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Chapter 3 - The allograft theory

3.5 A comparison between CTVT and DFTD allograft programs and undone science undone science

3.5.3 The DFTD research program

3.5.3.1 A chronology of DFTD studies

In 2003 and 2004 two early studies were published in relation to the genetic diversity of devils. At the time these were not related to DFTD but became important in later research. The studies were undertaken by Menna Jones and colleagues and published in the journal Molecular Ecology. Jones was also to become a key scientist in future DFTD research. The first paper was published in 2003 and according to the abstract

‘[devil] populations are impacted by habitat clearance and anthropogenic mortality and genetic studies could be of value in informing levels of genetic diversity, mating system, dispersal and effects of natural and anthropogenic landscape features on gene flow’.49 The study revealed ‘moderate genetic variability across the species range'.50 The second study, published in 2004, again investigated genetic diversity, finding that the northwestern population was the more genetically distinct.51 The abstract concluded with the observation that there appeared to be stronger population subdivisions within carnivorous marsupials such as devils than in their placental mammal equivalents.52

45 Loh RC, 2006, The Pathology of Devil Facial Tumour Disease in Tasmanian Devils (Sarcophilus harrisii), Master of Philosophy, Murdoch University, Perth, Western Australia

46 Tasmanian Government Department of Primary Industries, Water and the Environment, 2005, Research into the Tasmanian Devil Facial Tumour Disease (DFTD) Progress Report, Department of Primary Industry, Water and Environment, Hobart, Tasmania

47 Tasmanian Government Department of Primary Industries, Water and the Environment, 2005, Devil Facial Tumour Disease Update. Available at: http://www.dpiw.tas.gov.au/inter.nsf/Attachments/LBUN-6FC79N/$FILE/DFTDUpdate.Aug05.pdf last accessed 18 September 2007

48 AusVet Animal Health Services Pty Ltd, 2005, Tasmanian Devil Facial Tumour Disease Response, Technical Workshop 29-31 August 2005, Final Report to Department of Primary Industries, Water &

Environment, Tasmania, Hobart, Tasmania

49 Jones ME, Paetkau,D, Geffen E & Moritz C, 2003, Microsatellites for the Tasmanian devil (Sarcophilus Laniarius), Molecular Ecology Notes, Vol 3, pp 277-279 p 277

50 ibid.

51 Jones, ME, Paetkau, D, Geffen E & Moritz C, 2004, Genetic diversity and population structure of Tasmanian devils, the largest marsupial carnivore, Molecular Ecology, Vol 13, pp 2197-2209

52 ibid, p 2197

In 2005 Corey Bradshaw and Barry Brook53 published in the journal Ecography results of a study relating to DFTD that first suggested a connection between facial lacerations and transmission.54 In support of the connection they cited both Guiler’s55 and Kabat’s56 observations that ‘[a]gonistic [conflict] interactions often lead to severe facial lacerations [in devils] that may increase the transmission rate of pathogens between individuals’.57 The reference to Guiler is a link that is incomplete, while the Kabat reference is a personal communication. They state ‘[o]ur models are still constrained by the lack of an explicit spatial component incorporating movement of infected individuals from disease-source regions to unaffected areas’.58 These studies on genetic diversity, population dynamics and spatial movements of devils were to become the basis of the DFTD research program.

In 2006, along with the Pearse and Swift article published in February, a number of other articles were published, including a paper by Richmond Loh and colleagues in Veterinary Pathology on the definition of the devil cancer DFTD.59 They confirmed the tumours to be a ‘poorly differentiated malignant round cell neoplasm’, qualifying the statement with ‘the scarcity and primitive appearance of the desmosomes were not

53 Both are now Professors of Ecology Evolution and Landscape Science, The University of Adelaide.

Available at: http://www.adelaide.edu.au/directory/corey.bradshaw and

http://www.adelaide.edu.au/directory/barry.brook last accessed 10 December 2012

54 Bradshaw CJA & Brook BW, 2005, Disease and the devil: density-dependent epidemiological processes explain historical population fluctuations in the Tasmanian devil, Ecography, Vol 28(2), pp 181-190

55 Guiler ER, 1992, The Tasmanian devil, St David’s Park Publication with the online reference (?url_ver=Z39.88-

2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook&rft.genre=book&rft.btitle – this reference cannot be accessed.

