Resistance phenotypes and genotypes among multiple-antimicrobial-resistant Salmonella enterica subspecies enterica serovar Choleraesuis strains isolated between 2008 and 2012 from slaughter pigs in Okinawa Prefecture, Japan.

A total of 349 Salmonella enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) strains, which were isolated between 2008 and 2012 from 349 pigs at two slaughterhouses in Okinawa Prefecture, Japan, were investigated for antimicrobial susceptibility and the presence of antimicrobial resistance genes. All isolates were resistant to at least four antimicrobial agents. The antimicrobial agents for which isolates showed a high incidence of resistance were as follows: ampicillin (100%) and streptomycin (100%), followed by gentamicin (99.7%), oxytetracycline (99.7%), sulfamethoxazole/trimethoprim (99.4%), nalidixic acid (40.1%) and oxolinic acid (40.1%). All isolates were sensitive to cefuroxime, ceftiofur, colistin, fosfomycin, enrofloxacin, orbifloxacin and danofloxacin. The predominant resistance phenotypes and genotypes were: resistance to ampicillin, streptomycin, gentamicin, oxytetracycline and sulfamethoxazole/trimethoprim (58.5%, 204/349) and blaTEM-strA-strB-aadA1-aadA2-aacC2-tet (B)-sul1-sul2-dhfrXII-dhfrXIII (36.1%, 126/349). The quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE of the quinolone-resistant isolates (n=12) showed amino acid substitutions of Ser-83→Phe or Asp-87→Tyr in GyrA and Ser-107→Ala in ParC. To our knowledge, this is the first report on the molecular characterization of antimicrobial resistance among S. Choleraesuis strains in Japan.

Detection and phylogenetic analysis of hepatitis E viruses from mongooses in Okinawa, Japan.

Hepatitis E virus (HEV) infection has previously been reported in wild mongooses on Okinawa Island; to date however, only one HEV RNA sequence has been identified in a mongoose. Hence, this study was performed to detect HEV RNA in 209 wild mongooses on Okinawa Island. Six (2.9%) samples tested positive for HEV RNA. Phylogenetic analysis revealed that 6 HEV RNAs belonged to genotype 3 and were classified into groups A and B. In group B, mongoose-derived HEV sequences were very similar to mongoose HEV previously detected on Okinawa Island, as well as to those of a pig. This investigation emphasized the possibility that the mongoose is a reservoir animal for HEV on Okinawa Island.

Evaluation of the petrifilm aerobic count plate for enumeration of aerobic marine bacteria from seawater and Caulerpa lentillifera.

The enumeration and evaluation of the activity of marine bacteria are important in the food industry. However, detection of marine bacteria in seawater or seafood has not been easy. The Petrifilm aerobic count plate (ACP) is a ready-to-use alternative to the traditional enumeration media used for bacteria associated with food. The purpose of this study was to evaluate the usefulness of a simple detection and enumeration method utilizing the Petrifilm ACP for enumeration of aerobic marine bacteria from seawater and an edible seaweed, Caulerpa lentillifera. The efficiency of enumeration of total aerobic marine bacteria on Petrifilm ACP was compared with that using the spread plate method on marine agar with 80 seawater and 64 C. lentillifera samples. With sterile seawater as the diluent, a close correlation was observed between the method utilizing Petrifilm ACP and that utilizing the conventional marine agar (r=0.98 for seawater and 0.91 for C. lentillifera). The Petrifilm ACP method was simpler and less time-consuming than the conventional method. These results indicate that Petrifilm ACP is a suitable alternative to conventional marine agar for enumeration of marine microorganisms in seawater and C. lentillifera samples.

Survey of Japanese encephalitis virus in pigs on Miyako, Ishigaki, Kume, and Yonaguni Islands in Okinawa, Japan.

Serum specimens were collected from 125 pigs on Miyako Island, 112 pigs on Ishigaki Island, and 42 pigs on Kume Island from 2005 to 2007, and 54 pigs on Yonaguni Island from 2006 to 2007. Their sera were tested for Japanese encephalitis virus (JEV) antibody by hemagglutination inhibition (HI) assay. Five serum samples (4.5%) from Ishigaki Island were positive for HI antibody, and 4 of the 5 samples were positive for 2-mercaptoethanol- sensitive antibody (IgM Ab). All samples from Miyako, Kume, and Yonaguni Islands were negative for HI antibody. Our results indicate that JEV transmission activity was extremely low on Miyako, Ishigaki, Kume, and Yonaguni Islands. The JEV genome (JEV-RNA) was detected from the sera of one pig on Ishigaki Island. The partial gene of the E region (151 nt) was analyzed phylogenetically. The analysis showed that the new JEV-RNA belonged to genotype 3 and was closely related to JEV strains isolated in Taiwan from 1985 to 1996. It was suggested that JEV previously introduced from Taiwan had been maintained on Ishigaki Island.

[Investigation and culture of microbial contaminants of Caulerpa lentillifera (Sea Grape)].

