Early Attachment Sites for Shiga-Toxigenic Escherichia coli O157:H7 in Experimentally Inoculated Weaned Calves

Weaned 3- to 4-month-old calves were fasted for 48 h, inoculated with 10¹⁰ CFU of Shiga toxin-positive Escherichia coli (STEC) O157:H7 strain 86-24 (STEC O157) or STEC O91:H21 strain B2F1 (STEC O91), Shiga toxin-negative E. coli O157:H7 strain 87-23 (Stx⁻ O157), or a nonpathogenic control E. coli strain, necropsied 4 days postinoculation, and examined bacteriologically and histologically. Some calves were treated with dexamethasone (DEX) for 5 days (3 days before, on the day of, and 1 day after inoculation). STEC O157 bacteria were recovered from feces, intestines, or gall bladders of 74% (40/55) of calves 4 days after they were inoculated with STEC O157. Colon and cecum were sites from which inoculum-type bacteria were most often recovered. Histologic lesions of attaching-and-effacing (A/E) O157⁺ bacteria were observed in 69% (38/55) of the STEC O157-inoculated calves. Rectum, ileocecal valve, and distal colon were sites most likely to contain A/E O157⁺ bacteria. Fecal and intestinal levels of STEC O157 bacteria were significantly higher and A/E O157⁺ bacteria were more common in DEX-treated calves than in nontreated calves inoculated with STEC O157. Fecal STEC O157 levels were significantly higher than Stx⁻ O157, STEC O91, or control E. coli; only STEC O157 cells were recovered from tissues. Identifying the rectum, ileocecal valve, and distal colon as early STEC O157 colonization sites and finding that DEX treatment enhances the susceptibility of weaned calves to STEC O157 colonization will facilitate the identification and evaluation of interventions aimed at reducing STEC O157 infection in cattle.     

Trichinella spiralis: the influence of short chain fatty acids on the proliferation of lymphocytes, the goblet cell count and apoptosis in the mouse intestine

This study was carried out to determine the influence of short chain fatty acids (SCFA) on spleen and mesenteric lymph node lymphocyte proliferation, goblet cells and apoptosis in the mouse small intestine during invasion by Trichinella spiralis. BALB/c mice were infected with 250 larvae of T. spiralis. An SCFA water solution containing acetic, propionic and butyric acids (30:15:20 mM) was administered orally starting 5 days before infection and ending 20 days post infection (dpi). Fragments of the jejunum were collected by dissection 7 and 10 dpi, and were examined for apoptotic cells in the lamina propria of the intestinal mucosa, and for goblet cells. The proliferation index of the cultured spleen and mesenteric lymph node lymphocytes with MTT test was also determined. The orally administered SCFA solution decreased the proliferation of mesenteric lymph node lymphocytes in the mice infected with T. spiralis at both examination times, but did not influence the proliferative activity of the spleen cells. Seven dpi, both in the spleen and mesenteric lymph nodes, the highest proliferation index of concanavalin A (Con A)-stimulated lymphocytes was found in the group of uninfected animals receiving SCFA animals. This tendency could still be seen 10 dpi in the mesenteric lymph nodes but not in the spleen, where the proliferation index in this group had significantly decreased. In vitro studies revealed, that butyric and propionic acids added to the cell cultures suppressed the proliferation of Con A-stimulated mesenteric lymph nodes and spleen lymphocytes taken from uninfected and T. spiralis-infected mice. Acetic acid stimulated proliferation of splenocytes taken from uninfected mice but did not affect lymphocyte proliferation in mesenteric lymph nodes from uninfected or infected mice. Orally administered SCFA increased the number of goblet cells found in the epithelium of the jejunum 7 dpi, but this number had decreased 10 dpi. The number of apoptotic cells in the lamina propria of the intestinal mucosa of animals infected with the T. spiralis and receiving SCFA was also lower, particularly 10 dpi. The above results show that SCFA can participate in the immune response during the course of trichinellosis in mice.     

