K88 Antigen Research Articles

Crossed immunoelectrophoresis and chemical structural analysis used for characterization of two varieties of Escherichia coli K2 polysaccharide antigen.

Crossed immunoelectrophoresis has shown that the Escherichia coli polysaccharide K2 antigen exists in two forms, K2ab and K2a. In confirmation of this finding, chemical structural analysis showed that K2ab, in addition to the galactose, glycerol and phosphate groups common to K2ab and K2a, contained 0 acetyl groups. The contrast between the earlier and the present K2 designation is discussed and the superiority of the CIE technique for this type of analysis is underlined.

Passive protection of lambs against enteropathogenic Escherichia coli: role of antibodies in serum and colostrum of dams vaccinated with K99 antigen.

Lambs from suckling ewes vaccinated with the K99 antigen were resistant to challenge with K99-positive enteropathogenic Escherichia coli. Serum and colostrum from these ewes were compared with samples from control ewes to establish methods for monitoring vaccination and to determine the mechanism of protection. Vaccination stimulated production of K99 antibodies. These could be detected by an indirect haemagglutination test and a haemagglutination-inhibition test. Antiglobulin and gel-diffusion tests were less reliable. Experiments with brush-border cells from calf intestine showed that the antibodies were associated with anti-adhesive activity. The antibodies were predominantly IgG and did not neutralise the activity of heat-stable enterotoxin. It was concluded that neutralisation of the adhesive properties of the K99-positive E. coli by colostral antibodies significantly contributed to the resistance of the lambs from vaccinated ewes.

Evidence for two adhesive antigens on the K99 reference strain Escherichia coli B41.

Immunoelectrophoresis and haemagglutination studies with a K99 antigen extract from the reference strain Escherichia coli B41 (O101:K99) demonstrated both anionic and cationic mannose-resistant haemagglutinins for horse red blood cells. Neither haemagglutinin was produced by bacteria grown at 18 degrees C. Antibodies to the cationic haemagglutinin were demonstrated in antisera to all the K99-positive E. coli strains in every serogroup examined, but antibodies to the anionic haemagglutinin were only detected in antisera to E. coli strains from the O9 and O101 serogroups. Inhibition of E. coli B41 adhesion to calf brush borders and indirect immunofluorescent staining indicated that the anionic haemagglutinin also exhibited adhesive properties.

Breast milk lymphocyte response to K1 antigen of Escherichia coli.

Comparison milk and blood lymphocyte blastogenic responses to the K1 antigen of Escherichia coli and lipopolysaccharide (LPS) from E. coli O127,B8 were examined in 16 postpartum women by [3H]thymidine uptake. Rabbit hemolysincoated sheep erythrocyte monolayers were used to deplete macrophages from milk lymphocyte preparations and to enrich for T lymphocytes in order to make milk preparations more comparable to blood preparations. Response was defined as a stimulation index of greater than or equal to 2.0. There was no evidence of selective response to K1 antigen by milk lymphocytes, since both blood and milk lymphocytes responded in four women and neither blood nor milk lymphocytes responded in nine. Milk lymphocytes alone responded to K1 in one woman, whereas blood lymphocytes alone responded in two women. Additional nonpaired milk or blood cultures were available from three women. None of these responded to K1 antigen. Corresponding lymphocyte cultures were stimulated with LPS. A positive K1 response was always accompanied by an LPS response, and the LPS response correlated with the K1 response in 17 of 19 women. Stool cultures examined with an antiserum agar showed no correlation between the presence of K1 E. coli in the stool and milk or blood lymphocyte response to K1 antigen. In the system used here, no selectivity of response of breast milk lymphocytes to K1 antigen was noted.

Production of K99 antigen by enterotoxigenic Escherichia coli strains of antigen groups o8, o9, o20, and o101 grown at different conditions.

The production of the K99 antigen by enterotoxigenic Escherichia coli strains with various O antigens was investigated by means of slide agglutination tests, enzyme-linked immunosorbent assays, and hydrophobic interaction chromatography. The extent of K99 production appeared to be dependent on the nutrient medium, as well as on the incubation temperature. Minimal salt medium with glucose and semisynthetic Minca medium were the most suitable for K99 production. In complex media the production of K99 antigen was strongly reduced. Optimal amounts of K99 antigen were produced at 37 degrees C. At 30 degrees C, weak production of K99 antigen was detected by hydrophobic interaction chromatography of enzyme-linked immunosorbent assay. Slide agglutination tests were negative with cultures grown below 32 degrees C. The production of K99 antigen appeared to be related to the O antigen carried by the host strain, but it seemed to be independent of the absence or the presence of various K polysaccharide antigens. Under all conditions used, strains with antigen O101 produced about 10 times more K99 antigen than did strains with antigen O8, O9, or O20. Transfer of the K99 plasmid from wild-type strains of different O antigens to E. coliK-12 C600 confirmed that phenotypic expression of the K99 antigen is most probably related to the cell wall composition of the host.

