Kinya Yamamoto

Tetracycline-resistance genes of Clostridium perfringens, Clostridium septicum and Clostridium sordellii isolated from cattle affected with malignant edema

The minimal inhibitory concentrations (MICs) of 10 antimicrobial agents against a total of 33 isolates of Clostridium perfringens, Clostridium septicum and Clostridium sordellii from cattle affected with malignant edema in Japan was determined. The low MIC activities of benzylpenicillin confirm the place of benzylpenicillin as the antibiotics of choice for treatment of malignant edema. Five (22%) of 23 C. septicum strains, five (71%) of seven C. perfringens strains and all strains of C. sordellii showed resistance to oxytetracycline. These oxytetracycline-resistant strains carried tetracycline-resistance genes [tetA(P), tetA408(P), tetB(P) and tetM]. The sequences of the tetracycline-resistance genes of some C. septicum strains were completely or nearly completely identical to those of strains belonging to other clostridiual species. This is the first report of resistance of C. septicum to tetracycline.

A taxonomic study on erysipelothrix by DNA-DNA hybridization experiments with numerous strains isolated from extensive origins.

The purpose of this study was to clarify the taxonomic relationship between all the serovars and species of the genus Erysipelothrix by performing DNA‐DNA hybridization experiments, the customary criterion for separation of bacterial genospecies. A total of 93 strains were isolated from a wide variety of sources, including pigs affected with acute or chronic erysipelas, other diseased animals, healthy animals, fish, retail meats, and environmental materials from throughout the world during the period 1958 to 1996. The present data on phenotypic characterization and DNA relatedness values demonstrate that 24 strains (96%) of E. tonsillarum are avirulent for swine, whereas 39 strains (66%) of genomic E. rhusiopathiae induced generalized or local urticarial lesion in swine after intradermal inoculation. This observation suggests that genomic E. tonsillarum has little etiological significance. Three minor groups contained several strains which exhibited minimal association with each type strain of E. rhusiopathiae and E. tonsillarum. In conclusion, it was confirmed that members of the E. rhusiopathiae and E. tonsillarum groups resemble each other in regard to many phenotypic characteristics, but differ in their ability to produce acid from saccharose and in their pathogenicity for swine. The genus Erysipelothrix certainly contains two main species: E. rhusiopathiae and E. tonsillarum.

Antimicrobial Susceptibilities of Erysipelothrix rhusiopathiae Isolated from Pigs with Swine Erysipelas in Japan, 1988–1998

The susceptibility to 21 antimicrobial agents of 214 strains of Erysipelothrix rhusiopathiae isolated from pigs affected with swine erysipelas in Japan between 1988 and 1998 was determined. Ampicillin, cloxacillin, benzylpenicillin, ceftiofur, tylosin, enrofloxacin and danofloxacin were the most active agents [minimum inhibitory concentrations (MICs); < or = 0.025-0.78 microgram/ml], followed by cefazolin, virginiamycin, tiamulin, chloramphenicol, florphenicol and oxolinic acid (MICs; 0.1-25 micrograms/ml). Activity was poor or absent with kanamycin and sulfadimethoxine. Strains resistant to dihydrostreptomycin, erythromycin, clindamycin, lincomycin, oxytetracycline and doxycycline were detected. The susceptibilities to dihydrostreptomycin and oxytetracycline tended to decrease. Investigation of the differences in antimicrobial susceptibility of the 214 strains according to their serotypes, sources, isolation years and regions, showed that the strains resistant to dihydrostreptomycin were most frequently found in the strains of serotype 1a and in strains from septicaemic cases. Strains resistant to oxytetracycline were detected in all serotypes and all sources, and most of the strains resistant to erythromycin were detected in the strains of serotype 2. The frequency of strains resistant to dihydrostreptomycin gradually increased from 1988 to 1996, but then decreased between 1997 and 1998. The frequency of strains resistant to oxytetracycline was remained more than 38% from 1988 to 1998. It was suggested that the strains resistant to dihydrostreptomycin and oxytetracycline were distributed over almost all districts of Japan.

