PCR-Based Quantification of Borrelia burgdorferi Organisms in Canine Tissues over a 500-Day Postinfection Period

Abstract

ABSTRACT Borrelia burgdorferi infection in beagle dogs was studied quantitatively with skin punch biopsy samples and blood samples collected at 4- and 2-week intervals, respectively, over a 500-day period. Thereafter, 25 tissue samples of each dog were collected for further analysis. Starting at day 120 after tick challenge, 12 dogs were treated with antibiotics (azithromycin, ceftriaxone, or doxycycline) for 30 consecutive days. Four dogs received no antibiotic therapy. Quantification of B. burgdorferi DNA was done with an ABI Prism 7700 Sequence Detection System with oligonucleotide primers and a fluorescence-labeled probe designed to specifically amplify a fragment of the ospA gene of B. burgdorferi strain N40. All 16 dogs became infected with B. burgdorferi after tick challenge. In skin biopsy samples, spirochete numbers peaked at day 60 postinfection (<1.5 × 10 6 organisms per 100 μg of extracted DNA), at the same time when clinical signs of arthritis developed in 11 of 16 dogs, and decreased to almost undetectable levels during the following 6 months. The number of B. burgdorferi organisms detected in skin biopsy samples was inversely correlated with the antibody levels measured by enzyme-linked immunosorbent assay. Antibiotic treatment reduced the amount of detectable spirochete DNA in skin tissue by a factor of 1,000 or more. At the end of the experiment, B. burgdorferi DNA was detectable at low levels (10 2 to 10 4 organisms per 100 μg of extracted DNA) in multiple tissue samples regardless of treatment. However, more tissue samples of untreated dogs than of antibiotic-treated dogs were positive, and tissue samples of untreated dogs also were positive by culture. Only 1.6% of 576 blood samples of all dogs were positive for B. burgdorferi by PCR.