Increased Intestinal Epithelial Cell Turnover and Intestinal Motility in Gymnophalloides seoi-Infected C57BL/6 Mice

The changing patterns of goblet cell hyperplasia, intestinal epithelial cell turnover, and intestinal motility were studied in ICR and C57BL/6 mice infected with Gymnophalloides seoi (Digenea: Gymnophallidae). Whereas ICR mice retained G. seoi worms until day 7 post-infection (PI), C57BL/6 mice showed a rapid worm expulsion within day 3 PI. Immunosuppression with Depo-Medrol significantly delayed the worm expulsion in C57BL/6 mice. Goblet cell counts were increased in both strains of mice, peaking at day 1 PI in C57BL/6 mice and slowly increasing until day 7 PI in ICR mice. In C57BL/6 mice infected with G. seoi, newly proliferating intestinal epithelial cells were remarkably increased in the crypt, and the increase was the highest at day 1 PI. However, in ICR mice, newly proliferating intestinal epithelial cells increased slowly from day 1 to day 7 PI. Intestinal motility was increased in G. seoi-infected mice, and its chronological pattern was highly correlated with the worm load in both strains of mice. Meanwhile, immunosuppression of C57BL/6 mice abrogated the goblet cell proliferation, reduced the epithelial cell proliferation, and suppressed the intestinal motility. Goblet cell hyperplasia, increased intestinal epithelial cell turnover, and increased intestinal motility should be important mucosal defense mechanisms in G. seoi-infected C57BL/6 mice.     

Presence and Characterization of Shiga Toxin-Producing Escherichia coli and Other Potentially Diarrheagenic E. coli Strains in Retail Meats

To determine the presence of Shiga toxin-producing Escherichia coli (STEC) and other potentially diarrheagenic E. coli strains in retail meats, 7,258 E. coli isolates collected by the U.S. National Antimicrobial Resistance Monitoring System (NARMS) retail meat program from 2002 to 2007 were screened for Shiga toxin genes. In addition, 1,275 of the E. coli isolates recovered in 2006 were examined for virulence genes specific for other diarrheagenic E. coli strains. Seventeen isolates (16 from ground beef and 1 from a pork chop) were positive for stx genes, including 5 positive for both stx₁ and stx₂, 2 positive for stx₁, and 10 positive for stx₂. The 17 STEC strains belonged to 10 serotypes: O83:H8, O8:H16, O15:H16, O15:H17, O88:H38, ONT:H51, ONT:H2, ONT:H10, ONT:H7, and ONT:H46. None of the STEC isolates contained eae, whereas seven carried enterohemorrhagic E. coli (EHEC) hlyA. All except one STEC isolate exhibited toxic effects on Vero cells. DNA sequence analysis showed that the stx₂ genes from five STEC isolates encoded mucus-activatable Stx2d. Subtyping of the 17 STEC isolates by pulsed-field gel electrophoresis (PFGE) yielded 14 distinct restriction patterns. Among the 1,275 isolates from 2006, 11 atypical enteropathogenic E. coli (EPEC) isolates were identified in addition to 3 STEC isolates. This study demonstrated that retail meats, mainly ground beef, were contaminated with diverse STEC strains. The presence of atypical EPEC strains in retail meat is also of concern due to their potential to cause human infections.Escherichia coli is an important component of the intestinal microflora of humans and warm-blooded mammals. While E. coli typically harmlessly colonizes the intestinal tract, several E. coli clones have evolved the ability to cause a variety of diseases within the intestinal tract and elsewhere in the host. Those strains that cause enteric infections are generally called diarrheagenic E. coli strains, and their pathogenesis is associated with a number of virulence attributes, which vary according to pathotype (54). Currently, diarrheagenic E. coli strains are classified into six main pathotypes based on their distinct virulence determinants and pathogenic features, including enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC)/Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusively adherent E. coli (DAEC) (37).Among diarrheagenic E. coli strains, STEC strains are distinguished by the ability to cause severe life-threatening complications, such as hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) (30). Other symptoms of STEC infection include watery diarrhea, bloody diarrhea, and hemorrhagic colitis (HC). STEC strains that cause HC and HUS are also called EHEC. Although individuals of all ages are at risk of STEC infection, children younger than 5 years of age and the elderly are more likely to suffer from severe complications (51). Outbreaks and sporadic cases of STEC infections have been reported frequently worldwide.The pathogenesis of STEC infection in humans is not fully understood. The major virulence factors implicated in STEC infection are potent Shiga toxins, which are classified into two groups: Stx1 and Stx2 (23). Additional factors that contribute to virulence have also been described, including intimin (encoded by the eae gene), an outer membrane protein involved in the attachment of E. coli to the enterocyte, and EHEC hemolysin (encoded by EHEC hlyA), which acts as a pore-forming cytolysin and causes damage to cells (41).The first STEC O157 infections were reported in 1982, when E. coli O157:H7 was involved in outbreaks associated with two fast food chain restaurants in the United States (44). Since then, ever-increasing numbers of cases and outbreaks due to STEC O157 have been reported worldwide. Although non-O157 STEC strains have also been associated with human cases and outbreaks, few laboratories have been looking for them, and their potential in causing human infections may be underestimated (2). Recently, though, the significance of non-O157 STEC strains as human pathogens has become more recognized. In the United States alone, there were 23 reported outbreaks of non-O157 STEC infection between 1990 and 2007 (10).Shiga toxin-producing E. coli can be transmitted through different routes, including food and water, person-to-person contact, and animal-to-person contact (9). Most human infections are caused by consumption of contaminated foods (16). Domestic and wild ruminant animals, in particular cattle, are considered the main reservoir of STEC and the main source for contamination of the food supply. Retail meats derived from animals could potentially act as transmission vehicles for STEC and other diarrheagenic E. coli strains. However, there is limited information about STEC contamination in retail meats, and fewer data exist about the presence of other diarrheagenic E. coli strains in retail meats. In the present study, we investigated 7,258 E. coli isolates from four types of meat samples (beef, chicken, pork, and turkey) collected during 2002 to 2007 to assess STEC contamination of retail meats. In addition, the presence of other potentially diarrheagenic E. coli strains was examined by detecting specific virulence determinants among E. coli isolates collected in 2006.     

