups of piglets. However, Enterococcus spp. was primarily detected in the diarrheic piglets. Furthermore, adherent bacteria were detected in 37 % diarrheic and 14 % non-diarrheic piglets. These bacteria were identified as E. coli and Enterococcus spp. and their presence in the intestinal mucosa was associated with histopathological changes. Conclusions The

  17. Prosthetic valve endocarditis. Experience with porcine bioprostheses.

    PubMed

    Sett, S S; Hudon, M P; Jamieson, W R; Chow, A W

    1993-03-01

    Prosthetic valve endocarditis remains an infrequent but serious complication of cardiac valvular replacement. Prosthetic valve endocarditis was diagnosed in 56 (1.8%) of 3200 patients in whom one or more porcine bioprostheses were implanted between 1975 and 1988. Of the 56 patients with prosthetic valve endocarditis, there were 40 men and 16 women, with a mean age at initial implantation of 57 years (27 to 81 years). Of the 56 patients, 6 were initially treated for native valve endocarditis. There were 8 cases of early prosthetic valve endocarditis (defined as occurring less than 60 days after initial surgical intervention) and 48 cases of late prosthetic valve endocarditis (occurring after 60 days). The overall mortality rate of the 56 patients was 32% (18 patients). Of the 8 patients with early prosthetic valve endocarditis, 6 (75%) died. Of the 48 patients with late prosthetic valve endocarditis, 12 (25%) died. The predominant organisms were Staphylococcus epidermidis (12 cases), Streptococcus viridans (8 cases) and Staphylococcus aureus (7 cases). The presence of hemodynamic compromise, including congestive heart failure, septic embolism, persistent sepsis, and echocardiographic evidence of vegetations, dictated the mode and timing of the addition of surgical intervention to medical therapy. The survival rate for medically and surgically treated patients with late prosthetic valve endocarditis was 91% (20 patients); none of the patients with early prosthetic valve endocarditis survived (all had severe hemodynamic compromise). We analyzed 18 factors for the prediction of early and late death. The predictors of death by univariate analysis for both early and late prosthetic valve endocarditis were age, diagnosis time, renal status, sepsis, management mode, fever, dental procedures, and dental prophylaxis. The predictors by multivariate analysis were age, diagnosis time, renal status, and management mode for early prosthetic valve endocarditis, and only diagnosis

  18. An in-depth comparison of the porcine, murine and human inflammasomes; lessons from the porcine genome and transcriptome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Emerging evidence suggests that swine are a scientifically acceptable intermediate species between rodents and humans to model immune function relevant to humans. The swine genome has recently been sequenced and several preliminary structural and functional analysis of the porcine immunome have been...

  19. Mouse retrovirus mediates porcine endogenous retrovirus transmission into human cells in long-term human-porcine chimeric mice

    PubMed Central

    Yang, Yong-Guang; Wood, James C.; Lan, Ping; Wilkinson, Robert A.; Sykes, Megan; Fishman, Jay A.; Patience, Clive

    2004-01-01

    Porcine endogenous retrovirus (PERV) is a potential pathogen in clinical xenotransplantation; transmission of PERV in vivo has been suggested in murine xenotransplantation models. We analyzed the transmission of PERV to human cells in vivo using a model in which immunodeficient NOD/SCID transgenic mice were transplanted with porcine and human lymphohematopoietic tissues. Our results demonstrate, we believe for the first time, that human and pig cells can coexist long-term (up to 25 weeks) without direct PERV infection of human cells. Despite the transplantation of porcine cells that did not produce human-tropic PERV, human cells from the chimeric mice were frequently found to contain PERV sequences. However, this transmission was due to the pseudotyping of PERV-C (a virus without human tropism) by xenotropic murine leukemia virus, rather than to de novo generation of human-tropic PERV. Thus, pseudotyping might account for the PERV transmission previously observed in mice. The absence of direct human cell infection following long-term in vivo coexistence with large numbers of porcine cells provides encouragement regarding the potential safety of using pigs that do not produce human-tropic PERV as source animals for transplantation to humans. PMID:15343388

  20. Hydrogen Sulfide Donor Protects Porcine Oocytes against Aging and Improves the Developmental Potential of Aged Porcine Oocytes

