Validation of an Indirect Immunofluorescence Assay and Commercial Q Fever Enzyme-Linked Immunosorbent Assay for Use in Macropods.

Kangaroos are considered to be an important reservoir of Q fever in Australia, although there is limited knowledge on the true prevalence and distribution of coxiellosis in Australian macropod populations. Serological tests serve as useful surveillance tools, but formal test validation is needed to be able to estimate true seroprevalence rates, and few tests have been validated to screen wildlife species for Q fever. In this study, we modified and optimized a phase-specific indirect immunofluorescence assay (IFA) for the detection of IgG antibodies against Coxiella burnetii in macropod sera. The assay was validated against the commercially available ID Screen Q fever indirect multispecies enzyme-linked immunosorbent assay (ELISA) kit (IDVet, Grabels, France) to estimate the diagnostic sensitivity and specificity of each assay, using Bayesian latent class analysis. A direct comparison of the two tests was performed by testing 303 serum samples from 10 macropod populations from the east coast of Australia and New Zealand. The analysis indicated that the IFA had relatively high diagnostic sensitivity (97.6% [95% credible interval [CrI], 88.0 to 99.9]) and diagnostic specificity (98.5% [95% CrI, 94.4 to 99.9]). In comparison, the ELISA had relatively poor diagnostic sensitivity (42.1% [95% CrI, 33.7 to 50.8]) and similar diagnostic specificity (99.2% [95% CrI, 96.4 to 100]) using the cutoff values recommended by the manufacturer. The estimated true seroprevalence of C. burnetii exposure in the macropod populations included in this study ranged from 0% in New Zealand and Victoria, Australia, up to 94.2% in one population from New South Wales, Australia.

Tissue distribution of Coxiella burnetii and antibody responses in macropods co-grazing with livestock in Queensland, Australia.

Coxiella burnetii, the causative agent of Q fever, is a zoonotic bacteria of global public health significance. The organism has a complex, diverse, and relatively poorly understood animal reservoir but there is increasing evidence that macropods play some part in the epidemiology of Q fever in Australia. The aim of this cross-sectional survey was to estimate the animal- and tissue-level prevalence of coxiellosis amongst eastern grey (Macropus giganteus) and red (Osphranter rufus) kangaroos co-grazing with domestic cattle in a Q fever endemic area in Queensland. Serum, faeces and tissue samples from a range of organs were collected from 50 kangaroos. A total of 537 tissue samples were tested by real-time PCR, of which 99 specimens from 42 kangaroos (84% of animals, 95% confidence interval [CI], 71% to 93%) were positive for the C. burnetii IS1111 gene when tested in duplicate. Twenty of these specimens from 16 kangaroos (32%, 95% CI 20% to 47%) were also positive for the com1 or htpAB genes. Serum antibodies were present in 24 (57%, 95% CI 41% to 72%) of the PCR positive animals. There was no statistically significant difference in PCR positivity between organs and no single sample type consistently identified C. burnetii positive kangaroos. The results from this study identify a high apparent prevalence of C. burnetii amongst macropods in the study area, albeit seemingly with an inconsistent distribution within tissues and in relatively small quantities, often verging on the limits of detection. We recommend Q fever surveillance in macropods should involve a combination of serosurveys and molecular testing to increase chances of detection in a population, noting that a range of tissues would likely need to be sampled to confirm the diagnosis in a suspect positive animal.

Serological evidence for the presence of wobbly possum disease virus in Australia.

Wobbly possum disease virus (WPDV) is an arterivirus that was originally identified in common brushtail possums (Trichosurus vulpecula) in New Zealand, where it causes severe neurological disease. In this study, serum samples (n = 188) from Australian common brushtail, mountain brushtail (Trichosurus cunninghami) and common ringtail (Pseudocheirus peregrinus) possums were tested for antibodies to WPDV using ELISA. Antibodies to WPDV were detected in possums from all three species that were sampled in the states of Victoria and South Australia. Overall, 16% (30/188; 95% CI 11.0-22.0) of possums were seropositive for WPDV and 11.7% (22/188; 95% CI 7.5-17.2) were equivocal. The frequency of WPDV antibody detection was the highest in possums from the two brushtail species. This is the first reported serological evidence of infection with WPDV, or an antigenically similar virus, in Australian possums, and the first study to find antibodies in species other than common brushtail possums. Attempts to detect viral RNA in spleens by PCR were unsuccessful. Further research is needed to characterise the virus in Australian possums and to determine its impact on the ecology of Australian marsupials.

PCR-coupled sequencing achieves specific diagnosis of onchocerciasis in a challenging clinical case, to underpin effective treatment and clinical management.

This study demonstrates the utility of a PCR-based DNA sequencing approach to make a specific diagnosis of onchocerciasis in a returned traveller. Although a clinical diagnosis was not possible, the surgical excision of a suprascapular nodule from this patient, combined with an histological examination of this nodule and PCR-based sequencing of DNA from a nematode from this lesion solved the case. The analysis of DNA sequence data confirmed the presence of Onchocerca volvulus infection, supporting an effective treatment-clinical management strategy for the patient.