Proliferative Enterocolitis Associated with Dual Infection with Enteropathogenic Escherichia coli and Lawsonia intracellularis in Rabbits

Both enteropathogenic Escherichia coli (EPEC) and an obligate intracellular bacterium, previously referred to as an intracellular Campylobacter-like organism and now designated Lawsonia intracellularis, have been reported as causes of enterocolitis in rabbits. An outbreak of enterocolitis in a group of rabbits, characterized by an unusually high rate of mortality, was found to be associated with dual infection with EPEC and L. intracellularis. The EPEC strain was found to have eaeA gene homology but was negative for afrA homology. The absence of the afrA gene, which encodes the structural subunit for the AF/R1 pilus, indicates that this rabbit EPEC strain is distinct from the prototypic RDEC-1 strain. This finding suggests that rabbit EPEC strains widely reported in Western Europe, which lack AF/R1 pili, are also present in rabbits in the United States. Dual infection with these two pathogens in rabbits has not been previously reported and may have contributed to the unusually high mortality observed in this outbreak.     

Enteropathogenic Escherichia coli in Psittaciformes

A total of 103 Escherichia coli isolates from psittaciform birds were examined for the presence of genes coding for shigatoxin 1 (Stx1), shigatoxin 2 (Stx2) and for intimin (eae), using the polymerase chain reaction (PCR). Sixty-eight E. coli strains were isolated from necropsy cases and faecal samples, the other 35 were from 205 cloacal swabs from Psittaciformes with various conditions. All isolates were tested for enterohaemorrhagic E. coli-haemolysin (HlyEHEC), some also for Stx production, but there was no geno-typic or phenotypic evidence of Stx in any of them. Seven isolates, six from birds with diarrhoea, harboured the eae gene, three of them belonging to the O110:H6 serotype, one each to serotypes O153:H10, O131:H-, O63:H6 and Osp:H6. These seven eae-positive strains were negative for shigatoxin and HlyEHEC, and the hlyEHEC gene was not detectable by PCR. However, a PCR amplifying the enteropathogenic E. coli (EPEC)-specific bundle-forming pili structural gene bfpA detected four bfpA positive strains (three of serotype O110:H6, one O131:H-) among the seven eae positive strains, which classifies them as EPEC. Our findings suggest that shigatoxin-producing E. coli are uncommon, but that EPEC should be considered as potential pathogens in psittaciform birds, which may be a source of human EPEC infections.     

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

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.     

Effect of Prior Experimental Human Enteropathogenic Escherichia coli Infection on Illness following Homologous and Heterologous Rechallenge

Two studies of adult volunteers were performed to determine whether prior enteropathogenic Escherichia coli (EPEC) infection confers protective immunity against rechallenge. In the first study, a naive control group and volunteers who had previously ingested an O55:H6 strain were fed an O127:H6 strain. In the second study, a control group and volunteers who had previously ingested either the O127:H6 strain or an isogenic eae deletion mutant of that strain were challenged with the homologous wild-type strain. There was no significant effect of prior infection on the incidence of diarrhea in either study. However, in the homologous-rechallenge study, disease was significantly milder in the group previously challenged with the wild-type strain. Disease severity was inversely correlated with the level of prechallenge serum immunoglobulin G against the O127 lipopolysaccharide. These studies indicate that prior EPEC infection can reduce disease severity upon homologous challenge. Further studies may require the development of new model systems.

