Search

Background Sarcoptic mange causes significant animal welfare and occasional conservation concerns for bare-nosed wombats (Vombatus ursinus) throughout their range. To date, in situ chemotherapeutic interventions have involved macrocytic lactones, but their short duration of action and need for frequent re-administration has limited treatment success. Fluralaner (Bravecto®; MSD Animal Health), a novel isoxazoline class ectoparasiticide, has several advantageous properties that may overcome such limitations. Methods Fluralaner was administered topically at 25 mg/kg (n = 5) and 85 mg/kg (n = 2) to healthy captive bare-nosed wombats. Safety was assessed over 12 weeks by clinical observation and monitoring of haematological and biochemical parameters. Fluralaner plasma pharmacokinetics were quantified using ultra-performance liquid chromatography and tandem mass spectrometry. Efficacy was evaluated through clinical assessment of response to treatment, including mange and body condition scoring, for 15 weeks after topical administration of 25 mg/kg fluralaner to sarcoptic mange-affected wild bare-nosed wombats (n = 3). Duration of action was determined through analysis of pharmacokinetic parameters and visual inspection of study subjects for ticks during the monitoring period. Methods for diluting fluralaner to enable ‘pour-on’ application were compared, and an economic and treatment effort analysis of fluralaner relative to moxidectin was undertaken. Results No deleterious health impacts were detected following fluralaner administration. Fluralaner was absorbed and remained quantifiable in plasma throughout the monitoring period. For the 25 mg/kg and 85 mg/kg treatment groups, the respective means for maximum recorded plasma concentrations (Cmax) were 6.2 and 16.4 ng/ml; for maximum recorded times to Cmax, 3.0 and 37.5 days; and for plasma elimination half-lives, 40.1 and 166.5 days. Clinical resolution of sarcoptic mange was observed in all study animals within 3–4 weeks of treatment, and all wombats remained tick-free for 15 weeks. A suitable product for diluting fluralaner into a ‘pour-on’ was found. Treatment costs were competitive, and predicted treatment effort was substantially lower relative to moxidectin. Conclusions Fluralaner appears to be a safe and efficacious treatment for sarcoptic mange in the bare-nosed wombat, with a single dose lasting over 1–3 months. It has economic and treatment-effort-related advantages over moxidectin, the most commonly used alternative. We recommend a dose of 25 mg/kg fluralaner and, based on the conservative assumption that at least 50% of a dose makes dermal contact, Bravecto Spot-On for Large Dogs as the most appropriate formulation for adult bare-nosed wombats. Graphical abstract

Devil facial tumour 1 (DFT1) is a transmissible cancer clone endangering the Tasmanian devil. The expansion of DFT1 across Tasmania has been documented, but little is known of its evolutionary history. We analysed genomes of 648 DFT1 tumours collected throughout the disease range between 2003 and 2018. DFT1 diverged early into five clades, three spreading widely and two failing to persist. One clade has replaced others at several sites, and rates of DFT1 coinfection are high. DFT1 gradually accumulates copy number variants (CNVs), and its telomere lengths are short but constant. Recurrent CNVs reveal genes under positive selection, sites of genome instability, and repeated loss of a small derived chromosome. Cultured DFT1 cell lines have increased CNV frequency and undergo highly reproducible convergent evolution. Overall, DFT1 is a remarkably stable lineage whose genome illustrates how cancer cells adapt to diverse environments and persist in a parasitic niche., Tasmanian devils are threatened by devil facial tumour 1 (DFT1), a transmissible cancer clone that spreads between animals by biting. A new study of more than six hundred DFT1 tumour genomes reveals this cancer’s evolution and lineage dynamics over a fifteen year period.

