F41 antigen among porcine enterotoxigenic Escherichia coli strains lacking K88, K99, and 987P pili.

Colonial morphological variations among enterotoxigenic Escherichia coli which lack K88, K99, and 987P (3P-) were shown to be correlated with expression of several surface antigens, i.e., type 1 pili, F41 pili, and somatic and capsular antigens. Such correlations were established by electron microscopy, serology, and hemagglutination of cells derived from these specific colonial types. Identification of F41 produced by the 3P- enterotoxigenic E. coli strains was made by immunodiffusion in agarose gel, immunofluorescence microscopy, subunit molecular weight determination in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and hemagglutination of F41-piliated 3P- cells and purified F41 pili. Two of the 3P- enterotoxigenic E. coli strains were also shown to be virulent in conventional neonatal pigs and calves. Intense immunofluorescence staining by reference F41 serum of ileal villi of 3P- -infected animals indicated that F41 was expressed during the disease process.     

Susceptibility of porcine intestine to pilus-mediated adhesion by some isolates of piliated enterotoxigenic Escherichia coli increases with age.

Two porcine isolates of enterotoxigenic Escherichia coli (ETEC) (serogroup O157 and O141) derived from fatal cases of postweaning diarrhea and lacking K88, K99, F41, and 987P pili (4P- ETEC) were tested for adhesiveness to small-intestinal epithelia of pigs of different ages. Neither strain adhered to isolated intestinal brush borders of newborn (1-day-old) pigs in the presence of mannose. However, mannose-resistant adhesion occurred when brush borders from 10-day- and 3- and 6-week-old pigs were used. Electron microscopy revealed that both strains produced fine (3.5-nm) and type 1 pili at 37 degrees C but only type 1 pili at 18 degrees C. Mannose-resistant in vitro adhesion to brush borders of older pigs correlated with the presence of fine pili. These strains produced predominantly fine pili in ligated intestinal loops of both older and newborn pigs, but adherence was greater in loops in older pigs. Immunoelectron microscopic studies, using antiserum raised against piliated bacteria and absorbed with nonpiliated bacteria, of samples from brush border adherence studies revealed labelled appendages between adherent bacteria and intestinal microvilli. Orogastric inoculation of pigs weaned at 10 and 21 days of age indicated significantly (P less than 0.001) higher levels of adhesion by the ETEC to the ileal epithelia of older pigs than to that of younger ones. We suggest that small-intestinal adhesion and colonization by these ETEC isolates is dependent on receptors that develop progressively with age during the first 3 weeks after birth. Furthermore, our data are consistent with the hypothesis that the fine pili described mediate intestinal adhesion by the 4P- ETEC strains studied.     

Pilus Production, Hemagglutination, and Adhesion by Porcine Strains of Enterotoxigenic Escherichia coli Lacking K88, K99, and 987P Antigens

Three strains of enterotoxigenic Escherichia coli which adhered, colonized intensively, and caused disease in pig intestine, but which did not produce pili of the K88, K99, or 987P antigen types were designated 3P(-) ETEC. The 3P(-) ETEC caused mannose-resistant hemagglutination, adhered to porcine intestinal epithelial cells in vitro, and produced pili. However, most bacteria taken directly from the intestine of pigs infected with 3P(-) ETEC appeared to be nonpiliated. Two preparations were isolated from the 3P(-) ETEC. One (material A) contained pili, caused mannose-sensitive hemagglutination, and did not inhibit adhesion of whole bacteria to epithelial cells in vitro. The other (material B) had no demonstrable pili, caused mannose-resistant hemagglutination, and blocked ahesion of bacteria to epithelial cells in vitro. Antiserum against an acapsular mutant (K(-)) of one 3P(-) ETEC strain was absorbed to remove antibodies directed against somatic (O) antigen. The absorbed antiserum agglutinated all three 3P(-) ETEC strains grown in the K(-) form at 37 degrees C, but not when they were grown at 18 degrees C. The absorbed antiserum blocked the hemagglutinating activity of material B, but not of material A. It also reacted (via indirect immunofluorescence) with all of the 3P(-) ETEC when they were grown in pig intestine. The results were interpreted to indicate that: (i) the epithelial adhesive and mannose-resistant hemagglutinating activities of the 3P(-) ETEC strains may be mediated by an antigen contained in material B; (ii) this antigen either is not pilus associated or is associated with pili that are not demonstrable by the methods used here; (iii) the 3P(-) ETEC strains produce type 1 pili which do not mediate their adhesion to intestinal epithelium of pigs.     

