Transcriptional analysis of p30 major outer membrane protein genes of Ehrlichia canis in naturally infected ticks and sequence analysis of p30-10 of E canis from diverse geographic regions. (1/54)

Rhipicephalus sanguineus ticks transmit Ehrlichia canis, the etiologic agent of canine ehrlichiosis. In experimentally infected ticks, only p30-10 transcript was detected among 22 p30 paralogs encoding immunodominant major outer membrane P30 proteins of E. canis. The present study revealed transcription of p30-10 by E. canis in naturally infected ticks and sequence conservation of p30-10 genes for E. canis from diverse geographic regions.  (+info)

Cloning and characterization of an Ehrlichia canis gene encoding a protein localized to the morula membrane. (2/54)

A gene encoding a 23.5-kDa ehrlichial morula membrane protein designated MmpA was cloned by screening an Ehrlichia canis expression library with convalescent dog sera, which resulted in three positive clones. Sequence analysis of the insert DNAs from all three clones indicated an open reading frame with a size of 666 bp that encodes MmpA. The structural analysis of MmpA indicated that it is a transmembrane protein with extreme hydrophobicity. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated the presence of a single copy of the mmpA gene in E. canis and Ehrlichia chaffeensis but not in the human granulocytic ehrlichiosis agent. The mmpA gene was amplified, cloned, and expressed as a fusion protein. Polyclonal antibodies to the recombinant protein (rMmpA) were raised in rabbits. Western blot analysis of E. canis and E. chaffeensis lysates with the anti-rMmpA serum resulted in the presence of an MmpA band only in E. canis, not in E. chaffeenesis. Sera from dogs which were either naturally or experimentally infected with E. canis recognized the recombinant protein. Double immunofluorescence confocal microscopy studies demonstrated that MmpA was localized mainly on the morula membrane of E. canis. Since the morula membrane is the interface between the ehrlichial growing environment and the host cytoplasm, MmpA may play a role in bacterium-host cell interactions.  (+info)

Characterization of the major antigenic protein 2 of Ehrlichia canis and Ehrlichia chaffeensis and its application for serodiagnosis of ehrlichiosis. (3/54)

Canine monocytic ehrlichiosis, caused by Ehrlichia canis or Ehrlichia chaffeensis, can result in clinical disease in naturally infected animals. Coinfections with these agents may be common in certain areas of endemicity. Currently, a species-specific method for serological diagnosis of monocytic ehrlichiosis is not available. Previously, we developed two indirect enzyme-linked immunosorbent assays (ELISAs) using the major antigenic protein 2 (MAP2) of E. chaffeensis and E. canis. In this study, we further characterized the conservation of MAP2 among various geographic isolates of each organism and determined if the recombinant MAP2 (rMAP2) of E. chaffeensis would cross-react with E. canis-infected dog sera. Genomic Southern blot analysis using digoxigenin-labeled species-specific probes suggested that map2 is a single-copy gene in both Ehrlichia species. Sequences of the single map2 genes of seven geographically different isolates of E. chaffeensis and five isolates of E. canis are highly conserved among the various isolates of each respective ehrlichial species. ELISA and Western blot analysis confirmed that the E. chaffeensis rMAP2 failed to serologically differentiate between E. canis and E. chaffeensis infections.  (+info)

Sequence and expression analysis of virB9 of the type IV secretion system of Ehrlichia canis strains in ticks, dogs, and cultured cells. (4/54)

Ehrlichia canis virB9 was cloned and expressed. The sequences of virB9 from six geographic locations were identical. virB9 was transcribed by E. canis in dogs, ticks, and cell culture. Infected dogs had antibodies to recombinant VirB9, indicating that VirB9 was produced by E. canis in dogs and was antigenic.  (+info)

The relationship between the degree of thrombocytopenia and infection with Ehrlichia canis in an endemic area. (5/54)

Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis. In order to evaluate platelet counts as a screening test for E. canis in an endemic area, 217 whole blood samples from dogs were divided into three groups: 71 non-thrombocytopenic samples (group A, platelet counts greater than 200 000/mL) and 146 thrombocytopenic samples (less than 200 000/mL). The thrombocytopenic group was further divided into 62 with platelet counts between 100 000-200 000/mL (Group B) and 84 samples with less than 100 000 platelets/mL (Group C). All samples were examined for the presence of a segment of the Ehrlichia canis 16S rRNA gene using a nested polymerase chain reaction. Sixty-seven of the 217 samples (30.9%) were positive for the presence of the E. canis 16S rRNA gene; 53 (63.1%) of the group C samples and 13 (21%) of group B. Only one (1.4%) of the non-thrombocytopenic samples (Group A) was positive. These data support the concept that platelet counts may be a good screening test for canine monocytic ehrlichiosis, and that the magnitude of thrombocytopenia may increase the reliability of diagnosis.  (+info)

Comparison of simultaneous splenic sample PCR with blood sample PCR for diagnosis and treatment of experimental Ehrlichia canis infection. (6/54)

This report presents evidence that dogs recover from acute canine monocytic ehrlichiosis (CME) after 16 days of doxycycline treatment (10 mg/kg of body weight every 24 h). Blood PCR was as valuable as splenic aspirate PCR for early diagnosis of acute CME. Splenic aspirate PCR was, however, superior to blood PCR for the evaluation of ehrlichial elimination.  (+info)

Evaluation of antibiotic susceptibilities of Ehrlichia canis, Ehrlichia chaffeensis, and Anaplasma phagocytophilum by real-time PCR. (7/54)

We determined MICs of antibiotics against Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia canis by real-time quantitative PCR. The doubling times of the organisms were established: 19 h for E. chaffeensis, 26 h for A. phagocytophilum, and 28 h for E. canis. In comparison to the reference method for determining sensitivities, which uses Diff-Quick staining, our PCR assay was very sensitive and specific. We confirmed that doxycycline and rifampin are highly active against these bacteria and found variable susceptibilities to fluoroquinolones; A. phagocytophilum was susceptible, but E. canis and E. chaffeensis were only partly susceptible. Beta-lactam compounds, cotrimoxazole, macrolide compounds, and telithromycin showed no activity against any of the three organisms. Thiamphenicol was found to be more active than chloramphenicol. For the first time, we showed that these three species have numerous point mutations in their 23S RNA genes, with those at positions 754, 2057, 2058, 2059, and 2611 (Escherichia coli numbering) known to confer resistance to macrolide compounds in other bacteria. The role of each of these mutations in resistance to these drugs should be investigated in the future. Our study confirms previous reports that quantitative PCR is a reliable method for determining antibiotic susceptibility; therefore, it might be useful for screening new drugs.  (+info)

An immunoreactive 38-kilodalton protein of Ehrlichia canis shares structural homology and iron-binding capacity with the ferric ion-binding protein family. (8/54)

Ehrlichiae are tick-transmitted, gram-negative, obligately intracellular bacteria that live and replicate in cytoplasmic vacuoles, but little is known about iron acquisition mechanisms necessary for their survival. In this study, a genus-conserved immunoreactive ferric ion-binding protein (Fbp) of Ehrlichia canis was identified and its iron-binding capability was investigated. E. canis Fbp was homologous to a family of periplasmic Fbp's involved in iron acquisition and transport in gram-negative bacteria. E. canis Fbp had a molecular mass (38 kDa) consistent with those of Fbp's in other bacteria and exhibited substantial immunoreactivity in its native conformation. The predicted three-dimensional structure of E. canis Fbp demonstrated conservation of important Fbp family structural motifs: two domains linked with a polypeptide "hinge" region. Under iron-binding conditions, the recombinant Fbp exhibited an intense red color and an absorbance spectrum indicative of iron binding, and it bound Fe(III) but not Fe(II). Fbp was observed primarily in the cytoplasm of the reticulate forms of E. canis and Ehrlichia chaffeensis but was notably found on extracellular morula fibers in morulae containing dense-cored organisms. Although expression of Fbp is regulated through an operon of three functionally linked genes in other gram-negative bacteria, the absence of an intact fbp operon in Ehrlichia spp. suggests that genes involved in ehrlichial iron acquisition have been subject to reductive evolution.  (+info)