Devil facial tumor disease (DFTD) is renowned for its successful evasion of the host immune system. Down regulation of the major histocompatabilty complex class I molecule (MHC-I) on the DFTD cells is a primary mechanism of immune escape. Immunization trials on captive Tasmanian devils have previously demonstrated that an immune response against DFTD can be induced, and that immune-mediated tumor regression can occur. However, these trials were limited by their small sample sizes. Here, we describe the results of two DFTD immunization trials on cohorts of devils prior to their wild release as part of the Tasmanian Government’s Wild Devil Recovery project. 95% of the devils developed anti-DFTD antibody responses. Given the relatively large sample sizes of the trials (N = 19 and N = 33), these responses are likely to reflect those of the general devil population. DFTD cells manipulated to express MHC-I were used as the antigenic basis of the immunizations in both trials. Although the adjuvant composition and number of immunizations differed between trials, similar anti-DFTD antibody levels were obtained. The first trial comprised DFTD cells and the adjuvant combination of ISCOMATRIX™, polyIC, and CpG with up to four immunizations given at monthly intervals. This compared to the second trial whereby two immunizations comprising DFTD cells and the adjuvant combination ISCOMATRIX™, polyICLC (Hiltonol®) and imiquimod were given a month apart, providing a shorter and, therefore, more practical protocol. Both trials incorporated a booster immunization given up to 5 months after the primary course. A key finding was that devils in the second trial responded more quickly and maintained their antibody levels for longer compared to devils in the first trial. The different adjuvant combination incorporating the RNAase resistant polyICLC and imiquimod used in the second trial is likely to be responsible. The seroconversion in the majority of devils in these anti-DFTD immunization trials was remarkable, especially as DFTD is hallmarked by its immune evasion mechanisms. Microsatellite analyzes of MHC revealed that some MHC-I microsatellites correlated to stronger immune responses. These trials signify the first step in the long-term objective of releasing devils with immunity to DFTD into the wild.

Devil facial tumor disease (DFTD) is an emergent transmissible cancer exclusive to Tasmanian devils ( Sarcophilus harrisii) and threatening the species with extinction in the wild. Research on DFTD began 10 years ago, when nothing was known about the tumor and little about the devils. The depth of knowledge gained since then is impressive, with research having addressed significant aspects of the disease and the devils’ responses to it. These include the cause and pathogenesis of DFTD, the immune response of the devils and the immune evasion mechanisms of the tumor, the transmission patterns of DFTD, and the impacts of DFTD on the ecosystem. This review aims to collate this information and put it into the context of conservation strategies designed to mitigate the impacts of DFTD on the devil and the Tasmanian ecosystem.

Tasmanian devils (Sarcophilus harrisii) risk extinction from a contagious cancer, devil facial tumour disease (DFTD) in which the infectious agent is the tumor cell itself. Because devils are unable to produce an immune response against the tumor cells no devil has survived ‘infection’. To promote an immune response we immunized healthy devils with killed DFTD tumor cells in the presence of adjuvants. Immune responses, including cytotoxicity and antibody production, were detected in five of the six devils. The incorporation of adjuvants that act via toll like receptors may provide additional signals to break ‘immunological ignorance’. One of these devils was protected against a challenge with viable DFTD cells. This was a short-term protection as re-challenge one year later resulted in tumor growth. These results suggest that Tasmanian devils can generate immune responses against DFTD cells. With further optimization of immune stimulation it should be possible to protect Tasmanian devils against DFTD with an injectable vaccine.

Tasmanian devils have spawned two transmissible cancer clones, known as devil facial tumour 1 (DFT1) and devil facial tumour 2 (DFT2). DFT1 and DFT2 are transmitted between animals by the transfer of allogeneic contagious cancer cells by biting, and both cause facial tumours. DFT1 and DFT2 tumours are grossly indistinguishable, but can be differentiated using histopathology, cytogenetics or genotyping of polymorphic markers. However, standard diagnostic methods require specialist skills and equipment and entail long processing times. Here, we describe Tasman-PCR: a simple PCR-based diagnostic assay that distinguishes DFT1 and DFT2 by amplification of DNA spanning tumour-specific interchromosomal translocations. We demonstrate the high sensitivity and specificity of this assay by testing DNA from 557 tumours and 818 normal devils. A temporal-spatial screen confirmed the reported geographic ranges of DFT1 and DFT2 and did not provide evidence of additional DFT clones. DFT2 affects disproportionately more males than females, and devils can be co-infected with DFT1 and DFT2. Overall, we present a PCR-based assay that delivers rapid, accurate and high-throughput diagnosis of DFT1 and DFT2. This tool provides an additional resource for devil disease management and may assist with ongoing conservation efforts.

