Lectin binding profile of the small intestine of five-week-old pigs in response to the use of chlortetracycline as a growth promotant and under gnotobiotic conditions.

Antibiotics have traditionally been used for growth promotion in the pork industry; however, their use in animal feed has recently been limited because of human health concerns. The intestinal microbiota plays an important role in mediating many physiological functions such as digestion and animal growth. It was hypothesized that use of antibiotics as growth promotants and subsequent variations in intestinal microbiota induce significant changes in the intestinal glycoconjugate composition, which ultimately affects animal growth and disease susceptibility. The aim of this study was to characterize the lectin binding profiles of the ileum of weanling pigs in response to the absence of intestinal microbiota and to the use of the antibiotic chlortetracycline as growth promotant. Eighteen half-sib piglets obtained by cesarean section were divided into 3 treatment groups (n = 6) and maintained as control, antibiotic-fed, and gnotobiotic piglets until 5 wk of age. The glycoconjugate composition of the ileal tissues was examined by lectin histochemistry. Lycopersicon esculentum lectin, Jacalin, Pisum sativum agglutinin, Lens culinaris agglutinin (LCA), and Sambucus nigra lectin showed significant differences (P < 0.05) in binding intensities on the dome and villous epithelium between the treatment groups. Griffonia simplicifolia lectin I, Glycine maxi agglutinin, and Arachis hypogea agglutinin exhibited differences (P < 0.05) between treatment groups in lectin binding on goblet cells. Triticum vulgaris agglutinin, Pisum sativum agglutinin, and LCA showed significant differences (P < 0.05) in binding intensities on dome, corona, and follicular regions of the ileum among treatment groups of animals. Overall, ileal tissues from gnotobiotic piglets expressed significantly weaker (P < 0.05) lectin binding for many lectins compared with control and antibiotic groups. This suggests that the intestinal microbiota plays an important role in the expression of sugar moieties in the intestine. Lectins LCA, Phaseolus vulgaris Leucoagglutinin, and Maackia amurensis lectin II showed significant differences (P < 0.05) in lectin bindings between control and antibiotic-fed piglets. This indicates that chlortetracycline as a growth promotant induces biologically relevant changes in the lectin binding profile of the ileum. These findings will help in further understanding the role of the gut microbiota and the mechanisms of action of antibiotics as growth promotants in pigs.

Inhibition of adhesion of Escherichia coli k88ac fimbria to its receptor, intestinal mucin-type glycoproteins, by a monoclonal antibody directed against a variable domain of the fimbria.

Strains of enterotoxigenic Escherichia coli that express K88 fimbriae are among the most common causes of diarrhea in young pigs. Adhesion of bacteria to receptors on intestinal epithelial cells, mediated by K88 fimbriae, is the initial step in the establishment of infection. Three antigenic variants of K88 fimbriae exist in nature: K88ab, K88ac, and K88ad. K88ac is the most prevalent and may be the only variant of significance in swine disease. Each K88 fimbrial variant is composed of multiple antigenic determinants. Some of these determinants are shared among the three variants and may be referred to as conserved epitopes, whereas others are unique to a specific variant and may be referred to as variable epitopes. In this study, monoclonal antibodies (MAbs) specific to either variable or conserved epitopes of K88ac fimbriae were produced. The specificity of each MAb was tested by enzyme-linked immunosorbent and immunoblot assays. Fab fragments were prepared from these MAbs and were tested for their ability to block the binding of K88-positive bacteria and purified fimbriae to porcine enterocyte brush border vesicles and purified K88 receptors, respectively. The purified receptors were intestinal mucin-type sialoglycoproteins (IMTGP) isolated from porcine enterocytes (A. K. Erickson, D. R. Baker, B. T. Bosworth, T. A. Casey, D. A. Benfield, and D. H. Francis, Infect. Immun. 62:5404-5410, 1994). Fab fragments prepared from MAbs specific for variable epitopes blocked the binding of bacteria to brush borders and of fimbriae to IMTGP. However, those from MAbs specific for a conserved epitope did not. These observations indicate that the receptor-binding domain of a K88ac fimbria is contained, at least in part, within the antigenically variable epitopes of that fimbria. Epitope mapping for one of the MAbs, which recognizes a linear epitope on K88ac fimbriae, indicated that this MAb binds to the region from amino acid no. 64 to no. 107 on the major subunit of K88ac fimbriae.

