Introduction
Buruli, or Bairnsdale, ulcer (BU) is a chronic, necrotising and debilitating disease of skin and subcutaneous tissues caused by
Mycobacterium ulcerans (MU). BU is predominantly described and reported as a human disease, but cases are occasionally reported in Australian domestic and wild animals [
1,
2,
3]. Most human cases are reported in sub-Saharan Africa, however the incidence of BU appears to be declining in this region[
4]. Australia is the only high-income country reporting significant endemic human BU[
4]. The most significant disease focus is in Victoria, where cases have been increasing in number, severity and geographical distribution over the past decade[
5,
6], with suburban cases now frequently reported in the state’s two largest cities, Melbourne and Geelong.
BU appears to be both vector-borne and zoonotic in Victoria, with increasing evidence implicating certain species of mosquito and possum as MU vectors and reservoirs, respectively[
7,
8,
9]. Common ringtail (CRT,
Pseudocheirus peregrinus) and common brushtail (
Trichosurus vulpecula) possums, both of which are endemic and abundant throughout Victoria, can excrete significant amounts of viable MU into the environment via their faeces[
7]. Possums are also susceptible to clinical disease: a 2014 paper described clinical BU in 27 possums, most of which had cutaneous ulcers on the face (exposing bone in one case), limbs and/or tail[
10]. Pathology and/or the presence of MU was confirmed by PCR in some internal tissues including lung and liver[
10], however the welfare impacts of BU on the affected possums were not evaluated or discussed.
We conducted surveillance of free-living possums across Victoria as part of the NHMRC-funded ‘Beating Buruli in Victoria’ project led by the University of Melbourne’s Doherty Institute and the Melbourne Veterinary School (UniMelb AEC 22910; DELWP permits 10009447 and 10010257). Possums that died or were euthanised for any reason at two veterinary clinics in Werribee and North Melbourne, Victoria, between May 2021 and December 2022 were collected and stored at -20°C. Necropsies were conducted on thawed possums at the Melbourne Veterinary School. Findings were recorded on customised necropsy sheets (see
Supplementary document 1) that include a silhouette for mapping any cutaneous lesions indicative of BU, and categorisation of clinical BU as mild, moderate or severe, based on the World Health Organization’s BU disease classification for assessment of human cases[
11]. Sample collection included plain microbiological swabs of each possum’s oral cavity, cloaca, pouch (where applicable) and any skin lesions, and a duplicate set of tissue and organ sections (full list in
Supplementary document 1) and any dissected ulcerative skin lesions collected into formalin for histopathology, and into cryovials stored at -80° for PCR, culture and genomic analysis where indicated.
This case series describes the clinical and laboratory findings from four cases of severe BU in CRTs that were euthanised due to the welfare impacts of the disease.
Discussion
This report describes four cases of severe BU in CRTs from Melbourne’s inner suburbs, where BU is considered endemic. These possums, euthanised due to welfare implications of the disease, all exhibited severe deep ulceration of one or more paws with swelling, necrosis and destruction of skin and underlying tissue, and exposed carpal/tarsal bones in two cases. Histological examination of the cutaneous ulcerations revealed severe ulcerative necrotising pyogranulomatous dermatitis and cellulitis, with intralesional Gram-positive and acid-fast bacteria observed both superficially and deep within the lesions.
Systemic infections were also confirmed in all four possums, with MU detected by PCR in all tissue and internal organ samples collected during necropsy. Faeces collected from all four possums were also PCR positive for MU, implicating these possums as potential sources of environmental contamination for zoonotic transmission to humans. Mild to moderate hepatitis was observed in Cases 3 and 4, and pulmonary changes were observed in Cases 1 and 3. No other significant histopathological changes were noted in the other examined organs, and no acid-fast bacteria were observed in stained organ or tissue sections. This could indicate that the presence of MU in organs (confirmed by PCR) was not associated with pathological changes, or may reflect inadvertent sampling bias or the effects of freeze-thaw artifacts and autolysis that were present to varying degrees in all four possums. In future, conducting necropsies on fresh rather than frozen possums, and/or performing in-situ PCR or immunohistochemistry with an MU antibody on tissue blocks may better detect MU presence or absence within organs of PCR-positive possums.
Severe, chronic BU as described in these four cases would likely have affected all Five Domains[
13] of the possums’ welfare. Assessment of the size, severity and locations of the ulcerative cutaneous lesions indicated likely functional impairment of affected limbs, thereby impeding the possums’ normal behaviours, and restricting their ability to navigate their environments and obtain food and water. Despite the lack of histopathological changes detected in these cases as discussed above, the systemic distribution of MU bacteria throughout all the major internal organs, as reported here, may be associated with impaired function of cardiovascular, respiratory, digestive, reproductive and/or immune systems, which would further reduce animal mobility, fitness and welfare. The below average body condition scores of these four possums and the possible pancreatic atrophy observed in Case 2 indicate reduced ability to obtain sufficient nutrition, and could reflect systemic impacts of severe BU. Extensive ulcerative lesions, particularly of the oedematous type as observed in cases 3 and 4, may also cause chronic pain[
14], which could further affect the mental domain of severely affected animals. These findings suggest that BU may be an important cause of impaired possum health and welfare.