    PubMed Central

    Krejcova, Tereza; Smelcova, Miroslava; Petr, Jaroslav; Bodart, Jean-Francois; Sedmikova, Marketa; Nevoral, Jan; Dvorakova, Marketa; Vyskocilova, Alena; Weingartova, Ivona; Kucerova-Chrpova, Veronika; Chmelikova, Eva; Tumova, Lenka; Jilek, Frantisek

    2015-01-01

    Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H2S) in the process of porcine oocyte aging. H2S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H2S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H2S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H2S from a donor (Na2S.9H2O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H2S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H2S on MPF and MAPK activities were detected and the intracellular mechanism underlying H2S activity remains unclear, our study clearly demonstrates the role of H2S in the regulation of porcine oocyte aging. PMID:25615598

  1. In depth global analysis of transcript abundance levels in porcine alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide and causes considerable economic loss. Infection of the primary target cells, porcine alveolar macrophages (PAMs), by PRRSV causes significant changes in their function by mechanisms that are not under...

  2. In depth global analysis of gene expression levels in porcine alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide. Infection of the preferential target cells, porcine alveolar macrophages (PAMs), by PRRSV causes significant changes in their function by mechanisms that are not understood. Serial Analysis of Gene Ex...

  3. Effect of porcine reproductive and respiratory syndrome virus on porcine alveolar macrophage function as determined using serial analysis of gene expression (SAGE)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide and causes considerable economic loss. The main target of infection is the porcine alveolar macrophage (PAM). Infection of PAM by PRRSV causes significant changes in their function by mechanisms that a...

  4. Biophysical Characterization and Activity of Lymphostatin, a Multifunctional Virulence Factor of Attaching and Effacing Escherichia coli *

    PubMed Central

    Cassady-Cain, Robin L.; Blackburn, Elizabeth A.; Alsarraf, Husam; Dedic, Emil; Bease, Andrew G.; Böttcher, Bettina; Jørgensen, René; Wear, Martin; Stevens, Mark P.

    2016-01-01

    Attaching and effacing Escherichia coli cause diarrhea and typically produce lymphostatin (LifA), an inhibitor of mitogen-activated proliferation of lymphocytes and pro-inflammatory cytokine synthesis. A near-identical factor (Efa1) has been reported to mediate adherence of E. coli to epithelial cells. An amino-terminal region of LifA shares homology with the catalytic domain of the large clostridial toxins, which are retaining glycosyltransferases with a DXD motif involved in binding of a metal ion. Understanding the mode(s) of action of lymphostatin has been constrained by difficulties obtaining a stably transformed plasmid expression clone. We constructed a tightly inducible clone of enteropathogenic E. coli O127:H6 lifA for affinity purification of lymphostatin. The purified protein inhibited mitogen-activated proliferation of bovine T lymphocytes in the femtomolar range. It is a monomer in solution and the molecular envelope was determined using both transmission electron microscopy and small-angle x-ray scattering. Domain architecture was further studied by limited proteolysis. The largest proteolytic fragment containing the putative glycosyltransferase domain was tested in isolation for activity against T cells, and was not sufficient for activity. Tryptophan fluorescence studies indicated thatlymphostatin binds uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) but not UDP-glucose (UDP-Glc). Substitution of the predicted DXD glycosyltransferase motif with alanine residues abolished UDP-GlcNAc binding and lymphostatin activity, although other biophysical properties were unchanged. The data indicate that lymphostatin has UDP-sugar binding potential that is critical for activity, and is a major leap toward identifying the nature and consequences of modifications of host cell factors. PMID:26786100

  5. Long-term persistence and leaching of Escherichia coli in temperate maritime soils.

    PubMed

    Brennan, Fiona P; O'Flaherty, Vincent; Kramers, Gaelene; Grant, Jim; Richards, Karl G

