Ethics statement
The animal ethics committee (AEC) of CSIRO’s Australian Centre for Disease Preparedness approved all animal work under approval number AEC: ACDP22012, in accordance with the National Health and Medical Research Council Australian code for the care and use of animals for scientific purposes 8th edition (2013)40. As ringtail possums are a protected species in Victoria, Australia, this work was conducted under Department of Environment Land, Water and Planning research permit number DELWP: 10010501. This study is reported in accordance with ARRIVE guidelines.
Challenge material preparation and enumeration
M. ulcerans strain JKD8049, an Australian human clinical isolate collected in the Bellarine Peninsula in 2004, was used as the challenge material. This isolate has been extensively characterized for the purposes of standardizing challenge dosing, as described elsewhere 41. In brief, the organism was cultured at 30oC in Sauton’s media (with animal-free supplement) in an orbital shaker, mechanically de-clumped by filtration, and stored in 20% glycerol cryopreservative. Colony forming units (CFUs) were determined by spotting 20 µm replicates of the thawed and briefly vortexed sample onto 7H10 Middlebrook agar supplemented with oleic acid, albumin, dextrose and catalase (OADC) in serial dilution. Agar plates were cultured at 30oC in atmospheric conditions and CFUs were manually counted every two weeks.
Molecular testing
Molecular testing of all samples, except formalin-fixed paraffin-embedded (FFPE) samples, was conducted as described previously (Blasdell et al, 2022). In brief, samples were collected into 2ml tubes containing DNA/RNA Shield (Zymo, Irvine, CA) and a mixture of 2.3mm and 0.5mm zirconia/silica beads (Bio Spec Products, Bartlesville, OK). Samples were homogenized, clarified and total nucleic acid extracted using the Quick DNA/RNA MagBead Pathogen kit (Zymo), followed by testing with the IS2404 real-time PCR assay, the standard assay for molecular detection of MU 42. Samples producing a CT value < 40 (threshold 0.02) were considered positive. FFPE samples were extracted using the Quick DNA/RNA MagBead kit (Zymo) as per the manufacturer’s protocol for FFPE samples, followed by testing with the IS2404 real-time PCR assay, as above.
Histology
After fixation of the tissues in 10% neutral buffered formalin, they were trimmed, processed routinely (ASP 300S tissue processor, Leica Biosystems, Australia), and subsequently embedded into paraffin blocks. 4 µm thick tissue sections were then prepared and stained with haematoxylin-eosin and the Wade-Fite modification of the Ziehl-Neelsen staining method 43.
Sodium dodecyl-sulphate polyacrylamide gel electrophoresis of M. ulcerans
To prepare whole cell lysate, 4 mg of bacterial colony (grown on solid Sauton’s media with vegetable supplement) was collected into ZR BashingBead Lysis Tubes (0.1 & 0.5 mm; Zymo) containing 400 µL lysis buffer (PBS supplemented with 5% SDS and 1x Protease Inhibitor Cocktail (Abcam, Cambridge, UK)). Bacterial cells were homogenized using a mechanical bead beater device (Tissue Lyser; Qiagen, Hilden, Germany) twice at 6,800 rpm for 30 seconds. Tubes were centrifuged at 10,000 g for ten minutes and cleared lysate was removed into new tubes, with 1:10 and 1:100 dilutions prepared using PBS.
For sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE), undiluted MU whole cell lysate (15 µL) was added to 4x Sample Buffer (5 µL; Bolt LDS Sample Buffer; Thermo Fisher Scientific, Waltham, MA) and heated at 95°C for five minutes. Whole cell lysate samples (20 µL) and a protein standards ladder (5 µL; Bio-Rad, Hercules, CA) were run on NuPAGE 4–12% Bis Tris gels (Thermo Fisher Scientific) at 200 V for 35 minutes. Gels were either stained with Coomassie Blue (Thermo Fisher Scientific) and visualized using ChemiDoc gel visualization system (Bio-Rad) or used for Western Blot analysis.
