Masatake Muramatsu

DNA relatedness among Erysipelothrix rhusiopathiae strains representing all twenty-three serovars and Erysipelothrix tonsillarum.

The levels of relatedness among strains of Erysipelothrix rhusiopathiae (serovars 1 through 23 and type N) were estimated by performing DNA-DNA hybridization experiments with the type strains of E. rhusiopathiae and Erysipelothrix tonsillarum, which are the two Erysipelothrix species that have been described. Two distinct DNA relatedness groups were identified. The group 1 strains, representing serovars 1, 2, 4 through 6, 8, 9, 11, 12, 15 through 17, 19, and 21 and type N, exhibited more than 73% hybridization with the type strain of E. rhusiopathiae but less than 24% hybridization with the type strain of E. tonsillarum. Group 2 included serovar 3, 7, 10, 14, 20, 22, and 23 strains, and these strains exhibited more than 66% hybridization with the type strain of E. tonsillarum but less than 27% hybridization with the type strain of E. rhusiopathiae. Strains representing serovars 13 and 18 exhibited low levels of hybridization (16 to 47%) with both of the type strains, indicating that these serovars may be members of a new genomic species. The members of the E. rhusiopathiae and E. tonsillarum groups resembled each other in many phenotypic characteristics, but differed in their ability to produce acid from saccharose and in their pathogenicity for swine. Our results confirm that the genus Erysipelothrix contains two main genomic species, E. rhusiopathiae and E. tonsillarum, which can be differentiated into serovars.

Erysipelothrix tonsillarum sp. nov. Isolated from Tonsils of Apparently Healthy Pigs

A new species, Erysipelothrix tonsillae, is proposed for avirulent Erysipelothrix strains of serotype 7, which is frequently isolated from tonsils of apparently healthy pigs. This species is morphologically and biochemically indistinguishable from the Erysipelothrix rhusiopathiae strains characterized to date. Seven strains of E. tonsillae that belonged to serotype 7 were avirulent for swine, except for strain T-334, which induced a local urticarial lesion at the site of inoculation. The 50% lethal dose values in mice ranged from 1.0 CFU to 6.9 × 105CFU. The E. rhusiopathiae strains belonging to serotypes 2 (six strains), 6 (one strain), 11 (one strain), 12 (one strain), and 16 (one strain) induced generalized or local urticarial lesions after intradermal inoculation, except for strain T-312 of serotype 12, which showed 50% lethal dose values of ≤2.5 × 10 CFU in mice. The deoxyribonucleic acid base composition of E. tonsillae is 36 to 40 mol% guanine plus cytosine. Strains of this species have little deoxyribonucleic acid homology (15 to 43%) with the type strain of E. rhusiopathiae (ATCC 19414). The type strain of E. tonsillae is T-305T(ATCC 43339).

Sequence variation of the gC gene among pseudorabies virus strains

We have determined the nucleotide sequences of the major glycoprotein C (gC) gene of 3 pseudorabies virus (PRV) strains isolated in Japan, the USA, and Northern Ireland after gene amplification mediated by a polymerase chain reaction (PCR). The nucleotide and deduced amino acid sequence homologies among the strains were > 98% and > 97%, respectively. The restriction patterns of the amplified DNA fragments generated by restriction endonucleases SalI and SmaI revealed three genomic variations among the 15 PRV strains. The Japanese PRV isolates have identical restriction fragment patterns and differ from those of the non-Japanese isolates examined. Among 3 PRV strains with distinctive genotype each other, there is no significant difference in pathogenicity for the ddY mouse.

Nationwide survey of Salmonella prevalence in environmental dust from layer farms in Japan.

A nationwide survey was conducted to determine Salmonella prevalence in airborne dust from layer farms. Of the 4,090 layer farms in Japan, 203 were surveyed and 48 (23.6%) of these were positive for Salmonella. Salmonella isolation rates were higher in the eastern (24.3%), central (25.6%), western (23.9%), and southern (27.5%) prefectures than they were in the northern (13.3%) prefecture. We recovered 380 Salmonella isolates and identified 34 different Salmonella serovars. Salmonella Infantis was the most prevalent serovar (42 [11.1%] of 380), followed by Salmonella Agona (39 [10.3%] of 380), Salmonella Mbandaka (37 [9.7%] of 380), Salmonella Cerro (32 [8.4%] of 380), Salmonella Thompson (29 [7.6%] of 380), and Salmonella Braenderup (27 [7.1%] of 380). Of the 380 isolates, 273 (71.8%) were resistant to more than one antibiotic. Salmonella Infantis (41 [97.6%] of 42), Salmonella Agona (38 [97.4%] of 39), and Salmonella Mbandaka (34 [91.9%] of 37) showed the highest resistance rates. We found 18 different resistance patterns and the most common (179 [47.1%] of 273) was resistant to dihydrostreptomycin. One of the 13 Salmonella Hadar isolates was resistant to eight antibiotics. To investigate characteristics of Salmonella Agona, Salmonella Infantis, and Salmonella Mbandaka isolates across different prefectures, we performed pulsed-field gel electrophoresis by using XbaI and BlnI. The Salmonella Agona and Salmonella Mbandaka dendrograms were grouped into seven clusters, with 80 and 70% similarity, respectively. Because the Salmonella Infantis dendrogram showed low similarity, there is a possibility of genetic diffusion of this serovar across Japan. This report is the first to describe Salmonella contamination in airborne dust from layer farms in Japan. Our findings should be useful for future Salmonella infection monitoring and control.

