K88 Antigen Research Articles

In vivo properties of a cloned K88 adherence antigen determinant

An Escherichia coli strain of serotype O9:K36:H19 harboring the K88 recombinant plasmid pMK005 was able to efficiently colonize the small bowel of young piglets after oral infection. The strain expressed K88 antigen in vivo, and bacteria were detected in close association with the surface of the intestinal villi. Mice infected orally or intravenously with attenuated Salmonella typhimurium SL3261 harboring pMK005 were well protected against subsequent challenge with the highly virulent S. typhimurium SL1344. Anti-K88 antibodies were detected in the serum of mice immunized with SL3261(pMK005).

Colonization antigens, antibiotic resistance and plasmid content of enterotoxigenic Escherichia coli isolated from piglets with diarrhoea in galicia (North-Western Spain)

Escherichia coli colonies isolated from 50 diarrhoeic and 29 healthy piglets were investigated for several properties related to pathogenicity, such as production of heat-labile (LT) and heat-stable (STa) enterotoxins, presence of K88 and K99 colonization antigens, mannose-resistant haemagglutinating activity (MRHA), β-haemolysis and antibiotic resistance. The objective was to establish the toxic and adhesive abilities of E. coli strains that cause porcine diarrhoea in Galician farms. Fifty-seven colonies from 14 diarrhoeic piglets formed STa, while no STa + colony was detected from healty piglets. Thirty-four of the 57 STa + colonies were resistant to gentamicin. Sixteen representative STa + strains isolated from the 14 infected piglets were serotyped, investigated for plasmid content and examined by electron microscopy. Of these STa + strains, 15 belonged to serotype 0141:K85ab and carried on their surface the fimbrial antigen P987. The remaining representative STa + strain belonged to serotype 0101:K30 and was K99 +, being the only STa + strain with MRHA activity. All 15 STa + P987 + strains possessed a similar plasmid pattern, with three plasmids ranging in molecular weight from 33 × 10 6 to 74 × 10 6; nine of the gentamicin-resistant strains possessed an additional plasmid of molecular weight 16 × 10 6, which was absent in the six gentamicin-sensitive strains. Strains producing LT or K88 antigen were not detected. Forty-one MRHA + colonies were isolated at similar rates from both diarrhoeic and healthy piglets. Twelve of the 19 non-enterotoxigenic MRHA + strains of which the O-group was established, belonged to serogroups (01, 02, 07, 08, 09 and 075) typical of the human E. coli strains that cause extraintestinal infections. Finally, a statistically significant association between haemolytic and MRHA activities in porcine E. coli was found. In conclusion, it was found that STa + E. coli strains belonging to serotype 0141:K85ab:P987 are associated with porcine diarrhoea in Galicia. Additionally, no correlation between the isolation of non-ETEC MRHA + strains and diarrhoea was observed.

Colicin resistance in relation to pathogenicity factors in strains of Escherichia coli isolated from the intestinal tract of piglets

Three hundred and fifteen E. coli strains isolated from the intestine of piglets were examined for K-antigens 88 and 99, enterotoxin production and colicin resistance. Of these strains 308 belonged to one of 3 following different groups: Group 1: 0149, K88, producing heat-labile (LT) and heat-stable (ST) enterotoxins, group 2: 064, K99, producing ST, and group 3: variable O-antigens, no K-antigens or enterotoxin production. Almost 100 % of the E. coli strains were found to be resistant to colicins E1, E3, Ia, H and D+X. Resistance to colicins E2, B+M, V and K+X were found in 91.7 %, 43.8 %, 49.8 % and 62.2 % respectively. E. coli strains in group 1 were always (resistant to colicin E2, while about 87 % of the other strains were resistant to this colicin. E. coli strains in group 2 were more often resistant to colicin B+M, V and K+X (65 %, 94 %, 83 %) than strains in group 1 (37 %, 24 %, 64 %) and strains in group 3 (37 %, 52 %, 46 %). E. coli strains in group 2 showed a high degree of multiresistance, 45.1 % of the strains being resistant to all of the 9 colicins. About 10 % of the other strains were resistant to all of the 9 colicins. E. coli strains harbouring the enteropathogenicity factors K99 antigen and ST production, showed a higher degree of colicin resistance than both the E. coli strains with K88 antigen and ST and LT production, and the E. coli strains lacking enteropathogenicity factors.

