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1.  Molecular Survey of Babesia gibsoni Using Haemaphysalis longicornis Collected from Dogs and Cats in Japan 
A nationwide survey of Babesia gibsoni using Haemaphysalis longicornis collected from dogs and cats in Japan was conducted using molecular methods. A total of 1,341 H. longicornis, including 305 females, 14 males, 332 nymphs and 690 larvae (153 pools) from 44 prefectures, were examined by B. gibsoni-targeted PCR. Partial sequence analysis revealed that 12 of 13 positive samples sequenced, including samples from Tottori, Hiroshima, Yamaguchi, Tokushima, Ehime and Oita prefectures (all in western Japan), were identical to B. gibsoni, and 1 sample from Kyoto Prefecture was most closely related to a Babesia species recently detected from feral raccoons in Hokkaido. H. longicornis is a candidate for transmission vector tick of the new Babesia species.
PMCID: PMC4197166  PMID: 24920547
Babesia gibsoni; distribution; Haemaphysalis longicornis
2.  High Genetic Diversity of Anaplasma marginale Detected from Philippine Cattle 
A total of 658 cattle in 6 provinces in the Philippines were screened for Anaplasma marginale infection by using a diagnostic heat-shock operon (groEL) gene-PCR assay. The screening-positive samples were further tested using the major surface antigen protein 1a (Msp1a) gene-PCR assay. Screening PCR results showed 130 cattle (19.8%) were positive for the A. marginale infection. Subsequent amplification using the Msp1a gene only showed 93 samples (14.1%) to be positive. In addition, 37 tandem-repeat structures, including 20 novel structures, and 41 distinct genotypes were identified. Interestingly, multiple infections of 4 different genotypes were also observed in A. marginale-infected cattle. The present study demonstrated the prevalence and characterization of diverse genotypes of A. marginale in the Philippine cattle.
PMCID: PMC4143641  PMID: 24717413
Anaplasma marginale; cattle; groEL; Msp1a; Philippines
3.  Prevalence and Molecular Analyses of Hemotrophic Mycoplasma spp. (Hemoplasmas) Detected in Sika Deer (Cervus nippon yesoensis) in Japan 
Hemotropic mycoplasmas (hemoplasmas) are cell-wall deficient, epierythrocytic bacteria that cause infectious anemia in several mammalian species. The prevalence of hemoplasma species was examined by screening and species-specific PCR using blood samples collected from 51 sika deer in Hokkaido, Japan. Molecular analyses were performed for the 16S rRNA, 23S rRNA and RNase P RNA (rnpB) gene sequences. A total of 23/51 (45%) deer DNA samples were positive for hemoplasmas in the screening PCR. Using species-specific PCR, 12 and 17 samples were positive for ‘Candidatus Mycoplasma haemocervae’ and ‘Candidatus M. erythrocervae’, respectively. Sequencing and phylogenetic trees of those three genes indicate that the ‘Candidatus M. haemocervae’ and ‘Candidatus M. erythrocervae’ detected in Japanese deer are potentially different species from the cervine hemoplasma found in deer from America and Brazil.
PMCID: PMC4013367  PMID: 24270803
Cervus nippon yesoensis; hemoplasma; Japan; phylogeny
4.  The Phylogenetic Position of Anaplasma bovis and Inferences on the Phylogeny of the Genus Anaplasma 
The present study aimed to determine the complete citrate synthase (gltA) and heat-shock protein (groEL) gene sequences of Anaplasma bovis and to infer phylogenetic relationships within the genus Anaplasma. Multiple alignments from single and concatenated sequences of the 16S rRNA, gltA and groEL genes of the genus Anaplasma were subjected to phylogenetic analyses. Percent identities of A. bovis nucleotide sequences were found highest with A. phagocytophilum in gltA (65.4%) and groEL (79.8%). Single gene phylogenetic tree results assumed similar phylogenetic positions within the genus Anaplasma, except for A. bovis. However, consensus and concatenated sequence phylogenetic trees showed similar results, revealing 2 subgroups within the genus.