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.

Relative opsonic and protective activities of antibodies against K1, O and lipid A antigens of Escherichia coli.

The K1 Escherichia coli capsular antigen has been implicated as a virulence factor because of the frequency of isolation of K1 containing strains from certain invasive human infections. In the study of the interaction between K1 strains, normal human polymorphonuclear cells (PMNs) and fresh human serum, we have found varying susceptibility to phagocytosis and killing; thus, the in vitro opsonophagocytic and in vivo protective role of K1, somatic O and core glycolipid antibodies remain unclear. We have therefore examined strains of E. coli with defined susceptibility to phagocytosis by normal PMNs and sera and compared the effect of K1, somatic O and lipid A antibodies in opsonophagocytic tests and mouse protection experiments. K1 E. coli strains demonstrating relative resistance to phagocytosis and killing were effectively opsonized only with specific K1 capsular antisera. Similarly, K1 capsular antisera, but not anti-O or lipid A antisera, also provided protection in mice challenged with a LD100 of K1 E. coli that were "resistant" to phagocytosis. The ability of purified capsular antigens from Neisseria meningitidis group B and K1 "resistant" E. coli to inhibit the phagocytosis of a "sensitive" non-K1 and a K1 E. coli strain of "intermediate" susceptibility to opsonophagocytosis was also investigated. Purified K1 and group B capsular antigens were able to block specific capsular-antibody mediated opsonophagocytosis, yet these capsular antigens failed to inhibit the phagocytosis of non-K1 "sensitive" or K1 "intermediate" E. coli. These studies suggest that K1 antibodies are obligatory for the in vitro and in vivo opsonophagocytosis of "resistant" K1 E. coli and that the K1 antigen must remain in situ on the bacterial surface to exert an anti-phagocytic effect.

Detection of the K99 antigen ofEscherichia coliin calf faeces by enzyme‐linked immunosorbent assay (ELISA)

Summary An enzyme-linked immunosorbent assay (ELISA) is presented for the detection of the K99 antigen of Escherichia coli in calf faeces. False-positive reactions were not observed with K99-negative strains and with several viral antigens. Only bovine coronavirus caused slight positive reactions which could be eliminated by a blocking test. As compared with the conventional procedure for the detection of the K99 antigen, ELISA seemed to be at least as sensitive and had the advantage that samples could be stored at -20° C before testing. In addition many samples could be handled at the same time and the results became available quickly. By carrying out the assay as a blocking test, specific antibody against K99 in serum or colostrum could be detected and titrated.

Defective Activation of the Third Component of Complement in the Sera of Newborn Infants

The activation of the terminal complement components, C3--9, plays an important role in the host's defense against infection. In the present study, the ability of bacteria to activate the third component of complement (C3) in newborn serum was examined. A variety of bacteria were incubated in test sera at 37 degrees C for 30 min and the percent of available C3 that was activated was measured. Using one strain of Escherchia coli (no. 3), 32% (mean) of the available C3 was activated in sera from 18 newborns, as compared to 85% in sera from their mothers and 79% in sera from 13 normal adults (P less than 0.005). In contrast, using another strain of E. coli (N70), the percent of C3 activated in newborn sera (83%) was the same as in sera from their mothers (81%) or in sera from normal adults (73%). The defective activation of C3 in newborn sera by E. coli was not related to the presence of the K1 antigen. Newborn sera were also challenged with other bacterial species and the activation of C3 was deficient when tested with klebsiellae, but not with staphylococci or streptococci. The defect in newborn sera appeared to be due to a deficiency of a serum factor rather than to the presence of an inhibitor.

Escherichia coli diarrhoea in pigs with or without the K88 receptor

There was a high incidence of neonatal scours in 38 litters of pigs born at Compton in a four month period during 1978. The most important cause of the disease was an enteropathogenic Escherichia coli strain which possessed the K88 antigen. The Compton herd has been bred to produce pigs of three genotypes with respect to the presence or absence of the intestinal receptor for the K88 antigen. These are homozygous dominants (SS) and heterozygotes (Ss) susceptible to infection by virulent K88-positive E coli, and homozygous recessives (ss) resistant to the disease. The highest incidence of diarrhoea was in the susceptible progeny of resistant dams and susceptible sires. There was no K88 associated diarrhoea in resistant progeny or in susceptible progeny of susceptible dams.

Immunization of calves against enterotoxigenic colibacillosis by vaccinating dams with purified K99 antigen and whole cell bacterins.