The Tasmanian devil is under threat of extinction due to the transmissible devil facial tumor disease (DFTD). This fatal tumor is an allograft that does not induce an immune response, raising questions about the activity of Tasmanian devil immune cells. T and B cell analysis has been limited by a lack of antibodies, hence the need to produce such reagents. Amino acid sequence analysis revealed that CD4, CD8, IgM, and IgG were closely related to other marsupials. Monoclonal antibodies were produced against CD4, CD8, IgM, and IgG by generating bacterial fusion proteins. These, and commercial antibodies against CD1a and CD83, identified T cells, B cells and dendritic cells by immunohistochemistry. CD4+ and CD8+ T cells were identified in pouch young thymus, adult lymph nodes, spleen, bronchus‐ and gut‐associated lymphoid tissue. Their anatomical distribution was characteristic of mammalian lymphoid tissues with more CD4+ than CD8+ cells in lymph nodes and splenic white pulp. IgM+ and IgG+ B cells were identified in adult lymph nodes, spleen, bronchus‐associated lymphoid tissue and gut‐associated lymphoid tissue, with more IgM+ than IgG+ cells. Dendritic cells were identified in lymph node, spleen and skin. This distribution is consistent with eutherian mammals and other marsupials, indicating they have the immune cell subsets for an anti‐tumor immunity. Devil facial tumor disease tumors contained more CD8+ than CD4+ cells, but in low numbers. There were also low numbers of CD1a+ and MHC class II+ cells, but no CD83+ IgM+ or IgG+ B cells, consistent with poor immune cell infiltration. Anat Rec, 297:925–938, 2014. © 2014 The Authors. The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology Published by Wiley Periodicals, Inc.

Contagious cancers that pass between individuals as an infectious cell line are highly unusual pathogens. Devil facial tumor disease (DFTD) is one such contagious cancer that emerged 16 y ago and is driving the Tasmanian devil to extinction. As both a pathogen and an allograft, DFTD cells should be rejected by the host–immune response, yet DFTD causes 100% mortality among infected devils with no apparent rejection of tumor cells. Why DFTD cells are not rejected has been a question of considerable confusion. Here, we show that DFTD cells do not express cell surface MHC molecules in vitro or in vivo, due to down-regulation of genes essential to the antigen-processing pathway, such as β 2 -microglobulin and transporters associated with antigen processing. Loss of gene expression is not due to structural mutations, but to regulatory changes including epigenetic deacetylation of histones. Consequently, MHC class I molecules can be restored to the surface of DFTD cells in vitro by using recombinant devil IFN-γ, which is associated with up-regulation of the MHC class II transactivator, a key transcription factor with deacetylase activity. Further, expression of MHC class I molecules by DFTD cells can occur in vivo during lymphocyte infiltration. These results explain why T cells do not target DFTD cells. We propose that MHC-positive or epigenetically modified DFTD cells may provide a vaccine to DFTD. In addition, we suggest that down-regulation of MHC molecules using regulatory mechanisms allows evolvability of transmissible cancers and could affect the evolutionary trajectory of DFTD.

