title: Western and dot blotting analyses of Ehrlichia chaffeensis indirect fluorescent-antibody assay-positive and -negative human sera by using native and recombinant E. chaffeensis and E. canis antigens.
abstract: Human monocytic ehrlichiosis is an emerging infectious disease caused by Ehrlichia chaffeensis, a gram-negative obligatory intracellular bacterium closely related to E. canis. The immunoreactive recombinant fusion proteins rP28 and rP30 have become available after cloning and expressing of the 28- and 30-kDa major outer membrane protein genes of E. chaffeensis and E. canis, respectively. Western immunoblotting was performed to analyze the antibody responses of the 37 E. chaffeensis indirect fluorescent-antibody assay (IFA)-positive and 20 IFA-negative serum specimens with purified whole organisms, rP28, and rP30. All IFA-negative sera were negative with purified whole organisms, rP28, or rP30 by Western immunoblot analysis (100% relative diagnostic specificity). Of 37 IFA-positive sera, 34 sera reacted with any native proteins of E. chaffeensis ranging from 44 to 110 kDa, and 30 sera reacted with 44- to 110-kDa native E. canis antigens. The 28-kDa E. chaffeensis and 30-kDa E. canis native proteins were recognized by 25 IFA-positive sera. Fifteen IFA-positive sera reacted with rP28 by Western blot analysis, whereas 34 IFA-positive sera reacted with rP30 (92% relative diagnostic specificity), indicating that rP30 is more sensitive than rP28 for detecting the antibodies in IFA-positive sera. These 34 IFA-positive sera were positive by the dot blot assay with rP30, distinguishing them from IFA-negative sera. Except for three rP30-negative but IFA-positive specimens that instead showed an E. ewingii infection-like profile by Western immunoblotting, the results of Western and dot blot assays with rP30 matched 100% with the IFA test results. Densitometric analysis of dot blot reactions showed a positive correlation between the dot density and the IFA titer. These results suggest that rP30 antigen would provide a simple, consistent, and rapid serodiagnosis for human monocytic ehrlichiosis.
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210-1093, USA.
authors.forename: A
authors.initials: A
authors.lastname: Unver
authors.forename: Y
authors.initials: Y
authors.lastname: Rikihisa
authors.forename: N
authors.initials: N
authors.lastname: Ohashi
authors.forename: L C
authors.initials: LC
authors.lastname: Cullman
authors.forename: R
authors.initials: R
authors.lastname: Buller
authors.forename: G A
authors.initials: GA
authors.lastname: Storch
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D004285:Dogs; D004536:Ehrlichia / Q000276:immunology*; D018469:Ehrlichia chaffeensis / Q000276:immunology*; D016873:Ehrlichiosis / Q000175:diagnosis* / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins