Akemi Kojima

Antimicrobial Susceptibilities, SErogroups, and Molecular Characterization of Avian Pathogenic Escherichia coli Isolates in Japan

Abstract In total, 83 avian pathogenic Escherichia coli (APEC) isolates from avian colibacillosis during a period from 2001 to 2006 in Japan were investigated for serogroups, typical virulence factors, antimicrobial susceptibility, and genetic relatedness. The most common serogroup was O78 (30.1%); 80.7% of isolates harbored the iss gene and 55.4% of isolates harbored the tsh gene. Antimicrobial resistance of the isolates was found for ampicillin (77.1%), oxytetracycline (75.9%), kanamycin (36.1%), fradiomycin (33.7%), trimethoprim (25.3%), enrofloxacin (21.7%), and florfenicol (6.0%). Although multiple antimicrobial-resistant phenotypes (three or more antimicrobials) accounted for 54.2% of isolates, no isolate exhibited resistance to all agents tested. The fluoroquinolone-resistant isolates had point mutations in GyrA (Ser83→Leu, Asp87→Asn) and ParC (Ser80→Ile, Glu84→Gly). Of 18 enrofloxacin-resistant E. coli isolates, nine isolates belonged to serotype O78. In PFGE analysis, eight of the nine enrofloxacin-resistant O78 isolates were classified into an identical cluster. This suggests that a specific genotype of fluoroquinolone-resistant O78 APEC may be widely distributed in Japan. Susceptibilidad antimicrobiana, serogrupos y caracterización molecular de aislamientos de Escherichia coli patógena en Japón. Un total de 83 aislamientos de Escherichia coli patógena provenientes de casos de colibacilosis aviar en Japón durante los años 2001 al 2006, fueron investigados para determinar su serogrupo, la presencia de factores típicos de virulencia, su susceptibilidad antimicrobiana y su relación genética. El serogrupo mas común fue el O78 (30.1%), el 80.7% de los aislamientos contenían el gen iss y el 54.4% de los aislamientos contenían el gen tsh. Entre los aislamientos se observó resistencia antibacteriana contra la ampicilina (77.1%), oxitetraciclina (75.9%), kanamicina (36.1%), fradiomicina (33.7%), trimetropina (25.3%), enrofloxacina (21.7%) y florfenicol (6%). Aun cuando los fenotipos de resistencia múltiple (tres o mas antibióticos) representaban el 54.2% de los aislamientos, ningún aislamiento mostró resistencia a todos los agentes antimicrobianos evaluados. Los aislamientos resistentes a la fluoroquinolona presentaron mutaciones puntuales en los genes GyrA (Ser83- Leu y Asp87- Asn), así como en el gen ParC (Ser80-Ile, y Glu84-Gly). De 18 aislamientos de E. coli resistentes a la enrofloxacina, nueve aislamientos pertenecían al serotipo O78. En el análisis mediante electroforesis de campo pulsado, ocho de los nueve aislamientos O78 resistentes a enrofloxacina se clasificaron en el mismo grupo. Esto sugiere que un genotipo específico de E. coli patógena del serogrupo O78 resistente a las fluoroquinolonas puede estar ampliamente distribuido en Japón. Abbreviations: ABPC = ampicillin; APEC = avian pathogenic Escherichia coli; ERFX = enrofloxacin; FFC = florfenicol; FM = fradiomycin; KM = kanamycin; MIC = minimum inhibitory concentration; NCCLS = National Committee for Clinical Laboratory Standards; OTC = oxytetracycline; PCR = polymerase chain reaction; PFGE = pulsed-field gel electrophoresis; QRDR = quinolone-resistant determining region; TMP = trimethoprim; UT = untypeable

Phylogenetic positions of Clostridium novyi and Clostridium haemolyticum based on 16S rDNA sequences.

The partial sequences (1465 bp) of the 16S rDNA of Clostridium novyi types A, B and C and Clostridium haemolyticum were determined. C. novyi types A, B and C and C. haemolyticum clustered with Clostridium botulinum types C and D. Moreover, the 16S rDNA sequences of C. novyi type B strains and C. haemolyticum strains were completely identical; they differed by 1 bp (level of similarity > 99.9%) from that of C. novyi type C, they were 98.7% homologous to that of C. novyi type A (relative positions 28-1520 of the Escherichia coli 16S rDNA sequence) and they exhibited a higher similarity to the 16S rDNA sequence of C. botulinum types D and C than to that of C. novyi type A. These results suggest that C. novyi types B and C and C. haemolyticum may be one independent species generated from the same phylogenetic origin.

