Molecular survey of Rickettsia, Ehrlichia, and Anaplasma infection of domestic cats in Japan.

The prevalence of Rickettsia, Ehrlichia, and Anaplasma in 1764 DNA samples extracted from feline peripheral blood from all 47 prefectures in Japan was evaluated by screening real-time PCR, genus-specific PCR, and DNA nucleotide sequencing. The survey revealed that all cats were negative for Rickettsia infection. Two cats were positive for Ehrlichia or Anaplasma based on the screening PCR assay. Nucleotide sequence analysis of the partial 16S rRNA including the divergent region near the 3'-end revealed that the 2 positives were most similar to Anaplasma bovis with percent identities of 99.8% and 99.2%. This was the first detection of A. bovis DNA fragments in cats. Although these 2 cats showed stomatitis, both were also infected with feline immunodeficiency virus. The relationship between A. bovis carriage and clinical disease is not yet understood.

Detection of Anaplasma bovis DNA in the peripheral blood of domestic dogs in Japan.

The prevalence of Ehrlichia and Anaplasma in 1,427 dogs from 32 Japanese prefectures was evaluated by PCR and DNA nucleotide sequencing. PCR screening demonstrated that 18 dogs (1.3%) were positive for Anaplasmataceae. Sequence analysis revealed that 14 of the amplicons were most closely related to Wolbachia spp., symbionts of Dirofilaria immitis, whereas three were identified as Anaplasma bovis. The remaining amplicon could not be sequenced. Almost the entire sequence of 16S rRNA (1,452 bp) from one of the positive specimens was determined, and subsequent phylogenetic analysis confirmed that the detected sequence was that of A. bovis. This is the first detection of A. bovis DNA fragments in dogs. Species-specific nested PCR showed that 15 (1.1%) of the 1,427 dogs involved in this study were positive for A. bovis. The geographical distribution of these dogs ranged from Aomori Prefecture in northern Japan to Kagoshima Prefecture in the south. The relationship between A. bovis infection and clinical disease is not yet clearly understood.

An epidemiological survey of feline hemoplasma infection in Japan.

Hemoplasma (hemotropic mycoplasma) often causes hemolytic anemia in infected cats, especially those with immune suppression. An updated nationwide epidemiological survey of feline hemoplasmosis was conducted in Japan. Blood samples were collected from 1,770 outdoor-accessing cats from March to October 2008. The infections were molecularly detected by PCR analyses, which are able to distinguish Mycoplasma haemofelis (Mhf), `Candidatus M. haemominutum' (CMhm), and `Candidatus M. turicensis' (CMt) infections. Of the 1,770 cats, 468 cases (26.4%) revealed a single- or co-infection of feline hemoplasmas [Mhf alone, 42 cases (2.4%); CMhm alone, 280 cases (15.8%); CMt alone, 48 cases (2.7%); Mhf+CMhm, 28 cases (1.6%); Mhf+CMt, 6 cases (0.3%); CMhm+CMt, 50 cases (2.8%); Mhf+CMhm+CMt, 14 cases (0.8%)]. In addition, male gender, middle to old age, history of fight wounds, and feline immunodeficiency virus infection were shown to be risk factors for hemoplasma infection. Close attention must be paid to the acute onset of disease in feline practice because a prevalence of hemoplasma infection was detected even in clinically healthy cats.

An updated nation-wide epidemiological survey of feline immunodeficiency virus (FIV) infection in Japan.

An updated nation-wide epidemiological survey of feline immunodeficiency virus (FIV) infection was conducted in Japan. Blood samples were collected from 1,770 outdoor accessing cats from March to October 2008. Serologically, 410 cats (23.2%) were positive for anti-FIV antibody. Proviral DNA of the FIV env V3-V5 region isolated from 348 cases could be phylogenetically analyzed. The present study disclosed a geographic distribution of four subtypes (A, B, C and D) of FIV in Japan. Even though an FIV vaccine was introduced in Japan, we do not currently know whether this vaccine is effective against all strains of FIV in Japan or not. Therefore, close attention still has to be paid to epidemic and genotypic trends of FIV.

