MOST PROBABLE NUMBER METHOD COMBINED WITH NESTED POLYMERASE CHAIN-REACTION FOR DETECTION AND ENUMERATION OF ENTEROTOXIGENIC CLOSTRIDIUM-PERFRINGENS IN INTESTINAL CONTENTS OF CATTLE, PIG AND CHICKEN

The most probable number (MPN) method combined with a nested polymeras e chain reaction (nested PCR) for the detection and enumeration of ent erotoxigenic Clostridium perfringens in the intestinal contents of cat tle, pig and chicken was examined. Ten-fold serial dilutions of sample s were added to three tubes of enrichment medium, which were incubated at 37 degrees C for 20-24 hr, and the C. perfringens enterotoxin gene was detected by nested PCR from the enrichment culture without isolat ing the organism. The results obtained by this method with artificiall y contaminated intestinal contents were significantly correlated with those obtained by a plate count method. When the method was applied to the detection and enumeration of indigenous enterotoxigenic C. perfri ngens, the organism was found in two, two and three samples of 10 inte stinal contents of cattle, pig and chicken, respectively. Most of the positive samples contained fewer than 10 MPN/g of enterotoxigenic C. p erfringens, except one sample of chicken, which contained 1.5 x 10(2) MPN/g. The MPN method combined with nested PCR is easy to perform and may be a useful tool for the detection and enumeration of enterotoxige nic C. perfringens in intestinal contents.