Occurrence of K99 antigen on Escherichia coli isolated from pigs and colonization of pig ileum by K99+ enterotoxigenic E. coli from calves and pigs.

Several strains of enterotoxigenic Escherichia coli (ETEC) isolated from pigs were found to have an antigen (K99) previously reported only on strains of calf and lamb origin and which facilitates intestinal colonization in the latter two species. Several human ETEC were also tested for K99; however, none were positive. Each of four K99-positive ETEC strains of calf origin and one of pig origin produced K99 in pig ileum in vivo, adhered to villous epithelium in pig ileum, colonized pig ileum, and caused profuse diarrhea in newborn pigs. In contrast to the K99-positive strains above, four K99-negative ETEC from humans and chickens and one K99-positive ETEC from a calf either did not colonize pig ileum or did so inconsistently. When the K99-negative strains did colonize, they had little or no tendency to adhere to intestinal villi. These results are consistent with the hypothesis that K99 facilitates adhesion to and colonization of pig ileum by some ETEC.     

Monoclonal antibody passive hemagglutination and capture enzyme-linked immunosorbent assays for direct detection and quantitation of F41 and K99 fimbrial antigens in enterotoxigenic Escherichia coli.

Production of diarrhea in neonatal calves by enterotoxigenic Escherichia coli depends on its ability to attach to the epithelial cells of the intestine via surface adhesins called pili or fimbriae and to secrete enterotoxins. The most important of these fimbriae are designated K99 and F41. We produced and characterized a murine monoclonal antibody specific to F41. This monoclonal antibody and a K99-specific monoclonal antibody were used to develop sensitive and specific passive hemagglutination and capture enzyme-linked immunosorbent assays (ELISAs) for detection and quantitation of F41 and K99 antigens in E. coli cultures and culture supernatants. The capture ELISA systems exhibited excellent sensitivity and specificity, whereas the passive hemagglutination systems appeared to be oversensitive. The ability of the capture ELISAs to detect K99 and F41 fimbrial antigens in fecal specimens from calves was evaluated. Fimbrial antigens were detected in six of six specimens from scouring calves but not in four of four specimens from nonscouring calves.     

Monoclonal antibody enzyme-linked immunosorbent assay for identification of K99-positive Escherichia coli isolates from calves.

For Escherichia coli to produce diarrhea in animals it must possess the ability to attach to the epithelial cells of the intestine and to produce enterotoxins. Tests developed to differentiate pathogenic from nonpathogenic E. coli have relied on detection of adherence structures called pili or detection of the toxins. We utilized a monoclonal antibody to K99 pili in an enzyme-linked immunosorbent assay to detect the presence of K99 pili in E. coli isolated from calves. Twenty-three E. coli isolates that were known to be stable toxin positive were all shown to produce K99 pili. A 100% correlation also was shown between the presence of K99 antigen and production of stable toxin by E. coli isolates. Of the 251 isolates, 245 were negative by K99 enzyme-linked immunosorbent assay and stable toxin assay. The other six were positive on both tests. The enzyme-linked immunosorbent assay also was shown to be specific for K99 pili by antibody-blocking assays. The number of E. coli necessary for detecting K99 pili by enzyme-linked immunosorbent assay was determined to be 3.5 X 10(5) bacteria per ml.     

In vitro adhesion of Escherichia coli to porcine small intestinal epithelial cells: pili as adhesive factors.

