DISEASE MANAGEMENT

The uniqueness of DFTD, being a transmissible disease perpetuated by the transfer of live neoplastic cells (clonal cancer) between free-ranging Tasmanian devils, elimi­nates individual animal treatment as a means of DFTD control at a population level.

Tumour cells outside the animal are easily killed by air exposure and desiccation, as well as by chlorine disinfect­ants, ethanol and common detergent preparations used for cleaning and disinfection in disease control (unpublished).

5.1 Treatment

Treatment of diseased Tasmanian devils may be war­ranted if individuals are genetically important or have dependent young. Several treatments have been trialled and are discussed, but to date none have been effective. Other possible therapeutic targets (i.e. genes) have been proposed by Wright et al. (2017) (i.e. PAX3 and TLL1 genes) and Hayes et al. (2017) (i.e. ERBB3). Generally the application of therapeutic agents in the management of DFTD in free-ranging Tasmanian devil populations poses significant challenges and a great deal of work is still required.

5.1.1 Chemotherapy

Two important studies by Phalen et al. (2013, 2015) reported on the use of chemotherapeutic regimens used in domestic animals for the treatment of solid tumours and their application in the potential treatment of DFTD.

Vincristine was selected as a potential treatment for DFTD because of its effectiveness against a wide range of cancer types, in particular mesenchymal tumours, and its history of use in a range of species. Its efficacy against CTVT made it a good candidate for investigation.

At escalating dose rates from 0.05 to 0.136 mg/kg administered to Tasmanian devils (n = 8) with early stage (non-metastatic stage) disease, vincristine had no effect on the lesions and above a dose rate of 0.105 mg/kg pro­duced signs of toxicity, including anorexia, vomiting, diarrhoea and neutropenia.

1.8 mL/min per kg was slower in the Tasmanian devil than in humans, suggesting there may be pharmacody­namic effects rather than pharmacokinetic effects that allow Tasmanian devils to tolerate higher dose rates of vincristine before showing signs of toxicity compared with other animals (Phalen et al. 2013).

Doxorubicin and carboplatin were also trialled because of their activity against a variety of solid tumours, including soft tissue sarcomas such as Schwann cell tumours. Escalating doses of each were administered to Tasmanian devils with early stage tumours (i.e. doxoru­bicin 0.75-1.0 mg/kg and carboplatin 8-26 mg/kg) and maximally tolerated doses of doxorubicin (1.0 mg/kg) and carboplatin (20 mg/kg) were identified. Signs of tox­icity of doxorubicin were neutropenia, anorexia and weight loss and for carboplatin anorexia, weight loss and azotaemia. Neither drug was efficacious against DFTD in the trial (Phalen et al. 2015).

Tigilanol tiglate (USAN), previously known as EBC- 46, is a phorbol ester that acts as a protein kinase C regu­lator. It was identified by a company QBiotics (Taringa, Brisbane, Qld) from the seeds of a north Qld rainforest plant, the blushwood tree (Hylandia dockrillii), as a pos­sible phyto-therapeutic. It was experimentally trialled for the treatment of DFTD with very encouraging results. The product was injected into the solid tumours directly and produced discolouration (darkening) of the tumours within 20 min post-injection and then marked degenerative effects within 48 hr post-injection. Com­plete resolution of tumours after sloughing and wound healing was achieved in some animals with small tumours within 21 d post-injection. Non-cancerous tissue appeared to be unaffected and closed by third- intention healing after fibrous contraction.

5.1.2 Immunotherapy

During early pilot studies to confirm the transmissible nature of DFT1, some animals implanted with viable 1-cm cubed DFT1 grew tumours at the surgical sites at variable times but most commonly 4 mo post-implant. In one animal a postoperative tumour disappeared over 6 mo after reaching a diameter of 6 cm and reappeared at the same site, growing unabated until the cessation of the trial. Similarly, one animal injected SC with a suspension of 1 ? 10⁶ viable cells/mL of phosphate-buffered saline (PBS) cultured DFT1 cells grew a 1-cm diameter tumour, which disappeared completely without return over a 3-mo period (unpublished). There was enough evidence from this pilot that DFT1 could be transplanted success­fully, confirming its transmissible nature. There were also suggestions that DFT1 did not have a clearly defined biology (i.e. infected, established, grew in situ unabated, metastasised and killed the animal) once inside a new host. For ethical and welfare reasons the trial was not repeated on a greater scale. The mechanisms of limitation could be tumour related (i.e. rapid growth leading to necrosis as viable blood supply was exhausted) or that there was an immune response that was overcoming pos­sible immunosuppression masking effects of the tumour. Hamede et al. (2013) proposed three possible hypotheses for low DFTD prevalence in a population of Tasmanian devils monitored in the West Pencil Pine area of north­west Tas. The population was disease free up until 2012 (Hamede et al. 2012a) and subsequently showed reduced disease impact upon entry into the population. The three proposed hypothesis for reduced disease impact were: lower host susceptibility to DFT1 conferred by host genetic factors or environmental variables; lower rate of injurious biting and/or variable Tasmanian devil contact networks compared with other sites; or lower DFT1 viru­lence caused by changed DFT1 genetics and pathogenic­ity.

