Effect of amino acids on expression of K99 adherence pili by Escherichia coli.

Various common L-amino acids differed in their ability to suppress K99 production in E. coli. Strains of E. coli also varied in their sensitivity to amino acid-mediated suppression of K99. Alanine, methionine, leucine, and valine were the most suppressive amino acids. However, when compared to single amino acids, mixtures of these amino acids were frequently either less suppressive, non-suppressive, or even stimulatory for K99 expression.

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.

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.

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.

Comparison of seroagglutination, ELISA, and indirect fluorescent antibody staining for the detection of K99, K88, and 987P pilus antigens of Escherichia coli.

Seroagglutination (SAT), enzyme-linked immunosorbent assay (ELISA), and indirect fluorescent antibody staining tests (IFAT) were compared for reliability in the detection of pilus antigens K99, K88, and 987P of Escherichia coli. Test sensitivities were compared using mixtures of piliated bacteria of several strains diluted to a constant optical density with a nonpiliated strain. Relative sensitivities and specificities of the 3 tests were also compared using 55 E. coli strains that had previously been serotyped and characterized for pilus genes by DNA probe. Although specificity was not a serious problem with any of the tests, the SAT was relatively nonsensitive. The IFAT showed the greatest sensitivity of the 3 tests in detecting K88, K99, and 987P E. coli.

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.

Attaching and effacing Escherichia coli infections in calves, pigs, lambs, and dogs.

Attaching and effacing Escherichia coli (AEEC) adhere to mucosal epithelium in both small and large intestine and induce a distinctive lesion characterized by an irregular scalloped appearance of the epithelial layer. Infection with attaching and effacing E. coli was detected in 14 calves, 7 pigs, 2 lambs, and 3 dogs. Affected animals were from farms and kennels in South Dakota, Minnesota, Iowa, Nebraska, and Wisconsin. Ages of affected animals were calves, 2 days to 4 months; pigs, 1-6 weeks; lambs, 1 week; and dogs, 7-8 weeks. Clinical signs included diarrhea in all animals, but other nonenteric disease problems were present in some animals. Concurrent infection with other enteropathogens was detected in 9 calves and 5 pigs. Infection with AEEC appeared to be the sole cause of illness and death in some animals. There was evidence of intestinal hemorrhage in 5 of the calves and in all 3 dogs. Attaching and effacing lesions varied from small scattered foci to widespread involvement of large areas of intestinal mucosa. Verotoxin was produced by E. coli strains isolated from 9 calves, but not by strains from pigs, lambs, or dogs.

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.

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.

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).

Use of immunofluorescence, Gram's staining, histologic examination, and seroagglutination in the diagnosis of porcine colibacillosis.

An indirect fluorescent antibody (IFA) test of ileal impression smears for K88, K99, and 987P pilus antigens was compared with histologic examination and seroagglutination of Escherichia coli isolates for efficacy in determining colibacillosis in pigs. Histologic examination appeared to be more effective than the IFA test in revealing colonization of the ileum by bacteria. However, histologic examination revealed little about the nature of the colonizing bacteria. Correlation between bacterial adherence, as observed in histologic sections of ileum, and the presence of bacteria with adherence pili, as determined by IFA testing, was 91%. Results of seroagglutination for pilus antigens correlated with results of histologic examination in only 84% of the cases. Pilus antigens were not identified by IFA testing or seroagglutination in 5% of the cases in which adherent bacteria were observed in histologic sections of the small intestine. Because little preparation time was required for the IFA test, results were available within 2 hours of necropsy. In contrast to histologic examination, the IFA test made possible identification of the colonizing organism as E coli and revealed the type of pilus antigen present. Adherence pili were identified more frequently by IFA testing than by seroagglutination. Examination of Gram's-stained ileal impression smears was useful in screening for colibacillosis in pigs. Bacterial adherence was found in positive correlation with the number of gram-negative bacilli observed. Bacterial adherence rarely was observed in histologic sections of ileum when smears contained less than or equal to 10 gram-negative bacilli/1,000 X microscopic field. Each diagnostic test compared offered advantages and disadvantages over the other tests. Seemingly, concurrent use of several of these tests, rather than one, should be used in the diagnosis of porcine colibacillosis.

Fimbriae and enterotoxins associated with Escherichia coli serogroups isolated from pigs with colibacillosis.