Enteropathogenic Escherichia coli (EPEC) strains are one of several categories of pathogenic E. coli strains that cause diarrhea. EPEC infections are prevalent on six continents (5, 22–24, 28, 43). In many parts of the world, EPEC strains are the most common bacterial cause of diarrhea in infants (7, 21, 43). Disease due to EPEC can be severe, refractory to oral rehydration, protracted, and lethal (3, 14, 21, 45, 48).The pathogenesis of EPEC infection involves three distinct stages, initial adherence, signal transduction, and intimate attachment (12). Initial adherence is associated with the production of a type IV fimbria, the bundle-forming pilus (BFP) (20), that is encoded on the large EPEC adherence factor (EAF) plasmid (50). EPEC uses a type III secretion apparatus to export several proteins, including EspA, EspB, and EspD, that are required for tyrosine kinase-mediated signal transduction within the host cell (17, 25, 30, 31). This signaling leads to phosphorylation and activation of a 90-kDa protein that is a putative receptor for the bacterial outer membrane protein intimin (44). Intimin, the product of the eae gene, is required for intimate attachment of bacteria to the host cell membrane and for full virulence in volunteers (13, 26, 27). The interaction between EPEC and host cells results in the loss of microvilli and the formation of adhesion pedestals containing numerous cytoskeletal proteins (16, 33, 34, 39, 46). This interaction between bacteria and host cells is known as the attaching and effacing effect (40).One of the most striking clinical features of EPEC infections is the remarkable propensity of these strains to cause disease in very young infants. Rare reports of disease in older children and adults usually reflect common-source outbreaks that probably involve large inocula (47, 53). In contrast, in nosocomial outbreaks among neonates, EPEC spreads rapidly by person-to-person contact, apparently involving low inocula (54). The incidence of community-acquired EPEC infection is highest in the first 6 months after birth (4, 7, 21). EPEC infection is also more severe in younger children (8). Infants are more likely to develop diarrhea during the first episode of colonization with EPEC than they are during subsequent encounters (8). Whether the low incidence of EPEC diarrhea in older children and adults is due to acquired immunity or decreased inherent susceptibility is not known.The immune response to EPEC infection remains poorly characterized. It has previously been demonstrated that volunteers convalescing from experimental EPEC infection develop antibodies to the O antigen component of lipopolysaccharide (LPS) of the infecting strain, to intimin, and to type I-like fimbriae (13, 15, 29, 38). Antibodies to common EPEC O antigens are found more often in children of greater than 1 year in age than they are in younger children (42). Breast-feeding is protective against EPEC infection (2, 19, 43, 52). Breast milk contains antibodies against EPEC O antigens and outer membrane proteins and inhibits EPEC adherence to tissue culture cells (6, 9, 49).In an earlier study, it was reported that volunteers infected with EPEC developed antibodies to a 94-kDa outer membrane protein (38). Subsequently, it was determined that this antigen was intimin (26). Interestingly, the lone volunteer in that earlier study who did not have diarrhea after challenge with a wild-type EPEC strain had prechallenge serum antibodies to intimin. This led to the hypothesis that antibodies to intimin are protective against EPEC infection. To test this hypothesis and to test the more general hypothesis that EPEC infection induces protective immunity, two volunteer studies were performed. The first was a heterologous-challenge study performed in 1986, in which volunteers were infected with an O55:H6 EPEC strain and challenged, along with a naive cohort, with an O127:H6 EPEC strain. The second was a homologous-challenge study performed in 1991, in which veterans of a study comparing the virulence of a wild-type EPEC O127:H6 strain with that of an isogenic eae mutant (13) were rechallenged, along with a naive cohort, with the homologous wild-type strain. The availability of new purified antigens allowed us to analyze data from these studies in the context of humoral immune responses.     

Enteropathogenic Escherichia coli Serotypes and Endemic Diarrhea in Infants

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.     

Enteropathogenic Escherichia coli diarrhea in hospitalized children in Bangladesh.

The role of enteropathogenic Escherichia coli (EPEC) was evaluated in a group of children with endemic diarrhea admitted to Dhaka Shishu Hospital in Dacca, Bangladesh. EPEC was detected in fecal samples of 23% of 104 cases and 8% of 74 concurrent control children. The most commonly isolated EPEC strains were serogroups O20a, O20c:K61; O20a, O20b:K84; O26:K60; and O18a, O18c:K77. Except for O26:K60, these groups had not been reported from Bangladesh. On testing for enterotoxin production, only two strains (serogroups O26:K60, O18a, and O18c:K77) were enterotoxigenic. None was enteroinvasive as tested in the guinea pig conjunctivitis model. Our study supports the concept that EPEC may be an important cause of endemic diarrhea in Bangladesh.     

Type III Secretion-Dependent Hemolytic Activity of Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) was found to exhibit a type III secretion-dependent, contact-mediated, hemolytic activity requiring the EspA, EspB, and EspD secreted proteins. EspB and EspD display homology to pore-forming molecules. Our data suggest a mechanism to explain the requirement for all three Esp proteins in the transfer of EPEC proteins, such as Tir, into target cells.     

An Ultrastructural Study of Enteropathogenic Escherichia Coli Infection in Human Infants

Over the past 2 years, we have studied and treated 18 infants with protracted diarrhea due to an enteropathogenic Escherichia coli serogroup 0119. All patients had persistent stool escretion and jejunal overgrowth with this pathogenic E. coli. Jejunal biopsy revealed atrophy of villi with a chronic inflammatory cell infiltrate in the lamina propria. E. coli 0119 adhered to the luminal surface of enterocytes. Electron microscopy showed disappearance of glycocalyx and microvilli at the areas of bacterial adherence. Intracellular damage was indicated by dilatation of rough endoplasmic reticulum, mitochondrial changes, and cytoplasmic pallor. Similar changes in histology and ultrastructure occurred in ileal epithelial cells. Glandular crypt epithelium showed prominent subnuclear vacuolation and separation of lateral intercellular junctions throughout the small intestine. Rectal mucosal biopsy showed mucus depletion and irregular atrophy of the epithelium, with E. coli 0119 adherent to the luminal surface. Ultrasuctural damage paralleled that in the small intestine. E. coli 0119 causes damage to epithelial cells throughout the infant intestinal tract. This damage leads to atrophy of villi and a marked reduction in absorptive surface area, resulting in protracted diarrhea.     

