Prevalence of fimbrial antigens and enterotoxins in nonclassical serogroups of Escherichia coli isolated from newborn pigs with diarrhea

Ninety-nine nonclassical serogroups isolated from newborn pigs with neonatal diarrhea were tested for fimbrial antigens F4(K88), F5(K99), F6(987P), F41, and F165, and for heat-labile enterotoxin, heat-stable enterotoxin a (STa), heat-stable enterotoxin b, verocytotoxin, and cytolethal-distending toxin. Thirty-two strains, belonging mostly to serogroups O64:K"V142,", O9:K103, and O20:K101, were F5-positive and usually produced STa, although 5 strains produced only heat-stable enterotoxin b. Fifteen strains, belonging mostly to serogroups O64:K"V142" and O20:K101, were F41 positive and usually produced STa. Twenty-three stains, belonging mostly to serogroups O64:K"V142," O9:K103, and O20:K101, were F6-positive and usually produced STa. Several strains produced more than one fimbrial antigen, either F5 and F41, F5 and F6, F6 and F41, F6 and F165, or F5, F6, F41, and F165. None of the strains produced heat-labile enterotoxin, verocytotoxin, or cytolethal-distending toxin. The indirect immunofluorescence test was much more sensitive than was the slide agglutination test for the detection of each of the fimbrial antigens F5, F6, F41, and F165 on strains grown in vitro. The production of F6 by certain strains for which only a low proportion of cells were F6-positive in vitro, as demonstrated by immunofluorescence, was confirmed by experimental infection of newborn pigs.     

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.     

Oral immunisation of pigs with fimbrial antigens of enterotoxigenic E. coli: an interesting model to study mucosal immune mechanisms

The intestinal mucosal immune system can discriminate actively between harmful pathogenic agents and harmless food antigens resulting in different immune responses namely IgA production and oral tolerance, respectively. Recently, a pig model has been developed for studying intestinal mucosal immune responses in which F4 fimbrial antigens of enterotoxigenic Escherichia coli (F4 ETEC) are used as oral antigens. A unique feature of this model is that soluble F4 antigens can be administered to pigs which have a receptor for this fimbriae (F4R(+)) on their small intestinal villous enterocytes and pigs which do not have this receptor (F4R(-)). Oral administration of F4 to the F4R(+) pigs results in an intestinal mucosal immune response that completely protects the pigs against a challenge infection. In F4R(-) pigs such an intestinal mucosal immune response does not occur. However, a priming of the systemic immune system can be seen similar to the priming in pigs fed with the same dose of a food antigen, suggesting that F4 in F4R(-) pigs behaves as a food antigen. The fact that different mucosal immune responses can be induced with soluble F4, makes it an interesting model to study mucosal immune mechanisms in the pig.     

Effect of heat stable and heat labile Escherichia coli enterotoxins and cholera toxin in combination with theophylline on unidirectional sodium and chloride flux in the small intestine of weanling swine.

The effect of heat stable and heat labile Escherichia coli enterotoxins or cholera toxin in combination with theophylline on net water, sodium and chloride and unidirectional sodium and chloride fluxes was examined in acute isolated loops of jejunum of weanling swine. The effect of heat stable enterotoxin in combination with theophylline was determined in loops located in the proximal jejunum, while combinations of theophylline and either heat labile enterotoxin or cholera toxin were studied in the distal jejunum. In each situation the addition of theophylline resulted in an additive rather than a synergistic increment of intestinal secretory activity. This study implies that the intestinal adenyl cyclase system and enterotoxin induced intestinal secretion may not be directly related in the swine small intestine.     

The role of oral immunisation in stimulating Escherichia coli antibody of the IgM class in porcine colostrum.

Oral immunisation with Escherichia coli polysaccharide antigens provided a primary antigenic stimulus which facilitated the production of humoral IgM antibody following a single parenteral antigen dose. The peak antibody response of preparturient sows was manipulated to coincide with the period of colostrum formation so that high levels of IgM antibody were made available for neonatal defence. The characteristics of the immune response remained unchanged on reintroduction of the immunisation schedule for a second gestation period.     

Permeability properties of swine small intestine: effect of a heat stable Escherichia coli enterotoxin.

The permeability of weanling swine small intestine was estimated using measurements of filtration coefficients and equivalent pore size. Hypertonic solutions of mannitol, erythritol and urea were used to calculate reflection coefficients in the duodenum, mid jejunum and distal jejunum. Estimated effective pore radius was 6.4-7.4, 5.6-7.2 and 4.7-4.9A degrees in the three respective regions. Similarly the filtration coefficient induced by hypertonic solutions of mannitol decreased significantly in the distal jejunal segments. The results show an aboral gradient of decreasing permeability along the small intestine of the weanling pig. In situ incubation of loops in the proximal jejunum with a heat stable Escherichia coli enterotoxin for one hour did not significantly change the effective pore size as calculated from reflection coefficients of hypertonic solutions of erythritol and urea. However, the filtration coefficients of loops exposed to the enterotoxin were significantly greater than control loops with hypertonic solutions of erythritol and urea but not mannitol. This suggests the occurrence of a slight reduction in epithelial porosity. The results support the hypothesis that intestinal secretion induced by heat stable E. coli enterotoxin is not the result of an increased mucosal permeability.     