Regulation of Follicle-Stimulating Hormone Secretion by Estradiol and Dimeric Inhibins in the Infantile Female Rat

Abstract Plasma and ovarian levels of the dimeric forms of inhibin and plasma estradiol-17β were investigated and compared with changes in plasma gonadotropins from Postnatal Day (PND) 5 to PND 30 in the female rat. The inhibin subunit proteins were localized in follicular granulosa cells of the ovary. Plasma immunoreactive inhibin levels were low until PND 15 and increased thereafter. Plasma levels of inhibin B (α and βB subunits) remained very low until PND 15 and then increased by approximately 24-fold. In contrast, plasma levels of inhibin A (α and βA subunits) were relatively low and steady until PND 20, then increased by approximately 3-fold at PND 25. Changes in ovarian inhibin A and B levels closely resembled those in plasma levels. Plasma FSH levels were low at PND 10 but started to peak from PND 15 and remained high until PND 20, followed by a remarkable reduction at PNDs 25 and 30. This dramatic fall in FSH coincided with the rise of inhibin A. A significant inverse correlation was observed between plasma FSH and plasma inhibin A (r = −0.67, P < 0.0002), ovarian inhibin A (r = −0.48, P < 0.01), plasma inhibin B (r = −0.48, P < 0.05), and ovarian inhibin B (r = −0.54, P < 0.01). Plasma estradiol-17β levels were elevated from PND 5 through PND 15 , then fell sharply through PND 30. Plasma estradiol-17β was significantly and positively (r = 0.75, P < 0.0002) correlated with plasma FSH. Plasma LH rose to higher levels at PND 15 and tended to be lower thereafter. The inhibin α, βA, and βB subunits were localized to primary, secondary, and antral and large antral follicles, but the types of these immunopositive follicles varied with age. It appeared that, at PND 25 and afterward, all three subunits were mainly confined to large antral follicles in the ovary. We conclude that estradiol-17β likely is the major candidate in stimulation of FSH secretion in the infantile female rat. We also conclude that inhibin regulation of pituitary FSH secretion through its negative feedback in the infantile female rat begins to operate after PND 20. We suggest that this negative feedback is achieved by increases in plasma levels of the two dimeric forms, and that inhibin A appears to be the major physiological regulator of FSH secretion at the initiation of this mechanism. We also conclude that large antral follicles in the ovary are the primary source of these bioactive inhibins that are secreted in large amounts into the circulation after PND 20.

Diversity of Plasmid Replicons Encoding the blaCMY-2 Gene in Broad-Spectrum Cephalosporin-Resistant Escherichia coli from Livestock Animals in Japan

Broad-spectrum cephalosporin (BSC) resistance has increased in Escherichia coli isolates from broiler chickens in Japan since 2004. The purpose of this study was to understand the epidemiology of BSC-resistant E. coli in livestock animals. Among 3274 E. coli isolates from 1767 feces of apparently healthy animals on 1767 farms between 2004 and 2009, 118 ceftiofur (CTF)-resistant isolates (CTF MIC ≥4 μg/mL) were identified on 74 farms. After elimination of apparently clonal isolates from a single animal, 75 selected CTF-resistant isolates (62 isolates from 61 broiler chickens, 10 isolates from 10 layer chickens, two isolates from two cows, and one isolate from a pig) were characterized. The bla(CMY-2) gene was most frequently detected in 50 isolates, followed by bla(CTX-M) (CTX-M-2: six isolates; CTX-M-14: four isolates; CTX-M-25: two isolates; CTX-M-1: one isolate) and bla(SHV) (SHV-12: seven isolates; SHV-2, SHV-2a, SHV-5: one isolate each). In particular, 42 of 62 broiler chicken isolates harbored bla(CMY-2). Pulsed-field gel electrophoresis analyses using XbaI revealed divergent profiles among the BSC-resistant isolates. The incompatibility groups of bla(CMY-2) plasmids from 34 of the 42 broiler chicken isolates belonged to IncIγ (10 isolates), IncA/C (nine isolates), IncB/O (seven isolates) and IncI1 (six isolates), or were nontypeable (two isolates). Co-transmission of resistance to non-β-lactam antibiotics was observed in transconjugants with IncA/C plasmids, but not with IncI1, IncIγ, and IncB/O plasmids except for one isolate with IncB/O. Our findings suggest that the bla(CMY-2) gene is a key player in BSC-resistant E. coli isolates and that coselection is unlikely to be associated with the abundance of bla(CMY-2) plasmids, except for IncA/C plasmids.