Identification of an intestinal neutral glycosphingolipid as a phenotype-specific receptor for the K88ad fimbrial adhesin of Escherichia coli.

In this study, we identified a receptor for the K88ad fimbrial adhesin of Escherichia coli in neutral glycosphingolipid preparations from intestinal epithelial cells of K88ad-adhesive pigs, which was absent in preparations from K88ad-nonadhesive pigs. Neither K88ab nor K88ac adhesin variants bound to this neutral glycosphingolipid. Because this receptor is an intestinal glycosphingolipid that binds K88ad adhesin, it has been designated IGLad. Carbohydrate compositional analysis of a partially purified preparation of IGLad identified galactose, glucose, and N-acetylglucosamine in a ratio of 1.5:1.0:0.5 as the major monosaccharides. Preliminary characterization experiments using lectins showed that IGLad contains the terminal glycanic structure Galbeta1-4GlcNAc. Removal of terminal beta-linked galactose residues from IGLad decreased the recognition of IGLad by the K88ad adhesin, indicating that terminal beta-linked galactose is an essential component of the K88ad adhesin recognition site on IGLad. Studies with purified glycosphingolipid standards demonstrated that K88ad adhesin binds to neolactotetraosylceramide (nLc4Cer) (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) , lactotriosylceramide (GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) and lactotetraosylceramide (Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) . Based on these studies, IGLad appears to be nLc4Cer.

K88 adhesins of enterotoxigenic Escherichia coli and their porcine enterocyte receptors.

The three antigenic variants of the K88 fimbrial adhesin (K88ab, K88ac, and K88ad) of enterotoxigenic Escherichia coli (ETEC) each exhibit unique specificity with regard to their hemagglutination characteristics. The variants are also unique in the specificity of their binding to the brush borders of enterocytes isolated from pigs with different genetic backgrounds. Diversity in enterocyte binding specificity suggests the existence of several K88 receptors, expressed individually or in various combinations on porcine enterocytes. Three candidate receptors have been identified that may explain the adhesion of K88 fimbrial variants to various porcine enterocytes. These receptors are an intestinal mucin-type sialoglycoprotein (IMTGP), an intestinal transferrin (GP74), and an intestinal neutral glycosphingolipid (IGLad). The IMTGP binds K88ab and K88ac, but not K88ad. The GP74 binds K88ab, but not K88ac or K88ad, and the IGLad binds K88ad, but not K88ab or K88ac. Each of the candidate receptors has been found in brush borders that are adhesive for the fimbriae that bind the respective receptor. They have not been found in brush borders that are not adhesive for those same fimbriae. The presence of IMTGP was highly correlated with susceptibility of neonatal gnotobiotic pigs to ETEC expressing K88ab or K88ac.

Pathogenicity of an enterotoxigenic Escherichia coli hemolysin (hlyA) mutant in gnotobiotic piglets.

Pigs infected with hemolytic F4(+) strains of enterotoxigenic Escherichia coli often develop septicemia secondary to intestinal infection. We tested the hypothesis that inactivation of hemolysin would reduce the ability of F4(+) enterotoxigenic E. coli to cause septicemia in swine following oral inoculation. Inactivation of the hemolysin structural gene (hlyA) did not decrease the incidence of septicemia in the gnotobiotic piglet model.

Expression of mucin-type glycoprotein K88 receptors strongly correlates with piglet susceptibility to K88(+) enterotoxigenic Escherichia coli, but adhesion of this bacterium to brush borders does not.

