Samples
Large volume serum samples were available from six (3 male and 3 female) domestic short-haired cats experimentally infected at 10 months of age with Dirofilaria immitis by subcutaneous inoculation of third-stage larvae (L3) and confirmed to be infected by recovery of adult worms at necropsy or confirmation of histological lesions. Briefly, trickle infection of a total of 100 L3 of D. immitis was performed by subcutaneous inguinal inoculation of each cat a total of four times, on study days 7, 14, 21, and 28, with 25 L3 (Missouri strain) harvested shortly prior to inoculation from infected Aedes aegypti mosquitoes (Liverpool strain). Whole blood samples were collected from the jugular vein or, rarely, the cephalic vein, of each cat on days 84, 112, 140, 168, 196, and 224 directly into vacuum tubes containing either EDTA or no additive. Cats were cared for through Oklahoma State University’s Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal resources program throughout the study; all research procedures followed a detailed animal care and use protocol approved by Oklahoma State University’s Institutional Animal Care and Use Committee. Anti-coagulated whole blood was assayed for microfilaria by modified Knott’s test and by real-time PCR for Wolbachia spp. as previously described [11, 12]. For tubes without additive, blood was allowed to clot, the serum separated by centrifugation, placed into aliquots, and stored at −80°C until further use.
Antibody testing
Antibody testing was conducted using a commercial assay (Solo Step® FH, HESKA) according to manufacturer’s instructions, and by a fee-for-service reference laboratory (ANTECH Diagnostics) using a commercially available microtiter plate assay (Synbiotics Corporation, Zoetis). The reference laboratory tested each sample in triplicate and provided optical density (O.D.) results from each well as determined on spectrophotometry as well as corresponding positive and negative control sample wells.
Antigen testing
All antigen testing was conducted using commercial assays according to manufacturers’ instructions, with the exception that samples were tested before and after heat treatment on each assay. For heat treatment, serum samples were placed in a heat block at 103°C for 10 minutes, the resultant coagulum centrifuged, and the supernatant used in each commercial assay. Test kits evaluated before and after heat treatment included enzyme linked immunosorbent assay (ELISA) in microtiter plate formats (DiroCHEK®, Synbiotics Corporation, Zoetis; PetChek® Heartworm PF Antigen Test, IDEXX Laboratories, Inc.), membrane bound ELISAs (SNAP® Feline Heartworm® Test, IDEXX Laboratories, Inc.), and lateral flow immunochromatographic tests (WITNESS® HW, Synbiotics Corporation, Zoetis). In addition, O.D. readings were obtained by spectrophotometry before and after heat treatment for one of the microtiter plate assays (PetChek® Heartworm PF Antigen Test, IDEXX Laboratories, Inc.) according to manufacturer’s directions.
Comparison of test results
Optical density from microtiter plate assay (PetChek® Heartworm PF Antigen Test, IDEXX Laboratories, Inc.) and mean O.D. of triplicate antibody tests (ANTECH Diagnostics) for each cat for all days on which antigen was detected in any one cat and for cats on each of three individual study days (study days 168, 196, and 224) were compared using one-way ANOVA in Excel 2007 (Microsoft Office) with significance assigned at alpha = 0.05 [13]. An antigen/antibody ratio was also determined for each cat on days 168, 196, and 224 using O. D. and mean O. D., respectively.