Farm-Level Impact of Therapeutic Antimicrobial Use on Antimicrobial-Resistant Populations of Escherichia coli Isolates from Pigs

We investigated the farm-level impact of the use of several different antimicrobial agents on the population of antimicrobial-resistant commensal bacteria of animal origin to appropriately assess the release risk of resistance. This study was carried out based on the results of a survey on the history of antimicrobial drug use in 297 pig farms and antimicrobial susceptibility testing for the 545 Escherichia coli isolates (one or two isolates/pig/farm). A comparative analysis with the nonexposed herd revealed that ampicillin (ABPC) resistance in E. coli increased in the herds that were exposed to penicillin (relative risk [RR], 1.75) and penicillin-streptomycin (RR, 2.28); dihydrostreptomycin (DSM) resistance, in the penicillin-streptomycin-exposed herd (RR, 1.75); and trimethoprim (TMP) resistance in the methoprim-sulfonamide-exposed herd (RR, 2.10). On the other hand, ABPC and DSM resistances increased in the tetracycline-exposed herd (RR, 1.66 and 1.58, respectively); TMP resistance, in the penicillin-exposed herd (RR, 1.77); and oxytetracycline and kanamycin resistances, in the penicillin-streptomycin-exposed herd (RR, 1.28 and 2.22, respectively). These results demonstrated that the development of cross-resistance and coresistance, imposed by the therapeutic use of the antimicrobials studied, contributed the farm-level prevalence of antimicrobial-resistant E. coli and that the influence of coselection was characteristic to individual antimicrobial agents used.

Characterization of flagellin from Clostridium chauvoei

Differential centrifugation and cesium chloride-equilibrium centrifugation were used to purify the flagella from the strain Okinawa of the formalin-fixed Clostridium chauvoei. SDS-PAGE profile of purified flagella showed that a major protein band with a molecular mass of 46 kDa, corresponding to the flagellin monomer, and at least two minor protein bands with molecular masses of approximately 73 and 100 kDa were found. The amino acid composition of C. chauvoei flagellin was similar to the flagellin of Salmonella typhimurium and Bacillus subtilis. In addition, C. chauvoei flagellin monomer shared limited sequence homology with the N-terminal amino acid sequence reported for other bacterial flagellins. N-terminal sequences of two minor bands corresponded to the flagellin monomer, indicating that higher molecular mass bands were polymeric forms of the flagellin monomer.

Flagella based enzyme-linked immunosorbent assay for evaluation the immunity in mice vaccinated with blackleg vaccines

A quantitative enzyme-linked immunosorbent assay (ELISA) using a purified flagella of Clostridium chauvoei as the antigen was developed to measure the anti-flagellar titre in mice, and the relationship between the anti-flagellar titre and the immunogenisity of blackleg vaccine was investigated. When mice were immunized according to the present potency assay with the vaccine or vaccine derivatives, a good correlation was obtained between anti-flagellar titres and survival rates of mice. Anti-flagellar titre of the order 48 might be enough to protect against the challenge exposure of the C. chauvoei, and when this level was adopted as an alternative criterion to predict adequate potency, 97% of vaccine derivatives that induce the anti-flagellar titre of more than 48 passed the present potency test. These results suggest that the flagella based ELISA may be useful as an aid for evaluation of immunogenisity of blackleg vaccines.

Development of a two-site enzyme-linked immunosorbent assay for quantification of the flagellar antigen in blackleg vaccines

Abstract A quantitative two-site enzyme-linked immunosorbent assay (two-site ELISA) procedure using a protective monoclonal antibody against the flagella of Clostridium chauvoei has been developed to measure a flagellar concentration in blackleg vaccines. The two-site ELISA was highly specific for flagella of C. chauvoei , and had a detection limit of approximately 0.125 μg ml −1 of flagella. With this assay, the flagella level in blackleg vaccines could be measured and will be available for quality controls of vaccines.

PRODUCTION OF RECOMBINANT PORCINE TUMOR NECROSIS FACTOR ALPHA IN A BACULOVIRUS EXPRESSION SYSTEM

Porcine tumor necrosis factor alpha (TNF-alpha) was produced using a baculovirus system in Spodoptera frugiperda (SF21AE) cells. Cytotoxic activity was detected in supernatant of sonicated SF21AE cells infected with recombinant viruses. The recombinant protein was also demonstrated to be functionally active by its ability to cause apoptosis in Wehi 164 cells. Three distinct bands of 26, 17 and 14 kDa were revealed by Western blot analysis using anti-human TNF-alpha antibody. Moreover, the anti-human TNF-alpha antiserum significantly neutralized the cytotoxic activity of the supernatant of sonicated SF21AE cells infected with recombinant viruses.