Serological survey of Neospora caninum infection among dogs in Japan through species-specific ELISA.

A seroepidemiological survey of Neospora caninum infection among dogs in Japan was conducted using species-specific enzyme-linked immunosorbent assay with recombinant surface antigen (Nc-SAG1t). Among 1,206 dogs examined, 126 dogs (10.4%) from 30 prefectures from Hokkaido to Okinawa were positive to N. caninum infections, which were more frequently detected in females than males. Siberian Huskies showed the highest positive rate compared with the other breeds. Dogs with pyometra and diabetes mellitus showed the higher positive rates than dogs with other diseases or without diseases.

Epidemiological survey of Babesia gibsoni infection in dogs in Japan by enzyme-linked immunosorbent assay using B. gibsoni thrombospondin-related adhesive protein antigen.

A nationwide epidemiological survey of Babesia gibsoni infection in non-fighting dogs was conducted using an improved ELISA with recombinant B. gibsoni thrombospondin-related adhesive protein (BgTRAP). A total of 1206 dogs from 27 prefectures were examined and 128 (10.6%) tested positive. In the eastern part of Japan, 39 dogs out of the 559 (7.0%) examined were positive, while 89 dogs out of 647 (13.8%) tested positive in the western part of Japan. Although the percentage of dogs that tested positive was significantly (p=0.0001) lower in the eastern part compared to the western part of Japan, overall these results indicate that B. gibsoni infection of dogs has a widespread geographic distribution throughout the country. A history of tick infestation was identified as a significant risk factor for B. gibsoni infection (p=0.0091), while sex (p=0.9411), age (p=0.0920) and breed (p=0.0549) of dogs were not statistically significant risk factors. These results indicate that tick infestation is the most dominant risk factor for B. gibsoni infection of non-fighting dogs in Japan and suggest that other B. gibsoni transmission routes, such as fighting and transplacental transmission, may be less important.

Serological survey of Rickettsia japonica infection in dogs and cats in Japan.

Antibodies against Rickettsia japonica in 20 of 1,207 dogs and 5 of 584 cats in Japan were detected using immunofluorescence. Some antibody-positive animals were detected in Niigata and Kagawa Prefectures, areas in which Japanese spotted fever in human patients has never been identified. Some animals were positive for antibodies against other new Rickettsia species.

Detection of Borrelia garinii, Borrelia tanukii and Borrelia sp. closely related to Borrelia valaisiana in Ixodes ticks removed from dogs and cats in Japan.

Ticks removed from 1136 dogs and 134 cats all over Japan were examined for Borrelia infection by PCR and sequencing. The 5S-23S rDNA intergenic spacer of Borrelia was detected from two Ixodes persulcatus ticks from two dogs and two unidentified Ixodes spp. from another two dogs in Hokkaido, and two Ixodes granulatus ticks from two cats in Okinawa. Additional 2 I. granulatus from the same cats also showed positive. Sequence analysis of the PCR products revealed that the one from Hokkaido was similar to B. garinii, the three from Hokkaido to B. tanukii, and the four from Okinawa to a novel Borrelia sp. closely related to B. valaisiana. The data was confirmed by analysis of the flagellin gene sequence. Infected ticks carried by companion animals can be introduced into the human environment.

Epidemiological survey of Babesia gibsoni infection in dogs in eastern Japan.

To determine the distribution of Babesia gibsoni infection in dogs in the eastern part of Japan, an epidemiological survey of dogs suspected of having B. gibsoni infection was attempted using the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Thirty-five of 115 such dogs (30.4%) were positive by PCR and/or ELISA. The 35 positive dogs consisted of 28 Tosa dogs, 4 American Pit Bull Terriers, and 3 mongrel dogs in Aomori, Fukushima, Ibaraki, Gunma, Chiba, Tokyo, Kanagawa, and Nagano Prefectures. The positive dogs had a significantly lower rate of tick exposure and a higher rate of bites by other dogs. Twenty-two of 35 B. gibsoni-positive dogs were infected with hemoplasma, and the rate of infection was significantly higher than that of B. gibsoni-negative dogs.

