Transmissible cancer in Tasmanian devils: localized lineage replacement and host population response

Tasmanian devil facial tumour disease (DFTD) is a clonally transmissible cancer threatening the Tasmanian devil (Sarcophilus harrisii) with extinction. Live cancer cells are the infectious agent, transmitted to new hosts when individuals bite each other. Over the 18 years since DFTD was first observed, distinct genetic and karyotypic sublineages have evolved. In this longitudinal study, we investigate the associations between tumour karyotype, epidemic patterns and host demographic response to the disease. Reduced host population effects and low DFTD infection rates were associated with high prevalence of tetraploid tumours. Subsequent replacement by a diploid variant of DFTD coincided with a rapid increase in disease prevalence, population decline and reduced mean age of the population. Our results suggest a role for tumour genetics in DFTD transmission dynamics and epidemic outcome. Future research, for this and other highly pathogenic emerging infectious diseases, should focus on understanding the evolution of host and pathogen genotypes, their effects on susceptibility and tolerance to infection, and their implications for designing novel genetic management strategies. This study provides evidence for a rapid localized lineage replacement occurring within a transmissible cancer epidemic and highlights the possibility that distinct DFTD genetic lineages may harbour traits that influence pathogen fitness.     

The impact of disease on the survival and population growth rate of the Tasmanian devil

1. We investigated the impact of a recently emerged disease, Devil Facial Tumour Disease (DFTD), on the survival and population growth rate of a population of Tasmanian devils, Sarcophilus harrisii, on the Freycinet Peninsula in eastern Tasmania. 2. Cormack-Jolly-Seber and multistate mark-recapture models were employed to investigate the impact of DFTD on age- and sex-specific apparent survival and transition rates. Disease impact on population growth rate was investigated using reverse-time mark-recapture models. 3. The arrival of DFTD triggered an immediate and steady decline in apparent survival rates of adults and subadults, the rate of which was predicted well by the increase in disease prevalence in the population over time. 4. Transitions from healthy to diseased state increased with disease prevalence suggesting that the force of infection in the population is increasing and that the epidemic is not subsiding. 5. The arrival of DFTD coincided with a marked, ongoing decline in the population growth rate of the previously stable population, which to date has not been offset by population compensatory responses.     

Conservation Management of Tasmanian Devils in the Context of an Emerging, Extinction-threatening Disease: Devil Facial Tumor Disease

An emerging infectious facial cancer threatens Tasmanian devils with extinction. The disease is likely to occur across the range of the devil within 5 years. This urgent time frame requires management options that can be implemented immediately: the establishment of insurance populations, in captivity, wild-living on islands, and aiming for eradication in areas that can be isolated. The long-term options of the spontaneous or assisted evolution of resistance or development of a field-deliverable vaccine are unlikely to be available in time. The disease’s characteristic allograft transmission through intimate contact simplifies isolation of insurance populations and breaking transmission in suppression trials. Better knowledge of contact matrices in wild devils will help focus timing and demographic targets of removals. A metapopulation approach is needed that integrates captive and wild-living island and peninsula (disease suppression) populations to minimize the loss of genetic diversity over 50 years until either extinction and reintroduction can occur, resistance evolves or a field-deliverable vaccine is developed. Given the importance of the insurance populations and the low genetic diversity of devils, a conservative target for retention of 95% genetic diversity is recommended. Encouraging preliminary results of the first disease-suppression trial on a large peninsula show fewer late stage tumors and no apparent population decline. Limiting geographic spread or suppressing the disease on a broadscale are both unlikely to be feasible. Since the synergy of devil decline and impending fox establishment could have devastating consequences for Tasmanian wildlife, it is crucial to manage the dynamics of new and old predator species together.     

Relaxation of risk-sensitive behaviour of prey following disease-induced decline of an apex predator,the Tasmanian devil

Apex predators structure ecosystems through lethal and non-lethal interactions with prey, and their global decline is causing loss of ecological function. Behavioural changes of prey are some of the most rapid responses to predator decline and may act as an early indicator of cascading effects. The Tasmanian devil (Sarcophilus harrisii), an apex predator, is undergoing progressive and extensive population decline, of more than 90% in long-diseased areas, caused by a novel disease. Time since local disease outbreak correlates with devil population declines and thus predation risk. We used hair traps and giving-up densities (GUDs) in food patches to test whether a major prey species of devils, the arboreal common brushtail possum (Trichosurus vulpecula), is responsive to the changing risk of predation when they forage on the ground. Possums spend more time on the ground, discover food patches faster and forage more to a lower GUD with increasing years since disease outbreak and greater devil population decline. Loss of top–down effects of devils with respect to predation risk was evident at 90% devil population decline, with possum behaviour indistinguishable from a devil-free island. Alternative predators may help to maintain risk-sensitive anti-predator behaviours in possums while devil populations remain low.     

