Tsutomu Maruyama

Prevalence and contamination levels of Listeria monocytogenes in retail foods in Japan.

Retail foods in Japan were surveyed for the presence and contamination levels of L. monocytogenes. It was isolated from 12.2, 20.6, 37.0 and 25.0% of 41 minced beef, 34 minced pork, 46 minced chicken and 16 minced pork-beef mixture samples, respectively. MPN values were higher than 100/g in five (10.9%) minced chicken samples, but lower than 100/g in all minced beef, pork and pork-beef mixture samples. The organism was also isolated from 5.4% of the 92 smoked salmon samples at MPN values lower than 10/g, and from 3.3% of 213 ready-to-eat raw seafood samples at MPN values from lower than 0.3 to higher than 100/g. None of the 285 vegetable samples were contaminated with L. monocytogenes. These findings indicate that ready-to-eat raw seafoods are relatively high risk among the foods surveyed in this study.

Isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from Apparently Healthy Dogs and Cats

Recently, a number of cases of human infections due to Yersinia enterocolitica have been reported in many countries, and much attention has been given to the organism as one of the important etiologic agents of enteritis, appendicitis, septicemia, arthritis and others (12). In most of the sporadic cases or outbreaks, however, the source and the route of the infection remained unknown, since ecology of the organism has not yet been understood clearly. The authors studied previously the distribution of Y. enterocolitica in healthy humans, livestock, foodstaff and the natural environment (14, 15). Through these investigations, it was found that Y. enterocolitica was isolated from swine and rats at a high rate, and over 70% of the isolates were of the same serovars as those from human cases. These findings suggested the importance of swine as one of the infective sources for humans. In the present survey, distribution of Y. enterocolitica in dogs and cats, which have close relations to humans as pet animals, was investigated to obtain more extensive ecological informations of the organism. These animals were also examined for the presence of Y. pseudotuberculosis. A total of 704 stray or unwanted adult dogs and 373 unwanted adult cats obtained from the Tokyo Metropolitan Dog Pound Office were submitted to the survey. The dogs were examined between October 1974 and September 1976, and the cats between June 1975 and September 1976. Autopsies were made immediately after the sacrifice, and about 1 g each of the ileo-cecal contents, rectal contents and mesenteric lymph node was sampled. The content to be tested was suspended into 5 ml of sterile saline, and the mesentric lymph node was homogenized with 10 ml of sterile saline. A loopful of each specimen thus prepared was directly plated on both SS and MacConkey agar plates, and incubated at 25 C for 48 hr. Enrichment cultures were also made by inoculating 1 ml of each specimen into 20 ml of 1/15 M phosphate buffer solution (pH 7.6), and incubated at 4 C for 21 days. After the cold enrichment, a loopful of each culture was spread on the agar plates, and incubated at 25 C for 48 hr. Suspicious colonies grown on the agar plates were identified by the method described previously (14). The biovars of Y. enterocolitica were determined according to the scheme and method described by Wauters (11) . Summarized results are shown in Table 1. Y. enterocolitica was isolated from 42

Correlation between Epithelial Cell Infectivity In Vitro and O-Antigen Groups of Yersinia enterocolitica

Yersinia enterocolitica has been isolated from patients mainly with intestinal disorders throughout the world, including Japan (1-3, 7, 10), and the organisms are assumed to be one of the enteropathogenic bacteria for human beings. In our experiments to clarify this pathogenicity, it was observed that the pathogenic strains of the clinical isolates vigorously penetrated epithelial cells in vivo and in vitro, and severe enteritis analogous to that in man could be produced in rabbits by intraduodenal inoculation (5, 6). Based on these results which suggested that the penetration of the bacilli through epithelial linings of the intestinal mucosa was an essential factor for establishment of infection, Y. enterocolitica was considered to be an invasion type enteric pathogen such as Shigella flexneri and Salmonella typhimurium (4). Although Y. enterocolitica is at present divided into various O-antigen groups

Comparison of indirect haemagglutination test, gel-diffusion precipitin test, and enzyme-linked immunosorbent assay for detection of serum antibodies to Pasteurella multocida in naturally and experimentally infected rabbits

Enzyme-linked immunosorbent assay (ELISA), gel-diffusion precipitin test (GDPT), and indirect haem agglutination test (IHAT) were evaluated for the detection of antibodies to Pasteurella multocida in both naturally and experimentally infected rabbits. A total of 285 rabbit serum samples from 7 rabbit colonies were tested by ELISA, GDPT, and IHAT, and nasal cultures were taken coincidentally to use as the standard in the serological tests. There was better correlation (98.0%) between the results of ELISA and positive nasal culture than between the GDPT (86.3%) or IHAT (23.5%) and positive nasal culture. In addition, ELISA and GDPT were positive in 26 (11.1%) and 21 (9.0%) of 234 serum samples from nasal culture negative rabbits, respectively. In experimentally infected rabbits, antibodies detected by the ELISA and GDPT began to rise one to 3 weeks post-inoculation. IHAT did not detect antibodies. These results are discussed in terms of value to serodiagnosis of rabbit pasteurellosis.