56 Cited personal communication.

57 Bradshaw C & Brook B, 2005, Disease and the devil: dependent epidemiological processes explain historical population fluctuations in the Tasmanian devil, Ecography, Vol 2(2), pp 185-190, p 183

58 ibid, p 188

59 Loh R, Bergfeld J, Hayes D, O’Hara A, Pyecroft S, Raidal S & Sharpe R, 2006, The Pathology of Devil Facial Tumor Disease (DFTD) in Tasmanian Devils (Sarcophilus harrisii), Veterinary Pathology, Vol 43, pp 890-895

enough evidence to classify DFTD as a carcinoma’.60 In concluding they stated that the

‘[t]ransmissibility of the tumor cells per se must be assessed to ascertain whether it satisfies Koch’s postulates’.61 Koch’s postulates are four criteria formulated by Robert Koch and Friedrich Loeffler in 1884 to establish a causal relationship between an infectious microbe and a disease.62 This study appears not to have been undertaken. A second paper published by Loh and colleagues again, in Veterinary Pathology in 2006, confirmed DFTD was consistent with cells of neuroectodermal63 origin.64 Noting there was little agreement on the cell type and classification of the neoplasm of DFTD, they stated,

‘DFTD also shares some morphologic, immunohistochemical staining, and possibly epidemiologic features with canine [C]TVT, which is a round-cell tumor of the skin. However, [C]TVT is negative for S-100. Karyotyping by cytogenetic analysis has revealed complex chromosomal rearrangements in DFTD cells but the nature of the aneuploidy differed from that found in [C]

TVT: DFTD cells were hypodiploid and contained chromosomal deletions and 4 complex marker chromosomes whose derivation was uncertain (A. Pearse, personal communication).’65

In concluding they further stated,

‘[a]n alternative explanation for the sudden occurrence of DFTD in multiple geographic locations across Tasmanian could be the occurrence of multiple concurrent epidemics owing to an unknown etiology. An epidemiologic analysis of DFTD should clarify this and may shed insights into the possible etiopathogenesis of the disease’.66

60 Loh, R, Bergfeld, J, Hayes, D, O’Hara, A, Pyecroft, S, Raidal, S and Sharpe R, 2006, The Pathology of Devil Facial Tumor Disease (DFTD) in Tasmanian Devils (Sarcophilus harrisii), Veterinary Pathology, Vol 43, pp 890-895, p 894

61 ibid, p 895

62 Princeton University, Koch’s postulates. Available at:

http://www.princeton.edu/~achaney/tmve/wiki100k/docs/Koch_s_postulates.html last accessed 29 June 2013

63 Dorland’s Medical Dictionary for Health Consumers, 2007, Neuroectoderm - region of the early embryo that develops into the brain and spinal cord as well as into the peripheral nervous system, Saunders. Available at: http://medical-dictionary.thefreedictionary.com/neuroectoderm last accessed 29 June 2013

64 Loh R, Hayes D, Mahjoor,A, O’Hara,A, Pyecroft S & Raidal S, 2006, The Immunohistochemical Characterization of Devil Facial Tumor Disease (DFTD) in the Tasmanian Devil (Sarcophilus harrisii), Veterinary Pathology, Vol 43, pp 896-903

65 ibid, p 900

66 ibid, p 902

This article also called for transmission trials to test Koch’s postulates to confirm the allograft theory.