Pregnant cattle were either vaccinated subcutaneously with (i) a suspension of purified Escherichia coli K99 pili, (ii) a Formalin-killed whole cell bacterin containing enterotoxigenic E. coli strain B44 (O9:K30;K99:H-), or (iii) a bacterin containing six different strains of bovine enterotoxigenic E. coli (multiple-strain bacterin), or were left as nonvaccinated controls. After birth, calves were allowed to nurse their dams and, at 12 to 14 h of age, were challenged orally with 10(11) cells of enterotoxigenic E. coli strain B44. Colostral antibody titers were determined against K99, K30, and O9 antigens of B44. In the nonvaccinated control group, 9 of 10 calves developed diarrhea and died within 24 to 72 h. Similarly, all six calves in the multiple-strain bacterin group developed diarrhea and four died. In contrast to calves in the two groups mentioned above, calves nursing cows vaccinated with either purified K99 or the homologous whole cell bacterin were protected against fatal diarrhea. There was a highly significant correlation (P less than 0.0005) between protection against fatal diarrhea and K99, but not K30 or O9 colostral antibody titers. Vaccination of cows with either purified pili or whole cell preparations containing sufficient K99 antigen may provide a means of preventing enterotoxigenic colibacillosis in calves.

Enterotoxins, O-groups, and K88 Antigen in Escherichia coli from Neonatal Piglets With and Without Diarrhea

In a comparison between piglets (1-7 days old) with diarrhea and healthy piglets of the same age and 1 to 8 weeks old, 810 intestinal strains of Escherichia coli from 81 piglets from as many different herds in Sweden were investigated with regard to O-group, enterotoxicity, and possession of K88 antigen. A clear difference was found between the E. coli isolates from piglets with diarrhea and from representatives of healthy herds without diarrhea, with regard to (i) the homogeneity of strains in individual pigs and (ii) the distribution of O-groups, K88 antigen, and frequency of enterotoxicity. Strains from piglets with diarrhea showed a high frequency of O-group 149 (53%), enterotoxicity (61%), and K88 antigen (56%), while not more than 3% of the strains from healthy piglets of the same age harbored any of these characteristics. Of the isolates in O-groups 8, 64, and 115, 36% were enterotoxigenic. The corresponding data for O-group 149 and nontypable strains were 96 and 1%, respectively. Furthermore, K88 antigen was only found in O-groups 8 and 149. In O-group 149, 96% of the strains (n = 167) produced the K88 antigen as well as heat-labile enterotoxin. In contrast, strains producing heat-stable enterotoxin were mainly found in O-groups 8, 9, 64, 115, and 141. There was a significant difference in the frequencies of E. coli strains producing heat-labile enterotoxin between piglets with diarrhea and those without, whereas the pathogenic role of heat-stable enterotoxin-producing strains was less apparent.

Behavior of Escherichia coli K antigens K88ab, K88ac, and K88ad in immunoelectrophoresis, double diffusion, and hemagglutination.

Porcine enteropathogenic Escherichia coli strains were found to possess a variant of the K88 antigen provisionally termed K88ad. We propose to include this antigen into the international E. coli typing scheme. Ultrasonic extracts of field strains of E. coli possessing the K88ab, K88ac, or K88ad antigen and their E. coli K-12 K88+ transconjugants showed a specific K88 precipitation line in immunoelectrophoresis and double diffusion only when grown at 37 degrees C, but not when grown at 18 degrees C. By using agarose gels, K88ab, K88ac, and K88ad antigens showed anodic mobility in immunoelectrophoresis. When using Difco Noble agar gels, K88ad was not mobile or anodic, K88ab was cathodic; K88ac of 17 strains was cathodic and of 24 strains was anodic. The immunoelectrophoretic behavior of a K88 antigen (K88ab, K88ac, or K88ad) did not alter after transfer of the corresponding plasmid to E. coli K-12. Anodic and cathodic K88ac antigens could not be distinguished serologically. The differences between the results obtained in Noble agar gels and agarose gels are due to electro-endosmotic flow. We describe a procedure which increases the detection level of K88+ transconjugants in a mating mixture. It is based on the specific mannose-resistant attachment of K88+ cells to guinea pig erythrocytes.