Distribution and Impacts of Tasmanian Devil Facial Tumor Disease

The Tasmanian devil, Sarcophilus harrisii, is the largest extant marsupial carnivore. In 1996, a debilitating facial tumor was reported. It is now clear that this is an invariably lethal infectious cancer. The disease has now spread across the majority of the range of the species and is likely to occur across the entire range within 5 to 10 years. The disease has lead to continuing declines of up to 90% and virtual disappearance of older age classes. Mark-recapture analysis and a preliminary epidemiological model developed for the population with the best longitudinal data both project local extinction in that area over a timeframe of 10 to 15 years from disease emergence. However, the prediction of extinction from the model is sensitive to the estimate of the latent period, which is poorly known. As transmission appears to occur by biting, much of which happens during sexual encounters, the dynamics of the disease may be typical of sexually transmitted diseases. This means that transmission is likely to be frequency-dependent with no threshold density for disease maintenance. Extinction over the entire current range of the devil is therefore a real possibility and an unacceptable risk.     

Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil podoplanin

Monoclonal antibodies (mAbs) against not only human, mouse, and rat but also rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. PDPN is used as a lymphatic endothelial cell marker in pathological diagnoses. However, mAbs against Tasmanian devil PDPN (tasPDPN), which are useful for immunohistochemical analysis, remain to be developed. Herein, mice were immunized with tasPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/tasPDPN) cells, and hybridomas producing mAbs against tasPDPN were screened using flow cytometry. One of the mAbs, PMab-233 (IgG₁, kappa), specifically detected CHO/tasPDPN cells by flow cytometry and recognized tasPDPN protein by western blotting. Furthermore, PMab-233 strongly detected CHO/tasPDPN cells by immunohistochemistry. These findings suggest that PMab-233 may be useful as a lymphatic endothelial cell marker of the Tasmanian devil.     

Characterization of major histocompatibility complex class I and class II genes from the Tasmanian devil (<Emphasis Type="Italic">Sarcophilus harrisii</Emphasis>)

The Tasmanian devil (Sarcophilus harrisii) is currently threatened by an emerging wildlife disease, devil facial tumour disease. The disease is decreasing devil numbers dramatically and may lead to the extinction of the species. At present, nothing is known about the immune genes or basic immunology of the devil. In this study, we report the construction of the first genetic library for the Tasmanian devil, a spleen cDNA library, and the isolation of full-length MHC Class I and Class II genes. We describe six unique Class II beta chain sequences from at least three loci, which belong to the marsupial Class II DA gene family. We have isolated 13 unique devil Class I sequences, representing at least seven Class I loci, two of which are most likely non-classical genes. The MHC Class I sequences from the devil have little heterogeneity, indicating recent divergence. The MHC genes described here are most likely involved in antigen presentation and are an important first step for studying MHC diversity and immune response in the devil.     

Hormonal control of birth behavior in the Tasmanian devil Sarcophilus harrisii

In a number of marsupial species, females exhibit characteristic, stereotyped parturient behavior that facilitates the passage of the neonates to the pouch. In macropodids, this parturient behavior can be induced in non-pregnant females and males by treatment with either prostaglandin F2alpha (PGF2alpha) or oxytocin (OT). This study investigated the effects of PGF2alpha and OT on behavior of Tasmanian devils. Animals tended to sit or lie down quickly, with little vocalization, after treatment with PGF2alpha or OT, while after saline, the animals remained alert, seldom sat, and frequently vocalized. Hormone treatment caused increased respiration. Urogenital and pouch grooming, a characteristic element of parturient behavior in macropodids, was seen in only one devil after hormone treatment. However, no pouch or urogenital grooming was seen in videotape of a devil giving birth, so this may not be a feature of parturient behavior in this species. Overall behavior of males and females was very similar suggesting that the behavioral effects observed may be due to direct neural action of PGF2alpha or OT, rather than an indirect response to uterine or vaginal contractions caused by the hormones. This study is the first to demonstrate that OT results in PGF2alpha secretion as PGFM levels rose after OT injection.     

