Bovine recto-anal junction squamous epithelial (RSE) cell adhesion assay for studying Escherichia coli O157 adherence.

AIM: To develop a new adherence assay, using cattle recto-anal junction squamous epithelial (RSE) cells, for evaluating bacterial adherence to cells of bovine origin. METHODS AND RESULTS: Proof of concept was demonstrated using the human gastrointestinal pathogen Escherichia coli O157:H7, for which cattle are reservoirs. Adherence assays were conducted using both RSE and HEp-2 cells, in the presence and absence of D+Mannose. E. coli O157 specifically adhered in a type I fimbriae-independent manner to RSE cells in significantly higher numbers and also bound significantly higher numbers of RSE cells than diverse laboratory strains of nonpathogenic E. coli. CONCLUSION: The RSE cell adhesion assay output highly reproducible and interpretable results that compared very well with those obtained using the more extensively used HEp-2 cell adherence assay. SIGNIFICANCE AND IMPACT OF THE STUDY: The RSE cell adhesion assay provides a convenient means of directly defining and evaluating pathogen factors operating at the bovine recto-anal junction. The RSE cell adhesion assay further has the potential for extrapolation to diverse bacteria, including food-borne pathogens that colonize cattle via adherence to this particular anatomical site.

Dexamethasone depletes gammadelta T cells and alters the activation state and responsiveness of bovine peripheral blood lymphocyte subpopulations.

Administration of dexamethasone (DEX) to cattle is commonly used in models of stress-induced effects on host defense, including models investigating interactions of microorganisms with their host. Much less is known about the effects of DEX on the adaptive immune response in cattle compared with other species. The objective of the present study was to characterize subsets of circulating lymphocytes in calves before and 48 h after the onset of parenteral DEX treatment. Treatment significantly reduced the overall percentage of circulating lymphocytes and disproportionately depleted the population of gammadeltaTCR(+)/CD8alpha(-) cells. Analysis within the CD8alpha(+) population of T cells revealed that DEX treatment also reduced the CD8alpha(low) subset of gammadeltaT cells coexpressing the activation marker ACT-2(+). By contrast, DEX treatment did not affect the percentage of CD8alpha(low)/CD25(+) cells, indicating that cells with a special activation state were affected. Dexamethasone treatment reduced the number of gammadeltaT cells but increased the percentages of CD14(+) monocytes and activated CD25(+) cells (both CD4(-) and CD4(+)) in peripheral blood mononuclear cell (PBMC) preparations. Although DEX treatment reduced the overall proliferative capacity of PBMC, it enhanced the relative number of proliferating CD4(+) lymphocytes. Lower levels of mRNA for several Th-prototype cytokines (IL-2, IFN-beta, IL-4, transforming growth factor-beta) were detected in short-term PBMC cultures established from DEX-treated calves compared with PBMC cultures from control calves; the amount of il-10 transcripts, however, was unaffected. Results of the study reported here clearly show that DEX treatment does not uniformly suppress the bovine immune system but has differential effects on lymphocyte sub-populations and functions. This information must be considered when using DEX treatment as a bovine stress model.

Shiga-toxigenic Escherichia coli-inoculated neonatal piglets develop kidney lesions that are comparable to those in humans with hemolytic-uremic syndrome.

Kidney lesions similar to those in humans with hemolytic-uremic syndrome were observed histologically in 82 of 122 piglets inoculated intragastrically with Shiga-toxigenic Escherichia coli but not in 29 controls. The locations of lesions matched locations where Stx-2 binding and Gb3 (globotriasylceramide receptors for Stx) were identified immunohistochemically.

Escherichia coli O157:H7 causes more-severe systemic disease in suckling piglets than in colostrum-deprived neonatal piglets.

Our objective was to determine if suckling neonatal piglets are susceptible to enterohemorrhagic Escherichia coli (EHEC) O157:H7 disease. Surprisingly, EHEC O157:H7 caused more-rapid and more-severe neurological disease in suckling neonates than in those fed an artificial diet. Shiga toxin-negative O157:H7 did not cause neurological disease but colonized and caused attaching-and-effacing intestinal lesions.

Semi-automated fluorogenic PCR assays (TaqMan) forrapid detection of Escherichia coli O157:H7 and other shiga toxigenic E. coli.

