10-Hydroxy-2-decenoic acid, a natural product, improves hyperglycemia and insulin resistance in obese/diabetic KK-Ay mice, but does not prevent obesity.

10-Hydroxy-2-decenoic acid (10H2DA) is a fatty acid found in royal jelly (RJ). In healthy mice, it activates 5'-AMP-activated protein kinase (AMPK) and increases glucose transporter 4 (GLUT4) translocation. Therefore, we examined whether 10H2DA has a potential therapeutic effect against type 2 diabetes in obese/diabetic KK-Ay mice. 10H2DA (3 mg/kg body weight) was administered to female KK-Ay mice for 4 weeks by oral gavage. Phenotypes for body weight, plasma glucose by oral glucose tolerance test and insulin levels were measured. mRNA and protein levels were determined using qRT-PCR and Western blot analyses, respectively. Long-term administration of 10H2DA significantly improved hyperglycemia and insulin resistance in KK-Ay mice, but did not prevent obesity. 10H2DA increased the expression of phosphorylated AMPK (pAMPK) protein in skeletal muscles; however, this expression did not correlate with increased GLUT4 translocation. Furthermore, 10H2DA neither enhanced the expression of adiponectin receptor mRNA nor activated the insulin signaling cascade, such as GSK-3ß phosphorylation, in the liver. We found that 10H2DA-treated mice had a significant increase in the expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Pgc-1α) mRNA in skeletal muscles compared with non-treated group (P=0.0024). These findings suggest that 10H2DA is involved in the improvement of type 2 diabetes, at least in part via activation of Pgc-1α expression, but does not prevent obesity.

Isolation of the Thogoto virus from a Haemaphysalis longicornis in Kyoto City, Japan.

Ticks transmit viruses responsible for severe emerging and re-emerging infectious diseases, some of which have a significant impact on public health. In Japan, little is known about the distribution of tick-borne viruses. In this study, we collected and tested ticks to investigate the distribution of tick-borne arboviruses in Kyoto, Japan, and isolated the first Thogoto virus (THOV) to our knowledge from Haemaphysalis longicornis in far-eastern Asia. The Japanese isolate was genetically distinct from a cluster of other isolates from Africa, Europe and the Middle East. Various cell lines derived from mammals and ticks were susceptible to the isolate, but it was not pathogenic in mice. These results advance understanding of the distribution and ecology of THOV.

Detection of rickettsial DNA in ticks and wild boars in Kyoto City, Japan.

The tick is a well-known vector for arthropod-borne pathogens, such as tick-borne encephalitis, Lyme disease, Japanese spotted fever and severe fever with thrombocytopenia syndrome. It is therefore important to know the tick population and distribution in our environment and wild animals in order to prevent tick-borne diseases. Here, we report the results of tick surveillance from May to September 2011 at 14 geographical points and in 5 wild boars in Kyoto City, Kyoto prefecture, Japan. We collected 3,198 ticks comprising 5 tick species, Haemaphysalis (H.) longicornis, H. flava, H. kitaokai, Amblyomma testudinarium and Dermacentor taiwanensis. Interestingly, the proportion of tick species varied according to geographical region within the city. The ticks collected in the city were reported as potential vectors of pathogens, such as rickettsiosis. We detected rickettsial DNA by PCR in 71.1% of 201 ticks investigated. The ticks that carried rickettsiae were distributed across the whole the city. The sequences of PCR-amplified DNA fragments were determined and showed similarities to spotted fever group rickettsiae. Although their pathogenicity for animals including humans is still unclear, it is important to stay alert and pay attention to tick-borne diseases in order to ensure the safety of the citizens of the city as well as that of visitors.

Development of a novel protocol for generating flavivirus reporter particles.

Infection with West Nile virus (WNV), a mosquito-borne flavivirus, is a growing public and animal health concern worldwide. Prevention, diagnosis and treatment strategies for the infection are urgently required. Recently, viral reverse genetic systems have been developed and applied to clinical WNV virology. We developed a protocol for generating reporter virus particles (RVPs) of WNV with the aim of overcoming two major problems associated with conventional protocols, the difficulty in generating RVPs due to the specific skills required for handling RNAs, and the potential for environmental contamination by antibiotic-resistant genes encoded within the genome RNA of the RVPs. By using the proposed protocol, cells were established in which the RVP genome RNA is replicated constitutively and does not encode any antibiotic-resistant genes, and used as the cell supply for RVP genome RNA. Generation of the WNV RVPs requires only the simple transfection of the expression vectors for the viral structural proteins into the cells. Therefore, no RNA handling is required in this protocol. The WNV RVP yield obtained using this protocol was similar that obtained using the conventional protocol. According to these results, the newly developed protocol appears to be a good alternative for the generation of WNV RVPs, particularly for clinical applications.

A study on Borna disease virus infection in domestic cats in Japan.

