HOMO - Journal of Comparative Human Biology 66 (2015) 27–37
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Childhood bone tuberculosis from Roman Pécs, Hungary L. Hlavenková a,∗, M.D. Teasdale b, O. Gábor c, G. Nagy d, ˇ s e, A. Marcsik f, R. Pinhasi g, T. Hajdu h,i R. Benuˇ a Institute for History of Medicine and Foreign Languages, Charles University in Prague, U Nemocnice 4, 121 08 Prague 2, Czech Republic b Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland c Archaeological Museum, Janus Pannonius Museum, Széchenyi tér 12, H-7621 Pécs, Hungary d Lánc Street Outpatient Polyclinics, Lánc utca 2, H-7626 Pécs, Hungary e Comenius University in Bratislava, Faculty of Natural Sciences, Department of Anthropology, Mlynská dolina B2, 842 15 Bratislava 4, Slovakia f Retired associate professor, University of Szeged, Mályva utca 23, H-6771 Szeged, Hungary g Earth Institute and School of Archaeology, Belfield, University College Dublin, Dublin 4, Ireland h Eötvös Loránd University, Faculty of Science, Institute of Biology, Department of Biological Anthropology, Pázmány Péter sétány 1/c, H-1117 Budapest, Hungary i Hungarian Natural History Museum, Department of Anthropology, Ludovika tér 2, H-1083 Budapest, Hungary
a r t i c l e
i n f o
Article history: Received 21 March 2014 Accepted 6 October 2014
a b s t r a c t A child from a Roman necropolis in Pécs, Hungary (4th century CE) was initially diagnosed with severe spinal osteomyelitis. The postcranial skeleton displayed bone alterations in the lower thoracic and upper lumbar segments, including vertebral body destruction, collapse and sharp kyphosis, and additional multiple rib lesions, suggesting a most likely diagnosis of pulmonary and spinal tuberculosis. This study discusses a number of selected diagnoses in the context of our pathological findings, complementing the macroscopic examination with radiological and biomolecular analyses. © 2014 Elsevier GmbH. All rights reserved.
∗ Corresponding author. Tel.: +421 903961993. E-mail address: [email protected] (L. Hlavenková). http://dx.doi.org/10.1016/j.jchb.2014.10.001 0018-442X/© 2014 Elsevier GmbH. All rights reserved.
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Introduction Tuberculosis is a chronic infectious disease caused by bacteria from the Mycobacterium tuberculosis complex. Human tuberculosis is caused by the Mycobacterium tuberculosis and is transmitted from person to person through the inhalation of infectious aerosols produced by persons with active pulmonary disease. Mycobacterium bovis is the causative agent for bovine tuberculosis and its transmission from animals to humans occurs through direct exposure or consumption of contaminated meat and dairy products (Évinger et al., 2011; Waldron, 2009). Tuberculosis is an infection known to primarily affect the soft tissues of the body, spreading into the lungs, intestines, skin or lymph nodes. In limited cases, the disease can also involve the skeleton, producing lesions that may develop in any portion of the body and may differ in nature in both adults and sub-adults (Köhler et al., 2012; Lewis, 2011; Roberts and Buikstra, 2003; Steinbock, 1976). The spine is the most commonly affected site in all age groups. In children, involvement of the joints (especially the hip, knee and ankle), the short bones of the hands and the feet, the inner surfaces of the ribs and the endocranial surfaces is common and can be regarded as possible indicators of tuberculosis (Évinger et al., 2011; Lewis, 2011). Herein, we reassess an old case of a young individual from late Roman Pécs (4th century CE) showing signs consistent with tuberculous infection; a diagnosis that was not considered in the initial osteological investigation in the 1970s (Éry, 1973). Archaeological background The Roman necropolis in Pécs, situated in the south-western part of Hungary, was established outside the ancient city of Sopianae and was in use during the 2nd–5th century CE. It occupied a large territory with the oldest burials discovered in its eastern part. These cremations, dated to the 2nd–4th centuries CE, belonged presumably to the pagan inhabitants. Some 3rd–5th century skeletal inhumations were also sparsely located among them. The north-western burial area, which was founded in the 3rd or 4th century CE, contained mainly simple pit graves or tombs constructed of bricks that grouped around more elaborate familial monumental buildings with funerary chapels or relic shrines (Hudák and Nagy, 2009). Their owners were the highest ranking citizens of Sopianae with