Claire Hawkins, Senior Scientist at DPIPWE, and colleagues also published the results of a study in 2006.67 This study of devil population numbers was undertaken through regional spotlighting surveys and trapping studies to assess the decline in devil population numbers. They found ‘[n]o evidence for density dependence, or immunity, in DFTD’. They did find however, that in the northeast, prevalence remains high despite a reduction of 75-80% in the local population.68 They advised that the Devil Disease Project Team would continue to analyse and to investigate changes in DFTD distribution, spread and impact, to identify any relationship between population density and DFTD prevalence.69 They also found a significant decline (41%) in devil sightings since the first DFTD reports.

Hamish McCallum and Menna Jones, both part of the DPIPWE research team, also published a paper in PLoS Biology in October 2006, using DFTD as a case study for how to manage an emerging disease that is also a serious conservation threat.70 They posed a number of questions, while at the same time claiming that the ‘apparent spatial and temporal progression of the disease strongly suggests that it is infectious and that it is spreading’.71 The article by McCallum and Jones concludes with the observation that

67 Hawkins CE, Baars C, Hesterman H, Hocking GJ, Jones ME, Lazenby B, Mann D, Mooney N, Pemberton D, Pyecroft S, Restani M & Wiersma J, 2006, Emerging disease and population decline of an island endemic, Biological Conservation, Vol 131, pp 307-324

68 ibid, p 319

69 ibid.

70 McCallum H & Jones ME, 2006, To lose both would look like carelessness: Tasmanian Devil Facial Tumour Disease, PLoS Biology, Vol 4(10), pp 1671-1674

71 ibid, p 1671

‘[t]he question of the nature of the transmission dynamics …might be important…but it is unlikely to have much short- to medium-term impact on devising appropriate management strategies. Selective culling is likely to be far more effective … [however]

the likely key periods for disease transmission during the mating season are outside human control’.72 The focus of this paper appears to be possible conservation measures rather than an attempt to understand the devil cancer itself.

In 2007 a number of articles on DFTD were published including those that appeared in a special September issue of a new journal EcoHealth. In this issue with a special focus on the devil decline there were four articles by DPIPWE researchers and two supporting articles. A paper by Menna Jones and colleagues from DPIPWE was included, which was on the conservation management of the Tasmanian devils. It reported encouraging preliminary results of the first suppression trials on Freycinet Peninsula on the east coast of Tasmania.73 It however recognized that limiting spread or suppressing the disease on a large scale was not feasible. The trials on the peninsula were later abandoned.74 In the same issue McCallum and colleagues, including Jason Wiersma from the Forest Practices Board, had an article titled ‘Distribution and Impacts of Tasmanian Devil Facial Tumor Disease’.75 The abstract describes a mark-recapture analysis and a preliminary epidemiological model. The authors concluded ‘[a]s

72 ibid, p 1674

73 Jones, ME, Jarman PJ, Lees, CM, Hesterman H, Hamede, RK, Mooney NJ, Mann D, Pukk, CE, Bergfeld J & McCallum, H, 2006, Conservation Management of Tasmanian Devils in the Context of an Emerging, Extinction-threatening Disease: Devil Facial Tumour Disease, EcoHealth, Vol 4(3), pp 326-337 74 University of Tasmania, nd. Selective culling can’t save the devils. Available at:

http://www.utas.edu.au/tools/recent-news/news/selective-culling-cant-save-the-tasmanian-devil last accessed 9 December 2012

75 McCallum, H, Tompkins, DM, Jones, ME, Lachish,S, Marvanek, S, Lazenby, B, Hocking, G, Wiersma, J & Hawkins CE, 2007, EcoHealth, Vol 4(3), pp 318-325

transmission appears to occur by biting, much of which happens during sexual encounters’ and further speculates that this ‘means that transmission is likely to be frequency-dependent with no threshold density for disease maintenance’.76 It would appear from these modeling studies it had become accepted that stopping the spread of the devil disease was impossible. However, this again raises the question, referred to in previous studies, how does the disease spread in areas where there is severely reduced devil population numbers?

