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PubMed1972

Effect of water activity on enterotoxin A production and growth of Staphylococcus aureus.

Previous studies indicated that enterotoxin B production by staphylococci was strongly inhibited by slight reductions in water activity (a(w)) levels. Similar studies reported herein, employing an enterotoxin A-producing strain, indicated that this organism was capable of producing enterotoxin at a much lower a(w) level than that required for enterotoxin B production. Staphylococcal growth rates were slowed by decreased a(w) levels in all media tested; however, final cell counts did not drop below 10(8)/ml in the media with the lowest a(w) levels.

Bacteriological Techniques↗
PubMed1973

Amino acid requirements for the production of enterotoxin B by Staphylococcus aureus S-6 in a chemically defined medium.

A minimal chemically defined medium has been developed for growth (approximately 25 Klett units) and production of detectable enterotoxin B (approximately 5-6 mug/ml) by Staphylococcus aureus S-6. This medium contains monosodium glutamate as a source of carbon, nitrogen, and energy, three additional amino acids (arginine, cystine, and phenylalanine), six inorganic salts, and four vitamins. Increasing the concentrations of several amino acids in a series of defined media gave no increase in enterotoxin production. Apparently the limiting factor for growth and enterotoxin production in these media is the biosynthesis of one or more missing amino acids, rather than the concentration of the amino acids present in the media. An additional requirement for proline and valine was observed when glucose was added as the primary source of energy. When compared to complex media, our results indicated that the inhibitory effect of glucose on enterotoxin synthesis in defined media was less evident or totally absent.

Amino Acids↗
PubMed1975

Stability of 125I-labeled staphylococcal enterotoxins in solid-phase radioimmunoassay.

Staphylococcal enterotoxins, Types A, B, and C, were labeled with 1252 by the chloramine-T method at approximately two levels of specific activity, 40 and 4 muCi/mug of protein. Toxins labeled with high specific activity showed extensive dissociation of 125I when stored at different temperatures, including -23 C. In contrast, toxins labeled with low specific activity did not show any significant loss of 125I when stored at -23 C for as long as 2 months. Enterotoxins, whether labeled with high or low activities, formed aggregates immediately upon labeling. Aggregate formation increased in high-activity-labeled toxins on storage at -23 C, and low-activity-labeled toxins showed no significant increase in aggregate formation, even after 2 months at -23 C. The aggregated forms of the enterotoxins were either devoid of antigenic activity in solid-phase radioimmunoassay or they possessed significantly reduced antigenic activity. Thus, a decrease in binding of 1252-labeled enterotoxin to specific antibody in solid-phase radioimmunoassay results mainly from (i) loss of 125I on storage, and (ii) formation of aggregates with reduced antigenic activity.

Binding Sites, Antibody↗
PubMed1975

Double-antibody radioimmunoassay for staphylococcal enterotoxin C2.

A sensitive double-antibody radioimmunoassay for staphylococcal enterotoxin C2 is described. The assay procedure employs anti-rabbit gamma globulin, prepared in goats, to precipitate the antigen-antibody complex of enterotoxin C2 and anti-enterotoxin C2. The test is sensitive to 100 pg of enterotoxin.

Animals↗
PubMed2006

Significance of heat-stable and heat-labile enterotoxins in porcine colibacillosis in an additive model for pathogenicity studies.

Although heat-stable (ST) and heat-labile (LT) enterotoxins produced by enterotoxigenic Escherichia coli (ETEC) have been documented as important factors associated with diarrheal diseases, investigations assessing the contributions of individual enterotoxins to the pathogenesis of E. coli infection have been limited. To address the individual roles of enterotoxins in the diarrheal disease caused by K88-positive ETEC in young pigs, enterotoxin-positive and -negative isogenic E. coli strains were constructed by using pBR322 to clone and express LT and STb. Four strains, K88+ astA, K88+ astA/pBR322, K88+ astA STb+, and K88+ astA LT+, were constructed and subsequently included in gnotobiotic piglet challenge studies, and their pathogenesis was assessed. The results indicated that all K88+ isogenic strains were able to colonize the small intestines of piglets exhibiting the K88 receptor. However, only LT- and STb-positive strains caused appreciable diarrhea. Piglets inoculated with the K88+ astA LT+ strain became dehydrated within 18 h, while those inoculated with the K88+ astA STb+ strain did not, although diarrhea developed in several piglets. The changes in the blood packed-cell volume and plasma total protein of gnotobiotic piglets inoculated with the LT-positive strains were significantly greater than those of pigs inoculated with the K88 astA/pBR322 strain (P = 0.012, P = 0.002). Immunochemistry image analysis also suggested that LT enhanced bacterial colonization in a gnotobiotic piglet model. This investigation suggested that LT is a major contributor to the virulence of K88+ ETEC and that isogenic constructs are a useful tool for studying the pathogenesis of ETEC infection.