Three antigenic variants of the K88 fimbrial adhesin exist in nature, K88ab, K88ac, and K88ad. Enterotoxigenic Escherichia coli (ETEC) strains that produce these fimbriae cause life-threatening diarrhea in some but not all young pigs. The susceptibility of pigs to these organisms has been correlated with the adherence of bacteria to isolated enterocyte brush borders. Whether that correlation holds for multiple K88 variants and over a broad genetic base of pigs is unknown and was the impetus for this study. We also desired to examine the correlation of the expression of a porcine intestinal brush border mucin-type glycoprotein (IMTGP) which binds K88ab and K88ac with the susceptibility of piglets to K88(+) ETEC. Of 31 neonatal gnotobiotic pigs inoculated with K88ab+ or K88ac+ ETEC, 13 developed severe diarrhea, became dehydrated, and died or became moribund. Another pig became severely lethargic but not dehydrated. In vitro brush border adherence analysis was not possible for 10 of the severely ill pigs due to colonization by challenge strains. However, of the 17 pigs that did not become severely ill, 8 (47%) had brush borders that supported the adherence of K88ab+ and K88ac+ bacteria in vitro, suggesting a poor correlation between in vitro brush border adherence and piglet susceptibility to K88(+) ETEC. By contrast, the expression of IMTGP was highly correlated with susceptibility to K88(+) ETEC. Of the 12 pigs that produced IMTGP, 11 developed severe diarrhea. The other pig that produced IMTGP became lethargic but not severely diarrheic. Only 2 of 18 pigs that did not produce IMTGP became severely diarrheic. Colonizing bacteria were observed in histologic sections of intestines from all pigs that expressed IMTGP except for the one that did not develop severe diarrhea. However, colonizing bacteria were observed in histologic sections from only one pig that did not produce IMTGP. The bacterial concentration in the jejuna and ilea of pigs expressing IMTGP was significantly greater (P < 0.005) than that in pigs not expressing IMTGP. These observations suggest the IMTGP is a biologically relevant receptor for K88ab+ and K88ac+ E. coli or a correlate for expression for such a receptor.

Multiple receptors on porcine intestinal epithelial cells for the three variants of Escherichia coli K88 fimbrial adhesin.

We evaluated intestinal epithelial membrane preparations from five phenotypes of pigs, distinguished by the variant of K88 fimbrial adhesin (K88ab, K88ac, K88ad) which bind to their intestinal epithelial cells (A-all three variants, B-K88ab and K88ac, C-K88ab and K88ad, D-K88ad, and E-none of the variants), for the presence of K88 adhesin receptors. Intestinal brush border membranes were prepared from 20 animals (four from each phenotype). Brush border proteins, that had been separated using SDS-PAGE and transferred to nitrocellulose membranes, were overlaid with biotinylated K88 adhesin, 35S-labelled K88+ Escherichia coli, or biotinylated K88+ E. coli. Biotinylated K88ab and K88ac fimbrial adhesins and labelled E. coli expressing K88ab or K88ac adhesin bound to 210- and 240-kDa receptors in phenotype A and B, but not phenotype C, D, or E animals. In contrast, no phenotype-specific receptors were identified for the K88ad adhesin. Previously, purified K88ab and K88ac fimbriae were shown to block K88ad binding, but purified K88ad fimbriae were unable to block K88ab or K88ac binding in phenotype A animals. These results point to the existence of three K88 adhesin receptors to account for the observed phenotypes: (1) Receptor bcd binds all three variants and is found in phenotype A pigs, (2) Receptor bc (210- and 240-kDa receptors) binds K88ab and K88ac and is found in phenotype A and B pigs, and (3) Receptor d binds K88ad and is found in phenotype C and D pigs.

Characterization of the carbohydrate moiety of intestinal mucin-type sialoglycoprotein receptors for the K88ac fimbrial adhesin of Escherichia coli.

We have previously identified two mucin-type sialoglycoproteins from porcine intestinal epithelial cells with approximate molecular masses of 210 (intestinal mucin-type glycoprotein IMTGP-1) and 240 kDa (IMTGP-2) as receptors for the K88ab and K88ac fimbrial adhesins of Escherichia coli. These receptors are detected in intestinal brush border membrane preparations from pigs with adhesive phenotypes but not from pigs with nonadhesive phenotypes and are postulated to be important determinants of the susceptibility of pigs to K88ab+ and K88ac+ enterotoxigenic E. coli infections. Using exoglycosidase digestion studies, we have now determined that beta-linked galactose is an important component in the recognition of IMTGP-1 and IMTGP-2 by the K88ac adhesin. In addition, we observed a differential distribution of the K88ac adhesin binding activity of IMTGP-1 and IMTGP-2 along the crypt-villus axis, suggesting that receptor activity is dependent on the maturation state of the intestinal epithelial cells. Brush borders from immature intestinal epithelial cells possessed the highest concentrations of IMTGP-1 and IMTGP-2 receptor activity, with a progressive decrease in receptor activity as the cells mature. To characterize the differences in the carbohydrate moieties of IMTGP-1 and IMTGP-2, we developed a procedure for purifying the receptors, using phenol extraction followed by serial lectin affinity chromatography. Carbohydrate compositional analysis of the purified receptors indicated that the carbohydrate moieties of IMTGP-1 and IMTGP-2 consist of both N- and O-glycans containing galactose, glucose, sialic acid, mannose, N-acetylgalactosamine, N-acetylglucosamine, and fucose. The major difference between the two receptors is that IMTGP-2 contains a higher percentage of monosaccharides (mannose and glucose) commonly found in N-glycans.

Norepinephrine-induced expression of the K99 pilus adhesin of enterotoxigenic Escherichia coli.

This study examined whether the provision of norepinephrine, as would be encountered within the highly innervated gastrointestinal system, affected the growth rate of enterotoxigenic Escherichia coli (ETEC) and the expression of the K99 pilus adhesin virulence-related factor. The addition of norepinephrine to serum-containing medium resulted in a 3- to 7-fold increase in the growth rate of the K99+ ETEC strain B44 as compared to growth in vehicle supplemented medium or medium supplemented with normetanephrine, a norepinephrine metabolite that contains one more methyl group than norepinephrine. ELISA analysis revealed that K99 pilus adhesin expression was increased in norepinephrine supplemented culture as compared to normetanephrine and vehicle supplemented controls. This increase occurred from 9 to 15 hours of incubation which represented the exponential growth phase for the norepinephrine supplemented culture. These results indicate that addition of norepinephrine affects both ETEC growth and expression of a specific virulence factor.

Distribution of K88 Escherichia coli-adhesive and nonadhesive phenotypes among pigs of four breeds.

Enterotoxigenic Escherichia coli expressing K88 fimbrial adhesins often cause diarrhea in young pigs. However, some pigs are inherently resistant to colibacillosis, because they lack receptors on their epithelial cell brush borders to which the fimbriae bind. Phenotypic diversity with respect to the binding of E. coli expressing K88 of the three variant types (K88ab, K88ac, and K88ad) was reported by Bijlsma et al. (1982), and binding specificities for each phenotype were described: A (adhesive to all three variants), B (adhesive to K88ab and K88ac), C (adhesive to K88ab and K88ad), D (adhesive to K88ad) and E (nonadhesive). Because brush border adhesiveness has been correlated with disease susceptibility, swine K88 adhesive phenotypes are of significance in the control of enteric disease. To determine the prevalence of the various K88 adhesive phenotypes in the swine population in the Midwestern United States, we tested epithelial cell brush borders of 24 purebred pigs from each of four breeds (Chester White, Duroc, Hampshire and Yorkshire) for adhesiveness to each of the K88 variants. Four, 4-week-old pigs (the largest and smallest healthy female littermates from two litters) were collected from each of 24 farms. Brush border vesicles from the pigs were tested for ability to bind E. coli expressing each K88 variant. The five brush border adherence patterns described for phenotypes A-E were observed. In addition, brush borders from some pigs only bound K88ab + bacteria. Nearly three quarters of the pigs whose brush borders tested, were found to be phenotype A (43%) or phenotype E (28%). These were the most common phenotypes in each breed, except Hampshire, in which case phenotypes C (17%) and D (25%) were more common than E (8%). There appeared to be no relationship between the phenotype of a pig and its weight relative to its littermate.