The use of the fluorescent antibody test to detect Escherichia coli with K88 and 987P pilus antigens

Direct fluorescent antibody tests were used to detect Escherichia coli possessing K88 and 987P antigens. Identification of bacteria was accomplished on suspensions of organisms from clinical isolates, on frozen sections and impression smears from small intestine and on faecal smears. This assay makes possible the rapid identification of E. coli possessing K88 and 987P pilus antigens.

Screening of pig intestines for K88 non-adhesive phenotype by enzyme immunoassay

An adhesion test for binding of porcine brush border membranes to Escherichia coli cells that possess the K88 antigen (K88 +) has been developed using enzyme immunoassay procedures. K88 pilus protein or K88 + E. coli cells were immobilized in the wells of polystyrene microtitre plates. These plates were incubated in the presence of material obtained by scraping the villous surface of pig small intestines. Adhesion of membrane material to immobilized K88 was detected by adding rabbit anti-brush border IgG followed by urease-labelled sheep anti-rabbit IgG conjugate. Action of bound enzyme on urea/bromo-cresol purple substrate solution (pH 4.8) produced an intense colour change from yellow to purple, enabling the test to be read visually. This test enables simple, rapid testing of large numbers of intestial samples and gives results that agree well with the more cumbersome microscopic adhesion test for adhesion of K88 + E. coli to purified brush border membranes.

Unsupervised Chemical Pattern Recognition In Complex Mass Spectra

Developing a reliable method for obtaining chemical information on components of complex materials is a major goal of modern pyrolysis mass spectrometry. Unsupervised methods are necessary since pure reference spectra of components are often unavailable. Recent developments in unsupervised mixture analysis using examples from bacteria, jet fuels, and wood are given. Factor and discriminant analysis on 18 strains of Escherichia coli showed a clear differentiation between K1 and non-K1 antigen strains. Graphical rotation confirmed that the differentiation was based on the K1 antigen colominic acid. Binary and ternary mixtures of jet fuel components were used to numerically extract spectra of pure compound classes present in fuel formulations. Mathematically extracted spectra compared favorably to component spectra, which were not included in the data set. The variance diagram technique gave the same directions of the component axes as target rotation, a supervised technique. Variance diagram methods were applied to single samples of wood by recording mass spectra as a function of temperature, since the three major components of wood pyrolyze at slightly different temperatures. A variance diagram with factor 2 expanded showed clearly the three major wood components: cellulose, hemicellulose, and lignin.

Hygromycin A and methoxyhygromycin, novel inhibitors of K88 antigen synthesis of enterotoxic Escherichia coli strain.

During the screening for inhibitors of K88 antigen synthesis that suppress hemagglutination by E. coli E68 NIH 457-71, we found Streptomyces sp. No. 207 produced two active substances, one of which was identified as hygromycin A. Another new compound, named methoxyhygromycin, was very similar to hygromycin A but methoxyhygromycin had a methoxy group on the neoinosamine-2 moiety instead of the methylene dioxy group found in hygromycin A. Although hygromycin A and methoxyhygromycin had almost no effect on the growth of E. coli E68 NIH 457-71, they inactivated the hemagglutination activity of E. coli at low concentrations of 12.5 μg/ml and 25 μg/ml, respectively.

Identification of an inducible catabolic system for sialic acids (nan) in Escherichia coli.