Animals↗
PubMed1978

Solid-phase radioimmunoassay method for determination of Escherichia coli enterotoxin.

The development of a solid-phase radioimmunoassay procedure for the determination of Escherichia coli enterotoxin(s) is described. Radioiodinated E. coli enterotoxin with about three radioiodine atoms per toxin molecule is, by the criterion of electrophoresis, identical to the unlabeled toxin. Goat anti-E. coli-enterotoxin antibody was coupled to polystyrene tubes and served as a solified toxin binder in the reported procedure. Various conditions necessary for the optimization and standardization of the solid-phase method were established. With the help of this technique it was possible to determine E. coli enterotoxin released from a porcine E. coli strain into culture medium.

Bacterial Toxins↗
PubMed1978

Production of enterotoxin by Yersinia enterocolitica.

Forty-three strains of Yersinia enterocolitica isolated from children with gastroenteritis and 18 laboratory strains were examined for enterotoxin production by using the infant mouse, Y1 adrenal cell, and rabbit ileal loop assay systems. All clinical isolates and seven laboratory strains were found enterotoxigenic in the infant mouse model, but none of the strains produced toxin in the Y1 adrenal cell system. One enterotoxin-producing strain was also tested by the rabbit ileal loop assay, confirming the results of the infant mouse assay. The enterotoxin was heat-stable and as active in the 6-h rabbit ileal loop as in the 18-h assay, suggesting a similarity to the heat-stable enterotoxin of Escherichia coli.

Animals↗
PubMed1978

Isolation of specific and common antibodies to staphylococcal enterotoxins A and E by affinity chromatography.

Specific and common antibodies to staphylococcal enterotoxins A and E (SEA and SEE) were isolated from anti-SEA and anti-SEE antisera by affinity chromatography. Anti-SEA and anti-SEE antisera were passed through a column with the cross-reacting enterotoxin coupled to a CNBr-activated Sepharose 4B matrix. Specifically bound common antibodies were eluted with NaSCN. The isolated specific antibodies reacted only with the homologous enterotoxin, whereas the common antibodies gave a reaction of identify with both enterotoxins in double gel diffusion plates. The common antibodies had a higher titer against SEE than against SEA. The significance of the isolation of antibodies common to two separate protein molecules is discussed.

Animals↗
PubMed1978

Inhibition of the secretory activity of Escherichia coli heat-stable enterotoxin by indomethacin.

The effect of indomethacin on the net intestinal accumulation of fluid induced by Escherichia coli heat-stable (ST) enterotoxin in the infant mouse model was examined. Indomethacin, when administered with ST enterotoxin, caused a striking decrease in net intestinal fluid accumulation. This inhibition of ST activity was dose dependent with various concentrations of indomethacin (P less than 0.01). A significant inhibition of toxicity was also observed when indomethacin was given before (P less than 0.01) or after (P less than 0.02) ST enterotoxin challenge. No significant differences in fluid accumulation were observed between control mice treated with buffer alone and those challenged with only indomethacin. These data indicate that indomethacin markedly decreases the net intestinal fluid accumulation induced by E. coli ST enterotoxin. Further studies on the potential use of indomethacin in both the prophylaxis and the therapy of diarrheal diseases appear warranted.

Animals↗
PubMed1979

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.

Animals↗
PubMed1979

Isolation and partial characterization of two different heat-stable enterotoxins produced by bovine and porcine strains of enterotoxigenic Escherichia coli.

Heat-stable enterotoxins (ST-124 and ST-1261) have been isolated from two different enterotoxigenic Escherichia coli of bovine (124) and porcine (1261) origin. The enterotoxin preparations were isolated by ultrafiltration and ion-exchange chromatography and were both active in the suckling mouse test and pig ligated loop test in the nanogram range. The bovine (ST-124) enterotoxin was not stable to heating in its isolated form, and significant differences in amino acid composition were observed between the two enterotoxins. Although both toxins were active at similar levels in the suckling mouse and pig ligated loop tests, ST-124 lacked the ability to cause the profound secretory responses seen with ST-1261 in the weanling pig ligated loop.