A comprehensive study of 223 Escherichia coli isolates from pigs with colibacillosis included determination of O serogroups, detection of heat-labile enterotoxin, heat-stable enterotoxin (STa and STb), and identification of K88, K99, 987-P, F-41, and type 1 fimbriae. The incidence of the various E coli types among isolates of pigs of different ages was also determined. Escherichia coli bearing K88 fimbriae accounted for 48% of all isolates studied, were most often of serogroup O157, O149, or O8, and usually produced labile toxin alone or in combination with STa or STb. These E coli were commonly isolated from pigs in each age group studied (0 to 5 days, 6 to 10 days, 11 to 24 days, and greater than 24 days). Escherichia coli bearing 987-P accounted for 30% of the isolates, were most often of serogroup O141 or O20, and usually produced STa. Escherichia coli bearing K99 accounted for 13% of the isolates, usually were of serogroup O101 or O8, and almost always produced STa. Escherichia coli bearing 987-P or K99 were most often isolated from pigs less than 6 days of age. Fimbriae F-41, when identified, were usually on E coli of serotype O101:K99. Although infrequently found, type 1 fimbriae were on E coli of most of the serogroups identified in this study.

Evaluation of a live avirulent Escherichia coli vaccine for K88+, LT+ enterotoxigenic colibacillosis in weaned pigs.

Live, avirulent Escherichia coli vaccine strains were constructed and tested for efficacy in preventing colibacillosis in 4-week-old pigs. Either or both of 2 plasmids were inserted into avirulent E coli strain G58-1 (0101:NM). These plasmids were pPMC4, which encodes for LTb subunits of heat-labile enterotoxin, and pDHF1, which encodes for K88ac fimbriae. Litter- and weight-matched pigs were removed from sows when they were 10 days old and vaccinated orally with the constructed strains or with G58-1 (negative control vaccine) when they were 2 weeks old and 5 days later. All pigs were challenge-inoculated with virulent E coli strain 3030-2 (O157:K88, LT+, STb+) 2 weeks after the first vaccination. Only 1 pig vaccinated with G58-1/pPMC4/pDHF1 developed diarrhea and none died following challenge inoculation. Seventeen of 31 control pigs developed diarrhea and 11 died. Of 18 pigs vaccinated with G58-1/pDHF1 then challenge-inoculated with the virulent strain, 5 developed diarrhea and 2 died. Fifteen of 18 litter- and weight-matched controls developed diarrhea and 8 died. When compared with G58-1 (negative control), G58-1/pPMC4 afforded no protection to pigs challenge-inoculated with 3030-2.

Characterization of immune responses of cattle to erythrocyte stroma, Anaplasma antigen, and dodecanoic acid-conjugated Anaplasma antigen: humoral immunity.

Normal erythrocyte antigen (sonically disrupted erythrocyte stroma; SES) and two anaplasma antigens (sonically disrupted anaplasma antigen; SAA, and French pressure cell-disrupted anaplasma antigen; FAA) were prepared from normal and Anaplasma marginale-infected blood. Portions of SAA and FAA antigens were chemically modified by conjugation with dodecanoic acid (SAADA and FAADA). Eleven cattle were vaccinated with SES, SAA, SAADA, or FAADA. Five weeks later, the 11 cattle, together with 3 controls, were challenge exposed with A marginale. The anti-anaplasma antibody response and the antierythrocyte-antibody response (including the blood group isoantibody response) were evaluated. Only SAA-vaccinated cattle developed anti-anaplasma antibody before challenge exposure. Isoantibodies were developed by 1 of the 3 SAADA-vaccinated cows and 1 of the 2 FAADA-vaccinated cows, as well as by all 3 SAA-vaccinated cows. After challenge exposure, all cattle developed anti-anaplasma antibody and antierythrocyte autoantibody.

Characterization of the inclusion limiting membrane of anaplasma marginale by immunoferritin labeling.

Purified anti-erythrocytic membrane antibody (PAMA) was prepared from rabbit anti-bovine erythrocyte serum by an adsorption and elution technique, utilizing bovine erythrocytes. Lysed and washed anaplasma-infected erythrocytes were incubated with PAMA or control reagents. Specimens were then subjected to immunoferritin labeling with ferritin antiglobulin conjugate. Upon examination by electron microscopy, specimens incubated with PAMA showed heavy ferritin labeling of erythrocytic membranes and also the limiting membranes of anaplasmal inclusions. Anaplasmal initial bodies freed from their inclusion membranes were not labeled. Negative control specimens, incubated with normal rabbit serum or PAMA which had been absorbed with erythrocytes, did not show specific ferritin labeling. Intact bovine erythrocytes, which were used as a positive control of anti-bovine erythrocytic membrane specificity, were heavily ferritin-labeled. Avian erythrocytes, a negative control of specificity, remained unlabeled. The results of this study indicate that the limiting membrane of the anaplasmal inclusion is derived from the erythrocytic membrane.

Characterization of immune responses of cattle to erythrocyte stroma, Anaplasma antigen, and dodecanoic acid-conjugated Anaplasma antigen: cell-mediated immunity.

