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Information on the prevalence of <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic> and associated risk factors for canine heartworm antigen positivity\u2014and thus disease\u2014in Australia is scarce or outdated. The current reference method for <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic> diagnosis in dogs is<jats:italic> via<\/jats:italic> the detection of heartworm antigen in blood using commercially available microwell-based enzyme-linked immunosorbent assays (ELISAs). Heat treatment of canine plasma prior to testing has been suggested to increase test sensitivity. The aim of the current study was to estimate the prevalence of <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic> in dogs confined to shelters in Queensland, Australia. The impact of heat treatment on antigen test results was also assessed.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>Blood samples (<jats:italic>n<\/jats:italic>\u2009=\u2009166) were collected directly from dogs in seven shelters across Queensland (latitudinal span of approx.\u20091700\u00a0km) into EDTA blood collection tubes. A commercially available ELISA (DiroCHEK\u00ae) was used to detect canine heartworm antigen in untreated and heat-treated plasma. Whole blood was concurrently tested for the presence of microfilariae and <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic> DNA using a modified Knott\u2019s test and real-time PCR, respectively. Risk factors (age, gender, source, location) associated with the odds of positivity for canine heartworm were assessed using binary logistic regression models.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>A total of 16 dogs (9.6%; 95% confidence interval [CI]: 5.9\u201315.2%) were positive for canine heartworm based on combined test results. Heat treatment did not impact on the positivity of <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic> antigen within samples (Cohen\u2019s kappa =\u20090.98), but the optical density was significantly increased in paired plasma samples for <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic> antigen-positive samples (Wilcoxon matched-pairs signed rank test, two-tailed <jats:italic>P<\/jats:italic>\u2009&lt;\u20090.01). Location of the dog in a shelter in northern Queensland was the only risk factor significantly associated with the odds of a dog being more likely to be <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic> antigen positive (odds ratio: 4.39; 95% CI: 1.26\u201313.51). All samples positive for the modified Knott\u2019s test were also positive for <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic> DNA by PCR.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>This study demonstrated the presence of heartworm-positive dogs in shelters in Queensland, with positive animals significantly more likely to occur in northern Queensland than southern Queensland. Sustained testing for the presence of <jats:italic>D<\/jats:italic>. <jats:italic>immitis<\/jats:italic> microfilariae and antigen remain important diagnostic tools in areas with known and re-emerging canine heartworm activity.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Graphical Abstract<\/jats:title>\n                \n              <\/jats:sec>","DOI":"10.1186\/s13071-021-04896-y","type":"journal-article","created":{"date-parts":[[2021,8,9]],"date-time":"2021-08-09T08:03:24Z","timestamp":1628496204000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Multiple diagnostic tests demonstrate an increased risk of canine heartworm disease in northern Queensland, Australia"],"prefix":"10.1186","volume":"14","author":[{"given":"Jessica L.","family":"Panetta","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8787-6755","authenticated-orcid":false,"given":"Nichola Eliza Davies","family":"Calvani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bronwyn","family":"Orr","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aldo Gianfranco","family":"Nicoletti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9921-4986","authenticated-orcid":false,"given":"Michael P.","family":"Ward","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1459-9117","authenticated-orcid":false,"given":"Jan","family":"\u0160lapeta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,8,9]]},"reference":[{"key":"4896_CR1","doi-asserted-by":"publisher","first-page":"507","DOI":"10.1128\/CMR.00012-12","volume":"25","author":"F Simon","year":"2012","unstructured":"Simon F, Siles-Lucas M, Morchon R, Gonzalez-Miguel J, Mellado I, Carreton E, et al. 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