Validation of a species-specific probe-based qPCR for detection of Setaria yehi (Filarioidea: Onchocercidae) in Alaskan moose (Alces alces gigas)

Northern ungulates contend with Setaria yehi and Rumenfilaria andersoni, filarioid nematodes that are transmitted by ectoparasitic blood-feeding arthropods, which can result in animal and population level impacts. Setaria yehi microfilariae can be detected in fresh blood samples using a modified Knott's test, or by postmortem detection by genetic sampling or through the retrieval of adult specimens in the peritoneal cavity. In this study we validated a novel qPCR for detection of S. yehi DNA in blood samples of moose (Alces alces). Additionally, we compared quantitative values from modified Knott's test to detect both S. yehi and R. andersoni from both fresh and frozen blood samples. Species-specific primers targeting a 121-base pair fragment of the cytochrome oxidase c subunit 1 (cox1) of S. yehi, and a species-specific probe were designed. The qPCR had a detection threshold of 0.157 pg/μL of parasite DNA. We collected 166 blood samples from wild moose captured on the Kenai Peninsula, Alaska from 2019 to 2022. Matching blood aliquots were tested by modified Knott's test and subjected to DNA extraction for subsequent qPCR. Quantitatively, blood samples had an average S. yehi microfilaremia (mf) of 472.2 mf/mL (0–14,490 mf/mL) and R. andersoni of 72.9 mf/mL (0.0–5071.5 mf/mL). Qualitatively, 32.53% (n = 54) of samples tested positive for S. yehi in each of the tests, and 37.35% (n = 62) when both tests were combined, with very good agreement between the results from Knott's test and qPCR (kappa = 0.90). The validation of the qPCR test for S. yehi allows for faster, less labor-intensive diagnosis and epidemiological surveillance of this emerging parasite in moose and other cervid hosts.