Variation in virulence in the gnotobiotic pig model of O157:H7 Escherichia coli strains of bovine and human origin.

Escherichia coli strains of serotype O157:H7 have been incriminated in outbreaks and sporadic cases of food-borne illness, including diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Food-producing animals, particularly cattle, are believed to be reservoirs of the organism. Whether all strains of bovine origin pose human health risk is unknown and was the impetus for this investigation. We compared the virulence of ten SLT-I, SLT-II, and eae DNA probe-positive O157:H7 strains from cattle to 10 like strains associated with human diarrheal disease outbreaks for virulence in one day-old gnotobiotic pigs. All strains caused diarrhea, and only four pigs inoculated with either of two bovine strains failed to develop that condition. Signs of central nervous system disease, death, debilitation requiring euthanasia before the end of an eight day observation period, and/or encephalomalacia occurred in 32/42 pigs inoculated with the strains isolated from human beings, 13/39 pigs inoculated with strains from cattle, and 7/7 pigs inoculated with a positive control strain. More strains of human origin (9/10) than bovine origin (5/10) caused these effects. The results of this study indicate considerable variability in virulence of O157:H7 strains possessing the same known virulence determinants, and suggest that disease outbreaks tend to be caused by the more virulent of these strains.

A three-receptor model for the interaction of the K88 fimbrial adhesin variants of Escherichia coli with porcine intestinal epithelial cells.

Four phenotypes of pigs distinguished by the variant(s) of K88 fimbrial adhesin (K88ab, K88ac, K88ad) that bind to their intestinal epithelial cells (I-none of the variants, II-K88ad, III-K88ab and K88ac, and IV-all three variants) have been identified. We hypothesize that the differences between the phenotypes are defined by the presence or absence of K88 adhesin receptors. We propose a three-receptor model to account for the observed phenotypes: 1) Receptor bed which binds all three variants and is found in phenotype IV, 2) Receptor be which binds K88ab and K88ac and is found in phenotype III and IV, and 3) Receptor d which binds K88ad and is found in phenotype II. We have identified the be receptor activity as a pair of mucin-type sialoglycoproteins (210 and 240 kDa). Although neither the bcd nor d receptor has been identified biochemically, their presence has been established using both blocking and receptor localization studies. Blocking studies using phenotype IV brush borders demonstrated that K88ab and K88ac fimbriae block the binding of E. coli expressing any of the K88 variants, but K88ad fimbriae block only K88ad E. coli binding. These results indicate that two receptors (bcd and bc) exist in the phenotype IV animals. Receptor localization studies on intestinal sections from phenotype IV animals showed that K88ab and K88ac adhesin binding is continuous from the crypt to the tip of the villus. The binding of the K88ad adhesin binding is multifocal in phenotype IV pigs, but continuous from crypt to tip of the villus in sections of phenotype II pigs. These studies verify the presence of two receptors (bcd and bc) in phenotype IV animals, and indicate that the K88ad receptor in phenotype IV animals (bcd) is different than in phenotype II animals (d).

A PCR-based method of detection and differentiation of K88+ adhesive Escherichia coli.