A comparison of Listeria monocytogenes serovar 4b isolates of clinical and food origin in Japan by pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) patterns of 102 L. monocytogenes serovar 4b isolates from patients and foods examined in Japan were compared with 16 isolates from foodborne listeriosis episodes which occurred in North America or Europe. Using a combination of PFGE patterns with the restriction enzymes SmaI, ApaI, AscI and Sse8387I, 82 clinical isolates from Japan were categorized into 45 PFGE types: the largest group of 17 isolates (20.7%) were of the same PFGE type as cultures from the large foodborne outbreaks which occurred in California (1985) and Switzerland (1983-1987). Twenty cultures from foods on retail sale in Japan were classified into 12 PFGE types: four isolates were of three PFGE types also recognized among isolates of clinical origin from Japan, including the predominant clinical type.

RAPD- and actA gene-typing of Listeria monocytogenes isolates of human listeriosis, the intestinal contents of cows and beef

Seventy‐five L. monocytogenes isolates of human listeriosis, the intestinal contents of cows and beef were divided into 5 major clusters, 17 sub‐clusters and 28 minor clusters by typing using random amplification of polymorphic DNA (RAPD). According to their major RAPD category, L. monocytogenes isolates serotyped as 1/2b and 4b were distinguished from L. monocytogenes isolates of serovars 1/2a and 1/2c. Moreover serovar 4b was distinguished from serovar 1/2b by a difference in the RAPD sub‐cluster category. All L. monocytogenes were found to possess either actA gene Type I or n, and only one actA gene type was detected in each RAPD minor cluster. actA gene Type II was observed in 32.0%, 38.5 % and 18.9% of isolates from humans, cows and beef, respectively, and was detected more frequently in serovar 4b (46.9%) than in serovars 1/2a (22.2%), 1/2b (7.7%) and 1/2c (0.0%). Twenty (80%) of 25 human isolates fell within three minor RAPD types (II‐d (16%), V‐p‐1 (36%), V‐p‐2 (28%)). Two isolates from humans and beef were found to have the same RAPD type (Type IV‐k‐1), actA gene type (Type I) and serovar (1/2b). Our results suggest that only a few genotypes of L. monocytogenes are predominant in human listeriosis in Japan, although the human isolates were collected over a broad span of time and a wide geographical range. Our results also suggest that RAPD‐, actA gene‐ and sero‐typing can be useful for epidemiological analysis.

Usefulness in the Epidemiology of Food Poisoning Cases of Detection of Specific Restriction Endonucleases in Some Serotypes of Salmonella and Yersinia

Restriction endonucleases have been employed as an extremely important tool in recombinant DNA technology. To date, the occurrence of more than 100 restriction endonucleases with different specificities has been reported.2,8 Restriction endonucleases have been screened in this laboratory for pathogenic bacteria belonging to the Enterobacteriaceae in the hope that: (i) the detection of specific restriction endonuclease is found at a high frequency in the species; and (ii) new restriction endonucleases with novel specificities might be found in pathogenic bacteria, since screening of restriction endonucleases have been rarely carried out. Here, we report that four clinically important food-poisoning bacteria produce specific restriction endonucleases at high frequencies. Some of these are expected to be useful for recombinant DNA technology after cloning of their gene into Escherichia coli K-12.

Rapid Method for Detection of Pathogenic Yersinia Enterocolitica and Yersinia Pseudotuberculosis Using Enzyme Immunoassay

In this paper we describe a convenient method for the detection of virulent Yersinia strains using an enzyme - immunoassay (EIA). An antiserum was prepared against virulent plasmid-coded specific proteins of Y. enterocolitica serotype 03. Then, EIA was performed with the antiserum and proteins of various Yersinia strains of different origin. Positive reaction in EIA was observed when the proteins of virulent plasmid-bearing strains were tested. These strains were Y. enterocolitica serotypes 05:27, 08 and 09, and Y. pseudotuberculosis serotypes lb, 2a, 2b, 2c, 3, 4a, 4b, 5a, 5b, 6, 7 and 8. On the other hand, the reaction was negative when plasmid- cured strains of the pathogenic Y. enterocolitica and Y. pseudotuberculosis were tested, as well as non-pathogenic Y. enterocolitica, Y. frederiksenii, Y. intermedia and Y. kristensenii. The results clearly indicate the usefulness of the EIA method for the rapid detection of virulent Yersinia strains.