Interestingly, the presence of epidermal hyperplasia and multifocal lymphoid aggregates observed in the subcutis of Case 2′s left forelimb lesion and at least one of Case 4’s tail ulcers indicate that some level of lesion healing is possible in CRTs. This is a novel finding in this species; to date, spontaneous resolution of BU lesions has only been reported in one common and one mountain brushtail possum[
10], however the long-term outcomes of the lesions are unknown as both individuals were lost to follow up. Complete resolution of untreated BU lesions can occur in people, however the mechanism by which the host’s immune system is able to overcome the immunosuppressive effects of the mycolactone toxin produced by MU is unclear[
15]. The frequency, extent and mechanisms by which spontaneous lesion healing occurs in possums are yet to be determined.
The distribution of BU lesions may give an insight into the methods by which possums become infected with MU. The predominance of BU lesions occurring on the distal limbs and tails of these four possums aligns with previously published findings by Fyfe, et al. [
7] and O’Brien, et al. [
10], who also reported lesions on possums’ noses, lips and ears. These relatively less furred regions may be more susceptible to bites from MU
-harbouring mosquitoes. Paws and tails used for climbing may more frequently incur micro-traumas that are susceptible to bacterial invasion; similarly, faces and ears may receive more injuries during fights with other possums. Direct BU transmission from a possum was recently described in a Victorian man who developed BU on his finger several months after being bitten by a CRT[
16]. While ingestion of MU via contaminated food or water might explain the transient MU faecal positivity observed in some clinically unaffected possums[
7,
10], it is less likely to cause severe cutaneous ulcerations as observed in these four cases.
BU is not currently a notifiable animal disease in Victoria, and a low awareness of the disease among veterinarians means BU cases in animals are likely to be substantially under-recognised, particularly in free-ranging wildlife that are not regularly presented to veterinary clinics. State government-funded wildlife rescue organisation Wildlife Victoria reported 75 cases of ulcerative conditions in rescued possums between 2013-2022, with an upward trend observed since 2018 (excluding 2020/2021, potentially due to decreased reporting during the COVID-19 pandemic)[
17]. BU was included as a differential diagnosis for cutaneous ulcerations in possums in the recently updated Victorian Wildlife Disease Rehabilitation Guidelines[
18], however, more targeted educational outreach to veterinarians, wildlife carers and pet owners about BU in animals is needed.
At present, possums with severe cutaneous lesions presenting to veterinary clinics in BU-endemic areas in Victoria are typically euthanised without attempting diagnosis. Loss of limbs or limb function, untreatable infectious disease, and chronic ill health are all recognised conditions that may preclude successful rehabilitation and release of wild animals, and are all indicators for euthanasia[
19]. While human cases of BU are successfully treated with long courses of antibiotics, frequent dressing changes and/or surgery[
11,
15,
20], these interventions are neither established nor likely to be suitable for free-living wildlife including possums. Furthermore, such interventions are likely in themselves to have additional welfare impacts from stress associated with human handling, proximity and captivity[
18,
19].
Other zoonotic mycobacteria species are maintained in abundant small, free-ranging mammalian wildlife species in urban environments. Natural infections of red squirrels with lepromatous leprosy, caused by infection with
M. leprae and
M. lepromatosis, were recently confirmed in the UK[
21]. A small longitudinal field-based study conducted over two years described clinical leprosy in wild British squirrels as a chronic, progressive ulcerative disease; lesions appeared to undergo repeated ulceration-healing cycles, but usually progressed from mild-moderate lesions to severe lesions within six months[
21]. Bovine tuberculosis, caused by the related
M. bovis that can cause serious disease in cattle and people, is maintained in many free ranging wildlife species across the globe, including brushtail possums (
Trichosurus vulpecula) in New Zealand, and European badgers (
Meles meles) in the UK[
22]. While there is a wealth of international literature pertaining to the epidemiology, surveillance, diagnosis and control of bovine tuberculosis in various wild animal species, discussions are largely focused on the role of wildlife in maintenance of the pathogen in the transmission cycle, and the impacts of
M. bovis on the health and welfare of infected wild animals and populations are largely lacking.
Humane management strategies for prevention of MU infection and/or reduced severity of clinical BU in possums are needed. Aside from ethical considerations, culling wildlife to control infectious diseases has shown equivocal results and may counterintuitively increase both transmission and geographical distribution of pathogens[
23,
24]. Oral bait vaccination may be a feasible strategy for control of BU in possums. The
M. tuberculosis bacille Calmette-Guérin (BCG) vaccine has been successfully used in New Zealand for the control of bovine tuberculosis (
M. bovis) in brushtail possums[
25,
26], and early results indicate that BCG vaccination protects against BU in mice[
27]. A recent article[
28] proposes a series of laboratory and field-based studies to trial BCG vaccination against MU infection in possums, and to optimise palatability, delivery and efficacy of oral bait vaccine formulations targeted to ringtail and brushtail possums. Successful BU disease control in possums could have manifold benefits including reducing individual morbidity and mortality, decreasing disease transmission and protecting collective health and welfare of possum populations. Given the strong evidence implicating possums as zoonotic reservoirs of MU in Victoria [
9], successful control of BU in possums may also benefit public health.
This case series provides an important first step for integrating animal welfare into BU research, however, more research, development and educational outreach are needed. Longitudinal studies, involving repeated sampling of individual possums and assessment of a range of behavioural, immunological and other factors would enable a deeper understanding of the disease progression and welfare impacts of BU over time. Other key aspects of BU in possums yet to be addressed include identification of the source(s) of MU infection; determination of incubation periods and fatality rates in naturally infected animals; identifying risk or protective factors for MU infection and clinical disease; and exploring disease transmission dynamics both within and between possum populations, and the mechanisms and extent to which zoonotic and/or vector-borne transmission to humans occurs.