burial chambers that were decorated with early Christian symbols and frescoes (Kárpáti and Gábor, 2004). During the 1958 and 1970 excavations a larger Christian grave group was excavated at the northwestern part of the necropolis, chronologically dated between 320 and 350 CE (Fülep, 1984). Based on the archaeological evaluation of the artefacts we may presume that these 104 single, double and even multiple inhumations were of craftsmen, merchants or freedmen. Materials and methods The skeletal remains under study were recovered from grave L/74 from the north-western area of the necropolis, and are now in the collection of the Janus Pannonius Museum in Pécs (inventory number: 74.1.95). The skeleton is of a child that was buried in a simple earth grave with a W-E (head–feet) orientation, reflecting the Christian burial customs here. No grave goods were found, but signs of grave robbing were recorded in the area of lower extremities (Fülep, 1984). The bones were moderately preserved with some vertebrae, the sacrum, the sternum, the right clavicle, and several small bones of the hand and feet missing. The age estimation was based on dental eruption (Schour and Massler, 1941) and long bone diaphyseal lengths (Johnston, 1962), which were originally used by Éry (1973) in her study. The age at the time of death was around 9.5–10 years. Traditional macroscopic examination was carried out with an emphasis to determine skeletal features of tuberculosis and any other anomalies indicating the health status of the child. The bone lesions described in the original study (Éry, 1973) were diagnosed as severe osteomyelitis in the spine with destruction and fusion of the vertebrae. However, these observations did not associate the pathological changes with tuberculosis and some additional inflammatory changes and lesions that were present on the ribs have not been documented. We applied radiological and molecular methods to further test our observations.
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Fig. 1. Grave L/74: lateral view of the thoraco-lumbar spine showing sharp angular kyphosis due to vertebral destruction in T12. Fusion of zygapophyseal joints between T11 and T12 can be seen.
Biomolecular analysis DNA was extracted from two skeletal elements from this burial: a tooth root (Grave L/74 – mandibular left first molar; Hpath1) and rib bone (Grave L/74 – rib; Hpath2) following previously published ancient DNA (aDNA) extraction protocols (MacHugh et al., 2000; Yang et al., 1998), in a specialised aDNA laboratory in Trinity College Dublin. Illumina sequencing libraries were then prepared from the aDNA extracts following the protocol of Meyer and Kircher (2010) with modifications by SánchezQuinto et al. (2012), further the end-repair step was replaced with the NEBNext End Repair Module (E6050). Seventy base pair single-end Illumina DNA sequencing of the libraries was completed on an Illumina MiSeq at TrinSeq, St James’s Hospital Dublin. The sequencing reads produced from this analysis were aligned to the human (UCSC – hg19), M. tuberculosis (NC 000962.3) and Brucella melitensis (NC 003317.1, NC 003318.1) genomes using standard aDNA alignment settings (Schubert et al., 2012). MapDamage2 and PMDtools were used to assess the authenticity of the aligned DNA from both samples using standard settings (Jónsson et al., 2013; Skoglund et al., 2014). Results Pathological alterations were noticeable predominantly in the axial skeleton, where they affected the ribs and in the spine characteristic changes were registered in the lower thoracic and upper lumbar regions. A complete destruction of the vertebral body occurred in T12 and caused a vertebral collapse between T9 and L1 segments, which bodies were atrophied. This collapse resulted in a gibbus deformity, forming a sharp angular kyphosis of about 90◦ (Fig. 1). Anterior and lateral portions of the affected vertebrae were too damaged to display any traces of remodelling, new bone formation or ankylosis, but on the posterior side, the zygapophyseal joints between T11 and T12 were fused. On radiographs, no destructive and osteolytic lesions, sclerosis or other hidden changes were illustrated in the preserved vertebral bodies (Fig. 2). New bone formation was observed on twelve ribs from both sides of the chest (Fig. 3). Diffused periostitis appeared in slight to moderate form and was mostly located on the costal groove and
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Fig. 2. Radiographs showing the affected vertebrae from anterior and right lateral views.