Non-O157:H7 Shiga toxin (verocytotoxin)-producing Escherichia coli strains: epidemiological significance and microbiological diagnosis

Shiga toxin (Stx)-producing Escherichia coli (STEC) are important causes of diarrhoea and the haemolytic uremic syndrome (HUS). The most common STEC serotype implicated worldwide is E. coli O157:H7 that is diagnosed using procedures based on its typical phenotypic feature, the lack of sorbitol fermentation. In addition to E. coli O157:H7, a variety of non-O157:H7 STEC strains that usually ferment sorbitol and are thus missed by using the diagnostic protocol for E.coli O157:H7 have been isolated from patients. Among these sorbitol-fermenting (SF) non-O157:H7 STEC, SF E. coli O157:H⁻ and non-O157 STEC strains of serogroups O26, O103, O111 and O145 have emerged as significant causes of HUS and diarrhoea in continental Europe and have been associated with human disease in other parts of the world. Microbiological diagnosis of non-O157:H7 STEC strains is difficult due to their serotype diversity and the absence of a simple biochemical property that distinguishes such strains from the physiological intestinal microflora. Screening for non-O157:H7 STEC and their isolation from stools is presently based on the detection of Stx production or stx genes that are common characteristics of such strains. Molecular subtyping of the most frequent non-O157 STEC demonstrated that strains of serogroups O26, O103 and O111 belong to their own clonal lineages and show unique virulence profiles. SF STEC O157:H⁻ strains that have been isolated mostly in Central Europe represent a new clone within E. coli O157 serogroup which has its own typical combination of virulence factors. This revised version was published online in November 2006 with corrections to the Cover Date.     

Serotypes,intimin variants and other virulence factors of <Emphasis Type="Italic">eae</Emphasis> positive <Emphasis Type="Italic">Escherichia coli</Emphasis> strains isolated from healthy cattle in Switzerland. Identification of a new intimin variant gene (eae-η2)

Background  

Enteropathogenic Escherichia coli (EPEC) and Shigatoxin-producing Escherichia coli (STEC) share the ability to introduce attaching-and-effacing (A/E) lesions on intestinal cells. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports information on the occurrence of eae positive E. coli carried by healthy cattle at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated EPEC and STEC strains.     

