Motonobu Hara

Identification of thermo‐acidophilic bacteria isolated from the soil of several Japanese fruit orchards

Aims:  To investigate the occurrence and distribution of thermo‐acidophilic bacteria (TAB) associated with various commercial fruit crop soils in Japan and to assess their ability to produce the odorous phenolic compound, guaiacol.

Detection of feline herpesvirus 1 DNA by the nested polymerase chain reaction.

The thymidine kinase region of feline herpesvirus 1 (FHV 1) genome in ocular/nasal swabs from cats with clinical manifestations of upper respiratory disease was amplified by nested polymerase chain reaction (nested PCR). Two primer pairs were prepared for nested PCR. FHV 1 DNA in ocular/nasal swabs was extracted using instaGene-DNA purification matrix. Nested PCR for the FHV 1 culture supernatants was ten times as sensitive as single PCR. On comparing viral isolation with single PCR and nested PCR for the detection of FHV 1 in ocular/nasal secretions, of 5 samples that yielded infectious virus in cell culture, 3 (60%) were positive in single PCR and 5 (100%) were positive in nested PCR. When 22 ocular/nasal swabs that did not yield FHV 1 were assayed, 3 were negative in both single PCR and nested PCR, 2 were positive in both single and nested PCR and 17 were positive in only nested PCR. Thus, FHV 1 was detected in 19/22 (86.4%) by the nested PCR and in 2/22 (9%) by single PCR. These results show that nested PCR is 4.8 (24 positive samples/5 positive samples) times as sensitive as single PCR.

Isolation and Identification of Methylobacterium Species from the Tap Water in Hospitals in Japan and Their Antibiotic Susceptibility

Contamination of tap water by Methylobacterium species has become a serious concern in hospitals. This study was planned to examine the distribution of Methylobacterium species inhabiting tap water used in Japanese hospitals and antibiotic sensitivity of the isolates in 2004. Species identification of 58 isolates was performed based on the homology of a partial sequence of 16S rDNA. The dominant Methylobacterium species in hospital water were M. aquaticum and M. fujisawaense. To examine the biochemical properties of these isolates, a carbon source utilization was tested using an API50CH kit. The phenotypic character varied widely, and was not necessarily consistent with the results of phylogenic analysis based on the partial 16S rDNA sequence, suggesting that the biochemical properties are not suitable for identification of Methylobacterium species. The isolates were also subjected to antibiotic sensitivity tests. They were resistant to 8 antibiotics, but highly sensitive to imipenem (MIC90=1 μg/ml) and tetracycline (MIC90=8 μg/ml). These findings concerning the isolates revealed the presence of Methylobacterium species with resistance to multiple antibiotics in hospital tap water.

Analysis of the VP2 protein gene of canine parvovirus strains from affected dogs in Japan.

To clarify the evolution of canine parvovirus 2 (CPV-2) that has recently been epidemic in Japan, VP2 gene sequences at positions 3556-4166 were analyzed in 107 CPV-2 strains obtained from rectal swabs of diarrheic dogs from 2009 to 2011. CPV-2b (95 strains) was more frequently detected than CPV-2a (nine strains), while CPV-2c was not detected. Remaining three strains were identified as the original type CPV-2, which should be derived from vaccines. These findings are similar to the previous results involving Japanese strains, suggesting there has been no great change in the recent CPV-2 epidemic in Japan. This epidemic is the same as that in Taiwan. Furthermore, a 324-lle mutant, which has been reported in Korean and Chinese strains, was detected in 66.7% of CPV-2a strains.

Phylogenetic Analysis of Field Isolates of Feline Calcivirus (FCV) in Japan by Sequencing Part of Its Capsid Gene

The molecular epidemiology of the infectious disease caused by feline calcivirus (FCV) in Japan was investigated by analysing the phylogenetic relationship among 21 Japanese field isolates, including the F4 strain, and 30 global isolates. Parts of the capsid gene (B–F) of the isolates were amplified by RT-PCR, and the amino acid sequences were compared with those from the global isolates. Thirty-seven and 14 out of a total of 51 isolates were clustered into two distinct genogroups, I and II respectively, by UPGMA and NJ analysis. Seven of the 21 Japanese isolates (33%) fell into group I together with 30 global isolates, while the other 14 Japanese isolates (67%) belonged to group II. The bootstrap repetition analysis of groups I and II formed by the NJ method gave a value of 99.0%. The 14 latter Japanese isolates were clearly separated from the isolates in group I, and they were different from any previously known FCV, forming a new genogroup, which implies that this lineage has been confined to Japan. Comparing the amino acid sequences shared by groups I and II, the amino acid at position 377 in B region was asparagine (Asn or Asp (NH2)) in group I, while it was lysine (Lys) in all the strains in group II. Similarly, the amino acid at position 539 in the F region was alanine (Ala) or proline (Pro) in group I, while it was valine (Val) in group II; glycine (Gly) at position 557 in group I was serine (Ser) in Group II; and phenylalanine (Phe) or leucine (Leu) at position 566 in genogroup I was tyrosine (Tyr) in group II.