Dose-response investigation of oral ketoprofen in pigs challenged with Escherichia coli endotoxin

In order to determine the effective dose, the effects of orally administered ketoprofen were evaluated in pigs following intravenous challenge with Escherichia coli endotoxin. One hour after the challenge, five groups of pigs were treated with either tap water or ketoprofen (0.5 mg/kg, 1 mg/kg, 2 mg/kg or 4 mg/kg). The body temperature was measured and a total clinical score was calculated after assessing the general behaviour, respiratory rate and locomotion of the pigs. Thromboxane B(2) and ketoprofen concentrations were analysed from blood samples. Ketoprofen treatment significantly reduced the rectal temperature and total clinical scores, and lowered blood thromboxane B(2) concentrations when compared with the control group. Ketoprofen plasma concentrations were lower than previously reported in healthy pigs after similar doses. The appropriate dose of orally administered ketoprofen in pigs in this model is 2 mg/kg, as the higher dose of 4 mg/kg failed to provide an additional benefit.     

The effect of cholera toxin and heat labile and heat stable Escherichia coli enterotoxin on cyclic AMP concentrations in small intestinal mucosa of pig and rabbit.

The effect of cholera toxin, heat labile and heat stable Escherichia coli enterotoxin on mucosal cyclic AMP concentrations was determined on the proximal jejunum of weanling pigs and young rabbits. Ligated loops were injected with solutions containing no enterotoxin for control and either cholera toxin, heat labile or heat stable E. coli enterotoxin. The loops were drained after either two, four or six hours incubation at which time accumulated fluid was recorded and mucosal samples removed for determination of cyclic AMP concentration. In the rabbit, cholera toxin and heat labile, but not heat stable E. coli enterotoxin stimulated intestinal secretion while in the pig all three enterotoxins induced net fluid accumulation. Cholera toxin and heat labile, but not heat stable E. coli enterotoxin elevated rabbit mucosal cyclic AMP concentrations. In the pig these enterotoxins had no significant effect on mucosal cyclic AMP concentrations. The results are inconsistent with the hypothesis that the adenyl cyclase system is an essential step for enterotoxin induced intestinal secretion. The activation of intestinal adenyl cyclase by bacterial enterotoxins may only be an associated and not a necessary event for the stimulation of intestinal secretion.     

Evaluation of heat-labile enterotoxins type IIa and type IIb in the pathogenicity of enterotoxigenic Escherichia coli for neonatal pigs

Type II heat-labile enterotoxins (LT-II) have been reported in Escherichia coli isolates from humans, animals, food and water samples. The goal here was to determine the specific roles of the antigenically distinguishable LT-IIa and LT-IIb subtypes in pathogenesis and virulence of enterotoxigenic E. coli (ETEC) which has not been previously reported. The prevalence of genes encoding for LT-II was determined by colony blot hybridization in a collection of 1648 E. coli isolates from calves and pigs with diarrhea or other diseases and from healthy animals. Only five isolates hybridized with the LT-II probe and none of these isolates contained genes for other enterotoxins or adhesins associated with porcine or bovine ETEC. Ligated intestinal loops in calves, pigs, and rabbits were used to determine the potential of purified LT-IIa and LT-IIb to cause intestinal secretion. LT-IIa and LT-IIb caused significant secretion in the intestinal loops in calves but not in the intestinal loops of rabbits or pigs. In contrast, neonatal pigs inoculated with isogenic adherent E. coli containing the cloned genes for LT-I, LT-IIa or LT-IIb developed severe watery diarrhea with weight loss that was significantly greater than pigs inoculated with the adherent, non-toxigenic parental or vector only control strains. The results demonstrate that the incidence of LT-II appeared to be very low in porcine and bovine E. coli. However, a potential role for these enterotoxins in E. coli-mediated diarrhea in animals was confirmed because purified LT-IIa and LT-IIb caused fluid secretion in bovine intestinal loops and adherent isogenic strains containing cloned genes encoding for LT-IIa or LT-IIb caused severe diarrhea in neonatal pigs.     

Polyserositis in pigs due to generalized Escherichia coli infection.

A fibrinous polyserositis syndrome due to generalized Escherichia coli infection in pigs was observed in 13 out of 17 systematically monitored herds. The mortality rate was approximately 0.1% among liveborn pigs. The occurrence was usually sporadic but a minor enzootic was observed in one herd. Most of the affected pigs succumbed during first or second week of life but cases were observed throughout the suckling period. The clinical signs included marked depression, anorexia, rough haircoat, laboured respiration and death in two to five days. Predominant gross pathological lesions were signs of septicaemia and a voluminous, gelatinous and fibrinous exudate in the pleural, the pericardial and the peritoneal cavities. Frequently also a firbinous polyarthritis and a fibrinous pneumonia were present. The majority of the isolated invasive E. coli strains were nonhaemolytic. Serologically 11 different E. coli O groups were encountered. O group most frequently represented was 020. None of the examined E. coli strains belonged to the serogroups which frequently are associated with enteropathogenicity in pigs.     

Determination of K88 antigens and enterotoxins of Escherichia coli strains isolated from Polish piglets with diarrhea by the use of enzyme-linked immunosorbent assays.

We studied 103 Escherichia coli strains isolated from suckling and weaned piglets with diarrhea using different ELISA tests. K88 fimbrial antigen was determined by the slide agglutination test and the ELISA inhibition method. LT and STa enterotoxins were tested directly in the microtiter plates using monoclonal antibodies. It was found that 56.3% strains possessed K88 antigen, all of which were of the K88ac type. There was 100% correlation between the slide agglutination and ELISA tests. Of the 103 strains tested 68.9% produced LT or STa or both toxins. LT-positive strains were the most common ones in both groups of piglets. All K88-positive strains were enterotoxigenic and elaborated LT (56 strains) or LT and STa (2 strains); STb production was not determined in this study. Our ELISA tests were easy to perform, specific and can be used for determination of K88 and enterotoxins in E. coli strains isolated from piglets.