Philip Nicholls

Learning Overview: The goal of this presentation is to describe the principles of bone diagenesis and their potential application to forensic science, the distinction between biogenic and diagenetic chemical signals in bones, the correlation between macroscopic and chemical characteristics of bone diagenesis, and an example of trace element analysis in a set of archaeological bones submerged for a known length of time in a marine environment. Impact on the Forensic Science Community: This presentation will impact the forensic science community by describing how the establishment of a correlation between macroscopic alterations and trace element concentrations and distributions in bones recovered from a marine environment will prove essential for members of the forensic science community involved in taphonomic assessments. Bone diagenesis is the global effect of the physical, biological, and chemical transformations that bones undergo between death and discovery in the archaeological or geological record. Diagenetic transformations, macroscopic and microscopic, are influenced by the physics, chemistry, and biology of the depositional environment. In living organisms, chemical processes are affected by diet, mobility, and pathologies. While studies of the diagenetic modifications and chemical composition of buried bones are extensively featured in the scientific literature, geochemical signatures characteristic of underwater bone diagenesis have not been studied in detail. This study investigated whether a geochemical fingerprint of the interaction between 11 archaeological mammalian bones and seawater and/or marine sediment could be discerned. The analyzed mammalian bones belong to a museum collection of underwater archaeological materials excavated from four submerged shipwreck sites off the Western Australia coast: Batavia (1629), Vergulde Draeck (1656), Zeewijk (1727) and Rapid (1811). The underwater excavations were conducted between 1968 and 1980, and bones from the four wrecks had been submerged in seawater and/or sediment for 347, 316, 241, and 169 years, respectively. With one exception, all of archaeological bones were fragmented, some were also heavily stained, and in two samples, the damage to the protective cortical layer was particularly extensive. Bone trace element chemistry was compared to that of a modern sheep bone (Ovis aries). Laser ablation-inductively coupled plasma/mass spectrometry was undertaken across bones mounted in epoxy rounds. Cross-sectional spot transverses followed a path from the cortical layer (exterior) through the trabecular bone in the interior. In the modern sheep bone, several trace elements showed bulk concentrations close to, or at, the limit of detection (Chromium [Cr], Cobalt [Co], Nickel [Ni], Copper [Cu], Yttrium[Y], Rare-Earth Element [REE], Thorium [Th], and Uranium [U]). In contrast, in the submerged bones, Lithium (Li), Cr, Cu, and U were elevated relative to the modern sheep bone, whereas Rubidium (Rb) and Barium (Ba) were depleted. Normalized trace element patterns in modern bone were flat, whereas in the archaeological samples, the normalized trace element pattern in the only whole sample (from Batavia) was different from that of the damaged bones from the other wrecks. Most elements with altered bulk concentrations in the archaeological bones are non-essential to biological life (Cu being the exception), supported by their low concentration in the modern sheep bone. However, Ba is usually enriched in bone by reason of known para-physiological metabolic processes. Since Li, Cr, Cu, U, Rb, and Ba are present in seawater in very low concentrations (<1ppm), it is reasonable to assume that in the archaeological bones, the relevant increase in bulk concentrations of Li, Cr, Cu, and U is entirely diagenetic in origin, perhaps due to protracted chemical exchange with sediment. The depletion of bulk concentrations of Rb and Ba is also diagenetic in origin and can be explained by protracted exposure to seawater and sediment. Furthermore, since the structure of cortical bone is denser than that of trabecular bone, cortical bone is less susceptible to alteration. This is reflected in the flat normalized element distribution profiles in bones where the cortical layer is missing or heavily damaged. As a consequence, the bulk chemical composition resulting from diagenetic chemical exchange in bone appears to be more uniformly distributed if the cortical layer is heavily damaged or missing, as reflected by the flat normalized elemental distribution profiles. In the only undamaged sample, the profiles of Li, Ba, Magnesium (Mg), Strontium (Sr), and Rb showed a gradual decrease in concentration from the outer surface toward the interior of the cortical bone. The overall conclusion is that macroscopic diagenetic alterations influence elemental concentrations and patterns of elemental distribution in bones, and their analysis allows the reconstruction of different taphonomic pathways.

Since 2006 veterinarians from Perth Zoo have been working collaboratively with the Department of Environment and Conservation and Murdoch University to investigate the causes of dramatic population declines in free-ranging woylies in south west Western Australia. Investigation to date has included examining and collecting diagnostic samples from free-ranging and captive woylies, and from conspecific free-living vertebrate species. Blood samples are analysed for haematologic and biochemical parameters, along with evidence for specific pathogens (including Macropod Herpes Virus, Trypanosomes, Toxoplasmosis and Piroplasmosis). Obviously diseased animals undergo thorough veterinary examination and treatment at Perth Zoo. Full post mortem examinations are conducted on deceased animals, including histological examination of tissues, by university pathologists. Historical information from previous disease outbreaks or population declines in woylies has been collated. Temporal and spatial analysis of the complex data sets collected during field investigations facilitates understanding of the factors influencing population dynamics.