PMCID: PMC3982816  PMID: 24189581
16S rRNA gene; Anaplasma bovis; citrate synthase gene (gltA); heat-shock operon gene (groEL); phylogeny
5.  Zygomycotic Mediastinal Lymphadenitis in Beef Cattle with Ruminal Tympany 
A 9-month-old steer was autopsied due to recurrent ruminal tympany. A macroscopic examination found an enlarged caudal mediastinal lymph node, and a section of the lymph node revealed necrosis with marked calcification, similar to tuberculous lymphadenitis. Histopathologically, the lesion consisted of multiple coagulative necrotic foci and fibrosis with macrophage, lymphocyte, eosinophil and multinucleated giant cell infiltration. Non-uniform width hyphae were detected in the necrotic area and within the cytoplasm of the multinucleated giant cells, and they were found to be anti-Rhizopus arrhizus antibody positive in an immunohistochemical examination. Therefore, the steer was diagnosed with necrotic caudal mediastinal lymphadenitis due to zygomycetes infection, and inhibition of eructation by the enlarged lymph node was the likely cause of the ruminal tympany.
PMCID: PMC3979945  PMID: 24018826
cattle; necrotic lymphadenitis; ruminal tympany; zygomycosis
6.  Effect of Chronic Hemoplasma Infection on Cattle Productivity 
The present study evaluated the effect of hemoplasmosis on cattle productivity. Prevalence of bovine hemoplasma was examined by polymerase chain reaction (PCR) using whole blood samples collected from 93 breeding cows and their 71 calves in Hokkaido, Japan. Monthly milk production records and other clinical data were compared between Mycoplasma wenyonii (Mw)-infected, “Candidatus Mycoplasma haemobos” (CMh)-infected, co-infected and PCR-negative groups. Blood chemical parameters were obtained from the 93 cows and 64 calves. PCR results showed that 89.2% (83/93) of cows and 14.1% (10/71) of calves were positive for bovine hemoplasma. Based on productivity data obtained from the 93 cows, Mw-infected, CMh-infected and co-infected cows had significantly lower monthly milk yield compared to PCR-negative cows. Furthermore, decline in milk yield was prolonged in CMh-infected and co-infected groups. No significant differences were found for other clinical findings among the four groups. Calf birth weight tended to be lower for Mw-infected, CMh-infected and co-infected groups compared to the PCR-negative group. There were no significant differences in all blood parameters of cows and calves among the four groups. In addition, no significant differences were found in any parameter between hemoplasma-infected and PCR-negative calves.
PMCID: PMC3942926  PMID: 23676278
cattle; direct PCR; hemoplasma; productivity
7.  PCR Detection of Babesia ovata from Cattle Reared in Japan and Clinical Significance of Coinfection with Theileria orientalis 
Journal of Clinical Microbiology  2012;50(6):2111-2113.
We describe here the clinical significance of coinfection with Theileria orientalis and Babesia ovata in cattle. Anemia status in a herd of dairy cattle in Japan was investigated in relation to infection with these parasites. Our findings indicate that while B. ovata infection might not be the primary cause of anemia in the cattle, it may contribute to the clinical development of anemia in animals coinfected with both B. ovata and T. orientalis.
PMCID: PMC3372144  PMID: 22442312
8.  Enzyme-Linked Immunosorbent Assays Based on Neospora caninum Dense Granule Protein 7 and Profilin for Estimating the Stage of Neosporosis 
Neospora caninum is an intracellular protozoan parasite that causes bovine and canine neosporosis, characterized by fetal abortion and neonatal mortality and by neuromuscular paralysis, respectively. Although many diagnostic methods to detect parasite-specific antibodies or parasite DNA have been reported, to date no effective serodiagnostic techniques for estimating pathological status have been described. Our study aimed to elucidate the relationship between the parasite-specific antibody response, parasite activation, and neurological symptoms caused by N. caninum infection by using a recombinant antigen-based enzyme-linked immunosorbent assay. Among experimentally infected mice, anti-N. caninum profilin (NcPF) antibody was only detected in neurologically symptomatic animals. Parasite numbers within the brains of the symptomatic mice were significantly higher than those in asymptomatic animals. In addition, anti-NcPF and anti-NcGRA7 antibodies were mainly detected at the acute stage in experimentally infected dogs, while anti-NcSAG1 antibody was produced during both acute and chronic stages. Furthermore, among anti-NcSAG1 antibody-positive clinical dogs, the positive rates of anti-NcGRA7 and anti-NcPF antibodies in the neurologically symptomatic dogs were significantly higher than those in the non-neurologically symptomatic animals. Our results suggested that the levels of anti-NcGRA7 and anti-NcPF antibodies reflect parasite activation and neurological symptoms in dogs. In conclusion, antibodies against NcGRA7 and NcPF may have potential as suitable indicators for estimating the pathological status of neosporosis.
