The paleopathological, paleoradiological, histological, molecular and forensic investigation of a female mummy (radiocarbon dated 1451–1642 AD) provides circumstantial evidence for massive skull trauma affecting a young adult female individual shortly before death along with chronic infection by Trypanosoma cruzi (Chagas disease). The mummy (initially assumed to be a German bog body) was localized by stable isotope analysis to South America at/near the Peruvian/Northern Chilean coast line. This is further supported by New World camelid fibers attached to her plaits, typical Inca-type skull deformation and the type of Wormian bone at her occiput. Despite an only small transverse wound of the supraorbital region computed tomography scans show an almost complete destruction of face and frontal skull bones with terrace-like margins, but without evidence for tissue reaction. The type of destruction indicates massive blunt force applied to the center of the face. Stable isotope analysis indicates South American origin: Nitrogen and hydrogen isotope patterns indicate an extraordinarily high marine diet along with C4-plant alimentation which fits best to the coastal area of Pacific South America. A hair strand over the last ten months of her life indicates a shift to a more “terrestric” nutrition pattern suggesting either a move from the coast or a change in her nutrition. Paleoradiology further shows extensive hypertrophy of the heart muscle and a distended large bowel/rectum. Histologically, in the rectum wall massive fibrosis alternates with residual smooth muscle. The latter contains multiple inclusions of small intracellular parasites as confirmed by immunohistochemical and molecular ancient DNA analysis to represent a chronic Trypanosoma cruzi infection. This case shows a unique paleopathological setting with massive blunt force trauma to the skull nurturing the hypothesis of a ritual homicide as previously described in South American mummies in an individual that suffered from severe chronic Chagas disease.

Copyright: © 2014 Panzer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this report, we describe the interdisciplinary study of a previously unknown mummy brought in the 1900s to Bavaria, Germany, which is now housed in the Bavarian State Archeological Collection. Since no records were available on the origin, life and living conditions, we used a broad panel of techniques to unravel the “life story” of the female individual resulting in an intriguing observation with an unexpected paleopathological and forensic outcome.

The human remains (mummies and skeletons) from previous cultures represent an enormous bioarchive suitable for the reconstruction of living and disease conditions in past populations, including evidence for infectious diseases and violent trauma. The application of modern analytical techniques provides an increasing spectrum of information to be used. Accordingly, the recent significant advances of modern radiological techniques and molecular analysis of various biomolecules (ancient DNA, “proteomics”) and instable as well as stable isotopes (absolute dating, diet, localization of origin) provide an increasing body of information. To this regard, complete mummies are much more informative than mummy parts or only bones [1] . Fine examples for the potential of such studies have exemplarily been recently shown by Hawass et al. [2] , [3] in the case of the mummy of Pharaoh Tutanchamun and other royal mummies from ancient Egypt. Accordingly, any such study seems to be helpful for our understanding of the past.

Materials and Methods

The mummy is currently housed in the Bavarian State Archaeological Collection. It is part of the museum since 1970 when it was transferred from the Anatomical Institute of the Ludwig-Maximilians University. There is has been recorded first in 1904. The mummy has no specimen or particular identification number (there exists only the identification number of the Anatomical Institute of the Munich University, #817/1904, and there has been given no new number when transferred to the Bavarian State Archaeological Museum in 1970; since this is, however, the only mummy in that museum it can unambiguously be identified and accessed by other researchers). All studies were undertaken with the full consent of the museum and supervised by one of the co-authors (BH).

Whilst the mummy was recorded first in the Anatomic Institute, there is no written evidence available neither about the mummy’s origin nor its way to the Munich University. During World War II, the mummy suffered some damage (especially the loss of both lower legs) during bombing [4]. Due to the dark brown external appearance (Figure 1A), she was tentatively assumed to represent a “bog body” from a moor region in the close surroundings of Munich (e.g. from the so-called “Dachauer Moos”) [4]. Unusually for this assumption, however, were the good preservation of the outer shape and – even more remarkably – the well-preserved structure of bone tissue even in previous radiograms [4]. Furthermore, despite the missing lower legs (that seem to have been torn off more recently), the female is presented in a squat-position and wears long aesthetic plaits which again is highly unusual for individuals of past European populations (Figure 1B). In the 1970ies the mummy was donated to the Bavarian Archeological State Collection, Munich. Since its donation, the female body had been on exhibition for several decades in the collection.