Initial work investigated the Tasmanian devil’s ability to display cytotoxic antitumour responses and develop antibodies against DFT1 cells and foreign tumour cells. Two Tasmanian devils were given monthly injections of equal volumes of PBS containing gamma-irradiated DFT1 cells and Montanide adjuvant (Seppic, Puteaux, France) SC for four treatments. Neither animal produced a measurable cytotoxic or humoral response against the DFT1 cells. In contrast, the animals did produce cyto­toxic responses and antibodies against xenogenic K562 cells, when injected under a similar regimen. The cyto­toxicity appeared to occur through the activity of natural killer (NK) cells in an antibody-dependent manner; how­ever, classical NK cell responses, such as innate killing of DFT and foreign cancer cells, were not observed. The indication that devils may produce an antibody-depend­ent cell-mediated cytotoxicity to foreign cells presented a potential pathway to induce cytotoxic responses against DFT cell and further evidence that Tasmanian devils pos­sessed NK cell responses in blood (Brown et al. 2011).

Additional work on the nature of this possible NK response was undertaken using Tasmanian devil periph­eral blood mononuclear cells (PBMCs) treated with either the mitogen concanavalin A, the toll-like receptor (TLR) agonist polyinosinic:polycytidylic acid or recom­binant Tasmanian devil interleukin-2 (IL-2). All three activated the PBMCs to kill cultured DFT1 cells in vitro, indicating other possible pathways for inducing in vivo induction of an anticancer response in affected devils (Brown et al. 2016).

As discussed previously, the DFT1 mechanism of immune avoidance and allograft propagation is proposed to be a downregulation of antigenicity on the surface of the DFT1 cells; however, in two mouse transplant studies there was rejection and antibody production against the DFT by the murine hosts.

Pye et al. (2016a) describes possible immune responses of free-ranging Tasmanian devils in a closely repeat- monitored population situated in the north-west Tas. Tasmanian devils (n = 52) were subjected to repeat blood sampling between 2008 and 2014 and serum anti-DFT1 IgG antibodies were monitored. Using an agglutination assay against MHC-1 epitopes on the surface of DFT1 cells it was found that six animals from the population expressed anti-MHC-1 antibodies. These animals showed clinical signs of DFTD during the sampling period and four showed tumour regression after the IgG response was observed (4-15 mo). One animal remained tumour- free for 2 yr and was then found to be affected again at 5 yr of age, while another animal was tumour-free for the following 3 yr before it was lost to follow up. This work indicated that DFT1 biology within free-ranging Tasma­nian devils is variable and gives further hope for a possi­ble immune-mediated response to DFTD.

Patchett et al. (2016) investigated the potential of the small immunomodulatory molecule imiquimod to pro­duce TLR7 anticancer immunity effects similar to those observed in humans, as well as testing its capacity to pro­duce apoptosis in DFT cell lines via other TLR7-inde- pendent mechanisms.

Specific work seeking a vaccine solution was initiated by Kreiss et al. (2015) and furthered by Tovar et al. (2017) with variable results. A limited number of Tasmanian devils were used within each trial and the variable results produced and lack of statistical significances within the data makes the transition to application in free-ranging Tasmanian devil populations difficult, but encouraging.

Initially, six Tasmanian devils were used in a trial in which killed DFT1 cells plus Montanide ISA71VG adju­vant (Seppic, Puteaux, France) vaccine preparations were administered SC in an effort to assess the protective capacity of the vaccines, as well as characterising the immune response to the vaccine in order to explain the mechanisms of any protective immunity to DFT1 should it occur. The cultured DFT1 cells were killed by sonica­tion, freeze-thaw cycling or gamma irradiation. Cell via­bility was tested to ensure no live cells were administered. Two animals were initially vaccinated with freeze-thaw cell preparations plus adjuvant, with only one developing a measurable immune response (anti-DFT IgG produc­tion but no cytotoxicity response) and subsequent to live allograft transplants the animal that showed no immune response grew tumours at the site of injection and was euthanased. The animal that produced an immune response did not grow any tumours subsequent to vacci­nation and after a second live DFT challenge grew only small tumours that were surgically excised and the animal remained disease-free 490 d post-injection.

Subsequently, the remaining four animals were given vaccine preparations of sonicated or irradiated cells with adjuvant, producing variable antibody and cytotoxic responses. None of these devils was challenged with live tumours and so assessment of protection was not con­firmed (Kreiss et al. 2015).