Sonically disrupted normal erythrocyte stroma (SES) and two anaplasma antigens (sonically disrupted anaplasma antigen; SAA, and French pressure cell disrupted anaplasma antigen; FAA) were prepared from normal and Anaplasma marginale-infected blood. The SAA and FAA antigens were chemically modified by conjugation with dodecanoic acid (SAADA and FAADA). Significant (P less than or equal to 0.05) anti-anaplasma lymphocyte-transformation responses were obtained from all cattle given SAA, SAADA, or FAADA vaccines. Only cows given SAA developed anti-anaplasma antibody. Mild antierythrocyte lymphocyte-transformation responses were obtained from most vaccinated animals. Delayed hypersensitivity to erythrocyte antigen was not detected. The SAA-vaccinated cows had the highest degree of protection in that they developed a smaller percentage of parasitemia and had less severe anemia than did other cattle in the study. The SAADA- and FAADA-vaccinated cattle developed a good cell-mediated immune response, but poor humoral immune response and had lower parasitemias than did challenge-exposed controls; but they developed severe anemia. It is suggested that cellular and humoral mechanisms are essential for protection in anaplasmosis. Evidence of protection from clinical anaplasmosis was not observed in SES-vaccinated cows.

Influence of bovine intestinal fluid on the expression of K99 pili by Escherichia coli.

A study was conducted to determine whether intestinal fluid collected from various portions of bovine intestine differed in its effect on production of K99 pili by Escherichia coli. The small and large intestines of 7 calves, euthanatized 4 hours after a final feeding of milk, were divided into 6 to 9 segments from which intraluminal fluids were collected. Depending on the amount of fluid collected, up to 20 E coli strains that express K99 pili were grown on media prepared from the content of each specimen and then were tested for K99 pilus expression. In general, intestinal fluid from the most proximal small intestinal segments were more suppressive to K99 pilus expression than was fluid from more distal segments of small intestine. Only about 20% of the E coli test strains expressed K99 pili when grown on medium prepared from proximal small intestinal segmental fluid, whereas greater than 90% did when grown on medium prepared from distal small intestinal segmental fluid. Fluid from the large intestine varied considerably from calf to calf in its effect on K99 pilus expression. A correlation was found between K99 pilus expression and pH of the intestinal fluid, with the lower pH values (characteristic of proximal intestinal segmental fluid) being suppressive. The correlation between K99 pilus production and the pH of the medium was verified, using defined laboratory media adjusted to various pH values. Strains of E coli grown in medium at or below pH 5.5 failed to express K99 pili, whereas the same strains when grown in medium at or above pH 6.5 expressed K99 pili in abundance.

Combined rotavirus and K99 Escherichia coli infection in gnotobiotic pigs.

Fifty nine 3-day-old gnotobiotic pigs were randomly assigned to 4 experimental groups: 14 pigs were orally inoculated with rotavirus (RV), 14 were orally inoculated with enterotoxigenic Escherichia coli (ETEC), 18 were orally inoculated with both agents, and 13 were controls. Pigs inoculated with RV plus ETEC were given the RV inoculum at 3 days of age and then, 24 hours later, were given the ETEC inoculum. Three pigs inoculated only with RV, 3 pigs inoculated only with ETEC, 4 pigs inoculated with RV plus ETEC, and 3 pigs in the control group were euthanatized at 5 and 7 days of age. Two pigs in each of the 4 experimental groups also were euthanatized at 9 days of age. Intestinal segments from 6 sites in the small intestine were examined by virologic, bacteriologic, and histologic procedures. For 10 days after inoculation, the remaining pigs in each group were observed clinically to monitor severity and duration of diarrhea, mortality, and shedding of RV or ETEC. Pigs inoculated with the combined RV plus ETEC inoculum developed more severe diarrhea, compared with pigs inoculated with the single agents; all dually inoculated pigs died between 3 and 6 days after inoculation. There was no mortality in pigs inoculated with either RV or ETEC. Lesions were restricted to the small intestine in pigs inoculated with RV plus ETEC and in pigs inoculated with RV or ETEC. There was no difference in the severity of the villus atrophy between the dually inoculated pigs and pigs inoculated only with RV.(ABSTRACT TRUNCATED AT 250 WORDS)

Attaching and effacing Escherichia coli infection as a cause of diarrhea in young calves.

A form of enteric Escherichia coli infection was identified in 60 calves from 59 farming operations. The E coli responsible for these infections principally colonized the colon, inducing a distinctive lesion described as attaching and effacing. Hemorrhagic enterocolitis or blood in the feces was observed on 40% of the farms. Of affected calves, 86.6% were dairy calves (average age, 11.8 days). Forty-four calves were infected concurrently with other enteropathogens (cryptosporidia, rotavirus, coronavirus, enterotoxigenic E coli, bovine viral diarrhea virus, coccidia). Verotoxin-producing E coli was recovered from 31 calves; 8 were serotype O111:NM isolates, 3 were serotype O5:NM, and 1 was serotype O26:NM.