Mayumi Kijima

Horizontal Transmission of Salmonella enteritidis and Effect of Stress on Shedding in Laying Hens

Horizontal transmission of Salmonella enteritidis in laying hens and the short-term effect of stress on shedding were examined in 32 seven-month-old laying hens. Half were inoculated with 10(5) colony-forming units of S. enteritidis phage type 4, and the remaining half were left uninoculated to study horizontal transmission. Isolation of S. enteritidis from cecal droppings of all hens was attempted every morning. Uninoculated hens rapidly became infected through contaminated drinking water. Introduction of young chickens to the same rearing room and withdrawal of water and feed for 2 days coincided with a rapid increase in the shedding rate of S. enteritidis for a short period of time. The results showed that a short-term increase in the shedding rate of S. enteritidis is associated with short-term exposure to environmental stress.

Drug resistance and pulsed-field gel electrophoresis patterns of Lactococcus garvieae isolates from cultured Seriola (yellowtail, amberjack and kingfish) in Japan

Aims:  To investigate the existing antimicrobial susceptibility and genetic characteristics of Lactococcus garvieae isolates from cultured Seriola in Japan.

Characterization of Lactococcus garvieae isolated from radish and broccoli sprouts that exhibited a KG(+) phenotype, lack of virulence and absence of a capsule

Aims:  To identify Lactococcus garvieae isolates from radish and broccoli sprouts and compare them with virulent and less virulent mutant strains obtained from yellowtails with regard to KG phenotype, presence of a capsule and virulence towards yellowtails and mice.

National surveillance of Salmonella enterica in food-producing animals in Japan.

A total of 518 fecal samples collected from 183 apparently healthy cattle, 180 pigs and 155 broilers throughout Japan in 1999 were examined to determine the prevalence and antimicrobial susceptibility of Salmonella. The isolation rates were 36.1% in broilers, 2.8% in pigs and 0.5% in cattle. S. enterica Infantis was the most frequent isolate, found in 22.6% of broiler fecal samples. Higher resistance rates were observed against oxytetracycline (82.0%), dihydrostreptomycin (77.9%), kanamycin (41.0%) and trimethoprim (35.2%). Resistance rates to ampicillin, ceftiofur, bicozamycin, chloramphenicol and nalidixic acid were <10%. CTX-M-2 β-lactamase producing S. enterica Senftenberg was found in the isolates obtained from one broiler fecal sample. This is the first report of cephalosporin-resistant Salmonella directly isolated from food animal in Japan.

Molecular mechanism of the regulation of expression of plasmid-encoded mouse bacteremia (mba) genes in Salmonella serovar Choleraesuis

SummaryThe regulation of mouse bacteremia genes (mba genes) encoded by a 6.4 kb region on the 50 kb virulence plasmid (pKDSC50) of Salmonella serovar Choleraesuis was analyzed. The genes mba1, mba2, mba3, and mba4, are arranged in this order, and form a cluster located in the 6.4 kb mba region. We prepared four antibodies, each specific for an individual Mba protein, using synthetic peptides as antigens. Their amino acid sequences were deduced from the DNA sequence of the corresponding mba genes. Each Mba peptide antiserum was able to recognize the corresponding Mba protein produced by Escherichia coli carrying a recombinant plasmid containing individual mba genes. When the recombinant plasmid contained all four mba genes (pMKD601), three Mba proteins (Mba2, Mba3, and Mba4) were identified by Western blotting analysis using Mba antisera. These proteins could not be detected when the recombinant plasmid lacked mba1 (pMKD201). Three species of mRNA for mba2, mba3, and mba4 with different chain length were detected from pMKD601 by Northern blot hybridization, and two start sites were identified by primer extension assay. Gel mobility shift assays demonstrated that Mbal specifically bound to a fragment containing the start sites of mRNAs. The amino acid sequence of Mbal had significant homology to the LysR family of DNA binding proteins, possessing a characteristic helix-turn-helix DNA binding motif. The present study provides clear evidence to show that the Mba1 protein binds to the promoter region of mba2, and positively regulates the expression of mba2, mba3, and mba4 genes.

Classification and antimicrobial susceptibilities of enterococcus species isolated from apparently healthy food-producing animals in Japan.

Summary A total of 1024 enterococci were recovered from faeces of healthy animals from 178 cattle, 178 pig and 156 broiler farms. Enterococcus faecium was the predominant species recovered (35.8%), followed by E. faecalis (31.3%) and E. hirae (25.6%). Oxytetracycline resistance was most frequently found among E. faecalis (85.9%), E. faecium (58.8%) and E. hirae (48.1%). Resistance rates to almost all antimicrobials were higher in E. faecalis than E. faecium and E. hirae. Isolates from cattle were more susceptible to the antimicrobials studied than those from pigs and broilers. VanA‐ or VanB‐type vancomycin‐resistant enterococci have not been found since the ban of avoparcin use 5 years ago.

Drug resistance and random amplified polymorphic DNA analysis of Photobacterium damselae ssp. piscicida isolates from cultured Seriola (yellowtail, amberjack and kingfish) in Japan

Aims:  To investigate the antimicrobial susceptibility and genetic characteristics of Photobacterium damselae ssp. piscicida isolates obtained from cultured Seriola in Japan.

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.

Antigenic analysis of Clostridium chauvoei flagella with protective and non-protective monoclonal antibodies.

Five monoclonal antibodies (mAbs) directed against the flagellin of Clostridium chauvoei were used to analyse the structural and antigenic characteristics on the bacterial flagellar surface. Immune electron microscopy showed that three protective mAbs recognized the surfaced-exposed epitopes on the flagellar filament of this bacteria. In contrast, two non-protective mAbs recognized internal epitopes of the flagellar filament. These findings have been confirmed by ELISA using mAbs absorbed with whole cells of C. chauvoei possessing flagella. Competitive binding assays showed that protective mAbs indicated reciprocal competition, while each of the non-protective mAbs had topographically distinct epitopes. Moreover, immunoblotting analysis with cyanogen-bromide-cleaved flagellin showed that protective mAbs may preferentially recognize conformational epitopes, whilst one of the non-protective mAbs may recognize a linear and conformation-independent epitope in the flagellin of C. chauvoei.

Cloning and expression of a gene encoding the flagellin of Clostridium chauvoei

Clostridium chauvoei is a causative agent of blackleg and the major protective antigen of the organism is the flagellar protein. Using an Escherichia coli expression library of the C. chauvoei Okinawa strain, we isolated the fliC gene encoding the flagellin protein. DNA sequence analysis revealed an open reading frame of 413 amino acid residues with a calculated molecular mass of 43819Da. Comparison of the sequence with those of flagellins from other bacteria showed considerable homology in the N-terminal and C-terminal domains. The glutathione-S-transferase (GST)-flagellin fusion protein and the purified FliC protein after removing the GST part with thrombin reacted with both polyclonal antisera and the non-protective monoclonal antibody (Mab), Mo-114. However, the protective Mab, Mo-41, which may recognize its conformational epitope, failed to react with both the GST-flagellin fusion protein and the purified FliC. Furthermore, the GST-flagellin fusion protein and the purified FliC induced very little protective immunity in mice. These results suggested that a conformation-dependent epitope play an important role in the development of immunity against blackleg.