Ehrlichia canis gp200 Contains Dominant Species-Specific Antibody Epitopes in Terminal Acidic Domains▿

Species-specific antibody epitopes within several major immunoreactive protein orthologs of Ehrlichia species have recently been identified and molecularly characterized. In this study, dominant B-cell epitopes within the acidic (pI 5.35) ankyrin repeat-containing 200-kDa major immunoreactive protein (gp200) of Ehrlichia canis were defined. The E. canis gp200 gene (4,263 bp; 1,421 amino acids) was cloned and expressed as four (N-terminal, 1,107 bp; N-internal, 910 bp; C-internal, 1,000 bp; and C-terminal, 1,280 bp) overlapping recombinant proteins. The N-terminal, C-internal, and C-terminal polypeptides (369, 332, and 426 amino acids, respectively) were strongly recognized by antibody, and the major epitope(s) in these polypeptides was mapped to four polypeptide regions (40 to 70 amino acids). Smaller overlapping recombinant polypeptides (14 to 15 amino acids) spanning these regions identified five strongly immunoreactive species-specific epitopes that exhibited conformational dependence. The majority of the epitopes (four) were located in two strongly acidic (pI 4 to 4.9) domains in the distal N- and C-terminal regions of the protein flanking the centralized ankyrin domain-containing region. The amino acid content of the epitope-containing domains included a high proportion of strongly acidic amino acids (glutamate and aspartate), and these domains appear to have important biophysical properties that influence the antibody response to gp200

Enzyme-Linked Immunosorbent Assay with Conserved Immunoreactive Glycoproteins gp36 and gp19 Has Enhanced Sensitivity and Provides Species-Specific Immunodiagnosis of Ehrlichia canis Infection

Ehrlichia canis is the primary etiologic agent of canine monocytic ehrlichiosis, a globally distributed and potentially fatal disease of dogs. We previously reported on the identification of two conserved major immunoreactive antigens, gp36 and gp19, which are the first proteins to elicit an E. canis-specific antibody response, and gp200 and p28, which elicit strong antibody responses later in the acute phase of the infection. In this report, the sensitivities and specificities of five recombinant E. canis proteins for the immunodiagnosis of E. canis infection by an enzyme-linked immunosorbent assay (ELISA) were evaluated. Recombinant polypeptides gp36, gp19, and gp200 (N and C termini) exhibited 100% sensitivity and specificity for immunodiagnosis by the recombinant glycoprotein ELISA compared with the results obtained by an indirect fluorescent-antibody assay (IFA) for the detection of antibodies in dogs that were naturally infected with E. canis. Moreover, the enhanced sensitivities of gp36 and gp19 for immunodiagnosis by the recombinant glycoprotein ELISA compared to those obtained by IFA were demonstrated with dogs experimentally infected with E. canis, in which antibodies were detected as much as 2 weeks earlier, on day 14 postinoculation. gp36 and gp19 were not cross-reactive with antibodies in sera from E. chaffeensis-infected dogs and thus provided species-specific serologic discrimination between E. canis and E. chaffeensis infections. This is the first demonstration of the improved detection capability of the recombinant protein technology compared to the capability of the “gold standard” IFA and may eliminate the remaining obstacles associated with the immunodiagnosis of E. canis infections, including species-specific identification and the lack of sensitivity associated with low antibody titers early in the acute phase of the infection

Identification of a Glycosylated Ehrlichia canis 19-Kilodalton Major Immunoreactive Protein with a Species-Specific Serine-Rich Glycopeptide Epitope