Quantitative and qualitative evaluation of iNOS expression in turbot (Psetta maxima) infected with Enteromyxum scophthalmi

Enteromyxum scophthalmi is the causative agent of turbot enteromyxosis, an intestinal parasitisation that produces severe desquamative enteritis leading to a cachectic syndrome and eventually the death. It is well known the importance of the innate immune response against parasites in fish, with the release of antimicrobial substances such as reactive oxygen and nitrogen species, produced by the inducible nitric oxide synthase (iNOS). This enzyme is mainly found in phagocytes, but also in structural cells from the intestinal mucosa. The aim of this study was to characterize iNOS in intestine and lymphohaematopoietic organs (spleen and anterior kidney) of turbot by means of immunohistochemistry in order to assess the possible changes of this enzyme through the infection. The presence of the enzyme was evaluated in control and E. scophthalmi-infected turbot. The results showed immunoreactivity in the apical border of enterocytes and mild staining of goblet cells in both control and infected turbot although it was more evident and widespread in infected turbot compared to control. Moderate numbers of iNOS+ cells were present in the lamina propria-submucosa of fish which presented moderate and severe inflammatory infiltrates at this level. In spleen and kidney, iNOS+ cells were scattered through the parenchyma and, in severely infected fish, tended to be allocated near the vascular structures and melano-macrophage centres. The number of positive cells at the lymphohaematopoietic organs was significantly higher in infected turbot and increased as infection progressed. The increase in the expression of iNOS in the tissues of E. scophthalmi-infected turbot was more evident in individuals with severer lesions. The measurement of the levels of iNOS during turbot enteromyxosis reveals a possibly delayed response that would not able to eliminate the parasites but would exacerbate mucosal injury.     

Isolation and Characterization of a New T-Even Bacteriophage, CEV1, and Determination of Its Potential To Reduce Escherichia coli O157:H7 Levels in Sheep

Bacteriophage CEV1 was isolated from sheep resistant to Escherichia coli O157:H7 colonization. In vitro, CEV1 efficiently infected E. coli O157:H7 grown both aerobically and anaerobically. In vivo, sheep receiving a single oral dose of CEV1 showed a 2-log-unit reduction in intestinal E. coli O157:H7 levels within 2 days compared to levels in the controls.     