Semi-automated detection of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and non-O157:H7 Shiga toxin-producing E. coli (STEC) was achieved using fluorogenic polymerase chain reaction (PCR). These PCR assays were designed to amplify 80, 120 and 150 bp regions of virulence genes stx1, stx2 and eaeA, respectively, using specific primers. The fluorogenic probes were used for specific detection of amplified products of the stx1 and stx2 genes of STEC, and the eaeA gene of EHEC O157:H7. For multiplex PCR assay, the three sets of primers and fluorogenic probes were included in one reaction to simultaneously amplify and detect any of the three targeted virulence genes. In non-multiplex PCR assay, each of the three virulence genes was amplified and detected in independent reactions. The specificity of these assays was evaluated using suspensions of STEC and other bacterial species lacking stx1, stx2 and eaeA. The multiplex assay detected all STEC harbouring any combination of three virulence genes. Three non-multiplex PCR reactions identified types of Shiga toxin genes carried by a STEC and identified STEC as either EHEC O157:H7 or non-O157:H7 STEC. Sensitivity limits of these assays in beef and faeces inoculated with EHEC O157:H7 were 5.8 to 580 cfu and 1.2 to 1200 cfu, respectively. These assays can be completed within 8-10 h when performed simultaneously or within 13 h if the multiplex assay is used as an initial screen for detecting STEC and the non-multiplex assay is used for subsequent detection of stx1 and stx2 of STEC and eaeA of EHEC O157:H7

Pathogenesis of Escherichia coli O157:H7 in weaned calves.

Cattle are an important reservoir of Shiga toxin-producing Escherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) that cause diarrhea, hemorrhagic colitis, and hemorrhagic uremic syndrome in humans. One strategy for reducing human foodborne EHEC infections is to reduce the levels of EHEC in cattle. Bovine O157:H7 infection models will facilitate identification of virulence factors involved in bovine infections. O157:H7 cause severe diarrhea and attaching and effacing (A/E) mucosal lesions in colostrum-deprived neonatal (< 2 h) calves. We hypothesized that O157:H7 also cause A/E lesions in older calves, but these were not detected in earlier studies because intestinal levels of O157:H7 were too low (< 10(6) CFU/g of tissue) for detection of focally distributed microscopic lesions. Weaned 3- to 4-month-old calves were fasted 48 h, inoculated via stomach tube with 10(10) CFU of O157:H7 or nonpathogenic E. coli, necropsied 4 d pi and examined histologically. Calves inoculated with O157:H7 had higher intestinal levels of inoculated E. coli than control animals. The rectum was the major site of colonization. A/E lesions were seen in the rectum and cecum of calves with high levels of O157:H7. Weaned calves, like neonatal calves, are susceptible to intestinal damage induced by EHEC O157:H7. The rectum and cecum may be principal sites of EHEC O157:H7 colonization during the carrier-shedder state in cattle.

Escherichia coli O157:H7 requires intimin for enteropathogenicity in calves.

Enterohemorrhagic Escherichia coli (EHEC) strains require intimin to induce attaching and effacing (A/E) lesions in newborn piglets. Infection of newborn calves with intimin-positive or intimin-negative EHEC O157:H7 demonstrated that intimin is needed for colonization, A/E lesions, and disease in cattle. These results suggest that experiments to determine if intimin-based vaccines reduce O157:H7 levels in cattle are warranted.

Differentiation of F18ab+ from F18ac+ Escherichia coli by single-strand conformational polymorphism analysis of the major fimbrial subunit gene (fedA).

Toxin-producing Escherichia coli expressing F18 fimbriae colonizes the small intestines of weaned pigs and causes diarrhea, edema disease, or both. The F18 family is composed of two antigenic variants, F18ab and F18ac. Because many strains do not express F18 fimbriae in vitro, identification and differentiation of these two variants are difficult. Single-strand conformational polymorphism (SSCP) analysis is a rapid method for identifying genetic mutations and polymorphisms. The F18 major fimbrial subunit genes (fedA) of 138 strains were amplified by PCR, and genetic differences were detected by SSCP analysis. The SSCP analysis of the fedA gene differentiated F18ab+ strains from F18ac+ strains. Most strains classified as F18ab+ by SSCP analysis contained Shiga toxin 2e and enterotoxin genes. Most strains classified as F18ac+ by SSCP analysis contained only enterotoxin genes. The SSCP analysis was a useful method for predicting the antigenicity of F18+ E. coli and could also be used for analysis of other virulence genes in E. coli and other pathogenic bacteria.