Tovar et al. (2017) report on a subsequent trial of nine Tasmanian devils each receiving one of six different pro­tocols of vaccination ± immunotherapy. The aim was to further assess the devil immune response to DFT1 vac­cine preparation killed cells (sonicated, irradiated, freeze-thaw) plus Iscomatrix ™ adjuvant and to assess the effects of repeat application of various DFT1 cell prepara­tions either SC or intralesional. All but one animal devel­oped DFTD after the vaccination protocols. That single animal received a protocol that included interferon­gamma stimulation of DFT1 cells, which increases sur­face antigen expression. The study had variable treatments and low replications within each treatment. Only one animal receiving live DFT1 cells and not developing sub­sequent tumours suggests that immune-based treatment requires further investigation.

A vaccination trial of 33 Tasmanian devils released to the wild and recaptured (n=8) over a period two years post vaccination, indicated the animals had a measurable immune response to the vaccine but that it was not pro­tective as six of the eight developed DFTD (Pye et al. 2021). Further investigations of vaccine delivery continue with oral bait methods showing some promise as devils will accept baits, however no vaccine delivery has been undertaken utilising this method (Dempsey et al. 2023).

5.2 Population management

5.2.1 Culling free-ranging Tasmanian devil populations

During the course of the DFTD response in Tas., particu­larly after the formation of the working hypothesis and subsequent case definition of DFTD being a transmissible allograft, there was much discussion about whether ani­mals diagnosed with DFTD should be removed from the free-ranging population. There were arguments proposed on welfare grounds and in the context of infectious dis­ease control whereby all affected animals are removed from a population thus reducing the risk of contact between affected and unaffected animals. Equally, it was argued that this was an emerging disease in a free-rang­ing population and that there should not be any interfer­ence with the natural spread by removing affected animals. Given the threatened status of the Tasmanian devil a policy of removal by euthanasia of affected ani­mals from free-ranging populations once diagnosed, was established within the DPIPWE Save the Tasmanian Devil Program (STDP). Concurrently, a disease-free founder managed population was developed as an insur­ance in the event of extinction in the wild and adminis­tered by the Australian Zoo and Aquarium Association.

Several ecological model studies were undertaken to assess the effects of test and cull strategies on the progres­sion of DFTD through the free-ranging Tasmanian devil population.

Hamede et al. (2012a) simulated the progress of DFTD through free-ranging populations of Tasmanian devils in order to predict the possible extinction time if the disease was uncontrolled. Modelling a range of contact networks, topographies and latent period predictions, they tested the usefulness of modelling in the face of this unique dis­ease outbreak. The one value that limited exact predic­tions was the latency period of DFTD, which has extreme variability.

One study site on the Forestier peninsula of mainland Tas. provided an ideal opportunity to test in real time whether test and cull strategies might work for the whole of Tas. as a form of disease control. The peninsula was isolated by a bridge that was impervious to all but four animals in the years of the trial. A trapping, test and release or cull program was conducted from June 2004 to June 2008, with 448 animals captured and 145 diagnosed with DFTD euthanased. After analysis and modelling there was no evidence that selective culling of infected animals in this population either slowed the rate of dis­ease progression or reduced the population-level impact of DFTD. The only indication that removal of effected animals altered the disease progression was that the aver­age tumour volume in affected animals decreased. This may be an important welfare consideration over and above the disease progression and control aspects (Lachish et al. 2010).

Model predictions further to this initial field trial using susceptible, exposed and infectious (SEI) frameworks with no resistant class also showed that unless removals are very frequent there will be little effect on disease suppression through the population. The trapping and cull frequency rates at which the effect is brought are pre­dictably too high to be practical or effective (Beeton and McCallum 2011). More recent mathematical modelling considered a range of interactive strategies to control DTD in free-ranging Tasmanian devil populations and con­cluded that no one strategy used alone would eliminate the disease from the wild population (Drawert et al. 2022).

5.2.2 Zoo and sanctuary-based management and restocking

To prevent extinction of Tasmanian devils, several strate­gies and programs have been implemented by the STDP. The program has several priority projects that ultimately aim to maintain a genetically diverse self-propagating population of Tasmanian devils. The programs are part­nered by research groups, zoos and wildlife parks. Insur­ance populations, within Tas. and mainland Australia, have been established so that animals can be bred and used as founder populations (genetically diverse) for wild release programs (e.g. Maria Is.). These island popula­tions, as well as fenced extensive managed populations, are carefully monitored, thus allowing constant improve­ment in release strategies of animals bred in managed care so that survival of released animals is maximised. Free-ranging Tasmanian devil populations are also closely monitored for DFTD, changes in animal numbers and age of breeding at first conception. The STDP is ever­changing and best referred to at <http://dpipwe.tas.gov. au/wildlife-management/save-the-tasmanian-devil-pro- gram/about-the-program>.