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

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immitis and associated risk factors for canine heartworm antigen positivity-and thus disease-in Australia is scarce or outdated.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.

Reptile-associated Borrelia species in the goanna tick (Bothriocroton undatum) from Sydney, Australia.

BACKGROUND: Knowledge on the capacity of Australian ticks to carry Borrelia species is currently limited or missing. To evaluate the potential of ticks to carry bacterial pathogens and their DNA, it is imperative to have a robust workflow that maximises recovery of bacterial DNA within ticks in order to enable accurate identification. By exploiting the bilateral anatomical symmetry of ticks, we were able to directly compare two DNA extraction methods for 16S rRNA gene diversity profiling and pathogen detection. We aimed to assess which combination of DNA extraction and 16S rRNA hypervariable region enables identification of the greatest bacterial diversity, whilst minimising bias, and providing the greatest capacity for the identification of Borrelia spp. RESULTS: We collected Australian endemic ticks (Bothriocroton undatum), isolated DNA from equal tick halves using two commercial DNA extraction methods and sequenced samples using V1-V3 and V3-V4 16S rRNA gene diversity profiling assays. Two distinct Borrelia spp. operational taxonomic units (OTUs) were detected using the V1-V3 16S rRNA hypervariable region and matching Borrelia spp. sequences were obtained using a conventional nested-PCR. The tick 16S rRNA gene diversity profile was dominated by Rickettsia spp. (98-99%), while the remaining OTUs belonged to Proteobacteria (51-81%), Actinobacteria (6-30%) and Firmicutes (2-7%). Multiple comparisons tests demonstrated biases in each of the DNA extraction kits towards different bacterial taxa. CONCLUSIONS: Two distinct Borrelia species belonging to the reptile-associated Borrelia group were identified. Our results show that the method of DNA extraction can promote bias in the final microbiota identified. We determined an optimal DNA extraction method and 16S rRNA gene diversity profile assay that maximises detection of Borrelia species.