Prevalence and characteristics of Listeria monocytogenes in feces of black beef cattle reared in three geographically distant areas in Japan.

This study was conducted to determine the prevalence and characteristics of Listeria monocytogenes in the feces of black beef cattle reared in geographically distant areas in Japan. We surveyed 130 farms in the following three areas: northern (Hokkaido prefecture), central (Gifu and Mie prefectures), and southern (Oita, Miyazaki, and Kagoshima prefectures) areas and collected 1738 fecal samples. Our data showed the following isolation rate for each area: northern, 11.4% of 651; central, 2.8% of 572; and southern, 2.9% of 515, indicating that the isolation rate in the northern area was significantly higher than that in the central or southern areas (p<0.01). Moreover, serotyping of 996 isolates identified 1/2b as the most prevalent serotype (40.5%), followed by 1/2a (36.9%), 4b (21.6%), and 4ab (1.0%). In the northern area, multiple serotypes were isolated from 60% of L. monocytogenes-positive farms. In addition, multiple serotypes were isolated from individual fecal samples from 18 cattle. Pulsed-field gel electrophoresis (PFGE) characterization of 239 isolates detected 48 different PFGE types. We found that isolates from northern farms were genetically diverse compared to those from central and southern farms. Five isolates from human clinical cases and three isolates from animal clinical cases were identical to isolates from black beef cattle. Furthermore, the isolates from northern and central farms were characterized to possess epidemic clone II or III markers. We next showed that the isolates were susceptible to penicillin, ampicillin, amoxicillin, gentamicin, kanamycin, streptomycin, erythromycin, vancomycin, tetracycline, chloramphenicol, ciprofloxacin, and trimethoprim/sulfamethoxazole. Taken together, our survey provides crucial data regarding the prevalence and characteristics of L. monocytogenes in black beef cattle farms throughout Japan.

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.

Comparison of commercial enzyme-linked immunosorbent assay kits with agar gel precipitation and hemagglutination-inhibition tests for detecting antibodies to avian influenza viruses.

We evaluated the utility of 5 commercial enzyme-linked immunosorbent assay (ELISA) kits for detecting antibodies to avian influenza viruses. The sensitivities and specificities of the ELISA kits were compared with those of the agar gel precipitation (AGP) and hemagglutination-inhibition (HI) tests. The results suggest that some ELISA kits might not be suitable for monitoring during the early stages of avian influenza virus infections. Therefore, ELISA kits should only be used in conjunction with a profound knowledge about monitoring of avian influenza.

Detection of highly pathogenic avian influenza virus infection in vaccinated chicken flocks by monitoring antibodies against non-structural protein 1 (NS1).

H5 and H7 highly pathogenic avian influenza virus (HPAIV) represent a major global concern in poultries and human health. Avian influenza (AI) vaccines are available but not preferred for field applications, primarily because vaccination interferes with sero-surveillances of AIV infection. To overcome the problem, ELISA systems using non-structural protein 1 (NS1) of AIV as antigens (NS1-ELISA) have been developed to measure anti-NS1 antibodies that are raised in AIV-infected but not in vaccinated chickens. However, some AI-vaccinated chickens having a weak anti-virus immune response may subsequently be infected with AIV and spread the virus. This raises a concern for the validity of NS1-ELISA to detect AIV infection in previously vaccinated chickens. In this study, we developed NS1-ELISA and assessed its feasibility to detect HPAIV infection in chickens previously immunized with H5 or H7 AI vaccines. The results indicated that the NS1-ELISA could identify HPAIV infection in both unvaccinated and vaccinated chickens at 1 week after infection in correlation with results from time-consuming virus isolation tests. Taken together, the NS1-ELISA system would be valuable tool to define HPAIV infection when AI vaccine program is in place.

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.

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.

Seroepidemiologic survey of Coxiella burnetii and attempt to detect Coxiella DNA in aged non-laying chickens in a prefecture of Japan where poultry farming prospers.

The indirect fluorescent antibody test (IFAT) revealed seropositivity to Coxiella burnetii in aged non-laying chickens in poultry farms in a prefecture in the central part of Japan. Seropositivity was 7%, and antibody titers ranged from 16 to 64. No DNA fragment specific for C. burnetii was detected in the chickens by nested-PCR. The prevalence of C. burnetii infection in a prefecture of Japan in which poultry farming prospers was 7%.

Establishment of a potency test by ELISA for a rabies vaccine for animal use in Japan.

The ELISA we developed was able to determine the antigen content and was suitable for a potency test, and we described a relative potency assay method which determines the potency of test vaccines by comparing the ELISA value of a test vaccine to that of a reference vaccine. In the present study, we standardized the reference vaccine used for determining the potencies of test vaccines, and established a potency test by ELISA. We evaluated the proposed reference vaccine by the neutralizing antibody responses in dogs after vaccination, by the challenge protection test in guinea pigs (GP potency test), which is the earlier official potency test used in Japan, and by the NIH potency test, which is widely used throughout the world. The results showed that a 4-fold dilution of the proposed reference vaccine induced sufficient immunity in dogs. A 3-fold dilution of the proposed reference vaccine passed the GP potency test. The international units (IU) calibrated by the NIH potency test were 3.7 IU/dose. From the results and the WHO recommendation that veterinary rabies vaccines should have a potency of at least 1.0 IU/dose, we determined to dilute the proposed reference vaccine by 3 fold and regarded it as the reference vaccine. Finally, we confirmed that there is a good agreement between the results of the potency test by ELISA and the results of the GP potency test. The establishment of the potency test by ELISA has made it possible to monitor the potency in the production process and has contributed to the stable production of the vaccine.

Occurrence of Erysipelothrix rhusiopathiae in Retail Raw Pork.

A total of 52 strains of Erysipelothrix rhusiopathiae was isolated from 38 (33.9%) of 112 retail raw pork samples which were purchased in the Tama area of Tokyo, Japan, February to November 1988. These isolates were classified into 14 different serovars: 6 (19.2%); 8 (17.3%); 11 (15.3%); 2 (7.7%); 5, 10, and 14 (each 5.8%); 1b, 4, 9, 12, and N (each 3,8%), 13 and 17 (each 1.9%). Of 38 samples, 12 samples contained two or three different serovars and the remaining 26 samples contained one serovar. Fourteen of 23 isolates examined were highly virulent for mice. Of 43 isolates tested, two and eight were resistant to chloramphenicol and oxytetracycline.