PMCID: PMC3294613  PMID: 22258707
9.  Evaluation of Rickettsia japonica Pathogenesis and Reservoir Potential in Dogs by Experimental Inoculation and Epidemiologic Survey ▿  
Rickettsia japonica pathogenesis and reservoir potential in dogs were evaluated by both experimental inoculation and epidemiologic survey. In the experimental inoculation study, dogs 1 and 2 were pretreated with an immunosuppressive dose of cyclosporine 14 days before inoculation and became ill after exposure to R. japonica. Dogs exhibited clinical signs, including fever, anorexia, depression, and decreased water consumption, between 36 and 96 h after inoculation, but these signs disappeared spontaneously by 5 days after inoculation. Dogs 3 and 4 were not pretreated with cyclosporine, and no clinical signs were detected in them throughout the 14-day observation period. The control dog was clinically normal and had a normal rectal temperature throughout the study period. We attempted to detect rickettsial DNA from peripheral blood and aspiration samples from kidney and spleen by nested PCR, but all samples examined were negative. The control dog lacked detectable titers to R. japonica antigen on day 14, while positive antibodies to R. japonica were detected in all four experimentally infected dogs, with titers of 1:160 to 1:80. In the epidemiologic survey, 24 (1.8%) of the 1,363 dogs examined throughout Japan had antibodies against R. japonica, with titers of 1:40 or more. However, we observed neither clinical signs at the time of sample collection nor nested PCR results indicative of rickettsial infection in these dogs. In conclusion, dogs in Japan can be exposed to R. japonica, and infected dogs with immunosuppressive conditions can temporarily develop clinical symptoms, including fever, anorexia, depression, and decreased water consumption.
PMCID: PMC3019780  PMID: 20980481
10.  Purulent Necrotic Dislocation of the Hip Joint Associated with Umbilical Infection in a Foal 
Journal of equine science  2010;21(2):17-20.
A 42-day-old heavy draft horse fell into sudden astasia. Significant swelling and heat sensation of the left femoral region were observed. Because of a friction sound in the left hip, we supposed that the hip joint was dislocated or the hip bone was fractured. Computed Tomography (CT) examination showed that the left hip joint was dislocated and the left femoral head was disjunct. We carried out a pathological autopsy, and made a diagnosis of the foal as fracture of the hip bone and femoral head with suppurative umbilical arteritis. Pathologic changes in the umbilical artery and hind leg were completely unilateral, suggesting that left umbilical arteritis spread to the blood circulation, causing arthritis and dislocation of the hip bone.
PMCID: PMC4013954  PMID: 24833975
dislocation; foal; umbilical infection
11.  Effects of mosapride on motility of the small intestine and caecum in normal horses after jejunocaecostomy 
Journal of Veterinary Science  2009;10(2):157-160.
The purpose of the present study was to evaluate the prokinetic effects of mosapride with non-invasive assessment of myoelectrical activity in the small intestine and caecum of healthy horses after jejunocaecostomy. Six horses underwent celiotomy and jejunocaecostomy, and were treated with mosapride (treated group) at 1.5 mg/kg per osos once daily for 5 days after surgery. The other six horses did not receive treatment and were used as controls (non-treated group). The electrointestinography (EIG) maximum amplitude was used to measure intestinal motility. Motility significantly decreased following surgery. In the treated group, the EIG maximum amplitude of the small intestine was significantly higher than in the controls from day 6~31 after treatment. These findings clearly indicate that mosapride could overcome the decline of intestinal motility after jejunocaecostomy in normal horses.
PMCID: PMC2801111  PMID: 19461212
horse; ileus; mosapride; small intestine
12.  Serological Survey of Rickettsia japonica Infection in Dogs and Cats in Japan▿  
Clinical and Vaccine Immunology : CVI  2007;14(11):1526-1528.
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.
PMCID: PMC2168167  PMID: 17913859
13.  Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in Dogs in a Village of Eastern Sudan by Using a Screening PCR and Sequencing Methodologies 
Babesia and Hepatozoon infections of dogs in a village of eastern Sudan were analyzed by using a single PCR and sequencing. Among 78 dogs, 5 were infected with Babesia canis rossi and 2 others were infected with B. canis vogeli. Thirty-three dogs were positive for Hepatozoon. Hepatozoon canis was detected by sequence analysis.
PMCID: PMC1287771  PMID: 16275954
14.  Molecular Detection of a New Anaplasma Species Closely Related to Anaplasma phagocytophilum in Canine Blood from South Africa 
Journal of Clinical Microbiology  2005;43(6):2934-2937.