Escherichia coli O157:H7 Infection in Dutch Belted and New Zealand White Rabbits

Enterohemorrhagic Escherichia coli (EHEC) produce one or more types of Shiga toxins and are foodborne causes of bloody diarrhea. The prototype EHEC strain, Escherichia coli O157:H7, is responsible for both sporadic cases and serious outbreaks worldwide. Infection with E. coli that produce Shiga toxins may lead to diarrhea, hemorrhagic colitis, or (less frequently) hemolytic uremic syndrome, which can cause acute kidney failure. The exact mechanism by which EHEC evokes intestinal and renal disease has not yet been determined. The development of a readily reproducible animal oral-infection model with which to evaluate the full pathogenic potential of E. coli O157:H7 and assess the efficacy of therapeutics and vaccines remains a research priority. Dutch belted (DB) rabbits are reported to be susceptible to both natural and experimental EHEC-induced disease, and New Zealand white (NZW) rabbits are a model for the intestinal manifestations of EHEC infection. In the current study, we compared the pathology caused by E. coli O157:H7 infection in DB and NZW rabbits. Both breeds of rabbits developed clinical signs of disease and intestinal lesions after experimental infection. In addition, one of the infected DB rabbits developed renal lesions. Our findings provide evidence that both breeds are susceptible to E. coli O157:H7 infection and that both may be useful models for investigating EHEC infections of humans.Abbreviations: EHEC, enterohemorrhagic E. coli; HUS, hemolytic uremic syndrome; DB, Dutch belted; STEC, Shiga-toxin– producing E. coli; NZW, New Zealand whiteEscherichia coli O157:H7 is the prototype enterohemorrhagic strain of Shiga-toxin–producing E. coli (STEC), which cause food and waterborne outbreaks and sporadic cases of serious intestinal disease that manifest as diarrhea or hemorrhagic colitis (or both).^12,13,31 Enterohemorrhagic E. coli (EHEC) are a subset of STEC that, in addition to elaborating Shiga toxins, encode the locus of enterocyte effacement, whose products allow intimate attachment of the bacteria to the epithelium.^16,19 Children infected with STEC are more susceptible than adults and may subsequently develop hemolytic uremic syndrome (HUS) that is characterized by hemolytic anemia, thrombocytopenia, and kidney dysfunction or failure.²⁹ Shiga toxins are considered to be major determinants involved in the pathogenesis of these E. coli-induced infections. Indeed, the capacity of STEC to cause bloody diarrhea and HUS derives from the activity of the Stx.^8,25,30,40 The 2 types of Shiga toxins, Stx1 and Stx2, are quite similar in sequence and structure, although their polyclonal antisera do not crossreact.^7,38,39,42A vaccine is currently not available to protect humans from infection or disease caused by STEC. There is a need to define the pathogenic mechanisms by which STEC cause disease and to develop strategies for the prevention and treatment of STEC-mediated HUS. Achieving this goal would benefit from a small animal model that displays gastroenteritis or signs of HUS similar to those occurring in humans. Naturally infected DB rabbits mimic the clinical and pathologic signs (including diarrhea, hemorrhagic colitis, and HUS) produced by STEC in humans.¹¹ In addition, infant NZW rabbits become infected with EHEC and subsequently exhibit diarrhea and hemorrhagic colitis.^20,28,34,36 The current study compared DB and NZW rabbits for breed-specific differences in response to E. coli O157:H7 infection.     

Application of Bacteriophages To Control Intestinal Escherichia coli O157:H7 Levels in Ruminants

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios ≥10² terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10¹⁰ PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be ≥10². In addition, phages were maintained at 10⁶ PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.     

Mucosal Immune Responses of Mice Experimentally Infected with Pygidiopsis summa (Trematoda: Heterophyidae)

Mucosal immune responses against Pygidiopsis summa (Trematoda: Heterophyidae) infection were studied in ICR mice. Experimental groups consisted of group 1 (uninfected controls), group 2 (infection with 200 metacercariae), and group 3 (immunosuppression with Depo-Medrol and infection with 200 metacercariae). Worms were recovered in the small intestine at days 1, 3, 5, and 7 post-infection (PI). Intestinal intraepithelial lymphocytes (IEL), mast cells, and goblet cells were counted in intestinal tissue sections stained with Giemsa, astra-blue, and periodic acid-Schiff, respectively. Mucosal IgA levels were measured by ELISA. Expulsion of P. summa from the mouse intestine began to occur from days 3-5 PI which sustained until day 7 PI. The worm expulsion was positively correlated with proliferation of IEL, mast cells, goblet cells, and increase of IgA, although in the case of mast cells significant increase was seen only at day 7 PI. Immunosuppression suppressed all these immune effectors and inhibited worm reduction in the intestine until day 7 PI. The results suggested that various immune effectors which include IEL, goblet cells, mast cells, and IgA play roles in regulating the intestinal mucosal immunity of ICR mice against P. summa infection.     

Characterizing the Escherichia coli O157:H7 proteome including protein associations with higher order assemblies

Background

The recent outbreak of severe infections with Shiga toxin (Stx) producing Escherichia coli (STEC) serotype O104:H4 highlights the need to understand horizontal gene transfer among E. coli strains, identify novel virulence factors and elucidate their pathogenesis. Quantitative shotgun proteomics can contribute to such objectives, allowing insights into the part of the genome translated into proteins and the connectivity of biochemical pathways and higher order assemblies of proteins at the subcellular level.