Detection of rickettsial DNA in ixodid ticks recovered from dogs and cats in Japan.

DNA from ticks recovered from 1137 dogs and 133 cats from all over Japan were examined for Rickettsia infection by citrate synthase gene (gltA)-based PCR and partial nucleotide sequencing. A total of 91 dog tick samples and 18 cat tick samples showed a single band of the appropriate size in the nested PCR. Sequence analysis was successfully performed on 102 samples. DNA of Rickettsia japonica or closely related Rickettsia spp. strains were detected from 38 ticks in 16 prefectures mainly in western Japan. The other 33, detected from 13 prefectures including Hokkaido and Okinawa, were found to be Rickettsia helvetica or closely related strains. A total of 29 DNA that showed highest homology with Rickettsia akari or closely related strains were detected in 19 prefectures, widespread throughout Japan. Rickettsia canada-like DNA was detected from Haemaphysalis sp. removed from a dog in Fukuoka, and ;Candidatus Rickettsia tarasevichiae'-like DNA was from Ixodes sp. removed from a dog in Hokkaido.

Detection of DNA of 'Candidatus Mycoplasma haemominutum' and Spiroplasma sp. in unfed ticks collected from vegetation in Japan.

DNA fragments of 'Candidatus Mycoplasma haemominutum', a feline heamobartonella pathogen, were detected from unfed Ixodes ovatus collected from vegetation in Hokkaido, Fukushima and Yamaguchi Prefectures, and unfed Haemaphysalis flava in Yamaguchi Prefecture. This finding suggests that ixodid tick is a possible vector of 'C. Mycoplasma haemominutum'. Spiroplasma DNA was also detected from unfed I. ovatus in Hokkaido, Fukushima and Yamaguchi Prefectures. The analysis of nucleotides sequence suggested that this Spiroplasma was distinct from registered species.

Detection of ehrlichial DNA in Haemaphysalis ticks recovered from dogs in Japan that is closely related to a novel Ehrlichia sp. found in cattle ticks from Tibet, Thailand, and Africa.

Tick DNA samples from dogs in Japan were examined for Ehrlichia infection by 16S rRNA gene-based PCR and sequencing. Three positive samples were detected from Haemaphysalis ticks, and higher levels of similarity (98.46 to 99.06%) were found to recently detected Ehrlichia spp. from cattle ticks in Tibet, Thailand, and Africa.

Epidemiological survey of Babesia species in Japan performed with specimens from ticks collected from dogs and detection of new Babesia DNA closely related to Babesia odocoilei and Babesia divergens DNA.

Detection and analysis of Babesia species from ticks recovered from dogs in Japan were attempted by PCR and nucleotide sequence analysis based on the 18S rRNA gene, respectively. A total of 1136 ticks were examined for Babesia DNA by 18S rRNA-based PCR and nucleotide sequencing. Partial sequences of Babesia canis vogeli DNA were detected from six ticks in Aomori, Nara, Hiroshima, Oita, and Okinawa Prefectures; and Babesia gibsoni Asia-1 DNA was also detected in four ticks in Osaka, Hiroshima, Miyazaki, and Okinawa Prefectures. Unique sequences of 1678 bp were also obtained from Ixodes ovatus ticks in Akita and Fukui Prefectures. The sequences were similar to those of Babesia odocoilei (97.7%) and Babesia divergens (97.6%). This is the first report of the detection of DNA belonging to this group in Japan.

Epidemiological survey of Anaplasma platys and Ehrlichia canis using ticks collected from dogs in Japan.

Detection of Anaplasma platys and Ehrlichia canis in ticks recovered from dogs in Japan was attempted using a species-specific nested PCR based on the 16S rRNA gene. A total of 1211 ticks recovered from 1211 dogs from all over Japan were examined for A. platys and E. canis. Four tick samples from Fukushima, Miyazaki and Kagoshima Prefectures recovered from four different dogs showed a positive reaction for A. platys. Although the four dogs did not show any clinical signs and no blood examination data were available, it is possible that A. platys has already been spread widely in Japan. No positive reactions were observed in any ticks examined for E. canis.