Flow Cytometric Analysis for Enterotoxin Exposed on Clostridium Perfringens Spores.

Abstract

ABSTRACT. Flow cytometric method (FCM) with fluorescent-labeled anti-CPE antibody was applied to develop a rapid, specific, and convenient method to detect enterotoxin (CPE) exposed on the surface of spores of Clostridium perfringens. The results obtained indicate that FCM can specifically detect CPE exposed on C. perfringens spores for a short time. Thus, FCM is found to be a rapid, specific, and convenient assay method for detection of CPE exposed on C. perfringens spores, suggesting that it will be hopefully useful to diagnose food poisoning caused by C. perfringens. -KEY WORDS: Clostridium perfringens, enterotoxin, FCM.J. Vet. Med. Sci. 60(12): 1357-1359, 1998 Rabbit antiserum was purified by gel filtration on Sephacryl S-300 (Pharmacia, Uppsala, Sweden). Purified antibody was labeled by the methods of Williams and Chase [24]. Thirty µg of fluorescein isothiocyanate, isomer I (FITC, Wako Pure Chemicals, Osaka, Japan) was added to 1 mg of antibody and incubated at 25°C for 1 hr. Then the mixture was applied to gel filtration on Sephadex G-25 (Pharmacia, Uppsala, Sweden). Further purification of FITC-labeled antibody was carried out by ion-exchange chromatography on DEAE-cellulose (DE-52, Whatman Paper Ltd, Maidstone, England).Heat treatment of formalin-treated C. perfringens spores was carried out by incubating at 60°C for 30 min. The spores (10 8 ) were also treated with 200 µl of rabbit anti-CPE serum by incubating at 37°C for 10 min. Binding of FITC-labeled anti-CPE antibody (40 µl) to either formalintreated vegetative cells or spores (10 6 /ml) was done by incubating at 37°C for 10 min. After these treatment, the spores were washed in PBS by centrifugtlior at 10,000 × g for 10 min.For flow cytometric assay, test samples were prepared as follows: 0.4 ml of formalin-treated vegetative cells or spores (10 8 /ml) was mixed with 50 µl of FITC-labeled purified rabbit anti-CPE antibody for 10 min at 37°C. To study the specificity of FITC-labeled anti-CPE antibody, CPE exposed on C. perfringens spores was treated with unlabeled rabbit anti-CPE antibody prior to FITC-* CORRESPONDENCE TO: Dr. UEMURA, T., Laboratory of Veterinary Public Health, Department of Veterinary Science, College of Agriculture, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan.Clostridium perfringens is a common food bacterium distributed widely in human foods, especially meat and poultry products [20]. Type A strains of C. perfringens produce an enterotoxin (CPE) which is the causative factor of human food poisoning [19]. CPE is synthesized during sporulation of C. perfringens vegetative cells and the lysis of sporulated cells liberates CPE into the intestinal tract [7]. Since CPE involves in food poisoning outbreaks caused by C. perfringens, several methods for detection of CPE have been developed over the past few decades [5,12,18,21]. Although enzyme-linked immunosorbent assay (ELISA) is one of the common methods to detect CPE at the present time, it is not useful to analyze CPE exposed on the surface of spores of ...

Fulminant Massive Gas Gangrene Caused by Clostridium perfringens

“…Rabbit anti-C.P enterotoxin A IgG was used for Western blotting, erythroimmunoassay and flow cytometric analysis (8)(9)(10). C. perfringens usually appears as a short, plump, strongly gram-positive rod that possesses a central or subterminal spore.…”

Flow Cytometry for the Detection of Enterohaemorrhagic Escherichia coli O157 : H7 with Latex Beads Sensitized with Specific Antibody

“…The strains of E. coli used in this study were obtained from Dr T. Tsukamoto (Osaka Prefectural Institute of Public Health) and Dr T. Kamiki (Sakai Municipal Institute of Public Health). All strains used were those described previously (Kusunoki et al, 1998a). Before inoculation of these strains into different media, the seed culture was suspended in the pond water at the campus.…”

“…Although immunoassays ( Stuart and Corbel, 1982; Farmer and Davis, 1985; Lior and Borvzyk, 1987; Perry et al, 1988; Padhye and Doyle, 1991) and polymerase chain reaction (PCR) methods ( Levine et al, 1987; Feng, 1993; Hunk et al, 1995; Meng et al, 1996; Desmarchelier et al, 1998; Tsukamoto and Kawai, 1998; Wang and Reeves, 1998) are available for the detection of E. coli O157 : H7, they are extremely time‐consuming. To overcome such a problem, flow cytometry methods (FCM) have been applied to detect E. coli O157 : H7 ( Kusunoki et al, 1996, 1998b; Tortorello et al, 1997; Seo et al, 1998a,b), Clostridium perfringens ( Reseland et al, 1992; Kusunoki et al, 1998a), Salmonella typhimurium ( McClelland and Pinder, 1994), and Listeria monocytogenes ( Donnelly and Baigent, 1986). Immunomagnetic separation has recently been reported to be a very useful method to detect rapidly and extract bacteria directly from food matrix ( Chapman et al, 1994; Bennett et al, 1996).…”