Borna disease virus (BDV) infection causes neurological disease in cats. Here, we report BDV infection in 199 hospitalized domestic cats in the Tokyo area. BDV infection was evaluated by detection of plasma antibodies against BDV-p24 or -p40. BDV-specific antibodies were detected in 54 cats (27.1%). Interestingly, the percentage of seropositive cats was not significantly different among the three clinical groups, i.e., healthy (29.8%), neurologically asymptomatic disease (22.2%) and neurological disease (33.3%). The specific antibodies were present even in cats aged below one year. The seropositive ratio was constant, irrespective of age and sampling season. The present study suggests that additional factors are required for onset of Borna disease in naturally infected cats and that BDV is transmitted through vertical routes in cats.

Antimicrobial resistance in fecal Escherichia coli isolated from growing chickens on commercial broiler farms.

To investigate the effects of rearing practices of commercial broiler chickens on the incidence of antimicrobial resistance in commensal Escherichia coli isolates, fecal E. coli isolates obtained in 4 farms were screened for anitimicrobial resistance. Ten E. coli isolates were recovered from each of the fecal samples collected from 10 birds in the farms at the ages of 2 days, 14-17 days, and 47-50 days. In 2 out of the 4 farms, no antimicrobials were used during the rearing period. In the other two farms, following collection of the fecal samples at 14 and 15 days of age, oxytetracycline (OTC), sulfadimethoxine (SDMX), and tylosin were given to birds on one farm and SDMX was used in the other. Isolates resistant to ampicillin and OTC that were obtained from an untreated flock at different sampling times were closely related to each other by pulsed-field gel electrophoresis patterns (PFGE) of XbaI-digested chromosomal DNA. PFGE analysis together with in vitro conjugation experiments suggested that diversity of resistance phenotypes within a clone may be resulted from the acquisition and loss of R-plasmids in an untreated and a treated flock. The numbers of resistance phenotypes observed among fecal isolates increased during the growth of the chickens in all the farms. The results in the present study suggest that persistence of commensal E. coli strains resistant to antimicrobials even in the absence of antimicrobial administration. It is also hypothesized that horizontal transmission of resistance determinants resulted in the emergence of different resistance phenotypes in those farms.

Characterization of Escherichia coli strains obtained from layer chickens affected with colibacillosis in a commercial egg-producing farm.

The present study reports colibacillosis of layer chickens in a commercial egg-producing farm in western Japan. Three flocks of chicken at 18-21 weeks of age were affected during the initiation of egg lay. Postmortem examination revealed pericarditis, perihepatitis, airsacculitis, subcutaneous inguinal lesion, and injured cloaca. Escherichia coli was isolated from the lesions of the affected birds. Twenty-two of 26 E. coli isolates (84.6%) obtained from 18 birds in the 3 flocks showed pulsed-field gel electrophoresis (PFGE) patterns that were considered to be closely associated to each other and arbitrarily designated as pattern A. All the 22 isolates with the PFGE pattern A harbored the putative virulence genes, astA, iss, iucD, tsh, and cva/cvi. Additional 2 PFGE patterns (B and C) were also found in E. coli isolates obtained from the affected flocks and had the putative virulence genes in combinations different from those in the pattern A strains. The results suggested that certain E. coli virulence genes and host factors, such as initiation of egg lay may be associated with occurrence of colibacillosis.

Antimicrobial susceptibilities of Salmonella isolates obtained from layer chicken houses on a commercial egg-producing farm in Japan, 1997 to 2002.

Susceptibility to antimicrobial agents was examined for 325 isolates of Salmonella enterica serotypes Cerro, Infantis, Livingstone, and Montevideo isolated from layer houses on a commercial egg-production farm in the western region of Japan between 1997 and 2002. No antimicrobials were used for therapeutic purposes on the farm during this period. From 1.8 to 3.1% of the isolates were resistant to ampicillin, chloramphenicol, and tetracycline. Resistance to streptomycin and sulfisoxazole was found in 52.9 and 65.5%, respectively, of Salmonella Montevideo isolates and in 0 to 13.2% of the isolates of the other serotypes. All the streptomycin-resistant isolates of Salmonella Montevideo also exhibited resistance to sulfisoxazole. Salmonella Montevideo isolates were first isolated in 1998, and 80.0% of the isolates obtained in this year were resistant to streptomycin and sulfisoxazole. The results suggest that Salmonella Montevideo isolates that had already acquired resistance may have been introduced into the layer houses, although the route and vehicle of transmission were uncertain. The proportion of Salmonella Montevideo isolates resistant to streptomycin and sulfisoxazole significantly decreased (P < 0.01) from 79.5% for 1998 through 1999 to 37.3% for 2000 through 2002. This decrease probably was due to the fact that no antimicrobials were used on the farm. Among 10 isolates from different serotypes obtained from 1997 to 2002 that were resistant to ampicillin, two and five isolates harbored 42- and 63-kb R plasmids and identical DraI restriction enzyme digest patterns, respectively, and carried the blaTEM gene. The results suggest that the ampicillin resistance determinants were transferred among different serotypes of Salmonella in the layer houses.