Tsutomu Tanikawa

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.

Elevated Warfarin Metabolism in Warfarin-Resistant Roof Rats (Rattus rattus) in Tokyo

Wild roof rats (Rattus rattus) live in proximity to human habitats, and they may carry numerous pathogens of infectious diseases. Pest control is important for public health, and warfarin is a commonly used rodenticide worldwide. However, continual use of warfarin may cause drug resistance in rodents and lead to failure of their control, especially in urbanized areas. In warfarin-resistant rats, the warfarin level in plasma was significantly lower after oral administration than that in the control warfarin-sensitive rats. Warfarin is metabolized by cytochrome P450 (P450), and hydroxylation of warfarin by P450 isoforms was significantly higher in warfarin-resistant rats (2-fold). Western blot analysis indicated that the level of CYP3A2 expression in warfarin-resistant rats was significantly larger than in warfarin-sensitive rats. The NADPH-P450 reductase activities in resistant rats were 8-fold higher than those in sensitive rats. In vivo, the administration of the P450 potent inhibitor proadifen (SKF-525A) increased the mortality of warfarin in the warfarin-resistant roof rats. We concluded that the mechanism of warfarin resistance in Tokyo roof rats is caused by increased clearance of warfarin.

A New Loop-Mediated Isothermal Amplification Method for Rapid, Simple, and Sensitive Detection of Leptospira spp. in Urine

ABSTRACT We developed a new loop-mediated isothermal amplification (LAMP) method to detect rrs, a 16S rRNA gene of pathogenic Leptospira spp. in urine. The method enables detection of two leptospiral cells per reaction mixture following boiling of urine specimens. The sensitivity of this method is higher than that of culture or of flaB nested PCR.

Human leptospirosis cases and the prevalence of rats harbouring Leptospira interrogans in urban areas of Tokyo, Japan

Thirteen patients with leptospirosis were identified, as confirmed by laboratory analysis during the last 5 years in our laboratory, who came from urban areas of Tokyo, Japan. All of the patients came into contact with rats before the onset of illness. Seventeen per cent of Norway rats captured in the inner cities of Tokyo carried leptospires in their kidneys. Most of these rat isolates were Leptospira interrogans serovar Copenhageni/Icterohaemorrhagiae. Antibodies against these serovars and their DNA were detected in the patients. This suggests that rats are important reservoirs of leptospirosis, and that rat-borne leptospires occur in urban areas of Tokyo.

Phylogenetic relationships of rat lungworm, Angiostrongylus cantonensis, isolated from different geographical regions revealed widespread multiple lineages

We conducted a pilot survey of genetic variation of A. cantonensis using small subunit (SSU) ribosomal (r) RNA and mitochondrial cytochrome c oxidase subunit I (coxI) gene sequences. Two distinct SSU genotypes (G1 and G2) were identified among 17 individual A. cantonensis worms from 17 different geographical localities in Japan, Mainland China, Taiwan, and Thailand. The partial coxI sequences were determined for 83 worms from 18 different geographical localities from Japan, Mainland China, Taiwan, and Thailand. Phylogenetic analysis showed eight distinct coxI haplotypes (ac1 to ac8). In 16 out of 18 localities, only a single coxI haplotype was found. However, in two localities, two coxI haplotypes coexisted. The common haplotypes found were: haplotype ac1 (Tokyo, Chiba, Kanagawa, Amamioshima Island, and Taichung), haplotype ac2 (Ishikawa, Shenzhen, and Lianjiang), haplotype ac5 (the Okinawa and the Ogasawara Islands), and haplotype ac7 (Miyagi, Aichi, and Kanagawa). Each of these regions is separated from the others by high mountain ranges or oceans. In addition, the lower genetic variation and particular geographical distribution of A. cantonensis in each location could indicate a founder effect, which may have resulted from multiple independent origins, and suggests that haplotypes migrated from endemic areas via human-related transportation.

Detection of Streptobacillus spp. in feral rats by specific polymerase chain reaction.

Streptobacillus moniliformis is an etiological agent of rat‐bite fever and Haverhill fever in human infection. As the currently available methods for identifying the causative bacteria are not satisfactory, we attempted to establish them by PCR using newly designed primers for the 16S rRNA gene of S. moniliformis. We then determined the prevalence of Streptobacillus spp. in two species of feral rats that inhabit an urban region in Japan, because information on the prevalence of the bacteria in feral rats is obscure. The use of PCR with newly designed primers showed that an extremely high proportion of R. norvegicus harbored the bacteria (61/66, 92%), whereas the prevalence was only 58% in R. rattus (30/52). The nucleotide sequence analysis of the 16S rRNA gene of Streptobacillus spp. isolated from oral swabs of feral rats showed at least two different types of bacteria among isolates from R. norvegicus and R. rattus.

Dioctophyme renale (Nematoda: Dioctophymatoidea) in the abdominal cavity of Rattus norvegicus in Japan.