Detecting seroconversion of possums to M. ulcerans using Western Blot and Dot Blot analysis
To investigate seroconversion to MU, selected samples were subjected to either Western Blot (WB) or Dot Blot analysis. Due to the lack of commercially available ringtail possum-specific secondary antibodies, Staphylococcus aureus Protein A-HRP conjugate was used as a secondary antibody substitute in these experiments. Possum #2 was selected to test for seroconversion via WB. Separated MU whole cell lysate proteins were transferred from SDS-PAGE gel to Invitrogen iBlot 0.2 µm Nitrocellulose Transfer Packs (Thermo Fisher Scientific) using the iBlot 2 Transfer System (Thermo Fisher Scientific) and the following program: 20 V for one minute, 23 V for four minutes, 25 V for three minutes. Following transfer, membranes were washed (washing Buffer; PBS supplemented with 0.1% Tween 20) for five minutes at room temperature (RT) and then blocked (blocking buffer; PBS supplemented with 5% skim milk powder and 0.1% Tween 20) for one hour at RT. Membranes were washed twice before incubation with possum serum. Possum #2 pre-infection and necropsy serum samples were diluted to 1:30, 1:100 and 1:1000 in blocking buffer and incubated with membranes overnight at 4°C. Membranes were washed twice before incubation with S. aureus Protein A-HRP conjugate (1:2000 diluted in blocking buffer; Abcam) for one hour at RT. Membranes were washed twice then incubated with Pierce ECL Western Blotting Substrate (Bio-Rad) for one minute and visualized using ChemiDoc blot visualization system (Bio-Rad).
Dot Blot analysis was executed using MU whole cell lysate prepared as described above, using samples from Possum #3 as the primary probe. This method probes the entire whole cell lysate (WCL) sample, instead of separated proteins as in the WB. Bacterial lysate (Neat, 1:10 and 1:100 dilutions; 2 µL) was pipetted in duplicate onto nitrocellulose membrane and allowed to dry for one hour at 4°C. Membranes were blocked with 5% BSA in PBS-T (PBS supplemented with 0.05% Tween 20) for one hour at RT. Possum #3 pre-infection and necropsy sera samples were diluted with 0.1% BSA in PBS-T to 1:10 (necropsy serum only) and 1:100, and incubated with membranes overnight at 4°C. The no sera control was incubated with BSA/PBS-T containing no possum sera. Following incubation, the membranes were washed three times with PBS-T before incubation with Protein A-HRP conjugate (Abcam) diluted to 1:2000 in BSA/PBS-T for one hour at RT. Membranes were washed three times and blots visualized with Pierce ECL Western Blotting Substrate (Bio-Rad) and ChemiDoc blot visualization system (Bio-Rad).
Trapping of wild possums
Animals were sourced from private properties in the Greater Geelong region. Prior to trapping, ringtail possum fecal samples were collected from potential properties and screened by detection of the MU-specific insertion sequence IS2404 PCR for MU DNA 42. Trapping was only conducted at IS2404 PCR negative properties. Ringtail possums were trapped using cage-style possum traps (61 x 18.5 x 19 cm) set overnight and baited with peanut butter and oat balls as well as apple. Upon capture the next morning, animals were health-checked by a veterinary professional and if deemed suitable for the challenge experiment, were transferred to individual wooden nest boxes (52 x 25.5 x 23 cm), provided with additional food (apple) and placed in a quiet, secure and sheltered location while awaiting testing outcomes. Fresh, voided fecal samples produced by the animals overnight were collected and screened by IS2404 PCR for MU DNA. Only adult or subadult animals negative for MU DNA, over 350g in weight, with no dependent young, no significant injuries and determined as visibly healthy by a veterinary professional were selected for use in the challenge study. Healthy juvenile animals or adults with dependent young were released at the site of capture at dusk of the same day, as were animals found positive for MU DNA but otherwise visibly healthy. Suitable animals (as described above) were transferred later the same day to the LAF at the Australian Centre for Disease Preparedness. This was repeated until a total of six suitable animals had been captured.
