Tasmanian Devil

Devil Facial Tumor Disease has devastated the devil population and the species now faces extinction. No natural resistance has been found in the affected population, but recently an area of Western Tasmania (West Pencil Pine) has been identified where the disease is not spreading as rapidly as would be expected. The devils in this area have a slightly different genetic makeup to devils in the east of Tasmania where the disease has spread rapidly throughout the population. The area of West Pencil Pine offers hope that some devils may have some level of natural resistance to DFTD. This area was continually monitored by trapping the devils and taking repeated blood samples from individual devils which were then analysed to determine if there was any evidence of an immune response to DFTD. Forty devils were trapped on at least five occasions and blood samples from these devils were analysed. Of these 40 devils there were five devils that had evidence for anti-DFTD antibodies. This suggests that these five devils had been exposed to DFTD and their immune response was activated and therefore they were protected against the disease. This is encouraging evidence that there are some devils that can produce an immune response against DFTD.

The best possible scenario for the survival of the Tasmanian devil is the existence of devils that can naturally respond to DFTD. Such “DFTD-resistant individuals” would have a natural immune response and this could be detected in recently exposed devils by the presence of antibodies to DFTD in their serum.  Consequently, the major objective of the project was to identify devils that might be genetically resistant to DFTD.

The first aim of this proposal was to identify DFTD-specific tumor antigens. The second aim was to develop a technique that could be used to screen as many devils as possible for evidence for an immune response against DFTD. The third aim was to screen Tasmanian devils from the area of West Pencil Pine as this area is where the disease is not spreading as rapidly as other areas, and there is evidence for a difference in the genetics of some animals in this population. The fourth aim was to determine whether devils could produce an immune response against DFTD.

Aim 1: To identify DFTD-specific tumor antigens.

In associated collaborative research we determined that DFTD is of Schwann cell in origin (Murchison et al., 2010). Using this information we screened a number of markers that were expressed by Schwann cells and DFTD tumor cells to identify a specific marker that could be used as a diagnostic tool and potentially as a target antigen for any vaccine. This led to the identification of periaxin as a reliable tumour-specific marker. A total of 30 DFTD tumours (including 10 metastases) were screened and 100% of these tumours expressed periaxin. We also analysed eight non-DFTD tumors and none of these expressed periaxin. We have submitted this work for publication and the abstract is attached (Academic output section).

Aim 2: To develop a technique that could be used to screen as many devils as possible for evidence for an immune response against DFTD.

We developed an immunofluorescence procedure where we test the serum for the presence of anti-DFTD antibodies. Briefly, DFTD tumour cells were incubated with the serum of the devil to be tested and the binding is detected with a series of antibodies, which were then analysed using a flow cytometer. This technique has been refined as part of this project to increase the sensitivity and has been successfully used to screen serum samples. One of the difficulties with this technique is that it is labour-intensive and requires a large number of cultured tumor cells. In order to increase the sensitivity and develop a more rapid screening test we attempted to develop a sensitive ELISA procedure. We invested a substantial amount of time in refining this procedure and, although the sensitivity increased, the specificity decreased. This was most likely due to the non-specific staining of serum from the Tasmanian devil. To overcome this it was necessary to produce a more specific anti-devil immunoglobulin antibody. This has required a whole new molecular biology approach using modern technology. To this end, we have been working in partnership with Dr. Lynn Corcoran from the Walter and Eliza Hall Institute in Melbourne, who is producing a much more sensitive anti-devil antibody. Once this reagent is available, which we anticipate will be by the end of 2010, we will be able to rapidly screen multiple devil serum samples in order to detect evidence for devils that may be “naturally” resistant to DFTD.

Aim 3: To screen Tasmanian devils from the area of West Pencil Pine for evidence of “natural immunity” to DFTD.

In order to undertake this study it was necessary to obtain multiple blood samples from the same devil. As part of the routine trapping procedure, 40 Tasmanian devils were trapped on at least five different occasions over a period of greater than 12 months. If a devil was exposed to DFTD but naturally resisted the disease due to its immune response, evidence of antibodies in the devil’s serum would be apparent. The multiple samples were required to obtain a “background” level in which we could compare potentially positive responses.

We screened serial samples of Tasmanian devils from West Pencil Pine (WPP, north western area of Tasmania). DFTD has been present in this population since 2006, but fewer devils than we would expect have been infected with the disease. We are working with Mr. Rodrigo Hamede from the School of Zoology, UTas, who traps devils in WPP every three months and collects blood samples for our antibody screening. This collaboration represented a unique opportunity to monitor a population over time for an anti-DFTD response. Through the technique described above, we have tested several serum samples from approximately 40 individual devils from WPP. Five animals have been detected with a possible antibody response against DFTD.
This is encouraging evidence that supports the hope that some devils in the wild have the capacity to respond to DFTD without contracting this infectious cancer.

Aim 4: To determine whether devils could produce an immune response against DFTD.

For an effective anti-DFTD vaccine, many questions must be answered, including the safety of the inoculum, whether the host can produce an effective anti-tumor response, how many doses are necessary and for how long immunity lasts. We have immunised six Tasmanian devils in captivity with a control cell line (K562 cells, which is a human leukemia cell line) and analysed both humoral and cellular immune responses. The humoral response was measured by the flow cytometer technique described above. The cellular immune response is measured through the percentage of tumour cells that are actively killed by the lymphocytes of the immunised animal (using a radioactive chromium release assay). All devils immunised against K562 cells produced a strong humoral and cellular responses, indicating that devils are capable of killing tumor cells.

The next aspect to investigate was the response against DFTD tumor cells. This required laborious cell culture work to provide sufficient cells for our study. We immunised six devils with inactivated DFTD cells, but only weak responses were observed in these devils. Two additional devils were then immunised using a different preparation of DFTD tumor cells (disrupted DFTD tumor cells as opposed to inactivated cells) and some promising responses were obtained, indicating that the immune system of these devils responded to DFTD. Although these responses were encouraging, it is unlikely that the intensity of the responses would be sufficient to protect the devils against DFTD.

To extend this work we have developed collaborations with a two Biotechnology companies that use cutting edge technology for our vaccination trials.

Through a collaborative study (which involved support from this grant and other related grants) we determined that DFTD is of Schwann cell in origin. This knowledge can now be used to target a pre-diagnostic test.

Through our endeavors to develop a sensitive assay to determine whether wild Tasmanian devils can produce a response to DFTD, we have developed an important collaboration which will result in the development of more specific reagents. These reagents will then be used to develop a range of important bioassays for monitoring devils in the wild.

We have determined that Tasmanian devils can develop a cytotoxic response against cancer cells, but at best they will only produce a weak response against DFTD tumor cells. This is an important outcome as it provides direction for future vaccine-related work, such as modifying the DFTD tumor cells.

The most exciting and encouraging result was the data that suggested that five devils from West Pencil Pine appeared to have levels of antibodies that were much higher than expected. If this can be proven to be correct (one we develop a more sensitive assay as detailed above) it will provide supporting evidence that some Tasmanian devils have the immunological (and presumably genetic) capacity to naturally respond to DFTD. Should this be the case it provides great hope for the survival of the species.