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Two hundred ninety-six Escherichia coli isolates from feces or intestines of calves with diarrhea were hybridized with 7 gene probes. One probe (the eae probe) was derived from the eae gene coding for a protein involved in the effacement of the enterocyte microvilli by the group of bacteria called attaching and effacing E coli (AEEC), and 2 probes were derived from genes coding for the Shiga-like toxins (SLT) 1 and 2 produced by the verocytotoxic E coli (VTEC). The other 4 probes were derived from DNA sequences associated with the adhesive properties of enteroadherent E coli (EAEC) to cultured cells (the EAF probe for the localized adherence pattern, probes F1845 and AIDA-1 for the diffuse adherence pattern, and the Agg probe for the aggregative adherence pattern). Hybridization results for the eae probe were in agreement, for all but 1 of the 8 isolates, with previously published phenotypic results of microvilli effacement. The latter was previously reported as effacing the microvilli of calf enterocytes, but was eae probe-negative. Two classes of isolates hybridized with the eae probe. Members of a first class (60 isolates) additionally produced a positive signal with 1 or both of the SLT probes (VTEC-AEEC isolates). Isolates hybridizing with the eae and the SLT1 probes were the most frequent: 56 isolates (ie, 93% of all VTEC-AEEC). Members of the second class (10 isolates) failed to hybridize with either SLT probe (non-VTEC-AEEC isolates).(ABSTRACT TRUNCATED AT 250 WORDS)

Two DNA probes were used for the detection of CNF1- and CNF2-positive E coli strains in a collection of 553 E coli isolates from cattle, sheep, goats, pigs, horses, dogs, cats and poultry. CNF-positive E coli were frequently associated with septicaemia in cattle, dogs, and cats, with diarrhoea in calves, cats and dogs, and with abortion in bovine and porcine species. CNF2-positive strains were observed among adult healthy cattle. They were also found in cases of pneumonia, metritis, mastitis in cattle and in 1 case of metritis of a mare. The physiopathology induced by CNF-positive E coli strains remains to be elucidated. However, the impact of CNF strains on veterinary pathology is clear and the diagnosis of CNF-producing E coli should become routine in veterinary practice.

Virulence factors and phenotypes of 61 strains CNF1+ were investigated. Eighty-nine percent of the strains produced an aerobactin and were resistant to the bactericidal activity of sheep serum, both of which are properties of septicemic strains of E coli. None of the strains reacted either with DNA probes corresponding to the enterotoxins STaP, STb, LT-I and LT-IIa, or to the verotoxins VT-I and VT-II. None produced the adhesins K99, Att25 (FY or F17) and Att111. The great majority (93.4%) of the CNF1+E coli possessed both properties. These properties allow CNF1+ to be distinguished from CNF-E coli.

Colony hybridizations with DNA probes for 3 heat-stable (STaP, STaH, and STb) enterotoxins and 1 heat-labile (LT) enterotoxin and for 4 adhesins (K99, F41, K88, 987P) were performed on 870 Escherichia coli isolates to determine pathotypes prevalent among enterotoxigenic E coli (ETEC) isolated from cattle in Belgium. One hundred thirty-two E coli isolates (15.2%) hybridized with probes STaP, K99, and/or F41. The 5 other probes were not hybridized by E coli isolates. Therefore, only STaP enterotoxin and K99 and F41 adhesins were virulence factors of ETEC isolated from cattle. Two major pathotypes accounted for 95% of the ETEC: STaP+K99+F41+ (67.4%) and STaP+K99+ (27.3%). The last 5% of probe-positive isolates had STaP+, STaP+F41+, or K99+F41+ minor pathotypes. Of 12 American ETEC isolates also assayed, 7 were positive with STb and/or 987P probes (pathotypes STaP+STb+, STaP+ 987P+, or STaP+STb+987P+) and may be porcine- rather than bovine-specific enteropathogens. The remaining 5 American ETEC isolates belonged to 3 minor pathotypes (STaP+, STaP+F41+, and K99+F41+) also found among Belgian E coli isolates. Such isolates may be derivatives of STaP+K99+F41+ or STaP+K99+ ETEC after in vivo or in vitro loss of virulence genes and/or non-ETEC isolates, which have acquired virulence genes by in vivo transfer.