Escherichia coli K-12 and K-12 hybrid strains constructed to express a polysialic acid capsule, the K1 antigen, were able to efficiently use sialic acid as a sole carbon source. This ability was dependent on induction of at least two activities: a sialic acid-specific transport activity, and an aldolase activity specific for cleaving sialic acids. Induction over basal levels required sialic acid as the apparent inducer, and induction of both activities was repressed by glucose. Induction also required the intracellular accumulation of sialic acid, which could be either added exogenously to the medium or accumulated intracellularly through biosynthesis. Exogenous sialic acid appeared to be transported by an active mechanism that did not involve covalent modification of the sugar. Mutations affecting either the transport or degradation of sialic acid prevented its use as a carbon source and have been designated nanT and nanA, respectively. These mutations were located by transduction near min 69 on the E. coli K-12 genetic map, between argG and glnF. In addition to being unable to use sialic acid as a carbon source, aldolase-negative mutants were growth-inhibited by this sugar. Therefore, the intracellularly accumulated sialic acid was toxic in aldolase-deficient E. coli strains. The dual role of aldolase in dissimilating and detoxifying sialic acids is consistent with the apparent multiple controls on expression of this enzyme.

Nucleotide sequences of four variants of the K88 gene of porcine enterotoxigenic Escherichia coli

The nucleotide sequences of four variants of the Escherichia coli K88 antigen gene, K88ab1, K88ab2, K88ac, and K88ad, have been determined. The K88ab2 and K88ac sequences have not been reported previously. The K88ab1 sequence is very similar to that determined by other workers, but the K88ad sequence differs considerably from that described in a previous report. Comparison of the amino acid sequences inferred from the gene sequences revealed certain clusters of amino acid substitutions which have been correlated with areas of potential antigenicity in the mature proteins.

Pathological and microbiological findings in 38 lambs with watery mouth

Thirty-eight lambs with watery mouth were presented alive for necropsy. The most frequently found abnormality was enteritis, in 25 lambs. Bacteria were cultured from the gut contents of all lambs and bacteraemia was detected at high frequency at necropsy, though very much less frequently six hours before. Escherichia coli strains did not possess K99 antigen. It was concluded that absolute starvation and septicaemia were unlikely causes of the disease; that there is support for the hypothesis that reduced gut motility is an important early feature and, that if bacteria are involved in the aetiology, they may have a non-specific role.

Neuraminidase associated with coliphage E that specifically depolymerizes the Escherichia coli K1 capsular polysaccharide.

Plaque morphology indicated that the five Escherichia coli K1-specific bacteriophages (A to E) described by Gross et al. (R. J. Gross, T. Cheasty, and B. Rowe, J. Clin. Microbiol. 6:548-550, 1977) encode K1 depolymerase activity that is present in both the bound and free forms. The free form of the enzyme from bacteriophage E was purified 238-fold to apparent homogeneity and in a high yield from ammonium sulfate precipitates of cell lysates by a combination of CsCl density gradient ultracentrifugation, gel filtration, and anion-exchange chromatography. The enzyme complex had an apparent molecular weight of 208,000, as judged from its behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was dissociated by sodium dodecyl sulfate at 100 degrees C to yield two polypeptides with apparent molecular weights of 74,000 and 38,500. Optimum hydrolytic activity was observed at pH 5.5, and activity was strongly inhibited by Ca2+; the Km was 7.41 X 10(-3) M. Rapid hydrolysis of both the O-acetylated and non-O-acetylated forms of the K1 antigen, an alpha 2----8-linked homopolymer of N-acetylneuraminic acid, and of the meningococcus B antigen was observed. Limited hydrolysis of the E. coli K92 antigen, an N-acetylneuraminic acid homopolymer containing alternating alpha 2----8 and alpha 2----9 linkages, occurred, but the enzyme failed to release alpha 2----3-, alpha 2----6-, or alpha 2----9-linked sialic residues from a variety of other substrates.

Effect of Prepartum Vaccination with K99 Escherichia coli Vaccine on Maternal and Calf Blood Antibody Concentration and Calf Health