Canine DNA samples from South Africa were found to contain 16S rRNA gene nucleotide and citrate synthase gene nucleotide and deduced amino acid sequences that were most similar to Anaplasma phagocytophilum: 98%, 66%, and 69% similarity, respectively. This suggests that a new Anaplasma species closely related to A. phagocytophilum occurs in Africa.
PMCID: PMC1151900  PMID: 15956424
15.  Transcriptional Control of BubR1 by p53 and Suppression of Centrosome Amplification by BubR1 
Molecular and Cellular Biology  2005;25(10):4046-4061.
Elimination of the regulatory mechanism underlying numeral homeostasis of centrosomes, as seen in cells lacking p53, results in abnormal amplification of centrosomes, which increases the frequency of chromosome segregation errors, and thus contributes to the chromosome instability frequently observed in cancer cells. We have previously reported that p53−/− mouse cells in prolonged culture undergo genomic convergence similar to that observed during tumor progression; early-passage p53−/− cells are karyotypically heterogeneous due to extensive chromosome instability associated with centrosome amplification, while late-passage p53−/− cells are aneuploid yet karyotypically homogeneous and chromosomally stable. Moreover, they contain numerically normal centrosomes. Through the microarray analysis of early- and late-passage p53−/− cells, we identified the BubR1 spindle checkpoint protein, which plays a critical role in suppression of centrosome amplification and stabilization of chromosomes in late-passage p53−/− cells. Up-regulation of BubR1 augments the checkpoint function, which effectively senses the spindle/chromosome aberrations associated with centrosome amplification. We further found that BubR1 transcription is largely controlled by p53. In early-passage p53−/− cells, BubR1 expression is low and the checkpoint function in response to microtubule toxin is considerably compromised. In late-passage cells, however, regaining of BubR1 expression restores the checkpoint function to mitotic aberrations caused by microtubule toxin. Our studies demonstrate the molecular aspect of genomic convergence in cultured cells, providing critical information for understanding the stepwise progression of tumors.
PMCID: PMC1087701  PMID: 15870277
16.  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 
Journal of Clinical Microbiology  2004;42(3):1353-1355.
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.
PMCID: PMC356832  PMID: 15004117
17.  Culture and Phenotypic Characterization of a Wolbachia pipientis Isolate 
Journal of Clinical Microbiology  2003;41(12):5434-5441.
The recent isolation of Wolbachia pipientis in the continuous cell line Aa23, established from eggs of a strain of the Asian tiger mosquito Aedes albopictus, allowed us to perform extensive characterization of the isolate. Bacterial growth could be obtained in C6/36, another A. albopictus cell line, at 28°C and in a human embryonic lung fibroblast monolayer at 28 and 37°C, confirming that its host cell range is broader than was initially thought. The bacteria were best visualized by Diff-Quik and May-Grünwald-Giemsa staining. Proteins from 213 to 18 kDa with two major protein bands of 65 and 25 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By Western blotting with specific polyclonal mouse and rabbit antisera, dominant immunoreactive antigens were found at approximately 100, 80, and 30 kDa. The genome size was calculated to be 1,790 ± 17 kb by pulsed-field gel electrophoresis. The sequence of the citrate synthase gene (gltA) of W. pipientis was determined by gene walking. Its position in the phylogenetic tree constructed with gltA confirmed that found in a phylogenetic tree constructed with 16S rRNA genes and that it belongs in the α subgroup of the class Proteobacteria and that it is closely related to but independent from the genera Ehrlichia, Anaplasma, and Neorickettsia.
PMCID: PMC308996  PMID: 14662922
18.  Serotyping Isolates of Anaplasma phagocytophilum by Using Monoclonal Antibodies 
Ten mouse monoclonal antibodies (MAbs) that react with Anaplasma phagocytophilum (the human granulocytic ehrlichiosis agent) Webster isolates were developed. Seven different isolates of A. phagocytophilum were subtyped with these MAbs. Western blot analysis revealed that these MAbs reacted mainly with 41- to 46-kDa Msp2 proteins. Six MAbs reacted with all isolates. Four other MAbs reacted with human isolates from Wisconsin, but not with human isolates from New York or with animal isolates. Three different serotypes were identified. These features may lead to the development of other specific MAbs in order to provide tools for antigenic characterization of human isolates of A. phagocytophilum.
PMCID: PMC193879  PMID: 12965936
19.  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 
Journal of Clinical Microbiology  2003;41(8):3494-3498.
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 1,136 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 1,678 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.
