2. Materials and Methods
In August 2020, an 11-year-old, castrated male, Pitbull dog from McAllen, Hidalgo County, southern Texas, was presented to the veterinarian with a history of ocular irritation for approximately 2-3 weeks. It was diagnosed with a perforating corneal ulceration of the right eye. The dog was born in McAllen and had never left the Rio Grande River Valley, Texas. The animal tested antigen-positive for heartworm,Dirofilaria immitis , during pre-anesthetic work-up, despite somewhat compliant chemoprophylaxis with an ivermectin-based monthly product. An enucleation was performed, and the eye was fixed in 10% neutral buffered formalin and submitted to the Texas A&M Veterinary Medical Diagnostic Laboratory for histologic processing and examination.
Genomic DNA was extracted using a Qiagen FFPE Tissue DNA extraction kit (Qiagen, USA) according to manufacturer’s instructions. Polymerase chain reaction (PCR) was performed in 25 µL reactions containing 0.25μM of each primer, 1x GoTaq® Green Master Mix (Promega Corporation, Madison, Wisconsin, United States) and 2.5 µL of DNA template. The cytochrome oxidase c subunit 1 (cox1 ) gene was amplified using the forward primer COIintF: 5’-TGA TTG GTG GTT TTG GTA A-3’ and reverse primer COIintR: 5’-ATA AGT ACG AGT ATC AAT ATC- 3’ (Casiraghi, Anderson, Bandi, Bazzocchi, & Genchi, 2001; Otranto et al., 2015; Otranto et al., 2011),. The cycling conditions included an initial denaturation step at 95℃ for 2 minutes, followed by 40 cycles at 95°C for 45 seconds, 52℃ at 45 seconds, and 72℃ for 90 seconds, and a final extension step at 72°C for 5 minutes. PCR products were purified using E.Z.N.A.® Cycle Pure Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions, followed by Sanger sequencing. Phylogenetic analysis was performed in MEGA X using the Maximum Likelihood method and General Time Reversible, Gamma distributed as the best fit model (Kumar, Stecher, Li, Knyaz, & Tamura, 2018).