Further Characterization of Molecular Markers in Canine Dirofilaria immitis Infection

Dirofilaria immitis is a common filarial parasite found in dogs and cats in the Americas, with the pathophysiological consequences of the infection differing somewhat between these 2 host species. Recent research efforts have been focused on determining if the microRNAs (miRNAs) released from adult Dirofilariae have a role as markers for distinguishing the intensity of adult worm infection, as well as determining the presence of new infections. This study expands previous work on 2 nematode miRNAs, miR34 and miR-71, by addressing their ability to discriminate between low and high D. immitis adult worm intensities in dogs. Serum samples were collected from 13 dogs, 8 of which carried known numbers of adult D. immitis at autopsy in their hearts and pulmonary vessels. Three groups of canine sera were created based on D. immitis burden: "control" (0 worms; 5 animals), "low intensity" (10-18 worms; mean ± SD = 12.3 ± 4.4; 4 animals), and "high intensity" (41-72 worms; mean 62.5 ± 15.1; 4 animals) groups. A qPCR analysis was performed on each sample to measure plasma levels of miR-34 and miR-71; however, no significant differences were observed between these groups in terms of levels of miRNAs, so the low- and high-intensity samples were then combined into a single "infected" category and compared to the "non-infected" controls. Copy numbers of both miR-34 and miR-71 were significantly higher in infected compared to uninfected animals ( P = 0.015 and P = 0.027, respectively). The Ct values of expression compared with the adult worm intensity for each miRNA revealed that both miR-34 and miR-71 significantly discriminate between the infected and non-infected groups ( P value < 0.0001 for both). These findings support the contention that miRNA 34 and miRNA 71, which are filarial-specific miRNAs, can both serve as biomarkers for the presence of D. immitis infection in dogs, but at this point they do not appear to reflect the actual intensity of adult parasites present.