    2010-03-01

    Enteropathogen contamination of groundwater, including potable water sources, is a global concern. The spreading on land of animal slurries and manures, which can contain a broad range of pathogenic microorganisms, is considered a major contributor to this contamination. Some of the pathogenic microorganisms applied to soil have been observed to leach through the soil into groundwater, which poses a risk to public health. There is a critical need, therefore, for characterization of pathogen movement through the vadose zone for assessment of the risk to groundwater quality due to agricultural activities. A lysimeter experiment was performed to investigate the effect of soil type and condition on the fate and transport of potential bacterial pathogens, using Escherichia coli as a marker, in four Irish soils (n = 9). Cattle slurry (34 tonnes per ha) was spread on intact soil monoliths (depth, 1 m; diameter, 0.6 m) in the spring and summer. No effect of treatment or the initial soil moisture on the E. coli that leached from the soil was observed. Leaching of E. coli was observed predominantly from one soil type (average, 1.11 +/- 0.77 CFU ml(-1)), a poorly drained Luvic Stagnosol, under natural rainfall conditions, and preferential flow was an important transport mechanism. E. coli was found to have persisted in control soils for more than 9 years, indicating that autochthonous E. coli populations are capable of becoming naturalized in the low-temperature environments of temperate maritime soils and that they can move through soil. This may compromise the use of E. coli as an indicator of fecal pollution of waters in these regions. PMID:20038692

  6. Comparison of ruminant and human attaching and effacing Escherichia coli (AEEC) strains.

    PubMed

    Horcajo, Pilar; Domínguez-Bernal, Gustavo; de la Fuente, Ricardo; Ruiz-Santa-Quiteria, José A; Blanco, Jesús E; Blanco, Miguel; Mora, Azucena; Dahbi, Ghizlane; López, Cecilia; Puentes, Beatriz; Alonso, María Pilar; Blanco, Jorge; Orden, José A

    2012-03-23

    The presence of 12 genes associated with virulence in human attaching and effacing Escherichia coli (AEEC) was studied within a collection of 20 enterohemorrhagic E. coli (EHEC) and 206 atypical enteropathogenic E. coli (EPEC) isolated from ruminants. In addition, virulence genes and the clonal relationship of 49 atypical EPEC O26 strains isolated from humans and ruminants were compared to clarify whether ruminants serve as a reservoir of atypical EPEC for humans. A great diversity in the content of virulence gene was found. Thus, the espH, espG and map genes were detected in more than 85% of ruminant AEEC strains; the tccP2, espI, efa1/lifA, ehxA and paa genes were present in 50-70% of strains; and other genes such as tccP, espP, katP and toxB were detected in <25% of strains. EHEC strains contained more virulence genes than atypical EPEC strains. Our results suggest for the first time that the efa1/lifA gene is associated with diarrhea in newborn ruminants and that the AEEC strains with the H11 flagellar antigen are potentially more virulent than the non-H11 AEEC strains. Importantly, we identified a new intimin variant gene, eaeρ, in three ruminant atypical EPEC strains. The comparison of ruminant and human EPEC O26 strains showed that some ruminant strains possess virulence gene profiles and pulse-field gel electrophoresis pulsotypes similar to those of human strains. In conclusion, our data suggest that atypical EPEC is a heterogeneous group with different pathogenic potential and that ruminants could serve as a reservoir of atypical EPEC for humans. PMID:21958746

  7. Diffusely Adhering Escherichia coli Strains Induce Attaching and Effacing Phenotypes and Secrete Homologs of Esp Proteins

    PubMed Central

    Beinke, Christina; Laarmann, Sven; Wachter, Clemens; Karch, Helge; Greune, Lilo; Schmidt, M. Alexander

