Toxin preparations were obtained by growing Clostridium difficile VPI strain 10463 in 2-liter brain heart infusion dialysis flasks at 37°C for 3 days. The initial step of the purification scheme involved ultrafiltration through an XM-100 membrane filter. Two toxic activities, designated toxins A and B, were separated by ion-exchange chromatography on DEAE-NaCl gradients. Toxin A was purified to homogeneity by an acetic acid precipitation at pH 5.5. Other separation techniques, including CM Sepharose CL-6B, (NH4)2SO4 and acetic acid precipitations, and hydrophobic interaction chromatography, were examined in attempts to further purify toxin B. Although these methods failed to increase the specific activity of toxin B, they provided additional evidence that the two toxins are distinct molecules. The toxins are acid and heat labile and are inactivated by trypsin and chymotrypsin, but not by amylase. The molecular weight of toxin A, as estimated by gel filtration and gradient polyacrylamide electrophoresis, ranged from 440,000 to 500,000. The estimated molecular weight of toxin B was 360,000 to 470,000. MATERIALS AND METHODS Bacterial strain. C. difficile VPI strain 10463 was grown in 2-liter brain heart infusion (dialysis flasks for 72 h at 37°C (3). Chemicals and reagents. Column supports for ionexchange chromatography (DEAE-Sepharose CL-6B and CM Sepharose CL-6B), hydrophobic interaction chromatography, concanavalin A 4B affinity chromatography, and gel filtration (Sepharose 6B) were obtained from Pharmacia Fine Chemicals, Piscataway, N.J. Reagents for the inactivation studies were obtained from Sigma Chemical Co., St. Louis, Mo., and included trypsin (bovine pancreatic, type III), chymotrypsin (bovine pancreatic), bacterial amylase (type Ila), and soybean inhibitor (type IS). Polyacrylamide gels were prepared from the following chemicals: electrophoresis pure acrylamide (Bio-Rad Laboratories, Richmond, Calif.), electrophoresis pure sodium dodecyl sulfate (Bio-Rad), electrophoresispure N,N'-methylenebisacrylamide (Eastman Organic

Cattle with Johne's disease can shed live Mycobacterium avium subsp. paratuberculosis (MAP) in their milk, and MAP can survive under simulated commercial pasteurization conditions. In several studies conducted in the United Kingdom and Canada, MAP DNA has been detected in retail pasteurized milk samples; however, in one study in the United Kingdom viable MAP was identified in commercially pasteurized milk. A double-blind study involving two laboratories was undertaken to evaluate retail pasteurized whole milk in the United States. Marshfield Clinic Laboratories used solid culture medium (Herrold's egg yolk agar slants with mycobactin J and amphotericin B, nalidixic acid, and vancomycin), and TREK Diagnostic Systems, Research and Development used liquid culture medium (ESP culture system). Cultures at both laboratories were eonfirmed by PCR. A total of 702 pints of retail whole milk were purchased in three of the top five milk-producing states (233 from California, 234 from Minnesota, and 235 from Wisconsin) over a 12-month period and were tested for the presence of viable MAP. The criteria used for identifying samples as positive for viable MAP were similar to those followed by most laboratories (positive culture with PCR confirmation). The combined data from the two laboratories revealed the presence of viable MAP in 2.8% of the retail whole milk pints tested. Although the number of samples containing viable MAP was similar among states (P > 0.05), there was a seasonal effect on the presence of viable MAP in retail milk (P = 0.05). More MAP-positive samples were identified during the third quarter of the year (July through September). Of the 22 brands of retail milk tested, 12 (55%) yielded at least one sample positive for viable MAP.

Antibodies against Clostridium difficile toxin A were purified by affinity chromatography from antiserum prepared against crude C. difficile toxin preparations. The affinity-purified antibody preparation was free of detectable amounts of antibodies to other C. difficile antigens, as demonstrated by crossed immunoelectrophoresis, and specifically neutralized the cytotoxicity of toxin A. An indirect enzyme-linked immunosorbent assay (ELISA) was subsequently developed using the antibody preparation for the specific detection of toxin A. The ELISA, which could detect 1 ng (5 ng/ml) of toxin A, was used to quantitate the toxin in the culture supernatant fluids of strains of C. difficile. The ELISA values for toxin A closely correlated with the toxin A and B cytotoxic titers of the supernatant fluids. In addition, toxin A was detected by ELISA in human fecal specimens from persons with antibiotic-associated colitis, demonstrating that this toxin is produced during C. difficile colitis. Clostridium difficile, which has been implicat

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An animal model simulating intra-abdominal sepsis was produced by implanting large bowel contents into the pelvic region of rats. Bacteriological analysis of infected sites showed quantitative differences according to the stage of disease. During the initial, often lethal, peritonitis stage, Escherichia coli (mean concentration, 106/ml), enterococci (105) and Bacterioides fragilis (106) were always present. Blood cultures obtained during this phase were uniformly positive, with E. coli being the principal isolate. Animals that survived this early acute peritonitis stage developed indolent intra-abdominal abscesses. The major isolates in abscess contents were B. fragilis (108 7) and Fusobacterium (108.6); E.