Multiple antigen target approach using the Accuplex4 BioCD system to detect Borrelia burgdorferi antibodies in experimentally infected and vaccinated dogs.

The primary objective of our study was to optimize detection of serum antibodies to Borrelia burgdorferi using a new commercial automated fluorescence system (Accuplex4 BioCD system, Antech Diagnostics, Lake Success, New York). The system used multiple natural and artificial peptides-outer surface proteins (OspA, OspC, OspF), an outer membrane protein (P39), and a proprietary synthetic peptide (small Lyme peptide [SLP])-and the results were compared with a commercially available enzyme-linked immunosorbent assay that uses a proprietary peptide (C6). Sera from 4 groups were evaluated: dogs vaccinated with 1 of 3 commercially available vaccines (n = 18); dogs infested with adult Ixodes scapularis (black-legged tick; n = 18); dogs previously vaccinated and then infested with I. scapularis (n = 18); and dogs with B. burgdorferi infection that were then vaccinated (n = 14). All of the vaccines evaluated induced OspA responses. However, antibodies against OspF or C6 were not induced in any of the vaccinated dogs. Additionally, the OspF antibodies had 100% sensitivity and specificity when compared to antibodies against C6 peptide. In B. burgdorferi-infected dogs, antibodies against OspC and SLP were detected in serum sooner than antibodies against the other targets. Low levels of antibodies against OspA developed in 6 of 14 B. burgdorferi-infected, unvaccinated dogs and had the shortest duration compared to the other antibodies. Detection of antibody responses to multiple B. burgdorferi targets with this system can be used to help differentiate vaccinated dogs from exposed dogs as well as acute infection from chronic infection.

Detection of antibodies against Anaplasma phagocytophilum in dogs using an automated fluorescence based system.

Infection with Anaplasma phagocytophilum can cause significant illness in some dogs and accurate diagnostic assays are needed. The objectives of the study were to optimize an automated fluorescence system for the detection of antibodies against A. phagocytophilum in canine serum. Serum and blood was collected temporally from seven dogs inoculated parenterally with culture-derived A. phagocytophilum and from 36 dogs exposed to wild-caught, adult Ixodes scapularis for 7 days. The system was optimized using the samples from the parenterally inoculated dogs. The ability to detect antibodies against A. phagocytophilum in the I. scapularis exposed dogs by the automated system was compared with a diagnostic kit (ELISA) and an indirect fluorescent antibody assay (IFA). Each blood sample was also assayed for A. phagocytophilum DNA by polymerase chain reaction (PCR). Of the 36 dogs exposed to I. scapularis, A. phagocytophilum DNA was amplified from blood from 22 dogs by PCR with first positive results occurring on weeks 1 (seven dogs), 2 (nine dogs), 3 (four dogs), 4 (one dog), or 5 (one dog). PCR results were positive prior to detection of antibodies in any of the three antibody assays for 19 dogs. The automated fluorescence system and IFA detected antibodies against A. phagocytophilum earlier than the ELISA. In conclusion, A. phagocytophilum PCR assays on blood are indicated in dogs with suspected acute anaplasmosis if serum antibody assays are negative.

Use of an automated system for detection of canine serum antibodies against Ehrlichia canis glycoprotein 36.

Ehrlichia canis is the most common cause of monocytotropic ehrlichiosis in dogs around the world. The purpose of the present study was to validate a new automated fluorescence system (Accuplex4™ BioCD system; Antech Diagnostics, Lake Success, New York) to detect antibodies against the E. canis immunodominant glycoprotein 36 (gp36). Sera and blood samples (ethylenediamine tetra-acetic acid) were collected from mixed sex beagles ( n = 8) on days 0, 3, 7, 10, 14, 17, 21, 28, 42, 49, 56, 63, 70, 77, 84, and 98 after intravenous inoculation with culture-derived E. canis. Sera were assayed using the Accuplex4 BioCD system (Accuplex4), an E. canis indirect fluorescent antibody test (IFAT), and a commercially available kit. A complete blood cell count and a proprietary E. canis polymerase chain reaction (PCR) were performed on each blood sample. On the day thrombocytopenia was first detected for each dog, E. canis DNA was amplified from blood of all dogs. At those times, E. canis antibodies were detected in 7 of 8 dogs by the Accuplex4, 1 of 8 dogs by the commercial kit, and 4 of 8 dogs by IFAT. Ehrlichia canis DNA was amplified from blood before seroconversion in any antibody assay for 6 dogs. Antibodies against gp36 were detected by Accuplex4 within 3 days of PCR-positive test results and were detected up to 25 days sooner than the commercial kit. After starting doxycycline treatment, E. canis DNA was no longer amplified by PCR assay, but serum antibodies remained detectable by all assays.