Correlation between adherence of Erysipelothrix rhusiopathiae strains of serovar 1A to tissue culture cells originated from porcine kidney and their pathogenicity in mice and swine

Adherence of four virulent and four avirulent strains of Erysipelothrix rhusiopathiae, serovar 1a, to porcine kidney cell lines, PK-15 and ESK cells, was examined in an in vitro system. The virulent strains adhered well to the cells (range of means, 9.95 +/- 0.87-36.01 +/- 1.10 per cell). In contrast, the avirulent strains showed negligible adherence to the cells (range of means, 0.11 +/- 0.04-1.41 +/- 0.13 per cell). Pretreatment of bacteria with heat, trypsin, or antiserum resulted in a marked decrease in adherence. Scanning electron microscopic examination revealed that the bacteria attached directly to the microvilli of cells.

Prevalence and molecular epidemiological characterization of antimicrobial-resistant Escherichia coli isolates from Japanese black beef cattle.

We investigated the prevalence of antimicrobial-resistant Escherichia coli in Japanese black beef cattle from the three major production regions of Japan. We collected and examined 291 fecal samples from Japanese black beef cattle in Hokkaido, Chubu, and Kyushu. Of the 3,147 E. coli isolates, 1,397 (44.4%) were resistant to one or more antibiotics; these included 553 (39.8%) of 1,388 isolates from Hokkaido, 352 (54.4%) of 647 isolates from Chubu, and 492 (44.2%) of 1,112 isolates from Kyushu. The difference in resistance rates between the three regions was significant. The antibiotics with the highest rates of resistance were oxytetracycline and dihydrostreptomycin (35.8% each), followed by ampicillin (21.4%). Further, E. coli isolates from calves had higher resistance rates than those from growing cattle and mature cattle, and the calf isolates showed high rates of resistance to gentamicin (20.2%), enrofloxacin (9.4%), and ceftiofur (4.2%). In addition, the high degrees of similarity in the genotypes of the isolates and in the resistance patterns on each farm suggest that resistance bacteria and resistance genes were horizontally transferred. Most isolates, in each of the three regions, harbored resistance genes such as blaTEM, strA, strB, aphA1, aphAI-IAB, and catI. In contrast to the isolates from Kyushu, most of which harbored aacC2, tetB, and dfrA12, the isolates from Hokkaido and Chubu harbored a variety of resistance genes. Furthermore, the prevalence of genes for resistance to dihydrostreptomycin, gentamicin, chloramphenicol, and trimethoprim differed significantly between the regions. This is the first large-scale study describing and comparing antimicrobial-resistant bacteria from different regions in Japan. The results will contribute to improving food safety and promoting careful usage of antimicrobial agents.

Evaluation on the pathogenicity of Erysipelothrix tonsillarum for pigs by immunosuppression with cyclophosphamide or dexamethasone.

The pathogenicity of Erysipelothrix tonsillarum was evaluated in pigs immunosuppressed with cyclophosphamide (CY) or dexamethasone (DM). Animals were treated with 15 mg/kg CY (n=8, five injections), 1 mg/kg DM (n=8, nine injections) or left untreated (n=8). On the fifth day after the beginning of drug treatments, swine were inoculated with one of two E. tonsillarum serovar 7 strains (approximately 10(6) CFU per pig). In the CY-treated group, both circulating neutrophil and lymphocyte counts decreased, whereas in the DM-treated group, lymphocyte counts decreased but neutrophil counts increased. During the observation period, none of the CY- or DM-treated pigs developed clinical signs or gross lesions, as well as non-treated pigs. Growth agglutination antibody titres in all pigs remained unchanged. Our findings indicate that E. tonsillarum strains are avirulent for swine, regardless of immune status.

Differentiation between glycoprotein III gene-deleted vaccine and wild-type strains of pseudorabies virus by polymerase chain reaction (PCR).

One of the attenuated and genetically recombinant modified-live viral (MLV) vaccine strains currently used contains a deletion in its glycoprotein III (gIII) gene, while prototypic wild-type pseudorabies (WT-PR) viruses contain an intact gIII gene. A polymerase chain reaction (PCR) system differentiating, based on this difference, between the vaccine virus and prototypic WT-PR viruses was investigated. This PCR system utilized two consecutive stages. Primers for the first-stage PCR were designed so as to amplify of DNA fragments lengths in respect to the vaccine and WT-PR viruses. The second-stage PCR amplification for improving the sensitivity and specificity and for confirming of the sites deleted from the first-stage PCR products produced an all-or-none result: internal DNA fragments were derived from only WT-PR viruses but not from the vaccine virus. These PCR-amplified fragment length polymorphisms clearly distinguished the vaccine virus from WT-PR viruses. The vaccine and WT-PR viruses in mixtures were each identified in this PCR system. This PCR system may permit rapid and sensitive detection of PR viral gIII gene, analysis of the genotype of PR virus isolates, and also examination of the isolates for purity and identity.