    1998-01-01

    Recent epidemiological studies indicate that Escherichia coli strains which exhibit the diffuse-adherence phenotype (DAEC strains) represent a potential cause of diarrhea in infants. We investigated the interaction of DAEC strains isolated from diarrhea patients in Brazil and in Germany with epithelial cells in tissue culture. The investigated strains were identified as DAEC strains by (i) their attachment pattern, (ii) presence of genes associated with the Dr family of adhesins, and (iii) lack of genetic markers for other diarrhea-associated E. coli categories. Several clinical DAEC isolates were shown to secrete similar patterns of proteins into tissue culture medium. Protein secretion was found to be regulated by environmental parameters, namely, medium, temperature, pH, and iron concentration. DAEC strains secreting these proteins induced accumulation of actin and tyrosine-phosphorylated proteins at sites of bacterial attachment, leading to the formation of pedestals and/or extended surface structures. These changes were phenotypically similar to the attaching and effacing (A/E) lesions observed with enteropathogenic and some enterohemorrhagic E. coli strains carrying the locus of enterocyte effacement (LEE) pathogenicity island. Proteins homologous to the EspA, EspB, and EspD proteins, necessary for signal transduction events inducing A/E lesions, were identified by sequence analysis and cross-reaction of specific antibodies. However, initially nonadhering strains secreting these proteins induced signal transduction events only after prolonged infection. These results indicate that secretion of the Esp proteins alone is not sufficient for efficient signal transduction. This study further shows that some DAEC strains are likely to contain a homolog(s) of the LEE locus which may contribute to the pathogenic potential of DAEC. PMID:9453606

  8. High-level Multi-Resistant and Virulent Escherichia coli in Abeokuta, Nigeria.

    PubMed

    Akinduti, Paul Akinniyi; Aboderin, Bukola W; Oloyede, Rasaq; Ogiogwa, Joseph I; Motayo, Babatunde O; Ejilude, Oluwaseun

    2016-01-01

    Multi-resistant Escherichia coli (E. coli) strains co-harboring virulence genes is a cause of high morbidity in Abeokuta, Nigeria. This study was designed to determine some virulent factors among enteropathogenic E. coli in Abeokuta, Nigeria. Approximately non-repetitive 102 isolates of E. coli were recovered from clinical samples from two health facilities in Abeokuta. Biotyping using API and antibiotic susceptibility was determined, and eae and flic genes were assayed by PCR. Antibiotic resistance relatedness was performed by DendroUPGMA. Results showed that 48.0% and 52.0 % were intestinal and extra-intestinal E. coli, ampicillin recorded 100% resistance, amoxycilli/clavulanic acid 64.7%, cotrimoxazole 57.8% and 56.8% resistance against cefotaxime, at MIC >16 ug/mL, 100%, 57.8%, and 50% have MIC50 to ampicillin, tetracycline, and ceftazidime, while 74.5% and 48.0% have MIC90 to ampicillin and ceftazidime. Significant rates of 4.9%, 7.8%, and 9.8% flic, eae, and flic/eae genes were found in intestinal isolates, while 2.9%, 2.0%, and 3.9% were found in extra-intestinal (P < 0.05). Two major clades of the resistant isolates reveal significant antibiotic relatedness among intestinal and extra-intestinal isolates, at 54% resistance similarities with very high multi-antibiotic resistance index of 1.0 (MARI). A high rate of undetected virulent E. coli pathotypes with high resistance could trigger unprecedented morbidity and mortality, mostly among children and the elderly. PMID:26263915

  9. Biophysical Characterization and Activity of Lymphostatin, a Multifunctional Virulence Factor of Attaching and Effacing Escherichia coli.

    PubMed

    Cassady-Cain, Robin L; Blackburn, Elizabeth A; Alsarraf, Husam; Dedic, Emil; Bease, Andrew G; Böttcher, Bettina; Jørgensen, René; Wear, Martin; Stevens, Mark P

    2016-03-11

    Attaching and effacing Escherichia coli cause diarrhea and typically produce lymphostatin (LifA), an inhibitor of mitogen-activated proliferation of lymphocytes and pro-inflammatory cytokine synthesis. A near-identical factor (Efa1) has been reported to mediate adherence of E. coli to epithelial cells. An amino-terminal region of LifA shares homology with the catalytic domain of the large clostridial toxins, which are retaining glycosyltransferases with a DXD motif involved in binding of a metal ion. Understanding the mode(s) of action of lymphostatin has been constrained by difficulties obtaining a stably transformed plasmid expression clone. We constructed a tightly inducible clone of enteropathogenic E. coli O127:H6 lifA for affinity purification of lymphostatin. The purified protein inhibited mitogen-activated proliferation of bovine T lymphocytes in the femtomolar range. It is a monomer in solution and the molecular envelope was determined using both transmission electron microscopy and small-angle x-ray scattering. Domain architecture was further studied by limited proteolysis. The largest proteolytic fragment containing the putative glycosyltransferase domain was tested in isolation for activity against T cells, and was not sufficient for activity. Tryptophan fluorescence studies indicated thatlymphostatin binds uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) but not UDP-glucose (UDP-Glc). Substitution of the predicted DXD glycosyltransferase motif with alanine residues abolished UDP-GlcNAc binding and lymphostatin activity, although other biophysical properties were unchanged. The data indicate that lymphostatin has UDP-sugar binding potential that is critical for activity, and is a major leap toward identifying the nature and consequences of modifications of host cell factors. PMID:26786100