表皮生长因子受体检测方法

表皮生长因子受体(EGFR)是一种受体型酪氨酸激酶,在多种肿瘤中都有过表达,并与肿瘤的发生、浸润、转移、预后及患者生存期密切相关.多种方法可用于检测组织、血液、细胞中EGFR蛋白表达及其酪氨酸激酶活性,EGFR的检测将有助于肿瘤的靶向治疗.     

Immunity to Escherichia coli in Pigs: Adhesion of Enteropathogenic Escherichia coli to Isolated Intestinal Epithelial Cells

A method was developed to test for the ability of Escherichia coli to adhere to isolated intestinal epithelial cells. Of the E. coli tested, those having either K88ac or K88ab antigens adhered to the cells, and those which did not have these antigens did not. Since some enteropathogenic E. coli did not have the ability to adhere, it is assumed that adherence is not an essential factor of pathogenesis but rather should be considered an enhancement to the pathogenicity of some E. coli. None of the E. coli enteropathogens of cattle tested adhered to either pig or cattle cells. Similarly, human strains did not adhere to pig cells. Although the test system may not have been ideal for human or bovine E. coli, the results reported here suggest that adhesiveness is a property limited to porcine enteropathogenic E. coli carrying one of the K88 antigens. Adhesiveness is associated with the K88c or K88b antigens, and their adhesive ability is only neutralizable by the homologous antisera.     

Role of Lactosyl Glycan Sequences in Inhibiting Enteropathogenic Escherichia coli Attachment

Previously, we found that asialo-lactosamine sequences served as receptors for enteropathogenic Escherichia coli (EPEC) binding to Chinese hamster ovary (CHO) cells. In the present report, we have extended these earlier results by examining the ability of lactosamine- or fucosylated lactosamine-bovine serum albumin (BSA) glycoconjugates to inhibit EPEC, strain E2348/69, binding to HEp-2 cells. We found that, consistent with our previous findings with CHO cells, N-acetyllactosamine-BSA was the most effective inhibitor of EPEC localized adherence to HEp-2 cells, with Lewis X-BSA being the next best inhibitor. Further investigation revealed that coincubating EPEC E2348/69 with these BSA glycoconjugates alone caused a decrease in the expression of the bundle-forming pilus structural subunit (BfpA) and intimin by the bacteria. BfpA and intimin expression were reduced to the greatest extent by N-acetyllactosamine-BSA and Lewis X-BSA, respectively. These results suggest that the glycoconjugate inhibition of EPEC binding to HEp-2 cells might be achieved, wholly or in part, by an active mechanism that is distinct from simple competitive antagonism of receptor-adhesin interactions.     

Properties of the Enterotoxic Component in Escherichia coli Enteropathogenic for Swine

The enterotoxic component in sterile syncase broth filtrates of Escherichia coli strains 340 (O9:K.:NM) and P307 (O8:K87,88a,b:H19) was studied. The enterotoxic activity in both strains was retained by an ultrafiltration membrane with a molecular weight retention of 100,000 (XM-100A) and eluted from a Sephadex G-200 column in the void volume. The enterotoxic activity in strain 340 was resistant to heating at 75 C for 30 min, but the activity in strain P307 was destroyed by heating at 60 C for 30 min. The P307 Sephadex G-200 column eluate possessing the enterotoxic activity, when desalted, contained 45.8% carbohydrate and 9.3% protein, and had an ED(50) of 2.2 mg/rabbit ileal loop. Immunodiffusion studies showed that this material contained both endotoxin and acid-polysaccharide capsular material. The enterotoxic activity was acidlabile and was destroyed by Pronase, but was resistant to trypsin and eluted as a single peak in the void volume of a 4% agarose column. The enterotoxic component could not be separated from the endotoxin; in fact, the data indicated that the two components are closely associated and that the enterotoxic activity resides in material of a protein nature.     