The objective of this study was to develop a polymerase chain reaction (PCR)-based method to detect and differentiate among Escherichia coli strains containing genes for the expression of 3 antigenic variants of the fimbrial adhesin K88 (K88ab, K88ac, and K88ad). Five primers were designed that allowed detection of K88+ E. coli, regardless of antigenic variant, and the separate detection of the ab, ac, and ad variants. Primers AM005 and AM006 are 21 base pair (bp) oligomers that correspond to a region of the K88 operon that is common to all 3 antigenic variants. Primers MF007, MF008, and MF009 are 24-bp oligomers that matched variable regions specific to ab, ac, and ad, respectively. Using primers AM005 and AM006, a PCR product was obtained that corresponds to a 764-bp region within the large structural subunit of the K88 operon common to all 3 antigenic variants. Primer AM005 used with MF007, MF008, or MF009 produced PCR products approximately 500-bp in length from within the large structural subunit of the K88 operon of the 3 respective antigenic variants. Fragments were identified by rates of migration on a 1% agarose gel relative to each other as well as to BstEII-digested lambda fragments. This PCR-based method was comparable to the enzyme-linked immunosorbent assay and western blot test in the ability to differentiate between the antigenic variants. K88+ E. coli were differentiated from among laboratory strains and detected in ileal samples taken from cannulated pigs challenged with a known K88+ variant. K88+ E. coli were also detected from fecal swabs taken from newly weaned pigs, thus confirming that this PCR-based test could provide a convenient clinical assay for the detection of K88+ E. coli. Detection and differentiation of K88+ E. coli using general and specific primers was successful. PCR methods of detection should permit identification of K88+ antigenic variants regardless of the level of expression of the antigen.

Enterohemorrhagic Escherichia coli O157:H7 requires intimin to colonize the gnotobiotic pig intestine and to adhere to HEp-2 cells.

In a previous study, enterohemorrhagic Escherichia coli (EHEC) O157:H7 with a deletion and insertion in the eaeA gene encoding intimin was used to establish that intimin is required for the organism to attach to and efface microvilli in the piglet intestine (M. S. Donnenberg, S. Tzipori, M. L. McKee, A. D. O'Brien, J. Alroy, and J. B. Kaper, J. Clin. Invest. 92:1418-1424, 1993). However, in the same investigation, a role for intimin in EHEC adherence to HEp-2 cells could not be definitively demonstrated. To analyze the basis for this discrepancy, we constructed an in-frame deletion of eaeA and compared the adherence capacity of this mutant with that of the wild-type strain in vitro and in vivo. We observed a direct correlation between the requisite for intimin in EHEC O157:H7 colonization of the gnotobiotic piglet intestine and adherence of the bacterium to HEp-2 cells. The in vitro-in vivo correlation lends credence to the use of the HEp-2 cell adherence model for further study of the intimin protein.

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

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

Clinical signs and lesions in gnotobiotic pigs inoculated with Shiga-like toxin I from Escherichia coli.

Gnotobiotic pigs were used as a model to study the contribution of Shiga-like toxin I to natural disease caused by enterohemorrhagic Escherichia coli in calves and human beings. Eleven 2- to 7-day-old gnotobiotic pigs of either sex, obtained by closed hysterotomy, were injected intramuscularly with graded doses of partially purified Shiga-like toxin I derived from a lysogenized Escherichia coli strain. Four other gnotobiotic pigs were injected with a mock toxin preparation obtained from a nonlysogenized culture of the same E. coli strain. All toxin-injected pigs developed diarrhea, and three displayed signs of neurologic disease. Pigs either died or were euthanatized 2 to 4 days post-inoculation. Necrosis of muscle was grossly evident at the site of injection in all toxin-inoculated pigs. Hemorrhage in the lumen of the small and large intestines and blood in the feces were also evident in two toxin-inoculated pigs. Microscopically, severe necrotizing myositis at the injection site, multifocal encephalomalacia, and mucosal infarcts and hemorrhage in the small and large intestines were seen. In small vessels at lesion sites, endothelial cells were frequently swollen or necrotic. Pigs inoculated with mock toxin did not develop diarrhea or exhibit signs of neurologic disease, and the only apparent lesion was mild microscopic myositis at the injection site in 1/4 pigs. The results of this study indicate that Shiga-like toxin I causes vascular damage and ischemic necrosis in the intestines and brains of gnotobiotic pigs. These lesions are similar to those seen in the intestines of calves and human beings with hemorrhagic colitis and in the brains of human beings with thrombotic thrombocytopenic purpura.

