Comparative Strain Analysis of Anaplasma phagocytophilum Infection and Clinical Outcomes in a Canine Model of Granulocytic Anaplasmosis
ACVIM 2008
D.G. Scorpio1; J.S. Dumler1; N.C. Barat1; J.A. Anastasio1; Daryn Daniluk2; Kristen Caterina2; Melissa Beall2; Ramaswamy Chandrashekar2
1Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2IDEXX Laboratories, Westbrook, ME, USA

A pilot study was conducted to determine which human or canine strain of Anaplasma phagocytophilum would reproduce clinical infection normally anticipated in dogs and other susceptible mammals with naturally-acquired granulocytic anaplasmosis.

Six hounds were inoculated IV with 1 x 106 of either A. phagocytophilum Webster (human), 98E4 Minnesota (canine), or E06 California (canine) strain bacteria. A. phagocytophilum was propagated in HL-60 (human leukemia) cells in vitro for 3 of the dog infections. Since infection in this manner introduces the potential confounder of HL-60 cells, we also used infected autologous neutrophils in the remaining 3 dogs. Once infected, dogs were monitored daily and clinical findings recorded. Clinical parameters were scored using a numerical grading system from 0-3, paying particular attention to signs of lethargy, anorexia, petechiation, lymphadenopathy, and fever. CBC, serum chemistry, and serology (IFA and SNAP® 4Dx®) were conducted between days 0 and 60. DNA was isolated and quantitative PCR performed. Inflammatory cytokine aberrations were evaluated using serum samples on the Luminex multi-analyte platform.

The most prominent clinical signs observed were generalized lymphadenopathy and scleral injection, with one dog (98E4 strain in neutrophils) developing fever lasting 4 days (103.9F-104.1F). Biochemistry parameters remained normal throughout the infection, with no changes in hepatic or renal functions. Most notable changes occurred in leukocyte and platelet counts, with prominent and sustained leukopenia and thrombocytopenia occurring among all dogs. Anaplasma morulae were noted in blood smear preparations between days 10-11. All dogs seroconverted by day 10-15 (IFA), and by day 17-21 (SNAP® 4Dx®), and all dogs developed infection (positive through and including day 60) with A. phagocytophilum as determined by qPCR using the msp2 gene target. Cytokine analysis revealed a 10-fold increase in IL-2, IL-18, and TNFα in the dog infected with A. phagocytophilum 98E4 strain in neutrophils.

There is substantial evidence to demonstrate that all strains produced infection, although canine A. phagocytophilum 98E4 reproduced clinical signs, hematologic changes, and inflammatory cytokine elevations most consistent with granulocytic anaplasmosis; therefore this strain will be used in future studies of A. phagocytophilum infection in dogs. Since animal numbers were small, additional studies are currently being carried out for definitive interpretation of pilot study results.

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Diana Scorpio

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