Housing and acclimatization of possums
On arrival at the LAF, each nest box containing an individual animal was securely hung in a separate ‘aviary-style’ enclosure (1.5 m wide x 0.75 m deep x 1.9 m high) and the door to the nest box was opened. Within the enclosure animals were provisioned with water, various food items, several branches for climbing, a hanging basket as an alternative nesting location, and fresh foliage. All six animals were housed in separate enclosures in the same room, which was maintained at PC2 throughout the experiment. Animals were allowed to acclimatize for up to four weeks prior to experimental challenge. During acclimatization, animals were not physically handled by staff (except on health grounds) but were monitored overnight for activity and behavioral changes using infra-red motion capture cameras. Cages were swept out and water and treat items replenished daily, whilst fresh foliage was replaced every two to three days. This replenishment regime continued throughout the study.
Experimental M. ulcerans challenge study
As this was an observational study assessing whether ringtail possums could be experimentally infected with MU, no negative control animals were included to minimize animal use. A sample size of six animals were chosen to minimize animal use and so that consistent infection could be demonstrated in a majority of animals (i.e. >75%). This was based on the assumptions that (a) one animal might not acclimatize to captive conditions, leaving five animals, and (b) that challenge failure may occur in one of these remaining five animals, providing a success rate of 80%.
On day 0 of the MU challenge, study animals were anaesthetized (inhalant isoflurane in oxygen, delivered via face mask) and health checked with the latter comprising a physical examination, assessment of weight, a subjective judgement of body condition 44 and recording of rectal temperature. Baseline oral swabs, voided feces and blood samples were collected, and a temperature-sensitive microchip was implanted subcutaneously in the inter-scapula region for more accurate measurement of body temperature. The challenge site (dorsal surface of the mid-tail region) was then shaved and disinfected (70% ethanol) and animals were challenged intra-dermally with 0.2 ml of inoculum containing 506 CFU (95% confidence interval 463–551 CFU) of the Australian JKD8049 strain of MU. Animals were monitored for seven nights post-challenge using infra-red motion-sensor video cameras to check for any behavioral changes. These cameras were also used to monitor animals suspected to have health issues, reduced activity, or reduced food intake throughout the study.
Post-challenge, fresh voided fecal samples were collected daily from the floor of enclosures and tested by IS2404 PCR. Prior to the onset of clinical signs, animals were anaesthetized and health checked (as described above with the addition of microchip temperatures being recorded) once weekly. Oral swabs and blood samples were collected at this time and tested by IS2404 PCR, and the site of challenge was assessed for evidence of swelling, hyperemia, hair loss, surface erosion, or ulceration. Once changes were detected at the site of inoculation or adjacent to it, health checks and sample collection were increased to twice weekly, and swabs of the challenge site were also collected. Clinical signs were graded as mild, moderate or severe and are defined in Table 4. Animals were humanely euthanized by intravenous or intracardiac overdose with 325mg/ml pentobarbitone at a dose of 150mg/kg while deeply anaesthetized with Tiletamine/zolazepam (5-10mg/kg) once one or more moderate (or severe) clinical signs were seen. This method of euthanasia is best practice for euthanasia of possums (as developed by experienced Australian wildlife veterinarians) and complies with the Australian code for the care and use of animals for scientific purposes40. Necropsies were conducted to look for evidence of gross pathology. The tail was removed from each animal at the sacrum (i.e. root of the tail), the tissue decalcified, and a full thickness transverse section was taken. During necropsy, samples from a range of tissues were collected for molecular testing and histopathology. Where histology revealed evidence of pathology, but molecular results were negative, the FFPE samples used for histology were also subjected to molecular analysis. The variation in time to the development of swelling at the site of challenge, ulceration and euthanasia between study animals was assessed using descriptive statistics (i.e. mean, standard deviation).
Table 4
Grading of clinical signs in ringtail possums experimentally challenged with M. ulcerans
Severity | Clinical sign |
Mild | Subcutaneous swelling (nodule) +/- localised hair loss, superficial erosion at challenge site (‘pre-ulcerative’ lesion) |
Fecal or oral swab material positive for M. ulcerans DNA |
Moderate | Swelling enlarges and/or becomes ulcerated |
Additional ulcers developing distant from original lesion (which may be at different stages of development) |
Severe | Ulcer enlarges and/or extends more deeply with exposure of the underlying muscle seen |
Fluid accumulation around the margin of an ulcer |
Poor body condition score (i.e. a score of 2 or less) |
Reduced activity for > 2 nights |