AJVR, Vol 71, No. 10, October 2010 1195 C anine granulocytic ehrlichiosis, caused by Ehrlichia ewingii, is characterized by fever, lethargy, anorex- ia, weight loss, vomiting, diarrhea, lameness, neutro- philic polyarthritis, severe but transient thrombocyto- penia, and transient mild nonregenerative anemia with Evaluation of peptide- and recombinant protein–based assays for detection of anti–Ehrlichia ewingii antibodies in experimen- tally Thomas P. O’Connor, PhD; Jill M. Saucier, BS; Daryn Daniluk, BS; Brett A. Stillman, PhD; Regis Krah, PhD; Yasuko Rikihisa, PhD; Qingming Xiong, PhD; Michael J. Yabsley, PhD; Dustin S. Adams, DVM; Pedro Paulo V. P. Diniz, DVM, PhD; Edward B. Breitschwerdt, DVM; Stephen D. Gaunt, DVM, PhD; Ramaswamy Chandrashekar, PhD Objective—To evaluate microtiter-plate format ELISAs constructed by use of different diagnostic targets derived from the Ehrlichia ewingii p28 outer membrane protein for detection of E ewingii antibodies in experimentally and naturally infected dogs. Sample Population—Serum samples from 87 kenneled dogs, 9 dogs experimentally in- fected with anti–E ewingii, and 180 potentially naturally exposed dogs from Missouri. Procedures—The capacities of the synthetic peptide and truncated recombinant protein to function as detection reagents in ELISAs were compared by use of PCR assay, western blot analysis, and a full-length recombinant protein ELISA. Diagnostic targets included an E ewingii synthetic peptide (EESP) and 2 recombinant proteins: a full-length E ewingii outer membrane protein (EEp28) and a truncated E ewingii outer membrane protein (EETp28). Results—A subset of Ehrlichia canis–positive samples cross-reacted in the EEp28 ELISA; none were reactive in the EESP and EETp28 ELISAs. The EESP- and EETp28-based ELISAs detected E ewingii seroconversion at approximately the same time after infection as the EEp28 ELISAs. In a field population, each of the ELISAs identified the same 35 samples as reactive and 27 sam- ples as nonreactive. Anaplasma and E canis peptides used in a commercially available ELISA platform did not detect anti–E ewingii antibodies in experimentally infected dogs. Conclusions and Clinical Relevance—The EESP and EETp28 ELISAs were suitable for specifically detecting anti–E ewingii antibodies in experimentally and naturally infected dogs. (Am J Vet Res 2010;71:1195–1200) ineffective erythropoiesis. 1–3 Ehrlichia ewingii infection has been reported in dogs in several states, including Missouri, Oklahoma, North Carolina, and Virginia. 4–6 Amblyomma americanum, which is distributed through- out the southeastern and south central United States, is the only confirmed vector for the transmission of E ewingii. 4,7,8 Serologic tests such as the IFA have been used di- agnostically for detection of antibodies against several ehrlichial pathogens. However, because E ewingii has not been cultured in vitro, a sensitive and specific se- rologic test to detect E ewingii antibody in dogs is not presently available. Ehrlichia chaffeensis and Ehrlichia Received June 30, 2009. Accepted August 29, 2009. From the Department of Immunoassay R&D, IDEXX Laboratories In- corporated, 1 Indexx Dr, Westbrook, ME 04092 (O’Connor, Saucier, Daniluk, Stillman, Krah, Chandrashekar); the Department of Vet- erinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210 (Rikihisa, Xiong); Southeastern Cooperative Wildlife Disease Study, Department of Population Health, College of Veterinary Medicine (Yabsley, Adams), and War- nell School of Forestry and Natural Resources (Yabsley), University of Georgia, Athens, GA 30602; the Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606 (Diniz, Breitschwerdt); and Clinical Pathology Laboratory, School of Veterinary Medicine, Louisiana State Univer- sity, Baton Rouge, LA 70803 (Gaunt). Presented in part at the annual meetings of the American College of Veterinary Internal Medicine, Seattle, June 2007, and Montreal, June 2009. Address correspondence to Dr. O’Connor (tom-oconnor@idexx. com). ABBREVIATIONS EEp28 Recombinant full-length Ehrlichia ewingii outer membrane protein EESP Ehrlichia ewingii synthetic peptide EETp28 Recombinant truncated E ewingii outer membrane protein IFA Immunofluorescence assay OMP Outer membrane protein Unauthenticated | Downloaded 08/20/22 10:22 PM UTC