Detection of Dirofilaria immitis via integrated serological and molecular analyses in coyotes from Texas, United States

Abstract

Wild canids serve as reservoir for various vector-borne pathogens of veterinary and medical importance, including the canine heartworm, Dirofilaria immitis. In North and Central America, coyotes (Canis latrans) may be a relevant reservoir host for heartworm transmission. The objective of this study was to determine the occurrence of D. immitis in coyotes across Texas using integrated antigen detection test and molecular assays. Matching whole blood and serum samples were collected from 122 coyotes from different locations across the state of Texas, United States, encompassing nine counties. Collections occurred from February to April 2016, and December 2016. Samples were assessed serologically using a commercial microtiter plate ELISA (DiroCHEK®), and molecularly by conventional PCR targeting the cytochrome oxidase c subunit 1 (cox1) and NADH dehydrogenase subunit 5 (nad5) of the mitochondrial DNA, and via a TaqMan© probe-based real-time PCR protocol, also targeting a fragment of the cox1 gene. Overall, 12 (9.83%) samples tested positive when serological and molecular results were combined. Seven of 122 samples (5.73%) were antigen-positive, 8 (6.55%) were qPCR-positive, and 4 (3.27%) were positive using conventional PCR. Of 12 positive samples, 4 tested antigen-positive by DiroCHEK® but were negative in all molecular tests, another 4 tested positive by at least one of the molecular assays but tested negative by DiroCHEK®, and 3 samples tested positive by both antigen test and at least one of the molecular assays. Two samples (16.67%) tested positive on both the antigen test and both conventional PCR and qPCR. Our study confirmed the presence of D. immitis infection in coyotes from southern and northern Texas. The combination of serologic and molecular diagnostic tests was proven synergistic for the identification of D. immitis infections, including occult dirofilariosis, and revealed a more accurate picture of heartworm occurrence in the sampled coyotes.

Keywords: Heartworm; Immunodiagnostics; Real-time PCR; Vector-borne disease; Wildlife reservoir.

Graphical abstract

Fig. 1

Texas locations where samples were collected, including Webb, Maverick, Jim Hogg, Haskell, Throckmorton, Hutchinson, Roberts, Reeves, and Loving counties.