Differences in Hydrophobic Surface Characteristics of Porcine Enteropathogenic Escherichia coli With or Without K88 Antigen as Revealed by Hydrophobic Interaction Chromatography

Differences in Hydrophobic Surface Characteristics of Porcine Enteropathogenic <i>Escherichia coli</i> With or Without K88 Antigen as Revealed by Hydrophobic Interaction Chromatography

Cloning mapping and expression of the genetic determinant that encodes for the K88ab antigen

The K88 antigen, a plasmid-specified virulence factor of E. coli involved in porcine neonatal diarrhoea, is often found to be associated with the ability to metabolize raffinose (Raf). Plasmid pRI8801 (51 megadalton) was used to clone the determinants of K88 and Raf with the vector pBR322. K88 was found to be encoded by a 7.7 megadalton HindIII fragment. The expression was highly dependent on the orientation of the HindIII fragment within pBR322. By in vitro generation of deletions, the HindIII fragment was reduced in size to 4.3 megadalton. The expression of K88 by pRI8801 and the recombinant plasmids was studied using an enzyme-linked immunosorbent assay. Raf was found to be located on a 4.0 megadalton SalI fragment. A physical map of pRI8801 was constructed. The K88 antigen and Raf genes are not closely linked but separated by a stretch of DNA of about 20 megadalton.

Isolation and characterization of K88 antigens

Isolation and characterization of K88 antigens

Isolation and characterization of K88 antigens

Isolation and characterization of K88 antigens

Isolation of a mannose-specific lectin from Escherichia coli and its role in the adherence of the bacteria to epithelial cells

A high molecular weight protein aggregate, which agglutinates yeast cells, human epithelial cells and mouse lymphocytes, was isolated from extracts of Escherichia coli by differential centrifugation and gel filtration. The agglutination is specifically inhibited by d-mannose and its derivatives, the best inhibitor being p-nitrophenyl α- d-mannoside. Sodium dodecyl sulfate gel electrophoresis showed that the lectin consists of protein subunits with identical M r of ∼36500. The amino acid composition of the purified lectin is different from that reported for the type I pili protein, the K99 antigen and the major outer membrane protein Ia of E. coli. The protein appears to be located on the bacterial surface, and is probably involved in the mannose-specific adherence of E. coli to eukaryotic cells.

Passive Protection of Lambs Against Experimental Enteric Colibacillosis by Colostral Transfer of Antibodies From K99-Vaccinated Ewes

Pregnant ewes were vaccinated with partially purified, cell-free K99 antigen isolated from an enteropathogenic strain of Escherichia coli, strain B41 (O101:K99:NM), to induce passive immunity via the colostrum in their offspring against an oral challenge with heterologous "calf-lamb" enteropathogenic strains of E. coli B44. After sucking their dams, lambs were dosed orally with 7X10(10)-2.2 X10(11) organisms within 4--21 h of birth. One group of 10 lambs was dosed with cultures of the mucoid (O9:K30(A),K99:NM) form of strain B44 and another group of 10 lambs with the non-mucoid (O9:K99:NM) form; two groups of four control lambs from unvaccinated dams were similarly challenged. All four control lambs challenged with mucoid B44, loose feaces were detected in only two of the four control lambs and in none of the lambs from vaccinated dams. This suggests that the polysaccharide K antigen may contribute to the virulence of "calf-lamb" enteropathogenic strains that possess the K99 antigen. However, lambs passively immunised with colostrum from dams vaccinated with K99 antigen alone were protected against the production of enteric colibacillosis by oral challenge with EPEC strain B44.

Differences in hydrophobic surface characteristics of porcine enteropathogenic Escherichia coli with or without K88 antigen as revealed by hydrophobic interaction chromatography

Porcine enteropathogenic Escherichia coli strains possessing or lacking K88 antigen were studied by using hydrophobic interaction chromatography on cross-linked agarose gels with alkyl or aryl substituents (amphiphilic gels) to determine whether or not they possessed surface-associated hydrophobic properties. Strains with K88ab or K88ac antigen adsorbed to phenyl and octyl Sepharose gels in the presence of 4 M sodium chloride. This property correlated with phenotypic expression of K88 antigen. Cells grown at 37 degrees C but not those grown at 18 degrees C possessed hydrophobic adsorptive characteristics in addition to the property of mannose-resistant hemagglutination of guinea pig erythrocytes. Adsorption of K88-positive strains to gels with hydrophobic ligands was independent of O group and enterotoxicity. Strains lacking K88 antigen did not adsorb to the hydrophobically substituted derivatives of Sepharose and lacked mannose-resistant hemagglutinating characteristics. Neither the presence of additional polysaccharide K antigens nor nonhemagglutinating pili conferred the property of hydrophobic interaction on the strains. K88-positive bacteria had a lower electrophoretic migration rate than did K88-negative bacteria of the same serotype in free-zone electrophoresis. K88-positive bacteria also adsorbed strongly to hydrophobic ligands in the presence of 1 M ammonium sulfate, whereas K88-negative strains did not. These observations provide evidence for the suspected role of hydrophobic interaction in the adhesive properties of certain enteropathogenic strains of E. coli. Moreover, hydrophobic interaction chromatography provides convenient and rapid alternative means of screening strains for a property potentially associated with adhesiveness.