Stephen Pyecroft and colleagues, in the same issue of EcoHealth, claimed that cytogenetic analysis of tumour tissue, together with evidence from Major histocompatibility (MHC) gene analysis, provides ‘significant evidence to confirm the tumour is a transmissible neoplasm’.77 At the time, the ‘evidence’ on the MHC genes was unpublished.78 A further article, in the same issue, by Professor Woods and colleagues on the immune system of the Tasmanian devil claims there is evidence that the devil has a competent immune system and ‘the most likely explanation for devil-to-devil transmission of DFTD is that the tumor is not recognized by the devil-to-devil as “non-self”

because of the limited genetic diversity.’79 It concluded that ‘[w]ith 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”’.80 Also included in the issue was an Editorial by Andy Dobson

76 ibid, p 318

77 Pyecroft, SB, Pearse, AM, Loh R, Swift, K, Belov, K, Fox, N, Noonan, E, Hayes, D, Hyatt, A, Wang, L, Boyle, D, Church, J, Middleton D & Moore, R, 2007, Towards a Case Definition for Devil Facial

Tumour Disease: What is it? EcoHealth, Vol 4(3), pp 346-351

78 It would however be published by Siddle et al in Immunogenetics in the same month, August 2007.

79 Woods GM, Kreiss A, Belov K, Siddle HV, Obendorf DL & Muller KH, 2007, The Immune Response of the Tasmanian Devil (Sarcophilus harrisii) and Devil Facial Tumour Disease, EcoHealth, Vol 4(3), pp 338-345, p 338

80 ibid.

titled ‘Sympathy for the Devil’81 and an article by Peter Daszak and Aleksei Chmura titled ‘Cover Essay: John Gould and a Devil’s Despair’.82

In August 2007 PhD students Hannah Siddle, Claire Sanderson and their supervisor Katherine Belov published in Immunogenetics the results of the first genetic library for the Tasmanian devil.83 They claimed that the ‘MHC genes described here are …an important first step for studying MHC diversity and immune response in the devil’.84 This equivocal statement is the source, at the time unpublished, referred to above by Pyecroft et al in EcoHealth, claiming confirmation of the transmissibility of the DFTD tumour.

In October 2007 Siddle and Belov together with the DFTD research scientists published an article in PNAS on the MHC genes in the Tasmanian devil.85 They noted that ‘[t]he most common mechanism of immune evasion by tumors is down-regulation of classical cell surface MHC molecules’, which is the case for CTVT but not for the devil cancer.86 They claimed a lack of MHC diversity, verified by genotyping, provided a ‘conclusive link between a loss of MHC diversity and spread of a disease’.87 They further claimed

‘[h]ere we provide conclusive multilocus genetic evidence for the allograft theory of DFTD transmission, confirming that this disease is a clonal rogue cell line’.88 This

81 Dobson AP, 2007, Sympathy for the Devil, EcoHealth Vol 4(3), pp 241-243

82 Daszak P & Chmura A, 2007, Cover Essay: John Gould and a Devil’s Despair, EcoHealth, Vol 4(3), pp 367-368

83 Siddle HV, Sanderson C & Belov K, 2007, Characterization of major histocompatibility complex class I and class II genes from the Tasmanian devil, Immunogenetics Vol 59, pp 753-760

84 ibid, p 753

85 Siddle, HV, Kreiss A, Eldridge MDB, Noonan, E, Clarke, CJ, Pyecroft, S, Woods GM & Belov K, 2007, Transmission of a fatal clonal tumor by biting occurs due to depleted MHC diversity in a threatened carnivorous marsupial, PNAS, Vol 104(41), 16221-16226

86 ibid, p 16221

87 ibid.