Devil facial tumour disease (DFTD) is a recently emerged fatal transmissible cancer decimating the wild population of Tasmanian devils ( Sarcophilus harrisii ). Biting transmits the cancer cells and the tumour develops in the new host as an allograft. The literature reports that immune escape mechanisms employed by DFTD inevitably result in host death. Here we present the first evidence that DFTD regression can occur and that wild devils can mount an immune response against the disease. Of the 52 devils tested, six had serum antibodies against DFTD cells and, in one case, prominent T lymphocyte infiltration in its tumour. Notably, four of the six devils with serum antibody had histories of DFTD regression. The novel demonstration of an immune response against DFTD in wild Tasmanian devils suggests that a proportion of wild devils can produce a protective immune response against naturally acquired DFTD. This has implications for tumour–host coevolution and vaccine development.

Summary The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations. PaperClip, Graphical Abstract Highlights ► Whole-genome sequences of the Tasmanian devil and two distant cancer subclones ► The Tasmanian devil cancer lineage originated recently in a female devil ► The devil cancer genome is relatively stable despite ongoing evolution ► Clonal divergence and geographic spread elucidated through patterns of mutation, Whole-genome sequences of the Tasmanian devil and two devil cancer subclones suggest that the cancer first arose from a female devil and that the clone has subsequently genetically diverged during its spread across Tasmania.

Pathogen-driven declines in animal populations are increasingly regarded as a major conservation issue. The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction by devil facial tumor disease, a unique transmissible cancer. The disease is transmitted through direct transfer of tumor cells, which is possible because the genetic diversity of Tasmanian devils is low, particularly in the major histocompatibility complex genes of the immune system. The far northwest of Tasmania now holds the last remaining disease-free wild devil populations. The recent discovery of unique major histocompatibility complex genotypes in the northwestern region of Tasmania has raised the possibility that some animals may be resilient to the disease. We examined the differences in the epidemiology and population effects of devil facial tumor disease at 3 well-studied affected sites in eastern Tasmania and 1 in western Tasmania (West Pencil Pine). In contrast to the 3 eastern sites, there has been no rapid increase in disease prevalence or evidence of population decline at West Pencil Pine. Moreover, this is the only onsite at which the population age structure has remained unaltered 4 years after the first detection of disease. The most plausible explanations for the substantial differences in population effects and epidemiology of the disease between eastern and western sites are geographic differences in genotypes or phenotypes of devils and functional differences between tumor strains in the 2 regions. We suggest that conservation efforts focus on identifying whether either or both these explanations are correct and then, if resistance alleles exist, to attempt to spread the resistant alleles into affected populations. Such assisted selection has rarely been attempted for the management of wildlife diseases, but it may be widely applicable.

Devil facial tumor disease (DFTD) is a transmissible neoplasm that is threatening the survival of the Tasmanian devil. Genetic analyses have indicated that the disease is a peripheral nerve sheath neoplasm of Schwann cell origin. DFTD cells express genes characteristic of myelinating Schwann cells, and periaxin, a Schwann cell protein, has been proposed as a marker for the disease. Diagnosis of DFTD is currently based on histopathology, cytogenetics, and clinical appearance of the disease in affected animals. As devils are susceptible to a variety of neoplastic processes, a specific diagnostic test is required to differentiate DFTD from cancers of similar morphological appearance. This study presents a thorough examination of the expression of a set of Schwann cell and other neural crest markers in DFTD tumors and normal devil tissues. Samples from 20 primary DFTD tumors and 10 DFTD metastases were evaluated by immunohistochemistry for the expression of periaxin, S100 protein, peripheral myelin protein 22, nerve growth factor receptor, nestin, neuron specific enolase, chromogranin A, and myelin basic protein. Of these, periaxin was confirmed as the most sensitive and specific marker, labeling the majority of DFTD cells in 100% of primary DFTD tumors and DFTD metastases. In normal tissues, periaxin showed specificity for Schwann cells in peripheral nerve bundles. This marker was then evaluated in cultured devil Schwann cells, DFTD cell lines, and xenografted DFTD tumors. Periaxin expression was maintained in all these models, validating its utility as a diagnostic marker for the disease.