Fig. 3. Rib lesions in the form of diffused new bone formation were located on the visceral surfaces and costal grooves of several ribs, shown by arrows.
the visceral surfaces of the upper/middle portions of the ribs. More destructive changes, that might be suggestive of abscess formations, were detected on one left rib (Fig. 4). A smaller oval lesion was present on the upper rim that became thickened and enlarged in this area and another smooth erosive lesion was observable on the inner surface of the rib. One out of the examined ribs showed abnormalities of curvature (Fig. 4). Less pronounced changes were noted on lower limbs. Slight traces of periosteal reactions could be observed on the linea aspera of the right femur and in the two thirds of both tibiae (Fig. 5), affecting their posterior side. There was a slight enthesopathy in the left radial tuberosity, a very slight dental calculus and two carious lesions in deciduous molars in the mandible of the child. Apart from these findings, the skeletal remains produced no distinctive evidence of stress or malnutrition. Results of molecular examination A preliminary Illumina shotgun-sequencing screen of the aDNA extracts from Hpath1 and Hapth2 produced 4,124,047 raw reads (Hpath1 = 3,303,529, Hpath2 = 820,518) of which 0.46% aligned uniquely to the human genome. However, following a stringent PMDtools analysis (PMD score = 3) to discern authenticable endogenous ancient DNA (Skoglund et al., 2014) only 0.03% of the reads
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Fig. 4. Two large lytic lesions on the left rib, suggestive of the presence of paravertebral abscess formation, and the rib with abnormal curvature.
Fig. 5. Right tibia (middle portion): remains of periosteal new bone formation.
remained. This lack of authenticable endogenous data prevented any further human genomic analyses in this study. The raw reads were further aligned to the Mycobacterium tuberculosis and B. melitensis genomes. Only 0.001% of the reads aligned to the Mycobacterium tuberculosis genome post PMDtools filtering (PMD score = 3) precluding the testing of the osteological diagnosis. A similar number also aligned to the B. melitensis genome (0.001%). In both these cases the recovered reads are however, most likely
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to be sequences of environmental bacterium origin, which have aligned to the target genomes due to sequence homology. Discussion Alternative diagnostic options for the pathological changes discussed in this paper include brucellosis, pyogenic osteomyelitis, Scheuermann’s disease and fungal infections. Brucellosis may result in destruction of vertebral bodies, preferably affecting the lumbar and lumbosacral segments, but unlike in tuberculosis, vertebral collapse in brucellar spondylitis is rare, bony repair is often prominent and usually accompanied by osteophyte formation. Gibbus deformity is not usual (Aufderheide and Rodríguez-Martín, 1998; Matos et al., 2011; Ortner and Putschar, 1981). Moreover, while in children there is a tendency for the joints, especially the hip and knee, to be more involved than the spine (Lewis, 2011), no such changes were noticed. Although pyogenic osteomyelitis can lead to vertebral collapse and kyphosis, the infection usually affects a single vertebra and often involves neural arches and spinous process. Usually prominent new bone formation occurs (Mays and Taylor, 2003; Ortner and Putschar, 1981). Scheuermann’s kyphosis is more usual in juveniles and causes vertebral body destruction with possible fusion of the anterior vertebral aspects and kyphosis. However, the spinal curving is not angular and multiple anterior wedging of the vertebrae is a significant feature (Aufderheide and Rodríguez-Martín, 1998; Lewis, 2011). Various fungal infections such as blastomycosis, cryptococcosis or aspergillosis may eventually result in vertebral collapse, but mostly the posterior elements are involved and this contrasts to the pattern seen in our presented case (Aufderheide and Rodríguez-Martín, 1998; Canci et al., 2005; Suzuki and Inoue, 2007). On the multiple rib involvement, a specific diagnosis is difficult to establish. Rib lesions are not reliable indicators and several pleural diseases, including pulmonary tuberculosis, bacterial pneumonia or actinomycosis, may all be regarded as plausible causes resulting in the development of the similar type of rib lesions (Lambert, 2002; Masson et al., 2013; Waldron, 2009). According to the observations, the thoraco-lumbar pathologies suggest that tuberculosis is the most probable cause of death of the 9.5–10-year old child. The features described here bear a strong resemblance to the typical vertebral