Methodology/Principal Findings

We examined protein profiles in cell lysate fractions of STEC strain 86-24 (serotype O157:H7), following growth in cell culture or bacterial isolation from intestines of infected piglets, in the context of functionally and structurally characterized biochemical pathways of E. coli. Protein solubilization in the presence of Triton X-100, EDTA and high salt was followed by size exclusion chromatography into the approximate M_r ranges greater than 280 kDa, 280-80 kDa and 80-10 kDa. Peptide mixtures resulting from these and the insoluble fraction were analyzed by quantitative 2D-LC-nESI-MS/MS. Of the 2521 proteins identified at a 1% false discovery rate, representing 47% of all predicted E. coli O157:H7 gene products, the majority of integral membrane proteins were enriched in the high M_r fraction. Hundreds of proteins were enriched in a M_r range higher than that predicted for a monomer supporting their participation in protein complexes. The insoluble STEC fraction revealed enrichment of aggregation-prone proteins, including many that are part of large structure/function entities such as the ribosome, cytoskeleton and O-antigen biosynthesis cluster.

Significance

Nearly all E. coli O157:H7 proteins encoded by prophage regions were expressed at low abundance levels or not detected. Comparative quantitative analyses of proteins from distinct cell lysate fractions allowed us to associate uncharacterized proteins with membrane attachment, potential participation in stable protein complexes, and susceptibility to aggregation as part of larger structural assemblies.     

Carbohydrate patterns in the digestive tract of Sparus aurata L. and Psetta maxima (L.) (Teleostei) parasitized by Enteromyxum leei and E. scophthalmi (Myxozoa)

The influence of Enteromyxum spp. infections on the carbohydrate patterns of the digestive tract of gilthead sea bream (GSB) Sparus aurata L. and turbot (TB) Psetta maxima (L.) has been studied. Histochemical stainings to differentiate the types of mucins and lectin-binding assays to detect terminal carbohydrate residues were applied to histological sections of GSB and TB uninfected or infected by Enteromyxum leei and E. scophthalmi, respectively. The number of intestinal GC decreased in severely infected fish in both parasitoses, though changes in mucin patterns were limited to the decrease in the staining intensity for acidic mucins in infected GSB. The TB stomach and intestine lacked histochemically detectable acidic mucins, or sialic acid detectable by SNA, in contrast with their abundance in GSB. Glucose/mannose, fucose and GlcNAc residues were less abundant in both infected hosts with respect to uninfected fish. In contrast, D-Gal and D-GalNAc moieties (detectable by BSL I) increased in most parts of E. scophthalmi-infected TB while decreasing (oesophagus) or remaining unchanged (intestine) in E. leei-infected GSB. The decreasing in the expression of acidic mucins and of sialic acid detectable by SNA in E. leei-infected GSB is remarkable. Differences in the carbohydrate patterns between both hosts could aid to explain the differences in the severity of both enteromyxoses. In addition, the changes induced by Enteromyxum spp. infections in the digestive tract of GSB and TB suggest a role of terminal carbohydrate residues in the parasite–host interaction.     

Persistent Colonization of Sheep by Escherichia coli O157:H7 and Other E. coli Pathotypes

Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness in humans. Ruminants appear to be more frequently colonized by STEC than are other animals, but the reason(s) for this is unknown. We compared the frequency, magnitude, duration, and transmissibility of colonization of sheep by E. coli O157:H7 to that by other pathotypes of E. coli. Young adult sheep were simultaneously inoculated with a cocktail consisting of two strains of E. coli O157:H7, two strains of enterotoxigenic E. coli (ETEC), and one strain of enteropathogenic E. coli. Both STEC strains and ETEC 2041 were given at either 10⁷ or 10¹⁰ CFU/strain/animal. The other strains were given only at 10¹⁰ CFU/strain. We found no consistent differences among pathotypes in the frequency, magnitude, and transmissibility of colonization. However, the STEC strains tended to persist to 2 weeks and 2 months postinoculation more frequently than did the other pathotypes. The tendency for persistence of the STEC strains was apparent following an inoculation dose of either 10⁷ or 10¹⁰ CFU. One of the ETEC strains also persisted when inoculated at 10¹⁰ CFU. However, in contrast to the STEC strains, it did not persist when inoculated at 10⁷ CFU. These results support the hypothesis that STEC is better adapted to persist in the alimentary tracts of sheep than are other pathotypes of E. coli.     