The number of Tasmanian devils in the wild is rapidly declining owing to a transmissible cancer, devil facial tumor disease (DFTD). Although progress has been made to understand the spread of this disease, crucial research on the pathogenesis of DFTD has been limited because of the threatened status of the host species. Here, the authors describe the development of a NOD/SCID (nonobese diabetic / severe combined immunodeficiency) mouse model that reproduces DFTD and provides a much-needed model to undertake studies into this intriguing transmissible cancer. Histologically, the DFTD produced in NOD/SCID mice (xenografted DFTD) was indistinguishable from the DFTD identified in Tasmanian devils. At the protein level, all xenografted DFTD tumors expressed periaxin, a marker that confirmed the diagnosis of DFTD. The karyotype of DFTD in NOD/SCID mice reproduced similar chromosomal alterations as seen in diseased devils. Furthermore, each NOD/SCID mouse inoculated with cultured DFTD tumor cells developed tumors, whereas DFTD did not develop in any of the inoculated immune-competent BALB/c mice.

Tasmanian devil lymphoid tissues (thymus, spleen, and lymph node) from seven animals, including pouch young, juvenile, and adult devils, were investigated using histological and immunohistochemical techniques. Antibodies against the conserved intracytoplasmic portion of CD3 and CD79b (T- and B-cell markers, respectively) and MHC II were used to label immune cells. The thymus from the juvenile devils and the pouch young had CD3+ cells that were primarily located in the medulla of the organ. The spleen consisted of red and white pulp areas with characteristic lymphoid follicles with CD79b+ and MHC II+ cells and nonfollicular T-cell-dominated periarteriolar lymphoid sheaths. Peripheral lymph nodes presented three distinct regions, outer cortex and medulla (both with primarily CD79b+ and MHC II+ cells) and paracortex (mainly CD3+ cells). Tissue architecture and distribution of the immune cells were similar to that seen in eutherian mammals and other marsupials, indicating that the Tasmanian devil has all the structural elements necessary for effective adaptive immunity.

OBJECTIVE: As the platypus (Ornithorhynchus anatinus), the Tasmanian devil (Sarcophilus harrisi) and the eastern barred bandicoot (Perameles gunni) are currently at risk of serious population decline or extinction from fatal diseases in Tasmania, the goal of the present study was to describe the normal immune response of these species to challenge using the lymphocyte proliferation assay, to give a solid basis for further studies. METHODS: For this preliminary study, we performed lymphocyte proliferation assays on peripheral blood mononuclear cells (PBMC) from the three species. We used the common mitogens phytohaemagglutinin (PHA), concanavalin A (ConA), lipopolysaccharide (LPS) and pokeweed mitogen (PWM). RESULTS: All three species recorded the highest stimulation index (SI) with the T-cell mitogens PHA and ConA. Tasmanian devils and bandicoots had greater responses than platypuses, although variability between individual animals was high. CONCLUSION: For the first time, we report the normal cellular response of the platypus, the Tasmanian devil and the eastern barred bandicoot to a range of commonly used mitogens.

The Tasmanian devil (TD) (Sarcophilus harrisii) is under threat from devil facial tumour disease (DFTD), a cancer that is transmitted between devils by direct cell implantation. As no devil is known to have rejected the tumour allograft, an understanding of the immune status of this species is essential to help explain the unique infectious nature of this cancer. We analysed differential white blood cell counts, the phagocytic response of neutrophils as well as mitogen-induced lymphocyte proliferation. Devils analysed included healthy TDs kept in captivity, healthy devils from disease-free and diseased areas as well as diseased devils. Neutrophils isolated from the peripheral blood of healthy devils showed competent phagocytosis and peripheral blood mononuclear cells from healthy and diseased devils proliferated in response to Con A, PHA and PWM stimulation. Although a wide range of responses was observed and relatively high doses of mitogens were required, there was no significant difference between males and females, adults and juveniles or between normal and diseased animals, suggesting that transmission of DFTD is not a consequence of a severely impaired immune system. As lymphocytes from all TDs appear to require strong stimulation for activation, this threshold may contribute to all devils being susceptible to DFTD.