Escherichia coli strains with pili (K99 or 987P) known to facilitate intestinal colonization adhered in vitro to porcine intestinal epithelial cells. These strains adhered equally to both ileal and jejunal epithelial cells. A laboratory E. coli strain that has type 1 pili also adhered to porcine intestinal epithelial cells. When nonpiliated cells derived from 987P+, K99+, or type 1 pilus+ strains were used for in vitro adhesion assays, they failed to adhere. The attachment of piliated bacteria to epithelial cells was a saturable process that plateaued at 30 to 40 bacterial cells attached per epithelial cell. Competitive inhibition of bacterial cell attachment to epithelial cells with purified pili showed that only purified 987P competed against the 987P+ strain and only purified type 1 pili competed against the type 1 pilus+ strain. Competition between a K99+ strain and K99 was not consistently achieved. K99+, 987P+, and type 1 pilus+ bacteria could be prevented from adhering to epithelial cells by Fab fragments specific for K99, 987P, or type 1 pili, respectively. Fab fragments specific for non-K99 bacterial surface antigens did not inhibit adhesion of the K99+ strain. It is concluded that adhesion of E. coli to porcine intestinal epithelial cells in vitro is mediated by pili and that the epithelial cells used apparently had different receptors for different pili.     

Detection of enterotoxigenic K99 (F5) and F41 from fecal sample of calves by molecular and serological methods

Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of neonatal calf diarrhea. Almost all ETEC bacteria are known to adhere to receptors on the small intestinal epithelium via their fimbriae, (F5 (K99) and F41).This study was undertaken to investigate the phenotypic and genotypic screening of virulence genes in E. coli K99 and F41. During January 2008 to December 2009, 298 diarrheic neonatal calves at 1–30?days old were studied by multiplex PCR, isolation, and serological grouping. Of the 298 diarrheic samples, 268 E. coli were isolated, of which 16 samples (5.3%) were positive for having the F5 (K99) fimbrial gene by PCR while all of the E. coli isolates also carried F41 fimbrial genes. Twenty-five percent of the isolates were proven not to be toxigenic as they did not possess the STa enterotoxin gene.     

Adhesion of enterotoxigenic Escherichia coli to human small intestinal enterocytes.

An improved enterocyte adhesion assay has been used to examine a collection of 44 strains of enterotoxigenic Escherichia coli (ETEC) for their ability to adhere to the brush border of isolated human duodenal enterocytes. Fourteen strains showed good adhesion; in each case the ability to adhere correlated with the production of colonization factor antigen I or II (CFA/I or CFA/II) fimbriae. CFA/II-positive producing coli surface antigens 1 and 3 (CS1 and CS3), coli surface antigens 2 and 3 (CS2 and CS3), and only coli surface antigen 3 (CS3) each showed good adhesion. CS3-mediated brush border attachment of CFA/II-positive ETEC was demonstrated by electron microscopy with monospecific antibody and an immunogold labeling technique. One CFA/I-positive ETEC strain was nonadherent in the assay, as were ETEC producing type 1 somatic fimbriae. Five animal ETEC strains producing K88, K99, F41, and 987P fimbriae were slightly more adhesive than control strains, but adhesion was significantly less than that of CFA-positive ETEC. Twenty five human ETEC strains that lacked CFA/I and CFA/II were nonadherent, suggesting either that the surface antigens responsible for adhesion to human intestinal mucosa in these strains were not being produced or that mucosal receptors for these strains are present in regions of the small intestine other than the duodenum.     

Age-specific colonization of porcine intestinal epithelium by 987P-piliated enterotoxigenic Escherichia coli.