Development of a serology-based assay for efficacy evaluation of a lactococcicosis vaccine in Seriola fish

Lactococcicosis is an infection caused by the bacterium Lactococcus garvieae and creates serious economic damage to cultured marine and fresh water fish industries. The use of the assay currently applied to evaluate the potency of the lactococcicosis vaccine is contingent upon meeting specific parameters after statistical analysis of the percent survival of the vaccinated yellowtail or greater amberjack fish after challenge with a virulent strain of L. garvieae. We found that measuring the serological response with a quantitative agglutinating antibody against the L. garvieae antigen (phenotype KG+) was an effective method of monitoring the potency of lactococcicosis vaccines. Vaccinated fish had significantly higher antibody titers than control fish when the L. garvieae Lg2-S strain was used as an antigen. Furthermore, the titer of the KG + agglutinating antibody was correlated with vaccine potency, and the cut-off titer was determined by comparing the data with those from the challenge test. An advantage of the proposed serology-based potency assay is that it will contribute to reduced numbers of animal deaths during vaccine potency evaluations.

Pathogenicity for mice and swine of Erysipelothrix isolates from the tonsils of healthy cattle.

The pathogenicity of 79 Erysipelothrix isolates from bovine tonsils for mice and swine was determined. Five (6.3%) isolates were lethal for mice. These isolates belonged to serovars 1b (one isolate), 2 (2), 19 (1) and 21 (1). The 50% lethal dose values of the isolates ranged from 0.33 to 5x10(2) CFUs in mice. Twenty Erysipelothrix isolates (25.3%) were weakly virulent inducing only emaciation while 12 (15.2%) inducing emaciation and ruffled hair. In swine, clinical signs of varying severity were observed. Four isolates were virulent, capable of inducing localized or generalized urticarial lesions accompanied with a rise in body temperature after intradermal inoculation. One isolate each of serovars 1b, 2 and 19 was highly virulent, capable of inducing generalized urticarial lesions while another Erysipelothrix isolate of serovar 2 induced only a localized urticarial lesion at the site of inoculation. Another isolate of serovar 1b induced itching and irritation without obvious urticarial lesion at the site of inoculation. On the other hand, one isolate of serovar 21 and two other isolates of serovar 2 could not induce experimentally any clinical sign of erysipelas other than rise in body temperature. There was a rise in growth agglutination (GA) titer of serum in all the inoculated swine. These observations suggest that Erysipelothrix isolates from cattle are pathogenic for mouse and swine, and may also be pathogenic for other animals and humans.

Bovine whole-blood culture as a tool for the measurement of endotoxin activities in Gram-negative bacterial vaccines.

In order to analyze bovine immune reactions against the Gram-negative bacterial vaccine, bovine whole-blood culture was used to investigate the pro-inflammatory cytokine responses stimulated with lipopolysaccharides (LPS) extracted from Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Klebsiella pneumoniae. We also examined the interaction between LPS and aluminum hydroxide gel for endotoxin activity and pro-inflammatory cytokine responses of whole bovine blood. Alteration in the mRNA concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-10 in whole-blood culture at 4h after stimulation with different doses of LPS was observed and determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The mRNA concentrations of TNF-α and IL-1β changed in a dose-dependent manner and differed depending on the type of LPS. Limulus test revealed that endotoxin activity was remarkably reduced when aluminum hydroxide gel was added to LPS. In contrast, the mRNA concentration of TNF-α in whole bovine blood was enhanced by LPS mixed with aluminum hydroxide gel. These results suggest that bovine whole-blood culture can be utilized to detect endotoxin activity of Gram-negative bacterial vaccines. In addition, whole-blood culture offers several advantages, such as ease of performance, few preparation artifacts, and a physiological cell environment, for investigating bovine immune response compared with the Limulus test.

Pathogenic characterization of Erysipelothrix rhusiopathiae Met-203 type SpaA strains from chronic and subacute swine erysipelas in Japan

To characterize the Erysipelothrix rhusiopathiae Met-203 type surface protective antigen (Spa) A strains causing swine erysipelas in Japan, the nucleotide sequence of the hypervariable region of the spaA gene was determined in 80 E. rhusiopathiae (serotype 1a) isolates collected from pigs with chronic and subacute swine erysipelas in 14 prefectures in 2008–2014. In this study, 14 (17.5%) isolates were Met-203 type SpaA strains. We confirmed the pathogenicity of a Met-203 type SpaA strain in specific-pathogen-free pigs. In this experiment, the two challenged pigs displayed arthritis, urticaria and other clinical signs, but recovered within 10 days. Our results reveal the existence of the E. rhusiopathiae Met-203 type strains that have been causing chronic erysipelas in Japan.