Detection and genotyping of canine coronavirus RNA in diarrheic dogs in Japan

Abstract To clarify the prevalence of canine coronavirus (CCoV) infection in Japan, faecal samples from 109 dogs with diarrhoea were examined for CCoV RNA together with canine parvovirus type 2 (CPV-2) DNA. The detection rates of CCoV and CPV-2 for dogs aged less than 1year were 66.3% and 43.8%, while those for dogs aged 1year or older were 6.9% and 10.3%, respectively, which were significantly different (p <0.0001 and p =0.0003, respectively), indicating not CPV-2 but CCoV is an important diarrhoea-causing organism in juvenile dogs. Among the CCoV-positive dogs, 65.5% and 72.7% showed to be positive for CCoV types I and II, respectively, and simultaneous detection rate of both types was high at 40.0%. Furthermore, transmissible gastroenteritis virus (TGEV)-like CCoV RNA was detected from 8 dogs. These findings indicate that CCoV type I and TGEV-like CCoV are already circulating in Japan, though no reports have been presented to date.

Sensitivity of FCV to recombinant feline interferon (rFeIFN).

Feline calicivirus cause feline respiratory diseases, and inactivated and attenuated vaccines are available for its prevention. Moreover, the presence of vaccine breakdown strains (VBS) is problematic. In Japan, feline recombinant interferon (rFeIFN) has been used for its treatment. However the method of compare with each strains has not established. To examine the relationship between the breakdown vaccine strain and rFeIFN sensitivity, the sensitivity of 47 field isolates to rFeIFN was determined. The Log PDD50 values were normally distributed within the range 1.1–3.7, with a mean value of 2.3 ± 0.64. Since 68.3% of the PDD values fell in the range of the mean ± standard deviation, the values in the range 1.7–2.9, the lower values, and the higher values were defined as representing moderate, low, and high sensitivity, respectively. Among the 15 vaccine breakdown strains, strain Fukuoka9 showed a low sensitivity, but strains ML89, T58, and N74 were highly sensitive, showing no association with vaccine breakdown. The amino acid sequence changes specific to the low rFeIFN-sensitive Fukuoka-9 strain were found, suggesting that these sites are involved in rFeIFN sensitivity.

Characteristics of a Pink‐Pigmented Bacterium Isolated from Biofilm in a Cooling Tower in Tokyo, Japan

Strain K‐20, a Gram‐negative, non‐motile, non‐spore‐forming and strictly aerobic rod, which produces a pale pink pigment, was isolated from biofilm in a cooling tower in Tokyo, Japan. The taxonomic feature of the strain was studied using phenotypic tests and phylogenetic analysis. Phylogenetic analysis of 16S rRNA gene sequences showed that the strain was related to Roseomonas gilardii subsp. rosea, Roseomonas gilardii subsp. gilardii, Roseomonas cervicalis and Roseomonas mucosa at 94.3–94.6 sequence similarities. Growth occurred at 25–40 C and pH 5.0–10.0, optimal at 35 C and pH 7.0. Growth did not occur in the presence of ≧2% NaCl. The API 20NE identification system gave a positive result for urease, L‐arabinose, potassium gluconate, adipic acid, malic acid and trisodium citrate (API code number 0201465). The predominant fatty acids of strain K‐20 were C18:1Δ11 (50.8%) and C16:1 (17.2%). Cells contained ubiquinone 10 (Q‐10) as the major quinone and the G+C content was 72.0 mol%. Based on phenotypic, chemotaxonomic and phylogenetic data, it was assumed that strain K‐20 (=JCM 14634) is a novel species of the genus Roseomonas.

Phylogenetic Analysis of the Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum Based Upon 16s rRNA

The nucleotide sequences of 16s rRNA genes of Erysipelothrix rhusiopnthiae and Erysipelothrix tonsillarum were determined. The sequences are almost similar (99.8%) with only three nucleotides mismatched.

Properties of a calicivirus isolated from cats dying in an agitated state

In June 1993, two of five pet cats kept in Yokohama city in Japan suddenly became agitated and died. Feline calicivirus (FCV) was isolated from them. One strain (FCV-S) was isolated from the spinal cord, lung and tonsil of cat 1, another (FCV-B) from the ileum, medulla oblongata and cervical spinal cord of cat 2, and a third (FCVSAKURA) from the oral cavity of one of the three surviving cats which showed no clinical signs. These three strains were equally resistant to pH 3˙0 and serologically similar to each other, but distinct from strain F9. A genetic analysis, using a 208 base pair fragment from region E of the capsid, showed that FCV-Ari had a 70˙4 per cent nucleotide and 77˙3 per cent amino acid homology and FCV-F9 had a 68˙6 per cent nucleotide and 73˙9 per cent amino acid homology with the three strains, indicating that these two strains were genetically distinct from the three new isolates. Unvaccinated cats and cats which had been vaccinated against FCV-F9 developed watery diarrhoea but did not become agitated after the administration of FCV-S. The FCV-S strain did not induce signs of excitability after it was administered orally to specific pathogen-free cats.