Clonally transmissible cancers are somatic cell lineages that are spread between individuals via the transfer of living cancer cells. There are only three known naturally occurring transmissible cancers, and these affect dogs, soft-shell clams, and Tasmanian devils, respectively. The Tasmanian devil transmissible facial cancer was first observed in 1996, and is threatening its host species with extinction. Until now, this disease has been consistently associated with a single aneuploid cancer cell lineage that we refer to as DFT1. Here we describe a second transmissible cancer, DFT2, in five devils located in southern Tasmania in 2014 and 2015. DFT2 causes facial tumors that are grossly indistinguishable but histologically distinct from those caused by DFT1. DFT2 bears no detectable cytogenetic similarity to DFT1 and carries a Y chromosome, which contrasts with the female origin of DFT1. DFT2 shows different alleles to both its hosts and DFT1 at microsatellite, structural variant, and major histocompatibility complex (MHC) loci, confirming that it is a second cancer that can be transmitted between devils as an allogeneic, MHC-discordant graft. These findings indicate that Tasmanian devils have spawned at least two distinct transmissible cancer lineages and suggest that transmissible cancers may arise more frequently in nature than previously considered. The discovery of DFT2 presents important challenges for the conservation of Tasmanian devils and raises the possibility that this species is particularly prone to the emergence of transmissible cancers. More generally, our findings highlight the potential for cancer cells to depart from their hosts and become dangerous transmissible pathogens.

Devil facial tumour disease (DFTD) is a transmissible cancer that has brought the host species, the Tasmanian devil, to the brink of extinction. The cancer cells avoid allogeneic immune recognition by downregulating cell surface major histocompatibility complex (MHC) I expression. This should prevent CD8(+) T cell, but not natural killer (NK) cell, cytotoxicity. The reason why NK cells, normally reactive to MHC-negative cells, are not activated to kill DFTD cells has not been determined. The immune response of wild devils to DFTD, if it occurs, is uncharacterised. To investigate this, we tested 12 wild devils with DFTD, and found suggestive evidence of low levels of antibodies against DFTD cells in one devil. Eight of these devils were also analysed for cytotoxicity, however, none showed evidence for cytotoxicity against cultured DFTD cells. To establish whether mimicking activation of antitumour responses could induce cytotoxic activity against DFTD, Tasmanian devil peripheral blood mononuclear cells (PBMCs) were treated with either the mitogen Concanavalin A, the Toll-like receptor agonist polyinosinic:polycytidylic acid or recombinant Tasmanian devil IL-2. All induced the PBMC cells to kill cultured DFTD cells, suggesting that activation does not occur after encounter with DFTD cells in vivo, but can be induced. The identification of agents that activate cytotoxicity against DFTD target cells is critical for developing strategies to protect against DFTD. Such agents could function as adjuvants to induce functional immune responses capable of targeting DFTD cells and tumours in vivo.

One of the most remarkable aspects of Devil Facial Tumour Disease (DFTD) is its infectious nature, and for successful transmission it must avoid detection by the devil’s immune system. For this to occur, the devil either is severely immunosuppressed or factors produced by the tumor contribute to its avoidance of immune detection. An analysis of the devil’s immune system revealed the presence of normal-looking lymphoid organs and lymphoid cells. At a functional level the lymphocytes proliferated in response to mitogen stimulation. Subcutaneous injection of a cellular antigen produced a strong antibody response, providing compelling evidence that the devil has a competent immune system. Tumor cell analysis demonstrated that the tumor expresses the genes of the major histocompatibility complex; however, there was a limited diversity. Therefore, the most likely explanation for devil-to-devil transmission of DFTD is that the tumor is not recognized by the devil as “non-self” because of the limited genetic diversity. With its consistent morphology and relatively stable genome, this tumor would provide a reasonable target for a vaccine approach, provided the immune system can be coaxed into recognizing the tumor as “non-self.”

