<p>Escherichia coli is a natural inhabitant of the intestines of both humans and animals, but there are also several pathogenic types of E. coli which cause disease in humans.</p><p>Strains of enterohemorrhagic E. coli (EHEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of shigatoxin 1 and 2 or combination of these toxins. Other major virulence factors include EHEC hemolysin and intimin, the product of the eae gene that is involved in attaching and effacing adherence phenotype. EHEC has also been associated with uncomplicated diarrhea.</p><p>The capacity to control EHEC disease and to limit the scale of outbreaks is dependent upon prompt diagnosis and identification of the source of infection.</p><p>The principal reservoirs of EHEC are cattle and food products, which presumably have come into contact with domestic animal manure and/or are inadequately pasteurised, these are important vehicles of infection.</p><p>In the present study, the PCR technique with primers detecting the verocytotoxin genes was shown to be a possible method to screen for and identify EHEC.</p><p>In summary stx genes were detected in 16 samples of 228 sampels and the eae gene was detected in 2 samples using PCR.</p>

The purpose of this study was to investigate the occurrence of Verotoxin-encoding bacteriophages in mussels, cultured downstream the sewage treatment plant in Lysekil. Mussels were collected in three growing areas from April 2008 to March 2009. Real-time PCR was performed for detection of vtx1 and vtx2 genes and enrichment of bacteriophages on non Verotoxin-producing Escherichia coli O157: H7 was carried out. All samples in real-time PCR analysis were negative; no presence of Verotoxin-encoding phages was shown. No plaque was formed on blood agar base plates, indicating that no bacteriophages had been taken up by E. coli bacteria The levels of Verotoxin-encoding phages and E.coli outside the sewage treatment plant in Lysekil were not high enough to be able to form VTEC in mussels, indicating that the faecal contamination was low. This does not exclude the presence of other more common pathogens such as norovirus and campylobacter.

Escherichia coli is a natural inhabitant of the intestines of both humans and animals, but there are also several pathogenic types of E. coli which cause disease in humans. Strains of enterohemorrhagic E. coli (EHEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of shigatoxin 1 and 2 or combination of these toxins. Other major virulence factors include EHEC hemolysin and intimin, the product of the eae gene that is involved in attaching and effacing adherence phenotype. EHEC has also been associated with uncomplicated diarrhea. The capacity to control EHEC disease and to limit the scale of outbreaks is dependent upon prompt diagnosis and identification of the source of infection. The principal reservoirs of EHEC are cattle and food products, which presumably have come into contact with domestic animal manure and/or are inadequately pasteurised, these are important vehicles of infection. In the present study, the PCR technique with primers detecting the verocytotoxin genes was shown to be a possible method to screen for and identify EHEC. In summary stx genes were detected in 16 samples of 228 sampels and the eae gene was detected in 2 samples using PCR.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. <i>Escherichia coli</i> which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49].<p> Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy.<p> To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyers patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation <i>in vitro</i>. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2.<p> To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. <i>Escherichia coli</i> which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49].<p> Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy.<p> To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyers patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation <i>in vitro</i>. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2.<p> To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

The beef industry has made tremendous strides in reducing pathogen contamination on carcasses. Multiple antimicrobial interventions have been validated for their use during harvesting. Information in regards to cross-contamination with pathogens in the post-harvest environment is limited. Surrogate microorganisms for enteric pathogens are commonly used to validate antimicrobial interventions and might allow for the simulation of cross-contamination through the post-harvest environment. The purpose of this study was to determine how the post-harvest environment impacts the direct and indirect transmission of pathogens. This was achieved by using fluorescent protein-marked surrogate strains of Escherichia coli O157:H7 and Salmonella spp. from inoculated carcasses to the adjacent ones and to the equipment and facility in three different abattoirs. Thirteen hide-on carcasses were inoculated using a gelatin-based slurry containing three nonpathogenic fluorescent protein-marked strains of E. coli biotype I. In order to determine direct and indirect cross-contamination, inoculated and adjacent carcasses were sampled (300 cm2) during the harvesting process at different stages: after hide opening (AHO), prior to evisceration (PE), after evisceration (AE), after splitting (AS), and after final intervention (AFI). Environmental samples consisting of the floor, walls, and air were tested as well as personal equipment including gloves, boots, and aprons. Equipment including hand knives, air knives, meat hooks, hide puller and split saw were also sampled. Results showed evidence of cross-contamination between inoculated carcasses and the adjacent non-inoculated ones for all abattoirs. Although this occurred in all abattoirs, surrogate counts on carcasses were below detectable levels (<1.4 log CFU/cm2) after antimicrobial interventions. Surrogates were found in low levels for all environmental samples. However surrogate counts from equipment such as knives, split saws, meat hooks, and hide puller were more frequently detected (15 percent) than those found on the floor, air and walls samples (10 percent). In the case of aprons, boots, and gloves, the prevalence of countable surrogate samples was 7 percent.