PMCID: PMC179768  PMID: 12904344
20.  Determination of the Nucleotide Sequences of Heat Shock Operon groESL and the Citrate Synthase Gene (gltA) of Anaplasma (Ehrlichia) platys for Phylogenetic and Diagnostic Studies 
The 1,670-bp nucleotide sequence of the heat shock operon groESL and the 1,236-bp sequence of the citrate synthase gene (gltA) of Anaplasma (Ehrlichia) platys were determined. The topology of the groEL- and gltA-based phylogenetic tree was similar to that derived from 16S rRNA gene analyses with distances. Both groESL- and gltA-based PCRs specific to A. platys were also developed based upon the alignment data.
PMCID: PMC120055  PMID: 12204973
21.  Citrate Synthase Gene Sequence: a New Tool for Phylogenetic Analysis and Identification of Ehrlichia 
Journal of Clinical Microbiology  2001;39(9):3031-3039.
The sequence of the citrate synthase gene (gltA) of 13 ehrlichial species (Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia muris, an Ehrlichia species recently detected from Ixodes ovatus, Cowdria ruminantium, Ehrlichia phagocytophila, Ehrlichia equi, the human granulocytic ehrlichiosis [HGE] agent, Anaplasma marginale, Anaplasma centrale, Ehrlichia sennetsu, Ehrlichia risticii, and Neorickettsia helminthoeca) have been determined by degenerate PCR and the Genome Walker method. The ehrlichial gltA genes are 1,197 bp (E. sennetsu and E. risticii) to 1,254 bp (A. marginale and A. centrale) long, and GC contents of the gene vary from 30.5% (Ehrlichia sp. detected from I. ovatus) to 51.0% (A. centrale). The percent identities of the gltA nucleotide sequences among ehrlichial species were 49.7% (E. risticii versus A. centrale) to 99.8% (HGE agent versus E. equi). The percent identities of deduced amino acid sequences were 44.4% (E. sennetsu versus E. muris) to 99.5% (HGE agent versus E. equi), whereas the homology range of 16S rRNA genes was 83.5% (E. risticii versus the Ehrlichia sp. detected from I. ovatus) to 99.9% (HGE agent, E. equi, and E. phagocytophila). The architecture of the phylogenetic trees constructed by gltA nucleotide sequences or amino acid sequences was similar to that derived from the 16S rRNA gene sequences but showed more-significant bootstrap values. Based upon the alignment analysis of the ehrlichial gltA sequences, two sets of primers were designed to amplify tick-borne Ehrlichia and Neorickettsia genogroup Ehrlichia (N. helminthoeca, E. sennetsu, and E. risticii), respectively. Tick-borne Ehrlichia species were specifically identified by restriction fragment length polymorphism (RFLP) patterns of AcsI and XhoI with the exception of E. muris and the very closely related ehrlichia derived from I. ovatus for which sequence analysis of the PCR product is needed. Similarly, Neorickettsia genogroup Ehrlichia species were specifically identified by RFLP patterns of RcaI digestion. If confirmed this technique will be useful in rapidly identifying Ehrlichia spp.
PMCID: PMC88292  PMID: 11526124
22.  Analysis of the 16S rRNA Gene Sequence of Anaplasma centrale and Its Phylogenetic Relatedness to Other Ehrlichiae 
The nucleotide sequence of the Anaplasma centrale 16S rRNA gene was determined and compared with the sequences of ehrlichial bacteria. The sequence of A. centrale was closely related to Anaplasma marginale by both level-of-similarity (98.08% identical) and distance analysis. A species-specific PCR was developed based upon the alignment data. The PCR can detect A. centrale DNA extracted from 10 infected bovine red blood cells in a reaction mixture. A. centrale DNA was amplified in the reaction, but not other related ehrlichial species.
PMCID: PMC96043  PMID: 11238202
23.  Detection of Ehrlichia platys DNA in Brown Dog Ticks (Rhipicephalus sanguineus) in Okinawa Island, Japan 
Journal of Clinical Microbiology  2000;38(11):4219-4221.
Thirty-two Rhipicephalus sanguineus females collected from eight free-roaming dogs in Okinawa Island, Japan, were examined for ehrlichial DNA by 16S rRNA-based PCR and subsequent sequencing. Partial sequences of Ehrlichia platys 16S rRNA (678 to 679 bp) were detected in three ticks (9.4%) from two dogs. This is the first report of detection of E. platys in Japan, and also the first report of detection in ticks.
PMCID: PMC87567  PMID: 11060094

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