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).