Neonatal (less than 1-day-old), 3- and 7-day old, and older (3-week-old postweaning) pigs were challenged by intragastric inoculation with 987P-piliated (987P+) enterotoxigenic Escherichia coli (ETEC) 987. Neonatal pigs were colonized (i.e., there were greater than or equal to 10(8) CFU of test strain per 10-cm ileal segment) and developed diarrhea. Intestinal colonization and the incidence and severity of diarrhea were lower in 3- and 7-day old pigs than in neonates. Older pigs were not colonized and did not develop diarrhea following oral inoculation with five strains of 987P+ ETEC. Strain 987 (987P+) adhered in vitro to intestinal epithelial cell brush borders isolated from both neonatal (sensitive) and older (resistant) pigs. The in vivo growth and expression of 987P pilus by strain 987 in ligated ileal loops created in neonatal and older pigs were similar. The in vivo adherence of 987P+ ETEC to intestinal epithelium in ligated ileal loops in neonatal and older pigs was compared. In neonatal pigs, most of the bacteria were in layers associated with the villous epithelium. In older pigs, most of the bacteria were associated with mucus-like material in the intestinal lumen. We concluded that swine develop an innate resistance to 987P+ ETEC by 3 weeks of age. This resistance does not appear to be due to an absence of 987P-specific receptors in the intestines of the older pig or to an inability of 987P+ bacteria to grow and express pili in the older pig. We hypothesized that the resistance of older pigs to 987P-mediated disease is due to release of 987P-specific receptors into the intestinal lumen, where these receptors facilitate bacterial clearance rather than bacterial adherence to intestinal epithelium and colonization.     

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.     

Ultrastructural study of adhesion of enterotoxigenic Escherichia coli to erythrocytes and human intestinal epithelial cells

The adhesion to erythrocytes and human intestinal epithelial cells of enterotoxigenic Escherichia coli strains H10407, B2C, and H10407P, expressing colonization factor antigen I (CFA/I), CFA/II, and type 1 fimbriae, respectively, was examined by electron microscopy. CFA and type 1 fimbriae were visualized by negative staining in thin sections after en bloc staining with ruthenium red and by immune labeling with antisera raised against purified fimbriae. By negative and ruthenium red staining, CFA/I, CFA/II, and type 1 fimbriae were indistinguishable and appeared as approximately 7-nm-diameter hollow cylindrical structures up to 1.5 micron in length; strain B2C also produced 2- to 3-nm-diameter flexible fibrillar fimbriae. Bacteria producing CFA/I, CFA/II, and type 1 fimbriae adhered to and agglutinated human, bovine, and guinea pig erythrocytes, respectively; CFA/I and CFA/II also mediated attachment of bacteria to the brush border of isolated human duodenal enterocytes. Electron microscopy of agglutinated erythrocytes and enterocytes with adherent bacteria showed, in each case, that bacterial adhesion involved the formation of many interactions between the tips of fimbriae and receptors on the erythrocyte or enterocyte brush border membrane. Immune labeling allowed different fimbrial antigens mediating bacterial attachment to human enterocytes to be identified.     

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.     

F41 pili as protective antigens of enterotoxigenic Escherichia coli that produce F41, K99, or both pilus antigens.

Pigs suckling dams that have been vaccinated with pilus antigen are protected against challenge with enterotoxigenic Escherichia coli (ETEC) strains that express the same pilus antigen. However, some ETEC strains express more than one pilus antigen. Pregnant swine were vaccinated either with E. coli HB101 that harbored a recombinant plasmid coding for F41 expression (F41+) or with the HB101 parent strain that carries the pHC79 vector (F41-). Suckling pigs born to vaccinated dams were challenged with ETEC that expressed either K99, F41, or both pilus antigens. Production of F41 in vivo was demonstrated by immunofluorescence assay of sections of ileum and by seroconversion against F41 antigen by pigs challenged with F41+ and K99+ F41+ ETEC strains. The F41+ vaccine protected against challenge with an F41+ ETEC strain. In contrast, F41+ vaccination did not protect against challenge with K99+ or K99+ F41+ ETEC strains. The F41- vaccine did not protect against challenge with any strain used. The results indicate that K99+ F41+ ETEC strains produce F41 antigen in the small intestine during disease and that F41+ vaccination can be a protective antigen if the challenge strain expresses only F41 antigen, but that F41+ vaccination may not protect against strains that produce both K99 and F41 antigens.     