Evidence of Pathogenic Subgroups among Atypical Enteropathogenic Escherichia coli Strains

We describe the characterization of 126 atypical enteropathogenic Escherichia coli (aEPEC) isolates from 1,749 Brazilian children. Classic aEPEC strains were more frequently found in children with diarrhea than in controls (P < 0.001), showing their importance as acute diarrhea agents in our country. Only aEPEC strains carrying either the ehxA or paa gene were significantly associated with diarrhea.Enteropathogenic Escherichia coli (EPEC), one of the six E. coli diarrheagenic pathotypes, produces an adherence factor chromosomally encoded by the eae (EPEC attaching and effacing) gene located within the locus for enterocyte effacement (LEE) pathogenicity island (15, 16, 18).“Typical” EPEC strains contain, in addition to eae, the EPEC adherence factor (EAF) plasmid (4), which encodes the bundle-forming pili that mediate localized adherence to epithelial cells (9, 26). EPEC strains lacking the EAF plasmid have been designated “atypical” EPEC (aEPEC) (12). Whereas typical EPEC strains express only the virulence factors encoded by the LEE region and the EAF plasmid, aEPEC strains have additional virulence properties (11, 32).Recently Afset et al. (2) described several virulence genes associated with diarrhea in aEPEC isolates from Norwegian children. In their study, genes belonging to the pathogenicity island OI-122 (efa1/lifA, nleB, nleE, and sen), present in the enterohemorrhagic E. coli (EHEC) reference strain EDL933 (17), and the gene for long polar fimbriae (lpfA) (10), found in EHEC O113 strains, were particularly frequent. Other genes, such as the porcine A/E-associated gene (paa) (5) and the EHEC hemolysin gene (ehxA) (27), were also found to be associated with diarrheal disease.Other E. coli adhesion factors recently described include the protein ToxB, required for full adherence expression in EHEC O157:H7 (30); Iha, an adherence-conferring protein similar to Vibrio cholerae IrgA (29); Saa, an autoagglutinating adhesin identified in LEE-negative strains (22); and Spf, a sorbitol-fermenting EHEC O157 fimbria (8). In addition, we recently identified a diffuse adherence (lda) locus in an aEPEC strain of the O26 serogroup that codes for adherence to HEp-2 cells (25).In this report, we describe the prevalence and virulence profile of aEPEC strains isolated from diarrhea patients and control subjects in several cities of Brazil from 1999 through 2004.Stool specimens from 1,102 children under 2 years of age with diarrhea, presenting to the emergency room of public hospitals in seven cities representing different regions of Brazil, and 647 randomly selected children without any gastrointestinal symptoms from the same hospitals were studied. All specimens were investigated for the presence of enteric pathogens, such as diarrheagenic E. coli, Shigella species, Salmonella species, Yersinia enterocolitica, Campylobacter species, and rotavirus (24).Atypical EPEC strains were isolated, identified, and serotyped as described elsewhere (11). One isolate per subject was stored at −70°C.aEPEC strains were screened by colony blot hybridization with 16 different DNA probes representing a panel of toxin, adhesin, and OI-122 genes. Fourteen DNA probes were prepared by PCR amplification from prototype strains. The genes, primers, amplicon size, PCR conditions, and prototype strains are given in Table ​Table1.1. Two DNA probes were prepared by plasmid extraction using the method of Birnboim and Doly (7) and digestion with appropriate restriction endonucleases. The cdt probe was a 1,357-bp fragment from plasmid pCVD448 (28), and the cnf probe was a 335-bp fragment of pEOSW1 (20). The probes were then purified by gel extraction (23) and labeled with [α-³²P]dCTP, and colony hybridization assays were performed as described elsewhere (24).

TABLE 1.

PCR primers and conditions used in this study and sizes of PCR amplicons

Effects of Epidermal Growth Factor Administration on Repair of Acetic Acid-Induced Colonic Ulcerations in Rats

Abstract

The effect of subcutaneous and luminal epidermal growth factor (EGF) administration on acetic acid-induced colonic ulceration was determined in adult rats. Application of acetic acid to the distal colonic lumen caused epithelial denudation, mucosal ulceration and inflammation in the exposed segment. Re-epithelialization was detectable 5 to 7 days later, with near-complete resolution of the lesion by 14 days post-injury. Luminal EGF (1.6 mg/kg bw/day) or subcutaneous EGF (200 pgmglkg bwlday), administered for 4 or 6 days from the time of ulceration failed to enhance re-epithelialization of the acid-exposed segment. However, mucosal and submucosal thickening was attenuated 20–40% by subcutaneous EGF, reflecting a reduction in edema. Luminal EGF had a similar but less substantial effect in the submucosa, but was more effective at attenuating muscularis thickening adjacent to the lesion. In conclusion, administration of exogenous EGF for up to 6 days failed to enhance re-epitheliahation of acetic acid-induced colonic ulcerations but did attenuate the associated edematous response.