Multiple diagnostic tests demonstrate an increased risk of canine heartworm disease in northern Queensland, Australia

Abstract

Background Canine heartworm (Dirofilaria immitis) is a life-threatening infection of dogs with a global distribution. Information on the prevalence of D. immitis and associated risk factors for canine heartworm antigen positivity—and thus disease—in Australia is scarce or outdated. The current reference method for D. immitis diagnosis in dogs is via 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 D. immitis in dogs confined to shelters in Queensland, Australia. The impact of heat treatment on antigen test results was also assessed. Methods Blood samples (n = 166) were collected directly from dogs in seven shelters across Queensland (latitudinal span of approx. 1700 km) into EDTA blood collection tubes. A commercially available ELISA (DiroCHEK®) was used to detect canine heartworm antigen in untreated and heat-treated plasma. Whole blood was concurrently tested for the presence of microfilariae and D. immitis DNA using a modified Knott’s 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. Results A total of 16 dogs (9.6%; 95% confidence interval [CI]: 5.9–15.2%) were positive for canine heartworm based on combined test results. Heat treatment did not impact on the positivity of D. immitis antigen within samples (Cohen’s kappa = 0.98), but the optical density was significantly increased in paired plasma samples for D. immitis antigen-positive samples (Wilcoxon matched-pairs signed rank test, two-tailed P < 0.01). 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 D. immitis antigen positive (odds ratio: 4.39; 95% CI: 1.26–13.51). All samples positive for the modified Knott’s test were also positive for D. immitis DNA by PCR. Conclusions 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 D. immitis microfilariae and antigen remain important diagnostic tools in areas with known and re-emerging canine heartworm activity. Graphical Abstract

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

“…Even though molecular techniques present some limitation, these methods can provide a rapid, sensitive, and accurate diagnoses, and prevent the occurrence of false-positive results due to cross-reactivity. Several studies have reported the use of molecular approaches targeting conserved DNA regions in the diagnosis of numerous filarial nematodes ( Laidoudi et al, 2020a , 2020b ; Panetta et al, 2021 ; Rishniw et al, 2006 ). As expected, with the present study the real-time PCR technique had a higher positive rate than conventional PCR and seems to be an alternative diagnostic method for detecting D .…”

Exploration of the sensitivity to macrocyclic lactones in the canine heartworm (Dirofilaria immitis) in Australia using phenotypic and genotypic approaches

“…Canine heartworm disease is a potentially deadly cardiopulmonary disease caused by the mosquito-borne filarial nematode Dirofilaria immitis , which is distributed across many parts of the world ( Simón et al, 2012 ; Genchi and Kramer, 2020 ; Lau et al, 2021 ; Noack et al, 2021 ). Although the prevalence of D. immitis in Australia is low, the parasite is endemic to the country and has recently re-emerged in dogs in Queensland ( Nguyen et al, 2016 ; Orr et al, 2020 ; Panetta et al, 2021 ). For the past 30 years in Australia and the USA, the main strategy for canine heartworm prevention has been the administration of macrocyclic lactone (ML) anthelmintics ( Orr et al, 2020 ; Diakou and Prichard, 2021 ; Lau et al, 2021 ; Noack et al, 2021 ; Prichard, 2021 ).…”

Exploration of the sensitivity to macrocyclic lactones in the canine heartworm (Dirofilaria immitis) in Australia using phenotypic and genotypic approaches

“…The use of MLs over the past 30 years has successfully reduced the prevalence of canine heartworm infection (Orr et al, 2020; Lau et al, 2021). However, the re-emergence of D. immitis infection in Queensland, Australia and the confirmation of ML-resistant isolates in the USA poses an important question of whether ML-resistance exists in Australia (Blagburn et al, 2011; Pulaski et al, 2014; Bourguinat et al, 2015; Blagburn et al, 2016; Nguyen et al, 2016; Maclean et al, 2017; McTier et al, 2017b; Orr et al, 2020; Panetta et al, 2021). By considering microfilariae counts as the phenotype and genotypic tests adopted from the USA, this study did not unequivocally demonstrate the presence of ML-resistant D. immitis in Australia.…”

“…The eluted DNA was stored at -20°C until further use in the qPCR and next generation sequencing (NGS) assays. A qPCR targeting the partial canine glyceraldehyde3-phosphate dehydrogenase (GAPDH) region was performed to verify the presence of dog DNA using primers with a probe, as previously described (Orr et al, 2020;Panetta et al, 2021) (Supplementary Table S1). A diagnostic TaqMan probe qPCR targeting the cox1 gene was performed to detect D. immitis DNA using primers with a probe, as previously described (Panetta et al, 2021) (Supplementary Table S1).…”

“…Canine heartworm disease is a potentially deadly cardiopulmonary disease caused by the mosquito-borne filarial nematode Dirofilaria immitis , which is distributed across many parts of the world (Simón et al, 2012; Genchi and Kramer, 2020; Lau et al, 2021; Noack et al, 2021). Although the prevalence of D. immitis in Australia is low, the parasite is endemic to the country and has recently re-emerged in dogs in Queensland (Nguyen et al, 2016; Orr et al, 2020; Panetta et al, 2021). For the past 30 years in Australia and the USA, the main strategy for canine heartworm prevention has been the administration of macrocyclic lactone (ML) anthelmintics (Orr et al, 2020; Diakou and Prichard, 2021; Lau et al, 2021; Noack et al, 2021; Prichard, 2021).…”