Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in Ontario and we failed to identify cytotoxic activity in vitro in the type A isolates recovered.

Objective—To evaluate enterotoxin production, enterotoxin gene distribution, and genetic diversity of Staphylococcus aureus in milk obtained from cows with subclinical mastitis. Sample—Milk samples obtained from 350 cows (1,354 mammary glands) on 11 Wisconsin dairy farms. Procedures—Of 252 S aureus isolates obtained from 146 cows, 83 isolates (from 66 cows with subclinical mastitis) were compared genotypically by use of pulsed-field gel electrophoresis and via PCR identification of toxic shock syndrome toxin 1 (TSST-1) and classical S aureus enterotoxin genes (sea, seb, sec, sed, and see). Results—Among the 83 S aureus isolates, ≥ 1 enterotoxin genes were identified in 8 (9.6%). Enterotoxin gene distribution was as follows: TSST-1, 7 isolates (8.4%); sec, 5 isolates (6.0%); and sed, 2 isolates (2.4%). Enterotoxin genes sea, seb, and see were not identified. Twelve pulsotypes and 5 subtypes were identified among the 83 isolates; 5 of the 12 pulsotypes were represented by only 1 isolate. In cows of 1 herd, only a single S aureus pulsotype was detected; in cows on most other farms, a variety of pulsotypes were identified. One pulsotype was recovered from 4 farms (n = 23 cows) and another from 5 other farms (16). Isolates with an enterotoxin gene were represented by 6 pulsotypes. Conclusions and Clinical Relevance—S aureus classical enterotoxins and TSST-1 were rarely recovered from milk samples obtained from cows with subclinical mastitis in Wisconsin. Diverse pulsotypes of S aureus were detected within and among farms, indicating that different strains of S aureus cause subclinical mastitis in dairy cows.

To assess the role of enterotoxigenic (ETEC), verotoxigenic (VTEC), and necrotoxigenic (NTEC) Escherichia coli in cattle with diarrhea, 1,524 colonies of E coli isolated from 197 calves with diarrhea and from 112 healthy controls were investigated for production of heat-labile and heat-stable enterotoxins, verotoxins, and cytotoxic necrotizing factors (CNF1 and CNF2). The ETEC were isolated from only 2 (1%) calves with diarrhea and from 5 (4%) healthy controls. In contrast, VTEC and NTEC that produced CNF2 were frequently identified. The VTEC were isolated from 18 (9%) calves with diarrhea and from 21 (19%) healthy cattle (P < 0.05), whereas NTEC that produced CNF2 were detected in 39 (20%) ill calves and in 38 (34%) controls (P < 0.01). Therefore, VTEC and NTEC that produced CNF2 were isolated significantly more frequently from healthy than diseased calves. Serogroups to which VTEC belonged differed considerably from the O groups involved with NTEC. Although, VTEC belonged to 18 serogroups, only 4 (O26, O103, O113, and O157) accounted for 56% (25 of 45) of verotoxigenic strains. The NTEC that produced CNF2 belonged to 26 serogroups; however, 64% (69 of 108) were from 6 serogroups (O1, O3, O15, O55, O88, and O123). Our results are compatible with cattle being a reservoir of VTEC that are pathogenic for human beings and with ETEC being an unusual cause of bovine colibacillosis in Galicia (northwestern Spain). Furthermore, results of this study indicate that VTEC and NTEC that produced CNF2 may be part of the normal intestinal flora of cattle.

Two hundred ninety-six Eschericbia 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 hy- bridizing 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). Most isolates of these 2 classes belong to only 4 serogroups: O5, O26, O111, and O118. In addition to these 2 AEEC classes, a VTEC class (20 isolates) was observed. Such isolates were positive with 1 or both SLT probes, but were negative with the eae probe. All but 1 isolate belonged to serogroups not found among the AEEC isolates. Only 7 of all AEEC and VTEC isolates were positive with the EAF, the F1845, or the AIDA-1 probe, and none were positive with the Agg probe. On the other hand, 32 non-VTEC, non-AEEC isolates were positive with the F1845 probe only, 2 were positive with the EAF probe only, and 1 was positive with the AIDA-1 probe only, thus constituting a possible class of EAEC isolates from cattle. The eae gene and the gene coding for the SLT1 are, thus, associated in most AEEC isolates from cattle. The isolates with other hybridization results VTEC and EAEC isolates) need more work to be clearly defined.