Pathogenicity of Escherichia coli O157:H7 in the intestines of neonatal calves.

Cattle are an important reservoir of Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains, foodborne pathogens that cause hemorrhagic colitis and hemolytic uremic syndrome in humans. EHEC O157:H7 strains are not pathogenic in calves >3 weeks old. Our objective was to determine if EHEC O157:H7 strains are pathogenic in neonatal calves. Calves <36 h old inoculated with EHEC O157:H7 developed diarrhea and enterocolitis with attaching and effacing (A/E) lesions in both the large and small intestines by 18 h postinoculation. The severity of diarrhea and inflammation, and also the frequency and extent of A/E lesions, increased by 3 days postinoculation. We conclude that EHEC O157:H7 strains are pathogenic in neonatal calves. The neonatal calf model is relevant for studying the pathogenesis of EHEC O157:H7 infections in cattle. It should also be useful for identifying ways to reduce EHEC O157:H7 infections in cattle and thus reduce the risk of EHEC O157:H7 disease in humans.

Biological relationship between F18ab and F18ac fimbriae of enterotoxigenic and verotoxigenic Escherichia coli from weaned pigs with oedema disease or diarrhoea.

Comparative fimbrial expression and adhesion studies were made on enterotoxigenic and verotoxigenic E. coli (ETEC and VTEC) strains isolated from cases of porcine postweaning diarrhoea or oedema disease. F107(F18ab) fimbriae--monitored by polyclonal and monoclonal antibodies and by electron microscopy--were poorly expressed on most VTEC strains. In contrast, 2134P(F18ac) fimbriae were more readily detected on most ETEC strains. The F18ac strains adhered in vivo to ligated intestinal loops in weaned pigs while the F18ab strains did not adhere or adhered weakly. Similarly, the F18ac strains adhered to isolated intestinal brush borders in weaned pigs but the F18ab strains (except for the F107 reference E. coli) did not adhere or adhered weakly in vitro. Neither the F18ab nor F18ac strains adhered to brush borders from newborn pigs. In vitro adhesion of F18ab and F18ac strains was mannose resistant and receptors for F18 seemed to differ from receptors for K88(F4). It is concluded that the antigenic variants of F18 fimbriae (F18ab and F18ac) are biologically distinct. F18ab fimbriae are expressed poorly both in vitro and in vivo and are frequently linked with the production of SLT-IIv and serogroup O139, while F18ac are more efficiently expressed in vitro and in vivo and most often are linked with enterotoxin (STa, STb) production, and serogroups O141, O157.

Pathogenesis of O157:H7 Escherichia coli infection in neonatal calves.

Cattle have been implicated as an important reservoir of Shiga-like toxin-producing Escherichia coli (SLTEC) O157:H7, enterohemorrhagic E. coli (EHEC) that cause hemorrhagic colitis and hemorrhagic uremic syndrome in humans. Naturally- or experimentally-infected cattle can shed low levels of E. coli O157:H7 long-term, but little is known about the pathogenesis of E. coli O157:H7 infection in cattle. E. coli O157:H7 induce characteristic attaching and effacing (A/E) mucosal lesions in ceca and colons of 1-day-old gnotobiotic piglets and this model is used to study the pathogenesis of SLTEC infections. A/E lesions were not detected in histologic sections of the intestines from adult cattle or 3- to 14-week-old calves infected with E. coli O157:H7. Our objective was to determine if E. coli O157:H7 induce A/E lesions in neonatal calves. Colostrum-deprived calves (< 12-h-old) were bottle-fed with antibiotic-free milk replacer containing 10(10) colony forming units (CFU) of O157:H7 (SLT-I+, SLT-II+) or nonpathogenic E. coli, necropsied 18 h postinfection and their intestines examined histologically. Bacterial attachment, effacement of microvillous borders, and destruction of epithelium were observed in the intestines of the neonatal calves inoculated with E. coli O157:H7. No lesions were observed in calves inoculated with nonpathogenic E. coli. The distribution of intestinal lesions in neonatal calves resembled that in gnotobiotic pigs. Neonatal calves are apparently more susceptible to A/E lesions induced by E. coli O157:H7 than are older calves or adult cattle and provide a model for studying the pathogenesis of E. coli O157:H7 infections in cattle.

