Evaluation of heat-treating heartworm-positive canine serum samples during treatment with Advantage Multi® for Dogs and doxycycline

Molly D. Savadelis, Jennifer L. Roveto, Cameon M. Ohmes, Joe A. Hostetler, Terry L. Settje, Michael T. Dzimianski, Andrew R. Moorhead

The use of heat-treatment in canine and feline serum has been hypothesized to break the formation of antigen-antibody complexes, thereby freeing the heartworm antigen allowing for detection by commercially available heartworm antigen kits. While studies have analyzed the effect of heat-treating serum and plasma samples in the detection of heartworm antigen, these studies have not utilized necropsy verified results for validation. This study evaluated the use of heat-treating serum samples in experimentally infected dogs during adulticidal treatment in comparison with necropsy adult heartworm recovery. As part of a primary study, a total of 16 dogs were experimentally infected with 16 sexually mature adult heartworms using surgical transplantation, allocating 8 dogs in both the control and treated group. Treated dogs received 10 months of topical administration of Advantage Multi® for Dogs (10% Imidacloprid + 2.5% Moxidectin) every 4 weeks and 30 days of 10 mg/kg doxycycline BID. Blood samples were collected from all study animals prior to surgical transplantation of adult heartworms, on study days 0, 1, 3, 7, 14, 21, 28, and every 4 weeks thereafter for the duration of this study. Concentration of heartworm antigen was tested using the DiroCHEK® heartworm antigen test kit using serum samples both pre- and post-heat-treatment. Serum samples were heat-treated at 103 °C in a dry heat block for 10 min and centrifuging at 1818× g for 20 min. There were a total of 4 instances (days 56, 140, 224 and 252) in 3 treated dogs in which a serum sample converted from negative for the detection of heartworm antigen prior to heat-treatment to positive for the detection of heartworm antigen post-heat-treatment. At necropsy, these dogs had no adult heartworms recovered and were all negative on antigen testing prior to and after heat treatment. There was 100% accuracy in the detection of either no infection, or 1-2 adult heartworm infections using the DiroCHEK in serum samples with and without heat-treatment at the time of necropsy. The DiroCHEK accurately diagnosed all dogs with live adults recovered at necropsy as heartworm antigen positive and all those dogs with no live adults recovered at necropsy as heartworm antigen negative without the use of heat-treatment for samples taken on the day of necropsy. Therefore, these results indicate that the use of heat-treating serum samples did not provide data of any additional value in the diagnosis of heartworm-positive dogs receiving treatment in this study. Additionally, these results may indicate that the conversion of serum samples from negative to positive for the presence of heartworm antigen with heat-treatment may not always accurately diagnose live adult heartworm infections since no adult heartworms were recovered at necropsy for those dogs in which a conversion event occurred. These conversion events may be detecting residual antigen leftover after all adult worms have died or may even be detecting off- target antigens, which have been denatured during heat-treatment. While a necropsy was not performed at the time of the conversion events, no live adult worms were recovered from any of the dogs in which a conversion event occurred earlier in treatment.