Takashi Hiramune

Phylogenetic Positions and Assignment of Swine and Ovine Corynebacterial Isolates Based on the 16S rDNA Sequence

The nucleotide sequences of the 16S ribosomal RNA gene (rDNA) of swine and ovine corynebacterial strains were determined. The sequences of the strains that identified as Corynebacterium pseudotuberculosis by their biochemical characteristics were homologous with each other. The phylogenetic position of C. pseudotuberculosis strains was closest to C. ulcerans and next closest to C. diphtheriae. The nucleotide sequence of another swine isolate, SC8, was similar to that of a recently proposed species, C. seminale, and a non‐validated species, “C. glucuronolyticum,” with about 0.01 to 0.02 evolutionary distances. Analysis of the predicted secondary structure of the 16S rRNA molecule agreed with the close phylogenetic relationships between C. pseudotuberculosis and C. ulcerans and between C. seminale and strain SC8.

Plasmid profiles of Klebsiella pneumoniae isolated from bovine mastitis

Plasmids of Klebsiella pneumoniae strains isolated from mastitic milk of cows were detected, and the epidemiological significance of their profiles was investigated. Of 47 K. pneumoniae isolates, 41 (87.2%) possessed plasmid(s). Although the molecular size of the plasmids ranged from 1.9 to 140 megadaltons (Md), the 125 Md plasmid was predominant (31/41, 75.6%). There was a great diversity in the plasmid profiles, however, no correlation was found between the plasmid profiles and capsular types of Klebsiella or the source of strains (dairy farms), except for one farm. All isolates obtained from cows on that farm possessed the 125 Md plasmid and their capsular type was 9.

Spontaneous Urinary Calculus in Young LEW Rats Caused by Corynebacterium Renale

Spontaneous urinary calculus was observed in only 27% of female LEW rats among nine inbred strains maintained in the Institute for Animal Experimentation. The condition occurred between 3 and 9 weeks of age with loss of weight, enlargement of the os urethral externum, anuria and general marasmus. All of the affected rats died from anuria 3 to 4 days after the onset. The affected rats had one to ten infected stones or many fine crystals of sand, which consisted of magnesium ammonium phosphate, in the urinary bladder and/or urethra. Analysis of microorganisms revealed that Corynebacterium renale, known as a causative agent of bovine pyelonephritis, was the most probable candidate for producing infection stones. This is the first report that the rat is spontaneously infected with C. renale. This could be a useful animal model for human urinary calculus and also for C. renale infection in the field of veterinary science.

Isolation of Corynebacterium pseudotuberculosis from the cervical canal of clinically normal sows

Two isolates from the cervical canal of clinically normal sows were indistinguishable from Corynebacterium pseudotuberculosis in their biochemical properties and genetic homology by DNA-DNA hybridisation. Furthermore, three sheep inoculated with one of the isolates developed typical lesions of caseous lymphadenitis and gave antibody responses specifically to C pseudotuberculosis.

Capsule types of Klebsiella pneumoniae isolated from the genital tract of mares with metritis, extra-genital sites of healthy mares and the genital tract of stallions

A survey of K. pneumoniae was performed on cervical swabs, feces and nasal swabs of mares and on samples from the genital tract of stallions from 1980 to 1986 in south-western Hokkaido, Japan. K1 was the predominant type (79 of 88, 89.8%) in the metritis cases due to K. pneumoniae in mares of racing breeds. The same type was isolated from semen and swabs of the fossa glandis of 6 of 20 (30.0%) of the stallions of racing breeds. Heavily encapsulated and less heavily encapsulated K1 strains were isolated from the stallions. Mares bred to stallions carrying heavily encapsulated strains developed metritis, while those bred to stallions carrying less heavily encapsulated strains did not. K39 was isolated from cervical swabs solely from metritis-infected mares of draft breeds and not from any mares of the racing breeds examined. Untypable strains were isolated from cervical swabs in 7 of 88 (8.0%) metritis cases of mares of racing breeds and from semen in 7 of 19 (36.8%) stallions of racing breeds and they were predominant in feces (19 of 21, 90.5%) and nasal swabs (3 of 4, 75.0%) of healthy mares of racing breeds.

Distribution of Corynebacterium renale among apparently healthy rats

We examined the distribution of Corynebacterium renale, a causative agent of urinary calculus, in clinically normal rats at 6 animal facilities in Japan. Swabs of the vulva and vaginal vestibule or prepuce of the rats were cultured for isolation of the organisms. C. renale has been isolated at only one animal facility, where cases of urinary calculus were reported several years ago. In this facility, 32% of female (43/135) and 22% of male (18/82) rats, 4-28 weeks old, were positive for C. renale. In contrast, 92 female and 169 male rats at other facilities without a history of the disease were negative for the organisms.

Determination of serovar of autoagglutinating strain of Actinobacillus lignieresii by gel precipitation test.

The serotyping of Actinobacillus lignieresii isolated from lesions of actinobacillosis in cattle and sheep in England was reported by Phillips (6), who established the existence of six serovars of A. lignieresii by the agglutination test. Recently, it was noticed in Japan that serotyping by the agglutination test was impossible for some autoagglutinating strains of A. lignieresii isolated from lesions of affected cattle. No report, however, has been published on the serotyping of autoagglutinating strains of the organism. The present study deals with the serotyping of autoagglutinating strains of A. lignieresii performed by the gel precipitation test with Westphal-type antigen. A. lignieresii NCTC 10563 (serovar 1), NCTC 10565 (serovar 2), NCTC 10566 (serovar 3), NCTC 10567 (serovar 4), NCTC 10568 (serovar 5), and NCTC 10569 (serovar 6) were supplied through the courtesy of Dr. J.E. Phillips, of the Royal School of Veterinary Studies, Edinburgh, Scotland, as reference strains. In addition, autoagglutinating strains 33, 41-3, 62, 67, 118, and 183 isolated recently in Japan were employed in this study. To prepare precipitation antigens, each strain was cultured in brain heart infusion broth (Difco) at 37 C for 40 hr. The organisms grown were harvested by centrifugation at 6,000 rpm for 20 min at 4 C. The packed cells were washed twice with saline and resuspended in 50 ml of distilled water. Westphal-type antigens were prepared from the aqueous phase of hot phenol-water extraction , as described by Baker and Wilson (1). For chemical analyses of the antigens, the total carbohydrate content was estimated by the phenol-sulfuric acid method (2) with glucose as a standard, and the protein content by the method of Lowry et al (4) with bovine serum albumin as a standard. Qualitative sugar composition was determined by the method of chromatography described by Staneck and Roberts (7) using the thin layer silica gel plate Avicell SF (Funakoshi Co., Ltd., Tokyo) with a solvent system of n-butanol water-—pyridine-toluene (10: 6: 6: 1, by vol.). Agar gel for the precipitation test was prepared by the method of Kwapinski (3). The antisera used were hyperimmune sera against heated whole cells of reference strains of the six serovars described previously (5). The chemical properties of the antigens are shown in Table 1. The antigens