Prevalence of Carriage of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 among Slaughtered Adult Cattle in France

The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene (eae). The objective of this study was to evaluate the prevalence of bovine carriers of these “top five” STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples. O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls (P < 0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover, simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.     

STAT6 Expression and IL-13 Production in Association with Goblet Cell Hyperplasia and Worm Expulsion of Gymnophalloides seoi from C57BL/6 Mice

In intestinal helminth infections, Th2 immune respones are generally associated with mucin secretion for worm expulsion from the host intestine. In particular, IL-4 and IL-13 are the important cytokines related with intestinal mucus production via STAT6 signalling in nematode infections. However, this perspective has never been studied in Gymnophalloides seoi infection. The present study aimed to observe the STAT6 signalling and cytokine responses in C57BL/6 mice, a mouse strain resistant to infection with this trematode. The results showed that worm expulsion occurred actively during days 1-2 post-infection (PI), when goblet cells began to proliferate in the small intestine. The STAT6 gene expression in the mouse spleen became remarkable from day 2 PI. Moreover, G. seoi infection induced a significant increase of IL-13 from day 4 PI in the spleen of infected mice. Our results suggested that goblet cell hyperplasia and worm expulsion in G. seoi-infected mice should be induced by STAT6 signalling, in which IL-13 may be involved as a dominant triggering cytokine.     

Heligmosomoides polygyrus: Simple recovery of post-infective larvae from mouse intestines

Mice infected orally with third-stage larvae of Heligmosomoides polygyrus were killed at various times after infection. Their small intestines were removed, tied at each end and incubated at 37 C in dilute culture medium. When intestines were taken from mice infected for a period of between 1 and 7 days, a number of developing larvae comprising up to 20% of the infective dose emerged within 60 min through the intestinal wall into the medium. The recovery of emergent larvae was highest using intestines from mice infected 36 to 120 hr previously. The proportion of parasites emerging from the intestines of 48-hr-infected mice was similar for doses of 100 to 2400 larvae. Significantly fewer larvae emerged from the intestines of mice resistant to reinfection and challenged with third-stage larvae 36–72 hr before necropsy.     

Restoration of anthropometric, biochemical and histopathological alterations by Lactobacillus casei supplementation in Giardia intestinalis infected renourished BALB/c mice

The present study describes the in vivo ameliorating effect of Lactobacillus casei supplementation in renourished Giardia intestinalis infected BALB/c mice. It was observed that daily administration of probiotic 7 days prior to Giardia-infection to renourished mice, efficiently reduced the excretion of Giardia cysts and trophozoite counts, along with significant increased fecal lactobacilli counts compared with Giardia-infected mice. It was also observed that oral feeding of probiotic to renourished-Giardia-infected mice abrogated all the anthropometric and biochemical anomalies. Histologically, morphological and cellular alteration of microvillus membrane integrity revealed that probiotic administration further ameliorated the mucosal damage in renourished-probiotic-Giardia-infected mice compared to severe microvillus atrophy, oedematous, vacuolated epithelial cells and ileitis in renourished-Giardia and Giardia-infected mice. Thus, it is suggested that probiotic used as the functional food helps in restoration of anthropometric, biochemical alterations and atrophied gut by enhancing the goblet cells and reducing the giardiasis.     

Requirement of Purine and Pyrimidine Synthesis for Colonization of the Mouse Intestine by Escherichia coli