Devil facial tumor disease (DFTD) is a transmissible cancer that has killed most of the Tasmanian devil (Sarcophilus harrissii) population. Since the first case appeared in the mid-1990s, it has spread relentlessly across the Tasmanian devil’s geographic range. As Tasmanian devils only exist in Tasmania, Australia, DFTD has the potential to cause extinction of this species. The origin of DFTD was a Schwann cell from a female devil. The disease is transmitted when devils bite each other around the facial areas, a behavior synonymous with this species. Every devil that is ‘infected’ with DFTD dies from the cancer. Once the DFTD cells have been transmitted, they appear to develop into a cancer without inducing an immune response. The DFTD cancer cells avoid allogeneic recognition because they do not express MHC class I molecules on the cell surface. A reduced genetic diversity and the production of immunosuppressive cytokines may also contribute.

The Tasmanian devil (Sarcophilus harrisii) is under threat of extinction due to a fatal infectious neoplastic disease, named Devil Facial Tumour Disease. Tumours are transferred as allografts between animals and no effective immune response or host resistance to the disease has been detected, raising interest in the immune function of the species. To investigate whether Tasmanian devils had a competent humoral immune response, four devils were immunised with horse red blood cells (HRBC) either intraperitoneally or subcutaneously. Antibody responses were measured by direct and indirect haemagglutination assays for a period of up to 40 weeks. Primary responses were well defined, but secondary responses were prominent only in the devils immunised subcutaneously. All devils showed evidence for a memory antibody response following a booster given 32 weeks after the first injection and this was more evident with the subcutaneous route. Tasmanian devils tested were capable of mounting a humoral immune response against HRBC and the subcutaneous injection in the presence of the adjuvant Montanide was a safe and effective route.

Pathogen-driven declines in animal populations are increasingly regarded as a major conservation issue. The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction by devil facial tumor disease, a unique transmissible cancer. The disease is transmitted through direct transfer of tumor cells, which is possible because the genetic diversity of Tasmanian devils is low, particularly in the major histocompatibility complex genes of the immune system. The far northwest of Tasmania now holds the last remaining disease-free wild devil populations. The recent discovery of unique major histocompatibility complex genotypes in the northwestern region of Tasmania has raised the possibility that some animals may be resilient to the disease. We examined the differences in the epidemiology and population effects of devil facial tumor disease at 3 well-studied affected sites in eastern Tasmania and 1 in western Tasmania (West Pencil Pine). In contrast to the 3 eastern sites, there has been no rapid increase in disease prevalence or evidence of population decline at West Pencil Pine. Moreover, this is the only onsite at which the population age structure has remained unaltered 4 years after the first detection of disease. The most plausible explanations for the substantial differences in population effects and epidemiology of the disease between eastern and western sites are geographic differences in genotypes or phenotypes of devils and functional differences between tumor strains in the 2 regions. We suggest that conservation efforts focus on identifying whether either or both these explanations are correct and then, if resistance alleles exist, to attempt to spread the resistant alleles into affected populations. Such assisted selection has rarely been attempted for the management of wildlife diseases, but it may be widely applicable.