deformity, recognised in cases of spinal tuberculosis commonly known as Pott’s disease. The collapse and atrophy of vertebral bodies, marked kyphosis and slight ankylosis of the posterior parts in association with pulmonary manifestations support this diagnosis in the present dry bone specimen (Holloway et al., 2011, 2013; Ortner and Putschar, 1981). A preliminary shotgun aDNA screen of a tooth and rib bone from this burial using next generation sequencing, failed to provide sufficient data to test the osteological diagnosis. Given the technical difficulties of detecting pathogens in skeletal remains via this method (Campana et al., 2014), this result was not unexpected. Further analyses using genomic capture which has been shown to be successful in retrieving pathogen genomes from archaeological samples, could be conducted in the future (Bos et al., 2011; Bouwman et al., 2012). However these methods were outside the limits of this preliminary study. Tuberculosis is mainly a disease of childhood (Dawson and Robson Brown, 2012), when children usually contract the illness from adults suffering from chronic pulmonary disease. Its presence is also an indication of a reverse transmission within a population (Lewis, 2011). Thus, it is reasonable to suppose that the presented child case may not be a single occurrence of this infectious condition in Sopianae. Of the approximately 69 sub-adults, only one 7-year-old child from grave L/99 had nonspecific stress conditions. Some active new bone formation was evident on the anterior aspects of the third and fourth lumbar bodies, while remodelling was noted on the inner surface of three ribs. Yet, without a MTB DNA analysis, the aetiology of the spinal changes remains uncertain. Respiratory diseases seem to be a common health concern in this urban community, with at least fifteen adults and one juvenile displaying pulmonary lesions of unspecified origin. Other skeletal evidence pathognomonic of tuberculosis was not recognised in adult or non-adult remains, although it should be noted that typical skeletal features are usually present in only around 3–5% of all infected individuals (Roberts and Buikstra, 2003). Concerning the sub-adult archaeological skeletal record, cases of child tuberculosis from the Roman period are rarely discussed (e.g., Baxarias Tibau, 1997). Most recently, Lewis (2011) described seven
L. Hlavenková et al. / HOMO - Journal of Comparative Human Biology 66 (2015) 27–37 Table 1 Distribution of tuberculosis in Roman period samples from the Old World. Country Austria
Egypt and Nubia
Location
Period (CE)
Halbturn Linz
2nd–4th century Late Antique
Age (years)
40–60
Osteomyelitis tuberculosa in left carpal bones
Sayala (Nubia)
4th century
22–24
Spinal involvement
Strouhal (1989), Roberts and Buikstra (2003)
Alington Ave., Dorchester, Dorset
2nd–4th century
Early 20s
Destruction of seven thoracic vertebral bodies (T2–T8) with collapse in T2, kyphosis Spinal involvement
Stirland and Waldron (1990)
Mature Ancaster, Lincolnshire
2nd–4th century
Mature Young adult
England
Ashton, Northamptonshire
2nd–4th century
Early 20s 20s
Cirencester, Gloucestershire
2nd–4th century
17–25 Adult Adult
Poundbury, Dorset
1st–3rd century
10.6–14.5
10.6–14.5
10.6–14.5
2.6–6.5
10.6–14.5
2.6–6.5 1.0–2.5
Description
References
Lytic lesion L1
Wiltschke-Schrotta and Berner (1999) Wiltschke-Schrotta and Teschler-Nicola (1991)
Roberts and Buikstra (2003) Collapse of T9 and kyphosis Cox (1989), Anderson (2001) Multiple joint involvement Cox (1989), Anderson (2001) Cavitations in T11–T12, Stirland and Waldron lesions T5–L5 (1990) Collapse of T10 and partial Stirland and Waldron collapse of adjacent (1990) vertebrae Spinal and rib involvement Roberts and Buikstra (2003) Pott’s disease Farwell and Molleson (1993), Lewis (2011) Pott’s disease Farwell and Molleson (1993), Lewis (2011) Lytic lesions and active Lewis (2011) new bone formation on thoratic vertebrae, sacrum, ilium and ribs, dactylitis on metacarpals, periostitis on ulna and radius. New bone formation and lytic lesions on postcranium Active new bone formation Lewis (2011) on body of L4–L5 and S1. Lytic lesion S1, active new bone formation on one rib Profuse active new bone on Lewis (2011) long bones, possible osteomyelitis on right scapula Osteomyelitis of the Lewis (2011) mandible, active new bone formation on innominates and ribs Dactylitis on metatarsals Lewis (2011) and metacarpals, active new bone formation on all long bones, scapulae, pelvis and four ribs Active rib periostitis on Lewis (2011) visceral aspects Active new bone formation Lewis (2011) on visceral aspects of ribs, femora and tibiae
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Table 1 (Continued) Country