  10. Genetic Relatedness Among Escherichia coli Pathotypes Isolated from Food Products for Human Consumption in Cartagena, Colombia

    PubMed Central

    Amézquita-Montes, Zorangel; Tamborski, Maria; Kopsombut, Usa G.; Zhang, Chengxian; Arzuza, Octavio S.

    2015-01-01

    Abstract Foodborne pathogens are a leading cause of mild-to-severe gastrointestinal illnesses worldwide. Escherichia coli pathotypes have been known to cause gastrointestinal illnesses in children less than 5 years old in Colombia. However, insufficient information is available on the prevalence of E. coli contamination of food products and the kind of E. coli food product reservoirs. The two objectives of this study were designed to address this issue. The first objective was to ascertain coliform, E. coli, and pathogenic E. coli contamination of food products readily available for human consumption in Cartagena, Colombia. The second objective was to evaluate the relationship between pathogenic E. coli isolated from food products and those isolated from cases of diarrhea in children. Food product samples consisting of pasteurized milk, unpasteurized fruit juice, ground beef, cheese, and vegetables were obtained at four retail stores. The food samples were cultured in liquid media and tested for the presence of coliforms and E. coli. E. coli isolates were tested by polymerase chain reaction for the presence of pathogenic E. coli. Coliforms, E. coli, and E. coli intestinal pathotypes contamination were detected in 88.4%, 53%, and 2.1% of food product samples, respectively. Ground beef and cheese were the only food samples contaminated with E. coli intestinal pathotypes including enteropathogenic (EPEC), Shiga toxin–producing (STEC), and enterotoxigenic E. coli (ETEC). Closed multilocus sequencing typing relationships between diarrheagenic E. coli isolates from food products and from individuals with diarrhea suggest that food products readily available at public markets in Cartagena can transmit ETEC and possibly EPEC and STEC. We demonstrated that a high proportion of food products for human consumption available at public markets in Cartagena are contaminated with coliforms, E. coli, and E. coli intestinal pathogens. Furthermore, food products containing E. coli

  11. Characterization of Escherichia coli Isolates from Hospital Inpatients or Outpatients with Urinary Tract Infection

    PubMed Central

    Toval, Francisco; Köhler, Christian-Daniel; Vogel, Ulrich; Wagenlehner, Florian; Mellmann, Alexander; Fruth, Angelika; Schmidt, M. Alexander; Karch, Helge; Bielaszewska, Martina

    2014-01-01

    Uropathogenic Escherichia coli (UPEC) is the most common cause of community- and hospital-acquired urinary tract infections (UTIs). Isolates from uncomplicated community-acquired UTIs express a variety of virulence traits that promote the efficient colonization of the urinary tract. In contrast, nosocomial UTIs can be caused by E. coli strains that differ in their virulence traits from the community-acquired UTI isolates. UPEC virulence markers are used to distinguish these facultative extraintestinal pathogens, which belong to the intestinal flora of many healthy individuals, from intestinal pathogenic E. coli (IPEC). IPEC is a diarrheagenic pathogen with a characteristic virulence gene set that is absent in UPEC. Here, we characterized 265 isolates from patients with UTIs during inpatient or outpatient treatment at a hospital regarding their phylogenies and IPEC or UPEC virulence traits. Interestingly, 28 of these isolates (10.6%) carried typical IPEC virulence genes that are characteristic of enteroaggregative E. coli (EAEC), Shiga toxin-producing E. coli (STEC), and atypical enteropathogenic E. coli (aEPEC), although IPEC is not considered a uropathogen. Twenty-three isolates harbored the astA gene coding for the EAEC heat-stable enterotoxin 1 (EAST1), and most of them carried virulence genes that are characteristic of UPEC and/or EAEC. Our results indicate that UPEC isolates from hospital patients differ from archetypal community-acquired isolates from uncomplicated UTIs by their spectrum of virulence traits. They represent a diverse group, including EAEC, as well as other IPEC pathotypes, which in addition contain typical UPEC virulence genes. The combination of typical extraintestinal pathogenic E. coli (ExPEC) and IPEC virulence determinants in some isolates demonstrates the marked genome plasticity of E. coli and calls for a reevaluation of the strict pathotype classification of EAEC. PMID:24478469