The protective effects of egg yolk powder prepared from hens vaccinated with heat-extracted antigens from K99-piliated enterotoxigenic Escherichia Coli (ETEC) strain 431 were evaluated in a colostrum-fed calf model of ETEC-induced diarrhea caused by a heterologous strain (B44). The antibody powder was obtained by spray-drying the water-soluble protein fraction of egg yolks after removing the lipid and fatty components by precipitation with hydroxypropylmethylcellulose phthalate. A total of 16 colostrum-fed calves were studied to determine whether the orally administered antibody powder would prevent fatal bovine colibacillosis caused by a virulent ETEC strain. Clinical response of individual calves was monitored and evaluated in the context of these variables: fecal consistency score, intestinal colonization, weight loss, and mortality. Control calves that were treated with vehicle (milk with egg yolk powder from nonimmunized hens) had severe diarrhea and dehydration and died within 72 hours after infection was manifested. In contrast, calves fed milk containing egg yolk powder with antipili agglutinin titers of 1:800 and 1:1,600 had transient diarrhea, 100% survival, and good body weight gain during the course of the study. Results indicate that the orally administered egg yolk powder protected against ETEC-induced diarrhea in neonatal calves and that the protective components may have been the antibodies raised by vaccination of chickens against ETEC.

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.

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 strains 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.

In vitro effects of a mixture of Escherichia coli heat-stable enterotoxins (STa and STb) on isolated jejunum of 3-week-old male pigs were studied, using everted intestinal sac techniques. Heat-stable enterotoxins increased chloride secretion and chloride absorption in everted intestinal sacs. The increase of secretory flux was greater than that for absorptive flux. Vasoactive intestinal peptide (6 x 10-9M) increased chloride secretion, but had no effect on chloride absorption. Neither vasoactive intestinal peptide nor pilocarpine (10-5M) had additive effect to ST. Secretory effects of ST were not blocked by atropine 2 x 10-5M), clonidine (10-6M), or morphine (4.2 X 10-6M).

This study was designed to determine the prevalence of enterotoxigenic S. aureus in table eggs in El-Fayoum city, Egypt. A total of 250 table egg samples (75 Baladi hens’, 75 white farm hens’, 75 brown farm hens’ and 25 duck egg samples) were collected randomly from poultry farms, groceries, supermarkets, and street vendors in El-Fayoum city, Egypt. Each Baladi hen ҆s egg sample was represented by five eggs, while each farm hen ҆s and duck egg sample was represented by three eggs. The shells and contents of eggs were examined for the presence of Staphylococcus spp < /em>., coagulase (coa), and staphylococcal enterotoxins (Ses) genes. The obtained results revealed that the examined samples of shells and contents of Baladi hens ҆, poultry farms ҆ (white and brown), and ducks ҆ eggs were contaminated with Staphylococcus spp. with incidences of 24.0, 9.3, 5.3, 44.0, 8.0, 2.7, 1.3 and 12.0 %, respectively and coagulase-positive S. aureus with the incidences of 16.7, 14.3, 0.0, 18.2, 0.0, 0.0, 0.0 and 33.3 %, respectively. Enterotoxin profiling by PCR proved that two classical enterotoxin genes (Seb and Sed) were produced from three (42.86%) coagulase-positive S. aureus strains, as two Baladi hens’ ҆ eggshells produced Seb and one of the ducks ҆ egg contents produced Sed. The public health hazards of the isolated strains and enterotoxins had been discussed.

Three-week-old weaned and colostrum-deprived neonatal (< 1 day old) pigs were inoculated to determine the pathogenicity of 2 enterotoxigenic Escherichia coli isolates that do not express K88, K99, F41, or 987P adhesins (strains 2134 and 2171). Strains 2134 and 2171 were isolated from pigs that had diarrhea after weaning attributable to enterotoxigenic E coli infection. We found that both strains of E coli adhered in the ileum and caused diarrhea in pigs of both age groups. In control experiments, adherent bacteria were not seen in the ileum of pigs < 1 day old or 3 weeks old that were noninoculated or inoculated with a nonpathogenic strain of E coli. These control pigs did not develop diarrhea. Antisera raised against strains 2134 and 2171 and absorbed with the autologous strain, grown at 18 C, were used for bacterial-agglutination and colony-immunoblot assays. Both absorbed antisera reacted with strains 2134 and 2171, but not with strains that express K99, F41, or 987P adhesins. A cross-reaction was observed with 2 wild-type K88 strains, but not with a K12 strain that expresses K88 pili. Indirect immunofluorescence with these absorbed antisera revealed adherent bacteria in frozen sections of ileum from pigs infected with either strain. We concluded that these strains are pathogenic and express a common surface antigen that may be a novel adhesin in E coli strains that cause diarrhea in weaned pigs.