Pilus-mediated binding of bovine enterotoxigenic Escherichia coli to calf small intestinal mucins.

In this study we show that the adhesion to mucus of the enterotoxigenic Escherichia coli strains responsible for diarrhea in calves involves a bacterium-mucin recognition phenomenon in which the bacterial pili and specific mucus receptors carried by the glycoproteins (2,000 to 400 kilodalton) play a major role. An adhesion maximum was observed at a pH of less than 6 (4.75 to 5.25). The sialic acids and galactose appeared to be at least partly responsible for the attachment of K99 pili, whereas F41 pili preferentially recognized desialylated receptors. The attachment of different strains of E. coli characterized by the presence of the three main pili, K99, F41, and FY, known to be responsible for the binding of enterotoxigenic E. coli to the intestinal epithelium of the calf, was studied using Scatchard and Hill analyses. The attachment mechanism of bacteria carrying K99 pili showed positive cooperativity. FY and F41 pili recognized independent receptor sites, the first on sialylated mucus and the second on sialidase-treated mucus. Moreover, F41 pili were found to bind the native mucus according to a negative cooperativity phenomenon. Finally, the recognition sites carried by bacterial pilins may be saturated by some animal glycoprotein glycans which are therefore adhesion inhibitors.     

Glycoprotein glycans that inhibit adhesion of Escherichia coli mediated by K99 fimbriae: treatment of experimental colibacillosis.

Calf diarrhea due to infection by enterotoxigenic Escherichia coli was treated by administration of glycoprotein glycans derived from bovine plasma. The glycan moieties of the nonimmunoglobulin fraction of plasma mimicked the oligosaccharide moiety of intestinal receptors recognized by K99 pili. These glycoprotein glycans inhibited adhesion of E. coli K99+ ST+ to erythrocyte glycoconjugates in vitro, and they protected colostrum-deprived newborn calves against lethal doses of enterotoxigenic E. coli (10(10) bacteria). Adhesion of bacteria to the intestines (duodenum, jejunum, and ileum) was significantly reduced (by 2 orders of magnitude) in treated calves.     

Prevalence of Pilus Antigens, Enterotoxin Types, and Enteropathogenicity Among K88-Negative Enterotoxigenic Escherichia coli from Neonatal Pigs

Enterotoxigenic Escherichia coli (ETEC) that were isolated from neonatal pigs and that did not react in preliminary tests for pilus antigen K88 were subjected to additional tests for K88 and for pilus antigens K99 and 987P. Four such isolates produced K88, 9 isolates produced K99, 55 isolates produced 987P, and the remaining 43 isolates produced none of the three pilus antigens (3P⁻). Immunofluorescence tests of ileal sections from pigs were more sensitive for 987P detection than was serum agglutination of bacteria grown from the ileum. Most ETEC that produced K88, K99, or 987P were enteropathogenic (adhered to ileal villi, colonized intensively, and caused profuse diarrhea) when given to neonatal pigs. In contrast, only 3 of the 43 ETEC that produced none of the pilus antigens were enteropathogenic. The isolates were also tested for the type of enterotoxin produced. The K88⁺ isolates all produced heat-labile enterotoxin (LT) detectable in cultured adrenal cells (i.e., were LT⁺). None of the 987P⁺, K99⁺, or enterpathogenic 3P⁻ isolates produced LT. However (except for a single K99⁺ isolate), they all produced heat-stable enterotoxin detectable in infant mice (STa⁺). Sixteen isolates produced neither LT nor STa but did produce enterotoxin detectable in ligated intestinal loops of pigs (STb). Most of these LT⁻ STa⁻ STb⁺ isolates were also K88⁻, K99⁻, and 987P⁻ and non-enteropathogenic. One of them was K99⁺ and enteropathogenic. Our conclusions are as follows. (i) Most enteropathogenic ETEC from neonatal pigs produce either K88, 987P, or K99; however, there are some that produce none of the three antigens. (ii) Immunofluorescence tests for pilus antigens produced in vivo are recommended for the diagnosis of ETEC infections. (iii) Reports of LT⁻ STa⁻ STb⁺ swine ETEC are confirmed; furthermore, such isolates can be enteropathogenic.     