  12. Diarrheagenic Escherichia coli: Prevalence and Pathotype Distribution in Children from Peruvian Rural Communities.

    PubMed

    Acosta, Gonzalo J; Vigo, Natalia I; Durand, David; Riveros, Maribel; Arango, Sara; Zambruni, Mara; Ochoa, Theresa J

    2016-09-01

    Diarrheagenic Escherichia coli (DEC) are common pathogens of childhood gastrointestinal infections worldwide. To date, research tracking DEC has mainly been completed in urban areas. This study aims to determine the prevalence and pathotype distribution of DEC strains in children from rural Peruvian communities and to establish their association with malnutrition. In this prospective cohort, 93 children aged 6-13 months from rural communities of Urubamba (Andes) and Moyobamba (jungle) were followed for 6 months. Diarrheal and control stool samples were analyzed using multiplex real-time polymerase chain reaction to identify the presence of virulence genes of DEC strains. The overall isolation rate of DEC was 43.0% (352/820). Enteroaggregative E. coli (EAEC, 20.4%), enteropathogenic E. coli (EPEC, 14.2%), and diffusely aggregative E. coli (DAEC, 11.0%) were the most prevalent pathotypes. EAEC was more frequently found in Moyobamba samples (P < 0.01). EPEC was the only strain significantly more frequent in diarrheal than asymptomatic control samples (P < 0.01). DEC strains were more prevalent among younger children (aged 6-12 months, P < 0.05). A decline in height-for-age Z-score (HAZ) was observed in 75.7% of children overall. EAEC was more frequently isolated among children who had a greater HAZ decline (P < 0.05). In conclusion, DEC strains were frequently found in stool samples from children in rural communities of the highlands and jungle of Peru. In addition, children with a greater decline in their growth rate had higher EAEC isolation rates, highlighting the importance of this pathogen in child malnutrition. PMID:27382080

  13. Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells.

    PubMed

    Liu, Hao-Yu; Roos, Stefan; Jonsson, Hans; Ahl, David; Dicksved, Johan; Lindberg, Jan Erik; Lundh, Torbjörn

    2015-04-01

    Heat shock proteins (HSPs) are a set of highly conserved proteins that can serve as intestinal gate keepers in gut homeostasis. Here, effects of a probiotic, Lactobacillus rhamnosus GG (LGG), and two novel porcine isolates, Lactobacillus johnsonii strain P47-HY and Lactobacillus reuteri strain P43-HUV, on cytoprotective HSP expression and gut barrier function, were investigated in a porcine IPEC-J2 intestinal epithelial cell line model. The IPEC-J2 cells polarized on a permeable filter exhibited villus-like cell phenotype with development of apical microvilli. Western blot analysis detected HSP expression in IPEC-J2 and revealed that L. johnsonii and L. reuteri strains were able to significantly induce HSP27, despite high basal expression in IPEC-J2, whereas LGG did not. For HSP72, only the supernatant of L. reuteri induced the expression, which was comparable to the heat shock treatment, which indicated that HSP72 expression was more stimulus specific. The protective effect of lactobacilli was further studied in IPEC-J2 under an enterotoxigenic Escherichia coli (ETEC) challenge. ETEC caused intestinal barrier destruction, as reflected by loss of cell-cell contact, reduced IPEC-J2 cell viability and transepithelial electrical resistance, and disruption of tight junction protein zonula occludens-1. In contrast, the L. reuteri treatment substantially counteracted these detrimental effects and preserved the barrier function. L. johnsonii and LGG a