We collected 24 brown rats, Rattus norvegicus, in Kanagawa Prefecture in Japan and found one rat harboring a dioctophymatid nematode. A single male and a female worm were recovered from the abdominal cavity and were identified as Dioctophyme renale based on morphologic features and a BLAST DNA sequence analysis. We describe the morphological features of the adult worms and eggs from this extremely rare case of D. renale infection in a brown rat.

Streptobacillus ratti sp. nov., isolated from a black rat (Rattus rattus)

An indole-, oxidase- and catalase-negative, non-motile bacterium, strain OGS16T, was isolated from an oral swab of a feral black rat (Rattus rattus) in 2007 in Japan. It stained Gram-negative and had pleomorphic, rod-shaped, non-spore-forming cells. Based on 16S rRNA gene sequence analyses, strain OGS16T was assigned to the genus Streptobacillus, with 16S rRNA gene sequence similarities of 99.3, 99.0, 98.6 and 95.5% to the type strains of Streptobacillus moniliformis, Streptobacillus notomytis, Streptobacillus felis and Streptobacillus hongkongensis, respectively. Strain OGS16T could also be differentiated clearly from other species of the genus Streptobacillus by rpoB, groEL and recA nucleotide and deduced amino acid sequence analysis. DNA-DNA relatedness as obtained by average nucleotide identity was 89.10% between strain OGS16T and Streptobacillus moniliformis DSM 12112T. Chemotaxonomic and physiological data for strain OGS16T were congruent with results for other closely related members of the family Leptotrichiaceae, represented by highly similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis also proved suitable in discriminating strain OGS16T unequivocally from all currently described taxa of the genus Streptobacillus. On the basis of these data, we propose the novel species Streptobacillus ratti sp. nov., with the type strain OGS16T (=JCM 31098T=DSM 101843T). The G+C content of the DNA of the type strain is 25.9 mol% and the genome size is 1.50 Mbp.

Multiple-locus variable-number tandem repeat analysis of Leptospira interrogans and Leptospira borgpetersenii isolated from small feral and wild mammals in East Asia.

Leptospira spp. are the causative agents of a worldwide zoonosis, leptospirosis, maintained by various mammals. Each Leptospira serovar is frequently associated with a particular maintenance host, and recently, Leptospira genotype-host association has also been suggested to limit serovars to restricted areas. We investigated the molecular characteristics of L. interrogans and L. borgpetersenii which were isolated from small feral and wild animals in four East Asian states using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA using 11 loci was performed on 110 L. interrogans serogroups from Japan (79 strains of 5 serogroups from 3 animal species), Philippines (21; 3; 2), Taiwan (7; 2; 3), and Vietnam (3; 1; 1). A MLVA method using 4 loci for L. borgpetersenii was established and performed on 52 isolates from Japan (26; 3; 7), Philippines (13; 1; 2), and Taiwan (13; 1; 3). In L. interrogans, serogroups Autumnalis and Hebdomadis appeared more genetically diverse than serogroups Bataviae, Grippotyphosa, Icterohaemorrhagiae, Pomona, or Pyrogenes. The former serogroup strains with the exception of one Hebdomadis strain were isolated from Apodemus speciosus while all the latter serogroup strains with the exception of Grippotyphosa were isolated from Rattus norvegicus. L. borgpetersenii was isolated from at least 11 animal species while L. interrogans was isolated from five species, which might suggest a wider host range for L. borgpetersenii. Broad host preference in a single genotype was also observed, which colonized not only different species of the same genera but also multiple animal genera. This study demonstrates that there may be variability in the range of genetic diversity among different Leptospira serogroups, which may be attributed to maintenance host animals and environmental factors.

Novel revelation of warfarin resistant mechanism in roof rats (Rattus rattus) using pharmacokinetic/pharmacodynamic analysis

Roof rats (Rattus rattus) live mainly in human habitats. Heavy use of rodenticides, such as warfarin, has led to the development of drug resistance, making pest control difficult. There have been many reports regarding mutations of vitamin K epoxide reductase (VKOR), the target enzyme of warfarin, in resistant rats. However, it has been suggested there are other mechanisms of warfarin resistance. To confirm these possibilities, closed colonies of warfarin-susceptible roof rats (S) and resistant rats from Tokyo (R) were established, and the pharmacokinetics/pharmacodynamics of warfarin in rats from both colonies was investigated. R rats had low levels of warfarin in serum and high clearance activity. These rats can rapidly metabolize warfarin by hydroxylation. The levels of accumulation in the organs were lower than those of S rats. R rats administered warfarin showed high expression levels of CYP2B, 2C, and 3A, which play roles in warfarin hydroxylation, and may explain the high clearance ability of R rats. The mechanism of warfarin resistance in roof rats from Tokyo involved not only mutation of VKOR but also high clearance ability due to high levels of CYP2B, 2C and 3A expression possibly induced by warfarin.