Map showing the Mornington and Bellarine peninsulas, and locations of towns referred to in the text.

<p>Map showing the Mornington and Bellarine peninsulas, and locations of towns referred to in the text.</p

Map showing the distribution of collection sites for possum faeces, indicating the locations of positive samples for <i>Mycobacterium ulcerans</i> DNA by real-time IS<i>2404</i> PCR.

<p>The locations of positive ringtail faecal samples are shown as red ovals; positive brushtail samples are shown as black ovals: negative samples are shown in white. The dotted circle shows a significant non-random clustering of <i>Mycobacterium ulcerans</i> positive possum faecal samples identified by spatial scan statistics (<i>P</i><0.0001; 16/30 possum faecal sample positives within a circle of radius 0.42 km). 4/6 residential addresses of human BU cases and 2/3 non-resident addresses fall within the radius of the cluster identified above. Addresses of holiday houses were unavailable for 3 non-resident BU cases. The inset figure (bottom left) depicts a heat map showing possum faecal bacterial loads of samples within the black rectangle, estimated from IS<i>2404</i> real-time PCR signal strength, ranging from negative (dark blue) to ≥10⁶<i>M. ulcerans</i> per gram of faeces (red).</p

Clinical, Microbiological and Pathological Findings of <i>Mycobacterium ulcerans</i> Infection in Three Australian Possum Species

<div><p>Background</p><p>Buruli ulcer (BU) is a skin disease caused by <i>Mycobacterium ulcerans</i>, with endemicity predominantly in sub-Saharan Africa and south-eastern Australia. The mode of transmission and the environmental reservoir(s) of the bacterium and remain elusive. Real-time PCR investigations have detected <i>M. ulcerans</i> DNA in a variety of Australian environmental samples, including the faeces of native possums with and without clinical evidence of infection. This report seeks to expand on previously published findings by the authors' investigative group with regards to clinical and subclinical disease in selected wild possum species in BU-endemic areas of Victoria, Australia.</p><p>Methodology/Principal Findings</p><p>Twenty-seven clinical cases of <i>M. ulcerans</i> infection in free-ranging possums from southeastern Australia were identified retrospectively and prospectively between 1998–2011. Common ringtail possums (<i>Pseudocheirus peregrinus</i>), a common brushtail possum (<i>Trichosurus vulpecula</i>) and a mountain brushtail possum (<i>Trichosurus cunninghami</i>) were included in the clinically affected cohort. Most clinically apparent cases were adults with solitary or multiple ulcerative cutaneous lesions, generally confined to the face, limbs and/or tail. The disease was minor and self-limiting in the case of both <i>Trichosurus</i> spp. possums. In contrast, many of the common ringtail possums had cutaneous disease involving disparate anatomical sites, and in four cases there was evidence of systemic disease at post mortem examination. Where tested using real-time PCR targeted at IS<i>2404</i>, animals typically had significant levels of <i>M. ulcerans</i> DNA throughout the gut and/or faeces. A further 12 possums without cutaneous lesions were found to have PCR-positive gut contents and/or faeces (subclinical cases), and in one of these the organism was cultured from liver tissue. Comparisons were made between clinically and subclinically affected possums, and 61 PCR-negative, non-affected individuals, with regards to disease category and the categorical variables of species (common ringtail possums <i>v</i> others) and sex. Animals with clinical lesions were significantly more likely to be male common ringtail possums.</p><p>Conclusions/Significance</p><p>There is significant disease burden in common ringtail possums (especially males) in some areas of Victoria endemic for <i>M. ulcerans</i> disease. The natural history of the disease generally remains unknown, however it appears that some mildly affected common brushtail and mountain brushtail possums can spontaneously overcome the infection, whereas some severely affected animals, especially common ringtail possums, may become systemically, and potentially fatally affected. Subclinical gut carriage of <i>M. ulcerans</i> DNA in possums is quite common and in some common brushtail and mountain brushtail possums this is transient. Further work is required to determine whether <i>M. ulcerans</i> infection poses a potential threat to possum populations, and whether these animals are acting as environmental reservoirs in certain geographical areas.</p></div

Data for possums with confirmed clinical <i>M. ulcerans</i> infection.

<p>Data for possums with confirmed clinical <i>M. ulcerans</i> infection.</p

Statistical comparison of possums with (clinical cases) and without cutaneous lesions (subclinical and non-affected cases).

<p>Statistical comparison of possums with (clinical cases) and without cutaneous lesions (subclinical and non-affected cases).</p

Data for sub-clinically affected possums.

<p>Data for sub-clinically affected possums.</p

Lesions caused by <i>M. ulcerans</i> infection in a common ringtail possum (case 16).

<p>This animal had multiple affected sites characterised by ulceration and/or oedema. (Image C. McCowan/J. Fyfe).</p

Graphical representation of PCR results of faecal samples collected from sub-clinically affected common brushtail and mountain brushtail possums that were repeatedly trapped during the period of the study.

<p>In the majority of these individuals the gut carriage of <i>M. ulcerans</i> DNA was demonstrated to be transient.</p

(a): Photomicrograph of a skin lesion obtained from case 16.

<p>The lesion is characterised by proliferative epidermis overlying fibrotic dermal tissue admixed with inflammatory cells, and superficial crust composed of serous exudate and degenerate leukocytes overlying a necrotic base. (H&E stain)) (<b>b</b>): Demonstration of numerous acid-fast bacilli (AFB) in an ulcerated skin lesion of case 16 (modified ZN stain), (<b>c</b>): the liver lesions in this case contained rare AFB, mostly within macrophages (arrow) (modified ZN stain). (Images C. McCowan).</p

Relative real-time IS<i>2404</i> PCR signal from gut contents of possums.

<p>Relative real-time IS<i>2404</i> PCR signal from gut contents of possums.</p