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 gibsoni; distribution; Haemaphysalis longicornis
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.
Anaplasma marginale; cattle; groEL; Msp1a; Philippines
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.
Cervus nippon yesoensis; hemoplasma; Japan; phylogeny
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.
16S rRNA gene; Anaplasma bovis; citrate synthase gene (gltA); heat-shock operon gene (groEL); phylogeny
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.
cattle; necrotic lymphadenitis; ruminal tympany; zygomycosis
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
cattle; direct PCR; hemoplasma; productivity
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.
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.
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.
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.
dislocation; foal; umbilical infection
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.
horse; ileus; mosapride; small intestine
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.