Tasmanian devils (Sarcophilus harrisii) are on the verge of extinction due to a transmissible cancer, devil facial tumour disease (DFTD). This tumour is an allograft that is transmitted between individuals without immune recognition of the tumour cells. The mechanism to explain this lack of immune recognition and acceptance is not well understood. It has been hypothesized that lack of genetic diversity at the Major Histocompatibility Complex (MHC) allowed the tumour cells to grow in genetically similar hosts without evoking an immune response to alloantigens. We conducted mixed lymphocyte reactions and skin grafts to measure functional MHC diversity in the Tasmanian devil population. The limited MHC diversity was sufficient to produce measurable mixed lymphocyte reactions. There was a wide range of responses, from low or no reaction to relatively strong responses. The highest responses occurred when lymphocytes from devils from the east of Tasmania were mixed with lymphocytes from devils from the west of Tasmania. All of the five successful skin allografts were rejected within 14 days after surgery, even though little or no MHC I and II mismatches were found. Extensive T-cell infiltration characterised the immune rejection. We conclude that Tasmanian devils are capable of allogeneic rejection. Consequently, a lack of functional allorecognition mechanisms in the devil population does not explain the transmission of a contagious cancer.

The Tasmanian devil ( Sarcophilus harrisii ) is threatened with extinction because of a contagious cancer known as Devil Facial Tumor Disease. The inability to mount an immune response and to reject these tumors might be caused by a lack of genetic diversity within a dwindling population. Here we report a whole-genome analysis of two animals originating from extreme northwest and southeast Tasmania, the maximal geographic spread, together with the genome from a tumor taken from one of them. A 3.3-Gb de novo assembly of the sequence data from two complementary next-generation sequencing platforms was used to identify 1 million polymorphic genomic positions, roughly one-quarter of the number observed between two genetically distant human genomes. Analysis of 14 complete mitochondrial genomes from current and museum specimens, as well as mitochondrial and nuclear SNP markers in 175 animals, suggests that the observed low genetic diversity in today's population preceded the Devil Facial Tumor Disease disease outbreak by at least 100 y. Using a genetically characterized breeding stock based on the genome sequence will enable preservation of the extant genetic diversity in future Tasmanian devil populations.

Be-Deviled Cancer Recently, a deadly transmissible cancer has emerged in Tasmanian devils, the largest existing marsupial carnivore. This disease, devil facial tumor disease (DFTD), leads to the growth of large facial tumors that frequently metastasize to internal organs. DFTD is thought to be transmitted by biting, and leads to death of affected animals within months, usually by obstructing the animals' ability to feed. Consequently, in the last 10 years Tasmanian devil numbers have dropped by about 60%. There are no genetic tests, vaccines, or treatments available for this disease, and without intervention, models predict that DFTD could cause extinction of Tasmanian devils in the wild within 50 years. Several lines of evidence suggest that DFTD is transmitted as a clonal allograft, whereby the cancer cells themselves are the agents of tumor transmission. Murchison et al. (p. 84 ) examined this hypothesis in detail by genotyping 25 tumor-host pairs from around Tasmania at 14 microsatellite loci and at a variable mitochondrial polymorphism. DFTD tumors were indeed found to be genetically distinct from their hosts and almost completely genetically identical to one another, supporting the idea of transmission by allograft.

A fatal transmissible tumor spread between individuals by biting has emerged in the Tasmanian devil (Sarcophilus harrisii), a carnivorous marsupial. Here we provide genetic evidence establishing that the tumor is clonal and therefore foreign to host devils. Thus, the disease is highly unusual because it is not just a tumor but also a tissue graft, passed between individuals without invoking an immune response. The MHC plays a key role in immune responses to both tumors and grafts. The most common mechanism of immune evasion by tumors is down-regulation of classical cell surface MHC molecules. Here we show that this mode of immune escape does not occur. However, because the tumor is a graft, it should still be recognized and rejected by the host's immune system due to foreign cell surface antigens. Mixed lymphocyte responses showed a lack of alloreactivity between lymphocytes of different individuals in the affected population, indicating a paucity of MHC diversity. This result was verified by genotyping, providing a conclusive link between a loss of MHC diversity and spread of a disease through a wild population. This novel disease arose as a direct result of loss of genetic diversity and the aggressive behavior of the host species. The neoplastic clone continues to spread although the population, and, without active disease control by removal of affected animals and the isolation of disease-free animals, the Tasmanian devil faces extinction.