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larger image TIFF original image Download: Figure 1. Macroscopic aspect of the mummy. (A) Frontal view of the mummy which reveals typical squatting position (although the legs are broken off below both knees). (B) External appearance of the hair plaits which are fixed at their ends by tiny ropes of foreign material. (C) Detailed view of the mummy’s face. Note the transverse defect above the left eye. Both eyes are closed and covered by skin. The mouth is ovally opened, the frontal teeth are missing. https://doi.org/10.1371/journal.pone.0089528.g001

Due to the inconsistencies with a presumed Middle European origin, the collection decided to investigate the mummy in more detail. This work-up consisted of a concise macromorphological and anthropological investigation, a complete body computed tomography (CT) scan, analysis of stable and unstable isotopes, histological analysis of various tissue samples, molecular identification of parasitic ancient DNA (aDNA) and forensic reconstruction of injury.

Paleopathological and Anthropological Investigation In a first step, the mummy was subjected to a detailed anthropological and macroscopical inspection. As indicated before, a full body length could not directly be measured due to a lack of both lower legs. Form and shape of the hair/plaits were recorded. The estimation of body height was done by estimating body height from the (radiologically determined) maximum length of the femora and humeri according to the established data sets by Sjovold [5].

Paleoradiological Analysis The mummy was thoroughly investigated by full-body CT scan (64-row detector CT, LightSpeed VCT, General Electrics, Milwaukee, Wisconsin, USA) in supine position with slice thickness of 0.625 mm, interval of 0.625 mm, 120 kV and 200 mA in standard algorithm as previously done [6]. Additional three-dimensional and multi-planar reconstructions as well as maximum intensity projections were prepared on the attached workstation (ADW 4.4, General Electrics, Milwaukee, Wisconsin, USA).

Microscopical Study and Morphological Hair Analysis For the investigation of the bone and soft tissue structure, first a bone-cartilage sample was taken at the “open end” of the right broken leg. Here the right patella was easily accessible. The sample was subsequently divided into equal halves with one being directly embedded into Epoxy resin and a resin section (4 µm thickness) was cut for Toluidin blue staining. The second half was rehydrated with Ruffer solution, gently decalcified and embedded into paraffin for routine histological staining as previously done [7], [8]. The details of the histomorphological analysis are presented in Text S1. Furthermore, a full-thickness biopsy was taken from the rectum wall which was divided into one half for histology and one half for subsequent molecular analysis. The histological sample was rehydrated as indicated before. All histological samples were stained with H&E, connective tissue stains, PAS, Grockotts silver staining, Prussian blue and May- Grünwald-Giemsa stain [7]. Furthermore, the histological sample was subjected to an immunohistochemical analysis of particular parasites (Trypanosoma cruzi, antibody Acris Antibodies GmbH, Herford Germany). This analysis was performed according to the previously established protocols for immunostainings of rehydrated mummified tissue material [8] (details of tissue preparation and staining are given in Text S1). An analysis of the mummy’s hair, as well as fibre analysis of the small fibre bundles that fixed the hair at its ends was performed by light microscopy (and in case of the fixing fibers with scanning electron microscopy). The latter was kindly performed by Mrs. Kerstin Gonda, Bayerisches Landeskriminalamt, Munich, Germany. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Sequence Data of the T. cruzi amplification product. https://doi.org/10.1371/journal.pone.0089528.t001

Unstable and Stable Isotope Analysis Unstable isotope analysis of 14C-radiocarbon was done in order to evaluate the mummy’s actual living period. This was done by the Archeometry Laboratory of the University of Erlangen with calibrated dating evaluation. Stable isotopes were determined in a hair strand (nitrogen, carbon,) according to well established protocols. Details and background are presented in Text S2. In brief, the hair strand of approximately 10 cm length was carefully removed from the mummy’s scalp, the proximal end was marked, the sample carefully cleaned with distilled water and dissected into 2 cm long segments. The standardized procedure of O’Connell and Hedges [9] was strictly followed. Stable isotope analysis and concentration measurements of nitrogen and carbon were performed using isotope ratio mass spectrometry (IsoAnalytical, Crewe, UK).

Molecular Investigation of Psychoactive Substances In a further approach, we tested a strand of mummy hair for the presence of various psychoactive substances, particularly those frequently used in South American settings. Therefore, the strand was carefully dissected, cleaned, and analyzed by GC-MS according to routine testing [10]. Resulting peaks were identified by comparison of elution intervals. Accordingly, the presence of tetrahydrocannabinol, cocaine, heroine and many other psychoactive substances was tested. All tests were kindly performed by D. Thieme, IDAS Kreischa/Dresden, Germany. PPT PowerPoint slide

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larger image TIFF original image Download: Table 2. Stable isotope analysis of mummy hair. https://doi.org/10.1371/journal.pone.0089528.t002