88 Siddle, HV, Kreiss A, Eldridge MDB, Noonan, E, Clarke, CJ, Pyecroft, S, Woods GM & Belov K, 2007, Transmission of a fatal clonal tumor by biting occurs due to depleted MHC diversity in a threatened carnivorous marsupial, PNAS, Vol 104(41), 16221-16226, p 16224

finding was to be later found to be false, when it was revealed that tissue grafts between devils had been rejected, indicating that the lack of genetic diversity in the devils MHC was not responsible for the transmission of the cancer.89 Subsequently, in an interview with Rachel Carbonell on the Australian Broadcasting Commission’s The World Today, Kathy Belov stated ‘I suppose all of science is about testing hypotheses. In this case, it turns out our hypothesis wasn’t correct’.90 In the introduction to the program Eleanor Hall stated:

[s]cientists investigating the deadly facial tumours decimating the Tasmanian devil population have just disproved their original theory and are now in a race against time to identify the cause of the cancer.

Also in 2007 Shelly Lachish, Jones and McCallum published a study on the impact of DFTD on devil population growth.91 From their observations they found strong evidence that the rate of DFTD infection in the target population was increasing and that the epidemic was not declining. Meanwhile, they also state, ‘[a]t this site, DFTD prevalence remains high (33%) despite a reduction in population size from approximately 7 individuals per square kilometer to just 0.18 individuals.92 They conclude given this decline in population numbers ‘local population extinction seems likely’.93

89 All eastern devils tested in ‘in vivo’ allograft experiments – total of 8 animals – all showed host-graft or graft-host rejection. 22 March 2009 Kreiss & Woods laboratory notes (Appendix C).

90 Carbonell R, 2012, Tasmanian devil facial tumour theory debunked, Australian Broadcasting Commission, The World Today with Eleanor Hall. Available at:

http://www.abc.net.au/worldtoday/content/2012/s3523185.htm last accessed 9 December 2012

91 Lachish, S, Jones, M & McCallum H, 2007, The impact of disease on the survival and population growth rate of the Tasmanian devil, Journal of Animal Ecology, Vol 76, pp 926-936

92 ibid, p 935

93 ibid.

Authors independent of the DPIPWE also published a paper titled ‘Update on the devil facial tumour in Tasmania’ in the European Journal of Oncology in 2007.94 The authors were Neil McGlashan95 from the School of Geography, University of Tasmania (UTAS), David Obendorf, a veterinary pathologist, and Jack S Harington,96 a cancer researcher.

It reported on the forum of research scientists held in Hobart in February 2007 revealing that transmission experiments to support the allograft cell transfer theory had been attempted but the results had not been published. Stephen Pyecroft from the DPIPWE Mt Pleasant laboratory presented an abstract to the Forum on his transmission trials which stated ‘[t]rial animals injected with cell lines and receiving surgical implants of tumour tissue developed actively developing cancers at the treatment sites, to a variable degree’.97 No further studies have been undertaken.

In 2008 Obendorf and McGlashan published a paper, titled ‘Research priorities in the Tasmanian devil facial tumour debate’, in the European Journal of Oncology proposing that two aspects of the devil research were ‘under-rated and under-funded’ and called for further research into these areas. 98 The first was the possibility of immunogenic resistance to DFTD in a separate western devil population. The second, more importantly, sought an investigation into what is described as an ‘all but neglected’ area of research stating:

94 McGlashan ND, Obendorf DL & Harington JS, 2007, Update on the devil facial tumour in Tasmania, European Journal of Oncology, Vol 12(2), pp 75-80

95 Neil McGlashan was a former member of the staff of the Cancer Research Unit and a member of the International Geography Union’s Commission for Medical Geography.

96 Dr Jack Harington was a senior member of the Cancer Research Unit at the South African Institute for Medical Research Johannesburg South Africa (Source: Harington JS & McGlashan ND, 1976, Migrant Workers and Cancer Patterns in Southern Africa, Journal of Southern African Studies, Vol 3(1), pp 92-101 97 Pyecroft SB, 2007, Transmission trials: Devil Facial Tumour Disease, Devil Facial Tumour Disease, Senior Scientist’s Scientific Forum, 20-22 February 2007, University of Tasmania, Hobart

98 Obendorf DL & McGlashan ND, 2008, Research priorities in the Tasmanian devil facial tumour debate, European Journal of Oncology, Vol 13(4), pp 229-238

that the genesis and effective transmission of this disease was the fateful culmination in a cascade of anthropogenic land-use activities and can more specifically be linked to a toxin-related aetiology occurring in a wild, carrion-feeding marsupial…99

Both of these papers appear on the DPIPWE List of Publications as at July 2011.