Caulerpa lentillifera is a kind of edible seaweed, known as 'sea grape' or 'green caviar'. It is used in fresh salads. However, it is sensitive to low temperature and osmotic pressure, and is easily spoilt by storage in a refrigerator or washing with tap water. That is the reason why it is difficult to prevent food poisoning, especially due to Vibrio parahaemolyticus. In this study we investigated of marine bacteria and V. parahaemolyticus in C. lentillifera and cultured them in order to develop effective control of bacteria in commercial farms. The sixteen farms in the Okinawa Islands were investigated from August to September in 2006. A total of 176 samples were collected from eleven points during the cultivation processes and from the products. About 10(3) cfu/mL of marine bacteria were detected in the seawater used in the tank culture, but after cultivation of C. lentillifera the number had increased to about 10(6) cfu/mL. The number of marine bacteria in C. lentillifera did not change significantly through the process of planting to the final product (about 10(7) cfu/g). V. parahaemolyticus was detected in seawater from all processes and C. lentillifera was isolated from 56% of seawater, 25% of seed-stocks, and 18.8% of product samples, though but thermostable direct hemolysin gene was not detected from enrichment cultures or isolated V. parahaemolyticus strains. These results indicate that for prevention of food poisoning by V. parahaemolyticus in C. lentillifera, it is important to establish a suitable sterilization procedure for each process.

Survey of the antibody against japanese encephalitis virus in Ryukyu wild boars (Sus scrofa riukiuanus) in Okinawa, Japan.

Serum specimens were collected from 99 wild boars in the Northern area of the main Okinawa Island and from 27 wild boars on Iriomote Island in Okinawa Prefecture from 1997 to 2005. Sera were tested for Japanese encephalitis virus (JEV) antibody by hemagglutination inhibition assay and IgG enzyme-linked immunosorbent assay. Sixty-four samples (64.6%) in the Northern area and 1 sample (3.7%) from Iriomote Island were positive for the JEV antibody. The difference in seroprevalence between the Northern area and Iriomote Island was statistically significant (P < 0.01, chi2 test). This difference may be due to the lack of a pig farm on Iriomote Island, whereas wild boars in the Northern area may be infected with JEV, amplified on pig farms. It is likely that there has recently been an increase in the number of wild boars living close to humans in certain areas of Japan. This in turn increases the possibility that wild boars are infected with JEV, which is amplified on pig farms, and these infected animals may play a role in carrying JEV to other regions of the country.

Sporadic cases and an outbreak of leptospirosis probably associated with recreational activities in rivers in the northern part of Okinawa Main Island.

In the summer of 2003, sporadic cases and an outbreak of human leptospirosis probably related to recreation in rivers occurred in the northern part of Okinawa Main Island. Sixteen of 22 suspected cases were definitely diagnosed as leptospirosis by serological test or isolation. The infective leptospiral serovar in 14 cases was presumed to be Hebdomadis. Transmission was thought to occur by exposure to river water that was contaminated by the urine of infected animals. The findings indicate that recreation in rivers in this area is a significant risk factor for infection with leptospires.

Hepatitis E virus infection in wild mongooses of Okinawa, Japan: Demonstration of anti-HEV antibodies and a full-genome nucleotide sequence.

Hepatitis E virus (HEV), a single-strand RNA virus, has been recovered not only from human beings but also from various species of animals. Here we report our results suggesting that mongoose should be added to the list of reservoir animals of HEV. Of 100 mongooses we examined in Okinawa, Japan, 21 were thought to be positive for anti-HEV antibodies, among which one was definitely positive for HEV RNA. Full-genome sequencing of the HEV isolate revealed that it segregates to a unique subgroup within genotype III. Interestingly, this mongoose strain was closely related to a swine isolate previously reported from Okinawa, implicating the possibility of interspecies transmission between these animals.

New genomospecies related to Borrelia valaisiana, isolated from mammals in Okinawa archipelago, Japan.

In 2000 and 2001, a survey was conducted of Borrelia isolated from various mammals in the southernmost islands of Japan, including Okinawa (main island), Izena, Iriomote and Ishigaki. Isolates obtained from the ear tissues of Suncus murinus (10 isolates), Mus calori (four isolates), Rattus norvegicus (one isolate) and Crocidura watasei (one isolate), were characterized by RFLP of the 5S-23S rDNA intergenic spacer and sequence analysis of the intergenic spacer, 16S rDNA and flagellin gene. While these isolates showed identical RFLPs to Borrelia valaisiana found in Korea, Taiwan and the southern and central parts of China, their RFLP patterns differed from those of B. valaisiana found in European countries, and strain Am501 isolated from Ixodes columnae in Japan. It was found that these isolates clustered with each other on a phylogenetic tree based on flagellin gene and 16S rDNA sequences, but were relatively divergent from the European B. valaisiana and strain Am501. These findings suggest that these isolates found in East Asia should be classified as a new genomospecies of Borrelia burgdorferi sensu lato.

Evidence for widespread infection of hepatitis E virus among wild rats in Japan.

Sporadic cases of hepatitis E have been reported in industrialized countries, including Japan. The source of hepatitis E virus (HEV) in these patients is unknown, although zoonotic transmission has been suggested. To investigate whether or not rodents might be a reservoir of HEV, we conducted an epidemiological survey for the antibody to a recombinant capsid protein of HEV using serum samples from wild rodents in Japan. One hundred and fourteen of 362 (31.5%) Norway rats (Rattus norvegicus) and 12 of 90 (13.3%) black rats (Rattus rattus) were positive for anti-HEV IgG. In contrast, all of the sera from 55 mice were negative for anti-HEV IgG. The rate of antibody positivity increased with weight among Norway rats. Seropositive rats were found in all five districts surveyed in this study, but the prevalence of anti-HEV IgG in wild rats differed among these prefectures. Despite the fact that Japan is a non-endemic country of hepatitis E, widespread infection of HEV was observed among wild rats in Japan. Our results suggested that HEV or a closely related virus is circulating among wild rats in Japan.