2.2. Diagnostic testing

Diagnostic assays were performed by three different research teams, each masked to the others’ results. One team collected samples and performed the point-of-care testing at each shelter; a second team performed point-of-care and microtiter plate ELISA testing, point-of-care antibody testing, and submission of samples to a commercial laboratory for antibody tests; and a third group performed microfilariae PCR and analyzed sequence data obtained through an academic core facility.

Whole blood anticoagulated with EDTA was tested for D. immitis antigen at each shelter on the day of collection using a point-of-care assay (WITNESS® Heartworm Test Kit; Zoetis, Parsippany, NJ, USA). Feline blood samples were also tested for FeLV antigen and FIV antibodies at each shelter using a point-of-care assay (WITNESS® FeLV-FIV; Zoetis, Parsippany, NJ, USA). Serum was tested for HW antigen by a microtiter plate ELISA method (DiroCHEK® Heartworm Antigen Test Kit; Zoetis, Parsippany, NJ, USA) according to manufacturer’s instructions, using samples both before and after heat treatment. Heat treatment was performed by heating serum to 104 °C for 10 min, then centrifuging at 16,000 x g to separate the liquid phase to be used in the ELISA (Little et al., 2018). After performing these tests and assessing marked discordant canine results between the WITNESS® Heartworm Test Kit and the DiroCHEK post heat treatment, stored canine serum was tested for HW antigen by a second point-of-care assay (SNAP® Heartworm RT Test; IDEXX Laboratories, Inc., Westbrook, ME, USA) to further examine test comparison between the DiroCHEK post heat treatment and another point-of-care test (Table 1)

Percentage of heartworm positive tests in 100 dogs and 100 cats recently admitted to Florida animal shelters as strays.

NP, Not performed; Ag, Antigen; HT, Heat treatment; Di, Dirofilaria immitis; Mf, Microfilariae; Ab, Antibody.

Blood was refrigerated at 4 °C pending microfilariae testing within 4 days of collection. A wet mount and modified Knott’s test was performed on each sample. The wet mount was prepared by mixing 10 μL of blood with 10 μL of saline on a glass slide and placing a cover slip (22-mm square) over the mixture; the entire area under the coverslip was examined by microscopy for microfilariae using the 10x objective (100x magnification). The wet mount preparation was reported as positive if any microfilariae were observed, but specific identification was not attempted due to the inability to measure motile microfilariae length and width. The modified Knott’s test was performed as previously described (Zajac and Conboy, 2012). Briefly, 1 mL of whole blood was placed in a 15 mL centrifuge tube, 9 mL of 2 % formalin added, and the tube inverted several times to lyse the red blood cells. The mixture was centrifuged at 500 x g for 5 min, the supernatant decanted, and the pellet stained with 0.1 % methylene blue. The stained material was transferred to 2–4 microscope slides, a coverslip (24-mm x 60-mm) applied to each slide, and the entire pellet examined for microfilariae. For samples with a very large number of microfilariae evident on wet mount, the modified Knott’s test was performed on a smaller volume of blood (0.1−0.25 mL). All microfilariae present were counted, the number of microfilariae per mL calculated, the length and width of up to 10 microfilariae present in a sample recorded, and a preliminary morphologic identification made by comparison to published descriptions (Zajac and Conboy, 2012). Whole blood was held frozen at −20 °C for PCR testing to confirm the microfilariae species.

Microfilariae were identified in frozen blood samples by PCR and sequencing as previously described (Otranto et al., 2011). Briefly, total nucleic acid was extracted from 200 μL of whole blood with a commercial kit (DNeasy Blood & Tissue Kit; Qiagen, Hilden, Germany) and used as template in a PCR targeting a ∼ 330 bp fragment of the 12S rRNA gene. Amplification was confirmed on a 2% agarose gel; amplicons were column-purified and sequenced directly with an ABI 3730 capillary sequencer (Applied Biosystems;Foster City, California, USA) at the Oklahoma State University Molecular Core Facility (Stillwater, Oklahoma, USA). Electropherograms were verified by visual inspection, compared with all available sequences in GenBank (National Center for Biotechnology Information), and the GenBank accession numbers of sequences with the closest identity reported (NCBI National Center for Biotechnology Information, 2019).

Feline serum was tested for HW antibodies using a point-of-care kit (Solo Step® FH; Heska, Loveland, CO, USA) according to manufacturer’s instructions. Feline serum was also tested in triplicate by a commercial laboratory (Heartworm Antibody Feline; Antech™ Diagnostics, Fountain Valley, CA, USA).