Age-related resistance to 987P fimbria-mediated colonization correlates with specific glycolipid receptors in intestinal mucus in swine.

Strains of enterotoxigenic Escherichia coli that produce 987P fimbriae (987P+ strains) colonize the small intestines and cause diarrhea in neonatal (< 6-day-old) pigs but not in weaned pigs. However, 987P+ E. coli strains adhere in vitro to intestinal epithelial cells from pigs of both ages. Two intestinal components, designated 987R and 987M, bind 987P fimbriae (987P) on Western blots (immunoblots). We examined brush borders (BB) and intestinal washes (IW) from pigs to determine if they contain glycolipids which bind 987P. Total glycolipid extracts from BB and IW of 4-week-old pigs were separated on thin-layer chromatograms and overlaid with purified 987P. Bound 987P were detected with 987P-specific antiserum. 987P bound to at least one moiety in both BB and IW glycolipids and also bound to several purified glycolipids, including gangliotetraosylceramide, lactosylceramide (CDH), sulfatide (SFT), gangliotriaosylceramide, and galactosylceramide (listed in order of decreasing affinity). Strain 987, but not the isogenic 987P- strain I36, bound to these same glycolipids, indicating that the fimbriae contain the adhesin required for binding to these lipids. Glycolipids extracted from BB and IW isolated from 3- and 4-week-old pigs and from BB isolated from 1-day-old pigs contained similar amounts of glycolipids like CDH and SFT that bound 987P. Finally, 987P bound to CDH, SFT, and total BB glycolipids separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon, and these glycolipids had mobilities similar to that of 987M. Thus, 987M may contain 987P-binding glycolipids. We hypothesize that glycolipid receptors for 987P, most likely CDH or SFT, in the mucus of older pigs bind 987P and inhibit 987P- mediated intestinal colonization by preventing the attachment of 987P+ E. coli to 987P receptors on the intestinal epithelium.

A monoclonal antibody identifies 2134P fimbriae as adhesins on enterotoxigenic Escherichia coli isolated from postweaning pigs.

Fimbriae (pili) of enterotoxigenic Escherichia coli (ETEC), including K88, K99, 987P, and F41, are adhesins that facilitate intestinal colonization in neonatal pigs. K88 is also associated with some ETEC isolated from weaned pigs. Many ETEC isolates from weaned pigs do not express known adhesins and are termed 4P-. A novel bacterial adhesin, 2134P, was recently identified on two 4P- ETEC isolates from weaned pigs. In this study, we identified a 2134P-specific monoclonal antibody, mAb 6C7/C1, that blocked the binding of 2134P+ bacteria to intestinal epithelial cells. Indirect immunofluorescent antibody and immunoperoxidase assays using mAb 6C7/C1 confirmed that the 2134P adhesin is expressed in vivo by adherent bacteria in pigs challenge-exposed with 2134P+ ETEC. 2134P was detected on 31% of 189 postweaning diarrhea 4P- ETEC isolates from the National Animal Disease Center's culture collection by dot blot immunoperoxidase assays using mAb 6C7/C1. We conclude that 2134P is a bacterial adhesin and is an important virulence attribute of some ETEC that cause diarrhea in weaned pigs.

Identification of plasmid and chromosomal copies of 987P pilus genes in enterotoxigenic Escherichia coli 987.

A DNA probe derived from the subunit gene of a cloned 987P determinant was used to characterize the locations of the 987P genes in several Escherichia coli strains. We examined E. coli 987, a nonpiliated mutant of strain 987 (I36) that does not express 987P in vitro, and a variant of I36 that expressed 987P following growth in vivo. We found that plasmid and chromosomal copies of the 987P subunit gene could be differentiated in strain 987 by restriction endonuclease analysis and Southern blot hybridization. The nonpiliated mutant I36 had lost the plasmid copy but retained the chromosomal copy of the 987P gene. A 987P-piliated variant of I36, which did not contain the 987P plasmid, colonized and caused diarrhea in neonatal pigs similarly to wild-type 987. The plasmid that hybridized with the 987P probe was transferred from strain 987 to an E. coli K-12 strain by conjugation. We were unable to demonstrate expression of 987P by these transconjugants. The data suggest that the chromosomal and plasmid copies of the 987P genes may interact to influence 987P expression.