This study was conducted to determine the effectiveness of trimming the original external carcass surfaces from subprimals during fabrication on the reduction of surrogates for Escherichia coli O157:H7. Carcass sides from five cattle (n = 10 sides) were inoculated along the pattern hide opening before entering the blast chill cooler with a gelatin slurry containing a bacterial cocktail of three rifampicin-resistant, nonpathogenic E. coli Biotype I strains. Following a 48 h chill, sides were fabricated to produce eight subprimals (brisket, chuck, clod, rib, bottom round, top sirloin, short loin, and inside round). Microbiological samples were taken from the original carcass fat surface area, initial lean surface area, trimmed fat surface area (where applicable), and trimmed lean surface area (where applicable). Trimming of the external fat surfaces reduced (P < 0.05) microbiological counts on the newly exposed lean surfaces of all eight subprimals during fabrication. However, these data also indicated that fat and lean surfaces that were not initially exposed to contamination became contaminated during the fabrication process. Trimming external surfaces reduces levels of pathogens, but under normal fabrication processes, pathogens may still be spread to the newly exposed surfaces.

Effects of a 10% xylitol solution (X) on adhesion of Escherichia coli O157:H7 and Salmonella serotype Typhimurium to meat surfaces were examined utilizing three approaches. In Experiment 1, rifampicin-resistant strains of E. coli O157:H7 and S. Typhimurium were dispersed in xylitol or a peptone solution (containing approximately 8.9 mean log per ml of each pathogen) and used to inoculate beef outside round meat surfaces. Samples were then rinsed with water or not rinsed in a 2X2 factorial arrangement. No interaction existed between inoculum type and post-inoculation treatments (P > 0.84). Incubation of pathogens in peptone or xylitol had minimal impact on pathogen adhesion (P > 0.76). Rinsing reduced counts by approximately 0.5 log CFU/cm2 (P < 0.01). Experiment 2 meat samples received a pretreatment of a water rinse, xylitol, or no rinse, followed by inoculation with pathogens dispersed in peptone solution (containing approximately 8.6 log mean log per ml of each pathogen). Samples received a post-inoculation treatment of a water rinse, xylitol rinse or no rinse in a 3X3 factorial arrangement. No interactions between pre- and post-inoculation factors were observed for surface pathogen load (P > 0.50). Post-inoculation rinsing reduced counts by approximately 0.5 log CFU/cm2 (P < 0.01) with no difference between water and xylitol (P > 0.64). Experiment 3 carcass surfaces were inoculated with pathogens at an initial level of 5.5 log CFU/cm2 and received a hot (35°C) water wash, 2.5% L-lactic acid spray, 10% xylitol spray, lactic acid + xylitol or hot water + xylitol. Pathogen counts were taken at 0 and 24 h post treatment. Lactic acid treatments reduced Salmonella by 3.3 log CFU/cm2 at 0 h (P < 0.01) and by 2.6 log CFU/cm2 after 24 h (P < 0.02). Hot water treatments reduced Salmonella by 1.5 log CFU/cm2 at 0 h (P < 0.07). Xylitol did not minimize pathogens (P > 0.62) nor did it increase effectiveness of other treatments. These data indicate that xylitol is ineffective at preventing E. coli O157:H7 and S. Typhimurium adhesion to meat surfaces.

Cell-to-cell communication, termed quorum sensing, mediated by AI-2 like activity, has been reported to regulate the expression of a variety of genes in E. coli O157:H7. A previous study in our laboratory has shown that foods can contain compounds that can interfere with AI-2 signaling. The underlying hypothesis of our studies is that the autoinducer molecules such as AI-2 are involved in the virulence and survival of enteric bacterial pathogens on food and food ingredients. The influence of AI-2 like activity on the survival and expression of virulence genes (hha and yadK) in E.coli O157:H7 was studied when the organism was stored in different types of ground beef extracts such as: cooked, uncooked, and autoclaved. The survival was observed at refrigeration temperature, while change in gene expression was studied using real-time PCR. Higher survival was observed in the cell exposed to cell free supernatant (CFS) containing AI-2 like molecules, compared to the one which was exposed to heat degraded AI-2 like molecules. The survival of cells was higher when exposed to cooked ground beef extracts compared to uncooked and autoclaved ground beef extracts. Similarly, higher gene expressions of both hha and yadK genes were observed in cells that were exposed to cooked beef extract samples as compared to samples that wereuncooked or autoclaved. About a 2 fold higher gene expression for both hha and yadK gene was observed when cells were subjected to cooked ground beef extracts in the presence of AI-2 like molecules compared to the ones exposed to uncooked ground beef extracts in the presence of AI-2 like molecules. Likewise, 3-fold higher gene expression was observed for cells exposed to cooked ground beef extracts compare to autoclaved ground beef extracts in the presence of AI-2 like molecules. The results suggest that the survival and virulence of enteric bacterial pathogens such as E.coli O157:H7 can be influenced by the interaction of food components and autoinducers such as AI-2, that are involved in bacterial cell communications.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. Escherichia coli which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49]. Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy. To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyer’s patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation in vitro. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2. To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.