One hundred and two dry, pregnant Holstein cows were identified alternately as vaccinated or nonvaccinated (Group 0) animals. Vaccinated cows were scheduled for vaccination at 6 and 3 wk prior to expected calving date with Vicogen®, a commercial vaccine produced for the prevention of calf scours caused by entero-pathogenic Escherichia coli that possess the K99 antigen. Group 1 included cows that were less than 6 wk from freshening when the experiment started and, therefore, received only one vaccination and cows that received two vaccinations with less than 5 days between the second vaccination and freshening. Those cows with interval between the second vaccination and parturition greater than 5 days were classified as Group 2. Soon after birth, each calf was given 2 liters of colostrum from its dam. For at least 3 days, and longer when available, calves from control cows received pooled colostrum from control cows and calves from vaccinated cows received pooled colostrum from vaccinated cows.Anti-K99 antibody liters were determined by an agglutination test on blood from cows and calves and on colostrum. Other measurements were made by standard procedures. Results from Groups 0, 1, and 2 were cow blood titer at freshening 21, 355, 306; calf total plasma protein at 24h of age 6.45, 6.31, 6.22; calf packed cell volume at 24h of age 32.9, 30.0, 30.2; calf blood titer at 24h of age 34, 762, 1114; colostrum titer 74, 1637, 3404. For 93 calves, mortality was 10.6, 11.1, and 7.1%. Differences in scours scores and incidence of sickness were not significant.

Evaluation of a monoclonal antibody to the K99 fimbrial adhesin produced by Escherichia coli enterotoxigenic for calves, lambs and piglets

All the K99+ Escherichia coli grown at 37 degrees C stained strongly with a peroxidase labelled K99 monoclonal antibody using a direct immunoperoxidase staining procedure. There was no reaction when these bacteria were cultured at 18 degrees C or when K99- E coli were grown at either temperature. The binding of the monoclonal antibody to K99 antigen was inhibited by OK antisera to heterologous K99+ E coli but OK antisera to E coli producing adhesins other than K99 were without effect. Using the slide agglutination test the reactions of the monoclonal antibody were identical to those of a polyclonal antiserum to K99 when both were used in parallel to examine 100 K99+ E coli from at least 10 somatic O groups and 1308 K99+ E coli from at least 82 different somatic O groups submitted for routine serological typing in England or the, USA. The monoclonal antibody reacted with K99+ E coli in cryostat sections of the ileum from a piglet infected with E coli strain B44 (O9: K30, K99, F41) but there was no reaction with similar material from piglets infected by E coli strains 1751 (O101: F41), X177/81 (O9: K103, 987P) or Abbotstown (O149: K91, K88ac).

Colicin Production in Relation to Pathogenicity Factors in Strains of Escherichia Coli Isolated from the Intestinal Tract of Piglets

Three hundred and fifteen E. coli strains isolated from the intestine of piglets were examined for K-antigens 88 and 99, enterotoxin production and colicin resistance. Of these strains 308 belonged to one of 3 following different groups: Group 1: 0149, K88, producing heat-labile (LT) and heat-stable (ST) enterotoxins, group 2: 064, K99, producing ST, and group 3: variable O-antigens, no K-antigens or enterotoxin production. Almost 100 % of the E. coli strains were found to be resistant to colicins E1, E3, Ia, H and D+X. Resistance to colicins E2, B+M, V and K+X were found in 91.7 %, 43.8 %, 49.8 % and 62.2 % respectively. E. coli strains in group 1 were always (resistant to colicin E2, while about 87 % of the other strains were resistant to this colicin. E. coli strains in group 2 were more often resistant to colicin B+M, V and K+X (65 %, 94 %, 83 %) than strains in group 1 (37 %, 24 %, 64 %) and strains in group 3 (37 %, 52 %, 46 %). E. coli strains in group 2 showed a high degree of multiresistance, 45.1 % of the strains being resistant to all of the 9 colicins. About 10 % of the other strains were resistant to all of the 9 colicins. E. coli strains harbouring the enteropathogenicity factors K99 antigen and ST production, showed a higher degree of colicin resistance than both the E. coli strains with K88 antigen and ST and LT production, and the E. coli strains lacking enteropathogenicity factors.

Phenotypic modification of the reactivity in human serum of escherichia coli K1 solates

Three Escherichia coli strains producing the K1 antigen and shown to be resistant to the complement-mediated bactericidal action of human serum when grown in batch culture, were cultivated in the chemostat under conditions of carbon-, nitrogen-, magnesium- and phosphate-limitation. All strains were fully serum resistant when grown under carbon-limiting conditions but became phenotypically serum sensitive when limited by magnesium. One strain, belonging to serogroup O7:K1, also displayed serum sensitivity when nitrogen limited and showed an intermediate serum response when phosphate was used as the limiting nutrient.