Occurrence and characteristics of enterotoxigenic Escherichia coli isolated from calves with diarrhea.

Of 1,004 isolates of Escherichia coli obtained during the spring of 1975 in seven different states from calves with diarrhea, 124 isolates were enterotoxigenic based upon ability to cause distention of the calf ligated intestinal segment. Isolates of enterotoxigenic E. coli (ETEC) were obtained from calves in six of the seven states. ETEC were detected in calves in 118 of 355 herds in Montana during the 1974 and 1975 spring beef calving seasons. The occurrence and serotypes of ETEC isolated from calves in states outside Montana were similar to ETEC isolated from calves in Montana. One hundred and fourteen of the 124 isolates of ETEC were placed in one of six different groups upon agglutination in OK antiserum. Serotyping of 35 of the 124 isolates of ETEC indicated the following serotype for isolates in each group: group 1, O9:K35; group 2, O101:K30; group 3, O8:K85; group 4, O20:K?; group 5, O8:K25; and group 6, O101:K28. Determination of the presence of K99 antigen indicated that 28 of 35 isolates of ETEC had K99 antigen, whereas the antigen was not detected in any of the 10 isolates of non-ETEC studied.     

Colonization of porcine intestine by enterotoxigenic Escherichia coli: selection of piliated forms in vivo, adhesion of piliated forms to epithelial cells in vitro, and incidence of a pilus antigen among porcine enteropathogenic E. coli.

In contrast to K88-positive porcine enterotoxigenic Escherichia coli (ETEC), K88-negative porcine ETEC strains did not adhere to isolated intestinal epithelial cells in vitro. However, they did adhere to intestinal epithelium in vivo. Growth of one such ETEC (strain 987) in pig small intestine consistently yielded a greater percentage of piliated cells than did growth in vitro. This increase was demonstrable by electron microscopy, by change in colonial morphology, and by agglutination in specific antisera against the pili of strain 987. In contrast to the stored stock culture (which contained very few piliated cells), richly piliated forms of strain 987 did adhere to isolated intestinal epithelial cells in vitro. A series of porcine E. coli strains was tested for agglutinability in antiserum against the pili of strain 987, and several K88-negative ETEC strains were agglutinated. These data are consistent with the hypothesis that pili facilitate intestinal adhesion and colonization by K88-negative ETEC strains.     

Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene in enterotoxigenic E. coli strains isolated from piglets and calves with diarrhea.

Enterotoxigenic Escherichia coli (ETEC) strains isolated from piglets and calves with diarrhea were tested for the presence of the enteroaggregative E. coli enterotoxin 1 (EAST1) gene sequences by PCR and colony hybridization. The EAST1 gene was found in most porcine ETEC strains with adherence factor K88, especially in those elaborating heat-labile enterotoxin. One porcine ETEC strain with adherence factor K99 was also positive for the EAST1 gene. In contrast, 987P-positive (987P+) ETEC strains from piglets, K99+ ETEC strains from calves, and K99+ F41+ or F41+ ETEC strains from piglets and calves were negative for the EAST1 gene. The K88ab+ or K88ac+ ETEC strains tested possessed the EAST1 gene on a plasmid that was distinct from a K88-encoding plasmid. The EAST1 gene sequences of the K88+ ETEC strains were identical to each other and 99.1 and 98.3% homologous to the previously reported sequences of ETEC strains colonizing humans and enteroaggregative E. coli strains, respectively. The data indicate that the EAST1 gene is distributed among porcine ETEC strains in association with the adherence factor type.