Starting from racemic soman (1,2,2-trimethylpropyl methylphosphonofluoridate), the degradation of its four stereoisomers in human serum (25 degrees, pH 8.8), has been studied at the nM level. Phosphylation of serum proteins is eliminated by preincubation of the serum with soman. A capillary gas chromatographic method with nitrogen-phosphorous detection allows the separation of the diastereoisomers. The total hydrolysis (enzymatic and non-enzymatic) rate constants of the isomers can then be resolved indirectly on the basis of the important rate difference between P(+) and P(-) isomers. The enzymatic hydrolysis rate constants are obtained by subtracting, for each isomer, the spontaneous (non-enzymatic) rate constant from the total hydrolysis rate constant. The non-enzymatic part of the hydrolysis is obtained from experiments in serum ultrafiltrate (30,000 NMWL). Enzymatic hydrolysis of C(+) P(+) soman occurs so rapidly that only a lower limit of the rate constant can be given. The other enzymatic rate constants are 0.016 min-1 for C(+)P(-), 0.74 min-1 for C(-)P(+) and 0.028 min-1 for C(-)P(-).

DNA-DNA colony hybridizations with a ST1a probe were done on bovine enterotoxigenic Escherichia coli strains and compared with the suckling mouse test. The results show that one fourth (7/24) of the strains positive in the baby mouse test do not hybridize the ST1a probe and suggest the existence of another or other toxins among the bovine enterotoxigenic E. coli strains.

Colony hybridizations with 4 enterotoxins (STaP, STaH, STb, and LT) and 1 adherence factor (K99) gene probes were done on Escherichia coli isolated from calves. Agreement between the K99 probing and a serologic assay to detect the K99 antigen was 99% for the identification of K99+ and K99- isolates. Ninety-five of the isolates (22%) hybridized with at least 1 enterotoxin gene probe (Ent+ isolates), and 82 (19%), with the K99 gene probe. The majority of Ent+ isolates (85%) reacted with probes for STaP and K99 genes. The STaP gene was present by itself in 4 of the Ent+ isolates (4%) and with the STb gene in 6 of the Ent+ isolates (6%). Five of the Ent+ isolates (5%) carried the STb and LT genes, and none (0%) of the isolates carried the STaH gene. All but 2 of the isolates with the K99 gene also had the STaP gene. Twenty-eight isolates shown to produce STa enterotoxin in previous studies failed to hybridize with any of the enterotoxin gene probes. These 28 isolates were also phenotypically negative when the tests for enterotoxin production were repeated. These isolates probably lost their genes for enterotoxin production during storage in the laboratory.

Hybridizations were done on bovine enterotoxigenic Escherichia coli with 2 heat-stable (ST) enterotoxin gene probes from porcine and human origin (STp and STh). Of the baby mouse-positive isolates, 56 (53%) hybridized the STp probe and 50 (47%) did not. There was no isolate that hybridized the STh probe. Hybridization with the Stp probe was more frequent (P less than 0.005) for E coli isolated from calves that died before 2 weeks of age (49 STp-positive isolates of 77 [64%] isolates) than for E coli isolated from calves that died after 2 weeks of age (2 STp-positive of 21 [10%] isolates).

Four hundred twenty-nine isolates of Escherichia coli from calves were tested for the production of HeLa cell cytotoxin(s). Isolates that produced enough cytotoxin to be detected in culture supernatants of iron-depleted broth were considered to produce increased amounts of cytotoxins. Isolates also were tested for homology with a DNA probe for a gene that encodes localized adherence of human enteropathogenic E coli. Four isolates produced increased amounts of cytotoxin that was neutralized by Shiga antitoxin (toxin designated as Shiga-like toxin-I [SLT-I]). A 5th isolate produced increased amounts of cytotoxin (SLT+) that was not neutralized by the Shiga antitoxin, but was neutralized by antitoxin against a variant of SLT (toxin designated as SLT-II). None of the isolates hybridized with the probe for the localized adherence gene. Three of the SLT+ isolates belonged to human enteropathogenic E coli serogroups O26 and O111. All 5 of the SLT+ isolates were from calves with diarrhea, but none of the 5 SLT+ isolates contained genes for classic heat-labile or heat-stable enterotoxins, for K99 fimbriae, or for invasiveness; neither did any of them adhere to HeLa cells in culture. Three of the 5 SLT+ isolates had attaching and effacing activities when inoculated into ligated intestinal loops of rabbits. One of the isolates with attaching and effacing activity in rabbits was originally isolated from a calf with lesions characteristic of those produced by attaching effacing E coli (AEEC). Calves inoculated with this SLT+ AEEC isolate developed focal colonic lesions characteristic of those produced by AEEC, but did not develop diarrhea.(ABSTRACT TRUNCATED AT 250 WORDS)