Attachment of E. coli-bearing K88 antigen to equine brush-border membranes

Equine small intestinal brush-border membranes, from 40 adult horses were tested in vitro for the presence of receptors for the Escherichia coli adhesive antigens K88ab, K88ac and K99. Only K88-positive strains of E. coli adhered strongly to horse brush-border membranes. In contrast, a K88-negative mutant strain J 2, 2 K99-positive strains and 3 E. coli strains isolated from foals failed to adhere to horse brush-border membranes. Purified K88ac pili when reacted with equine brush-border membranes inhibited to a great extent the adhesion of K88-positive E. coli. Similarly, K88-positive E. coli previously reacted with K88 antibody, did not attach to equine brush-border membranes. Oral inoculation of 4 newborn foals with strains of K88-positive enterotoxigenic E. coli, producing either heat-stable or heat-labile enterotoxin, caused diarrhoea in 1 animal.

Influence of K99 on the phagocytosis and killing of K99-positive Escherichia coli by mouse peritoneal cells in vitro.

The expression of K99-plasmid encoded products was examined to assess their influence on phagocytosis and killing by mouse peritoneal cells. Strains of K99-positive Escherichia coli were cultured under varying conditions which either allowed or inhibited the expression of K99-plasmid products. The survival of phagocytosis by the bacteria was found to depend more on the expression of the K99 antigen than the expression of its associated adhesin.

Capsule reduces adherence of enterotoxigenic Escherichia coli to isolated intestinal epithelial cells of pigs.

Previous reports have demonstrated that heat-stable (A-type) capsule on piliated enterotoxigenic Escherichia coli enhances colonization of enterotoxigenic E. coli in the small intestine and enhances virulence of enterotoxigenic E. coli. In this report, four encapsulated enterotoxigenic E. coli strains and one encapsulated nonenterotoxigenic strain of E. coli and their nonencapsulated mutants were tested for adhesion to isolated intestinal epithelial cells or brush borders from neonatal pigs. The enterotoxigenic E. coli also expressed the K99 pilus antigen. The nonencapsulated mutants of the four enterotoxigenic E. coli adhered in higher numbers than did the encapsulated parental strains. Both the encapsulated and nonencapsulated forms of enterotoxigenic E. coli 431 grown at 18 degrees C (K99 production suppressed) adhered poorly to the isolated cells. The nonenterotoxigenic E. coli 1793 which does not express K99 antigen also adhered poorly in both encapsulated and nonencapsulated forms. Fab fragments of anticapsular immunoglobulin G failed to block the effect of capsule on adherence of strain 431. The results indicated that K99 was the principal mediator of in vitro adhesion of the enterotoxigenic E. coli strains and that capsule impedes the in vitro adhesion. They also suggested that the capsular enhancement of colonization by such strains in vivo probably is by some mechanism other than enhanced adhesion to epithelium.

Inhibition of adhesion of Escherichia coli K88 antigen by mammary secretions of susceptible and resistant sows

Anti-adhesive activities of colostrum and milk from genetically susceptible sows, which protected their susceptible offspring in an outbreak of neonatal diarrhoea caused by K88-positive Escherichia coli, were compared with the activities in mammary secretions of resistant dams that did not protect their susceptible progeny. There was significantly more anti-adhesive antibody in the secretions of susceptible sows than in resistant sows, both during the disease period, and 1 year later. Fractionation of colostrum by gel filtration and ion-exchange chromatography led to identification of the anti-adhesive antibodies as including both IgA and IgM.

Identification of Escherichia coli K1 antigen

We compared the use of bacteriophage sensitivity, seroagglutination with polyclonal antisera raised in rabbits or horses, seroagglutination with murine monoclonal antibody, and the serum agar precipitin technique for the detection of K1 capsular polysaccharide among clinical isolates of Escherichia coli obtained from blood stream infections. Some E. coli isolates failed to yield agreement among these tests, indicating that reliable detection of K1 antigen may require the use of multiple tests.