However, other papers by these authors, who raise the issue of competing hypotheses, do not appear. These include a paper published in 2005 by Harington and McGlashan titled ‘The Tasmanian Devil Facial Tumour Disease (DFTD) – a problem unresolved’ in Annals of the Australasian College of Tropical Medicine.100 In it they noted,‘[w]hilst no viral aetiology has yet been established, direct spread by biting and transfer of allograft cells is currently favoured speculation.’101 They further suggested that

‘[b]ecause of the lesion’s visual similarity with Kaposi’s sarcoma in humans, a form of Devil AIDS (DAIDS) or Devil HIV (DHIV) also merits consideration.’102

A further two papers were published in 2006 which are not cited in the DPIPWE List.

The first, a letter by McGlashan, Obendorf & Harington titled ‘Researching the Tasmanian devil facial tumour’, drew attention to the need to consider ‘[t]he capacity of highly toxic new-generation agents to be mutagenic, genotoxic or oncogenic needs consideration’. The second, by McGlashan, Obendorf and Harington, was again published in the European Journal of Oncology and titled ‘Aspects of the fatal malignant disease among the Tasmanian devil population’. In this paper the authors again raise the possibility that, as the Tasmanian devil is the top carnivore at the head of a native herbivorous marsupial food chain, the ‘role of bioaccumulated persistent

99 ibid, p 230

100 Harington JS & McGlashan ND, 2005, The Tasmanian Devil Facial Tumour Disease (DFTD) – a problem unresolved. Annals of the Australasian College of Tropical Medicine, Vol 6(2), p 34

101 Harington & McGlashan, 2005, p 34

102 Harington JS & McGlashan ND, 2005, The Tasmanian Devil Facial Tumour Disease (DFTD) – a problem unresolved. Annals of the Australasian College of Tropical Medicine, Vol 6(2), p 34

organic pollutants and possibly genotoxic chemicals requires investigation as do conventional infectious pathogens such as exogenous and endogenous viruses…’.103

The papers published prior to 2007 were also not cited in the EcoHealth issue in 2007 mentioned above. According to Obendorf the then DFTD Manager, Alistair Scott asked to see a draft of the first devil paper before submission to the journal as the Tasmanian government and its scientists had ‘a right to contact the journal’s editor and get the opportunity to referee or veto this paper’.104

Under pressure in 2008 a paper was published on a preliminary pilot study into the role of chemicals in the devil cancer. Walter Vetter and his colleague, Roland von der Recke both from the University of Hohenheim in Stuttgart, Germany, Robert Symons from the Australian National Measurement Institute and Stephen Pyecroft from the DPIPWE published a paper in Rapid Communications in Mass Spectrometry.105 This paper reported findings of residues of chemicals PBBs (flame retardants) and PBDEs in devil tissue. A full analysis of this paper and the lack of studies following these initial findings is given in Chapter 5.

In 2008 a number of studies were also published on the Tasmanian devil immune system. It has been shown elsewhere that CTVT cancer down regulates the dogs’

immune system in order to establish in the new host; this is not the case in the devil

103 McGlashan ND, Obendorf DL & Harington JS, 2006, Aspects of the fatal malignant disease among the Tasmanian devil population (Sarcophilus laniarius), European Journal of Oncology, Vol 11(2), pp 95-102, pp 95-96

104 Email from Obendorf to me (Appendix D).

105 Vetter W, Recke R, Symons R & Pyecroft S, 2008, Determination of polybrominated biphenyls in Tasmanian devils (Sarcophilus harrisii) by gas chromatography coupled to electron capture negative ion tandem mass spectrometry or electron ionization high-resolution mass spectrometry, Rapid

Communications in Mass Spectrometry, Vol 22, pp 4165-4170