2017 ACVIM Forum Research Abstract Program

Blood samples from dogs were collected with owner permission for use in a vector borne agent prevalence study in Mexico. After collection, the aliquot of blood was placed in EDTA, and then 300 lL was blotted onto filter paper (VWR Ò Grade 413), allowed to dry, and stored in individual sterile bags. The filter paper samples were stored at room temperature, and the EDTA blood was stored at -80°C until assayed at the central laboratory. A glyceraldehyde 3-phosphate dehydrogenase (GAPDH) quantitative PCR was used to estimate DNA yields in the different experiments. Different buffers and incubation periods were assessed in experiments using the entire filter paper in different washing procedures, and it was determined that an overnight PBS wash gave the best results (Method 1). In Method 2, a commercially available kit (QIAamp Ò DNA Blood Mini Kit Dried Blood Spot (DBS) protocol) was performed as per the manufacturer's instructions. Once Method 1 was optimized, 12 samples previously shown to be positive for E. canis DNA in blood by conventional PCR assay and 3 known negative samples were thawed and used to make 2 filter paper blots which were allowed to dry overnight before being subjected to DNA extraction Methods 1 and 2. The whole blood and filter paper derived DNA extracts were then assayed in the GAPDH qPCR and the Ehrlichia spp. PCR assay. A total of 11 whole blood samples were PCR positive with bands characterized as strong (n = 4), medium (n = 3), or weak (n = 4). One sample using Method 1 was excluded due to the washing of an incorrect blot.