{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,17]],"date-time":"2026-02-17T14:28:47Z","timestamp":1771338527775,"version":"3.50.1"},"reference-count":49,"publisher":"Public Library of Science (PLoS)","issue":"7","license":[{"start":{"date-parts":[[2024,7,17]],"date-time":"2024-07-17T00:00:00Z","timestamp":1721174400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100015476","name":"Kansas State University College of Veterinary Medicine","doi-asserted-by":"crossref","award":["T35OD029981"],"award-info":[{"award-number":["T35OD029981"]}],"id":[{"id":"10.13039\/100015476","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:sec id=\"sec001\">\n<jats:title>Background<\/jats:title>\n<jats:p>The increase in reports of resistance to macrocyclic lactones in the canine heartworm, <jats:italic>Dirofilaria immitis<\/jats:italic> is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic> isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux\u2014detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methods<\/jats:title>\n<jats:p>Our objective was to optimize the two assays further by testing various assay parameters in <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic> isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic> isolates\u2014JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2\u20134 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Results<\/jats:title>\n<jats:p>Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec004\">\n<jats:title>Conclusions<\/jats:title>\n<jats:p>These assays provide a greater understanding of the biochemical variation among isolates of <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic>, which can be exploited in the future to develop <jats:italic>in vitro<\/jats:italic> diagnostic tests capable of differentiating susceptible and resistant isolates.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pone.0307261","type":"journal-article","created":{"date-parts":[[2024,7,17]],"date-time":"2024-07-17T17:29:33Z","timestamp":1721237373000},"page":"e0307261","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Strain-level variations of Dirofilaria immitis microfilariae in two biochemical assays"],"prefix":"10.1371","volume":"19","author":[{"given":"Naomi","family":"Hampton","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vicki","family":"Smith","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew T.","family":"Brewer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3077-0201","authenticated-orcid":true,"given":"Jeba R. 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