Evidence that disease-induced population decline changes genetic structure and alters dispersal patterns in the Tasmanian devil

Infectious disease has been shown to be a major cause of population declines in wild animals. However, there remains little empirical evidence on the genetic consequences of disease-mediated population declines, or how such perturbations might affect demographic processes such as dispersal. Devil facial tumour disease (DFTD) has resulted in the rapid decline of the Tasmanian devil, Sarcophilus harrisii, and threatens to cause extinction. Using 10 microsatellite DNA markers, we compared genetic diversity and structure before and after DFTD outbreaks in three Tasmanian devil populations to assess the genetic consequences of disease-induced population decline. We also used both genetic and demographic data to investigate dispersal patterns in Tasmanian devils along the east coast of Tasmania. We observed a significant increase in inbreeding (F_IS pre/post-disease −0.030/0.012, P<0.05; relatedness pre/post-disease 0.011/0.038, P=0.06) in devil populations after just 2–3 generations of disease arrival, but no detectable change in genetic diversity. Furthermore, although there was no subdivision apparent among pre-disease populations (θ=0.005, 95% confidence interval (CI) −0.003 to 0.017), we found significant genetic differentiation among populations post-disease (θ=0.020, 0.010–0.027), apparently driven by a combination of selection and altered dispersal patterns of females in disease-affected populations. We also show that dispersal is male-biased in devils and that dispersal distances follow a typical leptokurtic distribution. Our results show that disease can result in genetic and demographic changes in host populations over few generations and short time scales. Ongoing management of Tasmanian devils must now attempt to maintain genetic variability in this species through actions designed to reverse the detrimental effects of inbreeding and subdivision in disease-affected populations.     

Contrasting patterns of variation in MHC loci in the Alpine newt

Major histocompatibility complex (MHC) genes are essential in pathogen recognition and triggering an adaptive immune response. Although they are the most polymorphic genes in vertebrates, very little information on MHC variation and patterns of evolution are available for amphibians, a group known to be declining rapidly worldwide. As infectious diseases are invoked in the declines, information on MHC variation should contribute to devising appropriate conservation strategies. In this study, we examined MHC variation in 149 Alpine newts ( Mesotriton alpestris ) from three allopatric population groups in Poland at the northeastern margin of the distribution of this species. The genetic distinctiveness of the population groups has previously been shown by studies of skin graft rejection, allozymes and microsatellites. Two putative expressed MHC II loci with contrasting levels of variation and clear evidence of gene conversion/recombination between them were detected. The Meal-DAB locus is highly polymorphic (37 alleles), and shows evidence of historical positive selection for amino acid replacements and substantial geographical differentiation in allelic richness. On the contrary, the Meal-DBB locus exhibits low polymorphism (three alleles differing by up to two synonymous substitutions) and a uniform distribution of three alleles among geographical regions. The uniform frequencies of the presumptively neutral Meal-DBB alleles may be explained by linkage to Meal-DAB . We found differences in allelic richness in Meal-DAB between regions, consistent with the hypothesis that genetic drift prevails with increasing distance from glacial refugia. Pseudogene loci appear to have evolved neutrally. The level of DAB variation correlated with variation in microsatellite loci, implying that selection and drift interplayed to produce the pattern of MHC variation observed in marginal populations of the Alpine newt.     

Allele-specific variation in the gene copy number of human cytosine 5-methyltransferase

Previously, we have identified two alternate allelic forms of cytosine 5-methyltransferase, 5-MT I and 5-MT II, specified by polymorphic fragments of 1.5 and 1.1 kb, respectively. In the presence study, a 0.8-kb genomic probe was prepared which was confirmed to be included within the polymorphic fragments. The 0. 8-kb probe hybridised with greater intensity to the 1.1-kb fragment than the 1.5-kb fragment. Densitometric analysis indicated that there is 1 copy of 5-MT associated with 5-MT I, whereas there may be 1-4 copies of the gene associated with the 5-MT II allele. Segregation studies demonstrated that the multiple copies of 5-MT II are inherited in a Mendelian fashion. These results allow novel approaches to investigating the underlying mechanisms of cytosine methylation and gene duplication.