Amino Acids↗
PubMed1979

Effect of chlorpromazine on the secretory activity of Escherichia coli heat-stable enterotoxin.

The effect of chlorpromazine on the net intestinal accumulation of fluid induced by Escherichia coli heat-stable (ST) enterotoxin in an infant mouse model was examined. Chlorpromazine, when administered with ST enterotoxin, caused a highly significant decrease in net intestinal fluid accumulation. The inhibition of ST activity was dose dependent with various concentrations of chlorpromazine (P less than 0.001). A significant inhibition of toxic activity was also observed when chlorpromazine was administered before (P less than 0.02) or after (P less than 0.05) ST enterotoxin challenge. No significant differences in fluid accumulation were observed between control mice treated with buffer alone and those treated with only chlorpromazine. These data indicate that chlorpromazine markedly decreases the net intestinal fluid accumulation induced by E. coli ST enterotoxin. Further studies on the potential use of chlorpromazine in both the prophylaxis and the treatment of diarrheal diseases appear warranted.

Animals↗
PubMed1979

Effects of heat-stable enterotoxin of Yersinia enterocolitica on ion transport and cyclic guanosine 3',5'-monophosphate metabolism in rabbit ileum.

Strains of Yersinia enterocolitica produce a heat-stable enterotoxin which is positive in the suckling mouse bioassay. Partial purification by a procedure previously worked out for heat-stable Escherichia coli enterotoxin yielded a substance which increases particulate guanylate cyclase activity and short-circuit current and inhibits active Cl-absorption in rabbit ileal mucosa. These effects of Y. enterocolitica enterotoxin are similar to those of heat-stable E. coli enterotoxin, suggesting a common mechanism of action.

Animals↗
PubMed1980

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.

Animals↗
PubMed1981

Effect of fractions of Ethiopian And Norwegian colostrum on rotavirus and Escherichia coli heat-labile enterotoxin.

Samples of colostrum from both Ethiopian and Norwegian women contained antirotavirus activities of immunoglobulin and non-immunoglobulin nature. No significant differences in rotavirus immunoglobulin A or in rotavirus-inhibiting activity were found between samples from the two countries. The non-immunoglobulin inhibitory activity was trypsin sensitive and heat stable (100 degrees C for 10 min). Escherichia coli heat-labile enterotoxin antibodies were measured in the colostrum samples by enzyme-linked immunosorbent assay. No E. coli enterotoxin-specific immunoglobulin A was detected, possibly due to the high background caused by the nonspecific adsorption of immunoglobulin A to the enzyme-linked immunosorbent assay plates in the absence of toxin. A total of 5 of 15 Ethiopian colostrum samples and 0 of 11 Norwegian colostrum samples neutralized the effect of E. coli heat-labile enterotoxin on YI adrenal cells. Both the Ethiopian and the Norwegian colostrum samples contained a non-immunoglobulin enterotoxin-inhibitory activity when the toxin was measured by enzyme-linked immunosorbent assay. This inhibitory activity was not trypsin sensitive, and extraction by chloroform-methanol indicated that the inhibitor was of a lipid nature.

Antitoxins↗
PubMed1982

Berberine inhibits intestinal secretory response of Vibrio cholerae and Escherichia coli enterotoxins.

Berberine, an alkaloid from the plant Berberis aristata, which has been known since ancient times as an antidiarrheal medication in India and China, inhibited by approximately 70% the secretory responses of the heat-labile enterotoxins of Vibrio cholerae and Escherichia coli in the rabbit ligated intestinal loop model. The drug was effective when given either before or after enterotoxin binding and when given either intraluminally or parenterally; it did not inhibit the stimulation of adenylate cyclase by cholera enterotoxin and caused no histological damage to intestinal mucosa. Berberine also markedly inhibited the secretory response of E. coli heat-stable enterotoxin in the infant mouse model. Although the mechanism of action of the drug is not yet known, these data provide a rationale for its apparent clinical usefulness in treating acute diarrheal disease.

Adenylyl Cyclases↗