Characteristics of Vero cytotoxin producing Escherichia coli associated with intestinal colonization and diarrhea in calves.

Isolates of Escherichia coli which produce Vero cytotoxin (VTEC) were obtained during 1983-1989 from calves raised in 5 north-central states of the USA. All of the calves experienced intestinal epithelial colonization by VTEC, diarrhea or both; twelve of the calves had bloody diarrhea. Twenty one isolates were serogroup O111 and the others were O103, O69, O45, 026, O5, or non-typable (4 isolates). All but one of the isolates hybridized with the CVD419 probe which identifies most VTEC strains. Thirty two isolates hybridized with the VT1 probe, 3 with both the VT1 and VT2 probes, and one with neither probe. The culture filtrate of the VT probe negative isolate was partially neutralized by SLT I monoclonal antibody. For the other isolates, the results of toxin neutralization by anti-SLT I and anti-SLT II monoclonal antibodies corresponded exactly with the VT1 and VT2 probe hybridization results. Three of the strains adhered in a localized manner to HEp-2 cells and Intestine 407 cells.

Immunocompromise in gnotobiotic pigs induced by verotoxin-producing Escherichia coli (O111:NM).

A verotoxin-producing Escherichia coli serotype O111:NM strain (strain 10049; verotoxin 1 positive) persistently infected experimentally inoculated gnotobiotic pigs, causing attaching-effecting intestinal lesions and chronic diarrhea. Experiments were performed to determine whether persistent infection might be associated with immunocompromise of the host of this organism. Pigs inoculated with this strain had a significant reduction in peripheral blood lymphocytes and lower antibody titers to sheep erythrocytes compared with control pigs. Compared with pigs given a verotoxin-negative pathogenic strain of the same serotype (O111:NM, strain 2430), pigs inoculated with the verotoxin-positive strain had lower peripheral lymphocyte counts and proliferative responses to concanavalin A, phytohemagglutinin, and pokeweed mitogens. The results of this study suggest that strain 10049 has an immunocompromising effect on gnotobiotic pigs.

Identification of two porcine brush border glycoproteins that bind the K88ac adhesin of Escherichia coli and correlation of these glycoproteins with the adhesive phenotype.

In this study, we identified two brush border glycoproteins (210 and 240 kDa) that bind both K88ac+ Escherichia coli and purified K88ac adhesin. The specificity of these binding glycoproteins for the K88ac adhesin was demonstrated in studies in which the binding of 35S-labeled K88ac+ E. coli and biotinylated K88ac adhesin to these glycoproteins was blocked in the presence of a 100-fold molar excess of unlabeled K88ac adhesin but not in the presence of the K99 adhesin. Pretreatment of adhesive brush borders with sodium metaperiodate destroyed both binding activities, indicating that the interaction between the K88ac adhesin and the binding glycoproteins requires the glycoprotein carbohydrate moiety. It was demonstrated previously that K88ac+ E. coli binds to adhesive brush borders but not to nonadhesive brush borders (R. Sellwood, R. A. Gibbons, G. W. Jones, and J. M. Rutter, J. Med. Microbiol. 8:405-411, 1975). In the present study, brush borders isolated from 10 different pigs were tested first for brush border adhesiveness and then for the presence of the binding glycoproteins. In all cases, the binding glycoproteins were detected only in the adhesive brush border preparations. These two binding glycoproteins may be the receptors used by K88ac+ ETEC to adhere to intestinal brush border cells. Their presence on adhesive brush borders and absence on nonadhesive brush borders may be the basis for resistance and susceptibility of pigs to K88ac+ ETEC infections.