To study the adaptation of an intestinal bacterium to its natural environment, germfree mice were associated with commensal Escherichia coli MG1655. Two-dimensional gel electrophoresis was used to identify proteins differentially expressed in E. coli MG1655 collected from either cecal contents or anaerobic in vitro cultures. Fourteen differentially expressed proteins (>3-fold; P < 0.05) were identified, nine of which were upregulated in cecal versus in vitro-grown E. coli. Four of these proteins were investigated further for their role in gut colonization. After deletion of the corresponding genes, the resulting E. coli mutants were tested for their ability to colonize the intestines of gnotobiotic mice in competition with the wild-type strain. A mutant devoid of ydjG, which encodes a putative NADH-dependent methylglyoxal reductase, reached a 1.2-log-lower cecal concentration than the wild type. Deletion of the nanA gene encoding N-acetylneuraminate lyase affected the colonization and persistence of E. coli in the intestines of the gnotobiotic mice only slightly. A mutant devoid of 5′-phosphoribosyl 4-(N-succinocarboxamide)-5-aminoimidazole synthase, a key enzyme of purine synthesis, displayed intestinal cell counts >4 logs lower than those of the wild type. Deletion of the gene encoding aspartate carbamoyltransferase, a key enzyme of pyrimidine synthesis, even resulted in the washout of the corresponding mutant from the mouse intestinal tract. These findings indicate that E. coli needs to synthesize purines and pyrimidines to successfully colonize the mouse intestine.The human gastrointestinal tract harbors a complex community of approximately 10¹⁴ microorganisms (11). Whereas the impact of intestinal bacteria on the host has been studied in detail, knowledge about the impact of host factors on gut microorganisms is still limited. Only a few proteomic studies have investigated the response of bacteria to the intestinal environment. The application of proteome analysis to the infant fecal ecosystem was hampered by the complexity of the microbial community: only 1 of 11 determined peptide sequences could be linked to an enzyme, the bifidobacterial transaldolase (8). In contrast, several metabolic pathways involved in carbon assimilation were demonstrated to be upregulated in Lactococcus lactis when investigated with mice monoassociated with this organism. YwcC, a phosphogluconolactonase probably involved in the pentose phosphate pathway, was found to be essential for the organism''s ability to colonize the mouse intestinal tract (15). Bifidobacterium longum was reported to express, after brief exposure to the gastrointestinal environment, several sets of proteins, including adhesion factors and metabolic genes (18). Comparative proteome analysis of Escherichia coli grown in vitro on LB or in the intestines of monoassociated mice indicated that E. coli utilizes a wider range of substrates in the mouse intestine than under in vitro conditions and is exposed to various forms of stress, including starvation (2).The present study was aimed at the identification of proteins essential for the colonization and persistence of E. coli in the gastrointestinal environment. The well-characterized nonpathogenic E. coli strain MG1655 was grown in vitro and its proteome compared with that of the same strain isolated from the ceca of monoassociated mice. To reduce the differences in nutrient availability between the in vivo and the in vitro situation, the in vitro cultures were grown anaerobically on a broth prepared from the mouse chow. Selected bacterial proteins undergoing the most prominent upregulation in the mouse cecum were identified and tested for their possible role in colonization. For that purpose, targeted deletion mutants were tested in competition with the wild-type (WT) strain with respect to their ability to successfully colonize the germfree mouse intestine. The results suggest that E. coli needs to synthesize purines and pyrimidines to colonize and persist in the mouse intestine.     

Development of microsporidia-infected Muscidifurax raptor (Hymenoptera: Pteromalidae) at different temperatures

Muscididfurax raptor, a pupal parasitoid of house flies and other filth flies, is commonly infected with the microsporidium Nosema muscidifuracis. To determine the effects of infection on developmental time, uninfected and infected adult M. raptor were allowed to parasitize pupae of the house fly (Musca domestica) for 24 h. Exposed pupae of the two groups (infected and uninfected) were held at 15, 20, 25, 30, 32, and 34 °C with 75–80% relative humidity. Development of infected M. raptor was significantly longer at all temperatures than that of uninfected parasitoids, resulting in approximately 7% extensions of developmental times. Uninfected females completed development in 14.6, 19.6, and 30.4 days at 30, 25, and 20 °C, respectively, compared with 15.8, 20.7, and 32.3 days for infected females at these temperatures. The differences in developmental times provided narrow windows for isolating large proportions of uninfected M. raptor females for disease management programs. This window was greatest at 20 °C; 61% of the uninfected females emerged by day 30, at which time only 10% of the infected females had emerged.