In 2002 a research group composed of scientists and veterinarians from the Department of Conservation and Land Management and Murdoch University started a collaborative research project to investigate a debilitating papillomatosis and carcinomatosis syndrome affecting the endangered western barred bandicoot (WBB; Perameles bougainville). One of the achievements of this project was the detection and characterisation of a novel viral agent in association with this disease, -the bandicoot papillomatosis and carcinomatosis vinus type 1 (BPCV1). This discovery was very exciting for our group because in addition to identifying a presumptive causal agent for this disease we had also discovered a novel virus with features intermediate between the Papillomaviridae and Polyomaviridae. As fate would have it, subsequent investigation of similar skin lesions in a southern brown bandicoot (lsoodon obesulus) led to the equally exciting discovery of a related novel virus, bandicoot papillomatosis carcinomatosis virus type 2 (BPCV2). This discovery confirmed , reinforced and complemented the research already undertaken with BPCV1, and supported the identification of 2 related viruses belonging to a unique group of double-stranded circular DNA viruses which may be the first 2 members of a new family of viruses. Having identified a probable cause of papillomatosis in western barred bandicoots and southern brown bandicoots, prophylactic and therapeutic modalities are now being investigated in an effort to find ways to prevent and/or treat these syndromes, thereby improving the welfare of these animals.

A putative novel papillomavirus has been implicated in a debilitating papillomatosis–carcinomatosis syndrome affecting endangered Western barred bandicoots (Perameles bougainville ). Multiply-rimed rolling circle amplification was employed to amplify circular DNA from lesional tissue. This was subsequently used to deduce viral genetic sequences. DNA probes were generated for the L1, L2 (major and minor capsid proteins) and large T-antigen-like (putative oncoprotein) open reading frames. A genomic probe was also constructed from the entire viral genome, randomly cut into fragments. These probes were labelled by nick translation with digoxigenin and annealed probes were detected using antidigoxigenin alkaline phosphatise-conjugated F(ab)2 fragments and visualized with exposure to precipitating BM Purple alkaline phosphatase substrate solution (all reagents for in situ hybridization: Roche Diagnostics, Indianapolis, IN, USA). Endogenous alkaline phosphatase activity, which was especially prominent in apocrine sweat gland tissue, was blocked with levamisole. There was positive nuclear staining of keratinocytes and sebocytes in lesional biopsies from wart-affected Western barred bandicoots sampled between 2000 and 2006 for all DNA probes tested. Overfixation, freezing and autolysis all reduced the sensitivity and specificity of this technique; however, with optimally fixed, well-preserved tissue samples, positive staining was obvious and reliable. These results confirm that genetic sequences from a putative novel papillomavirus are situated within the histological lesions of the Western barred bandicoot papillomatosis–carcinomatosis syndrome, providing further evidence to support the hypothesis that this syndrome has a viral aetiology.

Western barred bandicoots (Perameles bougainville) from five Western Australian colonies were examined during 2006. Bandicoots were trapped or netted, then examined under isoflurane (Isorrane: Baxter Healthcare, Old Toongabbie, NSW, Australia) general anaesthesia. Fleas, ticks and larval trombiculid mites (chiggers) were collected from bandicoots and their handling bags and preserved in ethanol. Chiggers were mounted and cleared in Hoyer’s medium. Fleas were boiled in potassium hydroxide, dehydrated through graded alcohols, cleared in methyl salicylate and mounted with Canada balsam. Ticks were identified using a dissecting microscope. Schoutedenichia emphyla chiggers were found on periscrotal, peripouch and aural skin in many P. bougainville from Dryandra. The ticks Haemaphysalis humerosa and H. ratti were collected from Bernier and Dorre Islands. In all, four flea species have been identified on P. bougainville. Echidnophaga gallinacea and Pygiopsylla tunneyi were common at Heirisson Prong and the Islands colonies, respectively. Xenopsylla vs. vexabilis was occasionally identified on bandicoots from Bernier Island and Heirisson Prong. Leptopsylla segnis and P. tunneyi were found on a bandicoot from Kanyana. No Austrochirus dorreensis (a fur mite) or Haemaphysalis lagostrophi were collected in 2006, despite previous literature reports. Apart from localized dermatitis with chigger infestation and mild blood loss from tick and flea attachment, the significance of ectoparasitism in P. bougainville remains unresolved. The potential role of these ectoparasites as disease vectors is under current investigation. This is the first report of S. emphyla, E. gallinacea, P. tunneyi, Xenopsylla vs. vexabilis and L. segnis in P. bougainville.