A total of 149 samples including 50 samples of seawater, 50 samples of sea mud and 49 shellfish obtained at 6 points in the Tokyo Bay areas between 1999 and 2004 were examined for Vibrio parahaemolyticus and other pathogenic vibrios. V.parahaemolyticus was isolated from all Samples. The number of V.parahaemolyticus in most sea mud samples were 10-100 times higher than that in seawater samples.NAG vibrio, V. fluvialis, V. mimicus, V. furnissii, and V. choleme O1 (cholera toxin negative) were isolated from 51 (34.2%), 21 (14.1%), 13 (8.7%), 8 (5.4%), and 2 (1.3%) of the samples, respectively. NAG vibrio was more frequently isolated from seawater samples than sea mud or shellfish samples. V.mimicus and V.fluvialis were more frequently isolated from the area where the salinity of the seawater was low.These findings show that V.parahaemolyticus and other pathogenic vibrios examind are widely distributed in the Tokyo Bay, but the distribution of different pathogenic vibrios varieds lightly by area. The most probable number of V.parahaemolyticus in seawater, sea mud and shellfish were compared by one-step and two-step enrichment culture methods.However, the results did not significantly differ between the two methods. Although V.parahaemolyticus strains belonging to serotype O3: K6 were isolated from 16 samples, only one was a tdh-positive strain and there were no trh-positive O3: K6 strains in seawater produce neither TDH nor TRH. Total of 7 tdh-positive strains, 6 trh-positive strains and 5 tdh and trh-positive str-late tdh- and/or trh-positive V.parahaemolyticus from the culture broth samples in which the tdh or trh gene was detected by PCR method.
In order to genotypically characterize Clostridium botulinum type A strains, we examined 23 strains from various origins (food-borne botulism, infant botulism, honey, honey-related materials and soil), by random amplified polymorphic DNA (RAPD) analysis and characterized the type A botulinum neurotoxin complex genes by PCR and PCR-RFLP analysis. By RAPD analysis, C. botulinum strains associated with infant botulism in Japan and a strain 804-1H, isolated from Brazilian honey, were clearly differentiated from the other strains examined.PCR and PCR-RFLP analysis were performed for the neurotoxin complex genes (type Aneurotoxin (BoNT/A), type B neurotoxin (BoNT/B), nontoxic-nonhemagglutinin (NTNH), hemaggulutinin (HA), and p47 protein). All of 23 strains tested gave positive amplification for BoNT/A and NTNH genes, and the PCR-RFLP analysis differentiated six strains associated with infant botulism in Japan, a strain from Brazilian honey and a strain AF84 (type 2) from other strains (type 1). For HA gene, the type 1 strains gave amplification while the type 2 strains gave no amplification. For BoNT/B gene, the type 1 strains (a strain Renkon and three American infant botulism strains) gave amplification (silent B strains). For p47 gene, the type 2 strains and the silent B strains were positive. The combinations of the PCR of HA, p47, and BoNT/B genes and the PCR-RFLP of BoNT/A, NTNH genes demonstrated three genotypes (A 1, A2, and A3) of neurotoxin complex genes. Thus, these PCR-based methods are useful for simple and rapid differentiation of the BoNT-producing C. botulinum strains.