Ehrlichia canis has a small subset of major immunoreactive proteins that includes a 19-kDa protein that elicits an early Ehrlichia-specific antibody response in infected dogs. We report herein the identification and molecular characterization of this highly conserved 19-kDa major immunoreactive glycoprotein (gp19) ortholog of the Ehrlichia chaffeensis variable-length PCR target (VLPT) protein. E. canis gp19 has substantial carboxyl-terminal amino acid homology (59%) with E. chaffeensis VLPT and the same chromosomal location; however, the E. chaffeensis VLPT gene (594 bp) has tandem repeats that are not present in the E. canis gp19 gene (414 bp). Consistent with other ehrlichial glycoproteins, the gp19 protein exhibited a larger-than-predicted mass (∼3 kDa), O-linked glycosylation sites were predicted in an amino-terminal serine/threonine/glutamate (STE)-rich patch (26 amino acids), carbohydrate was detected on the recombinant gp19 protein, and the neutral sugars glucose and galactose were detected on the recombinant amino-terminal polypeptide. E. canis gp19 composition consists of five predominant amino acids, cysteine, glutamate, tyrosine, serine, and threonine, concentrated in the STE-rich patch and a carboxyl-terminal domain predominated by cysteine and tyrosine (55%). The amino-terminal STE-rich patch contained a major species-specific antibody epitope strongly recognized by serum from an E. canis-infected dog. The recombinant glycopeptide epitope was substantially more reactive with antibody than the synthetic (nonglycosylated) peptide, and periodate treatment of the recombinant glycopeptide epitope reduced its immunoreactivity, demonstrating the importance of a carbohydrate immunodeterminant(s). The gp19 protein was present on reticulate and dense-cored cells, and it was found extracellularly in the fibrillar matrix and associated with the morula membrane, the host cell cytoplasm, and the nucleus

The genome of obligately intracellular Ehrlichia canis reveals themes of complex membrane structure and immune evasion strategies

Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, α-proteobacterium is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, and 17 putative pseudogenes, and a substantial proportion of non-coding sequence (27 percent). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences, and a unique serine-threonine bias associated with the potential for O-glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly G:C tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Proteins associated with pathogen-host interactions were identified including a small group of proteins (12) with tandem repeats and another with eukaryotic-like ankyrin domains (7)

The Genome of the Obligately Intracellular Bacterium Ehrlichia canis Reveals Themes of Complex Membrane Structure and Immune Evasion Strategies

Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, α-proteobacterium, is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, 17 putative pseudogenes, and a substantial proportion of noncoding sequence (27%). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences and a unique serine-threonine bias associated with the potential for O glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly(G-C) tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Genes associated with pathogen-host interactions were identified, including a small group encoding proteins (n = 12) with tandem repeats and another group encoding proteins with eukaryote-like ankyrin domains (n = 7)

Diagnóstico sorológico de erliquiose canina com antígeno brasileiro de Ehrlichia canis Serological diagnosis of canine monocytic ehrlichiosis with Brazilian antigen of Ehrlichia canis

O presente trabalho relata o isolamento de Ehrlichia canis em cultivo de células DH82 e posterior padronização da Reação de Imunofluorescência Indireta (RIFI). Leucócitos de uma cadela experimentalmente infectada com o isolado Jaboticabal de E. canis foram inoculados em cultivo de células DH82. A inoculação foi monitorada após a segunda semana, a cada 5-6 dias, através de exames citológicos e pela amplificação de um fragmento do gene dsb de Ehrlichia pela Reação em Cadeia pela Polimerase (PCR) para confirmação da infecção. A cultura apresentou-se positiva aos 27 dias pós-inoculação pela PCR e aos 28 dias pela citologia. No 33o dia pós-inoculação, observou-se 20% de células infectadas e, aos 53 dias, 60% de infecção. Atualmente, o isolado encontra-se estabelecido em células DH82, com várias passagens atingindo 90-100% de células infectadas entre 7-10 dias após a inoculação. Após o seqüenciamento do produto de PCR, o isolado apresentou-se 100% similar à seqüência correspondente de E. canis depositada no GenBank. As células infectadas foram utilizadas como antígeno para a padronização da RIFI para detecção da infecção em cães.<br>The present study describes a successful isolation of Ehrlichia canis and its establishment in DH82 cells, followed by the development of an Indirect Fluorescent Antibodies Test (IFAT). Leukocytes collected from an experimentally infected dog with the Jaboticabal strain of E. canis were used to inoculate a DH82 cell monolayer. Two weeks later, the inoculated culture was checked for infectivity, every 5-6 days by both cytological staining and PCR, targeting a fragment of the dsb gene. The cell culture showed to be infected by Ehrlichia on day 27 by PCR and on day 28 by cytological staining. By the day 33, the infection rate reached 20% and on day 53, 60%. Currently, the isolate is established in DH82 cells, with several passages reaching 90-100% of infected cells, within 7 to 10 days post inoculation. After sequencing, the amplicon was identical to other E. canis corresponding sequences available in the GenBank. DH82 infected cells were used to standardize an IFAT for the diagnosis of canine ehrlichiosis