France
Hungary
Location
Period (CE)
Age (years)
Description
References
Queensford Mill, Dorset Tolpuddle Hall, Dorset Towcester, Water Lane, Northamptonshire Victoria Rd, Winchester, Hampshire West Hall
2nd–4th century 2nd–4th century 2nd–4th century
Adult
Spinal involvement
Adult
Spinal involvement
30–40
Pott’s disease T2–T9
Roberts and Buikstra (2003) Roberts and Buikstra (2003) Anderson (2001)
2nd–4th century
Adult
Spinal and rib involvement
Holloway et al. (2011)
4th century
Northern France (site not specified)
4th–5th century
˝ Gyor
3rd–4th century
25–30
Visegrad-Diós
4th–5th century
56–60
Herculaneum
1st century
35–40
Italy
30–35
Mitza Salida (Sardinia) Rome, Nomentana way
1st–3rd century 1st–2nd century
Rome
2nd–3rd century
Adult
Marvelè
2nd–3rd century
30–35
Lithuania
Verˇsvai
Spain
Prat de la Riba, Tarragona
Adult 25–35
3rd–4th century
3rd century
Roberts and Buikstra (2003)
15–17
4 individuals, details not available
Roberts and Buikstra (2003)
Tuberculous spondylitis in upper thoracic vertebrae (T2–T5) with slight anterior kyphosis Vertebral lesions T9–T11
Hajdu et al. (2012)
Lytic cavitation T11 and active new bone formation T12–L1, lytic lesions on ribs and eroded sternal xiphoid apophysis Collapse of L2 and active new bone formation L5, lytic lesions on ribs Pott’s disease
Capasso and Di Tota (1999)
Anterior scalloping T6–T9, fusion of T7–T8, resorptive lesion T10 Pulmonary calcification
Merczi (2001)
Capasso and Di Tota (1999) Manos and Floris (2005) Canci et al. (2005)
Cucina et al. (1999)
Pott’s disease, destruction in the head of one rib, thickening of the metatarsal bones Elbow and wrist joint lesions Tuberculous spondylitis in lower thoracic vertebrae with gibbus formation
Jankauskas (1999)
Cervical and thoracic involvement
Baxarias Tibau (1997)
Jankauskas (1999) Jankauskas (1999)
probable cases of tuberculosis found at the Roman Poundbury Camp in England, involving active new bone formation and lytic lesions in the spine, rib periostitis, diffuse active new bone formation on long and flat bones, osteomyelitis in the mandible or dactylitis on metacarpals. Such a wide range of lesions was not detected in Sopianae, in which the diagnosis of tuberculosis shows certain similarities with some cases documented in Hungarian or Portuguese skeletal series. A case of a child, 10–11 years old, with tuberculous spondylitis has been reported from a cemetery in Bácsalmás Homokbánya, dated to the 16th–17th century CE (Molnár et al., 2005). A second case of a 12-year-old child, displaying tuberculous spondylitis, was discovered at the cemetery of the São Miguel Church, Portugal, which was in use from 13th/14th to 19th centuries CE. The pulmonary form of tuberculosis was also noted in the same individual (Matos et al., 2011).
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Contemporary osteological and written evidence from the Old World suggests that tuberculosis was a relatively widespread and common disease in ancient Roman populations. The majority of cases currently reported in the paleopathological literature (Table 1) are from Roman Britain (also see: Conheeney, 2000; Steele and Mays, 1999), though the disease is known to had been present in other parts of Europe as well, emerging in France, Spain, Italy, Austria and Lithuania. In Hungary, two cases of bone tuberculosis, dated to the Roman period, have been reported previously. Hajdu et al. ˝ a small 3rd–4th century settlement located near a military facility (2012) described a case from Gyor, in Arrabona, where hypertrophic and reactive bone formation and slight angulation were seen in the upper thoracic spine of a young adult woman. Another female individual from Visegrad-Diós, dated between 340 and 430 CE, displays vertebral lesions possibly of tuberculous origin (Merczi, 2001). The presented case from Pécs is the first case of childhood tuberculosis identified in Roman Hungary. It also yields new evidence on the distribution of tuberculosis in the Roman world and among different social groups living in urban areas. For the studied population of Sopianae, osteological findings and chemical results revealed that part of the urban community, particularly women and children, had experienced inadequate nourishment (Éry, 1973). Such stress markers were absent in the skeletal remains of this child, indicating that he was very likely well provided for by his family and largely spared periods of nutritional and physical stress. However, little information is available about the living conditions and occupation of this population. Given the status of the city as an administrative centre of the Valeria province, it is likely that a number of factors, including increased urbanisation, overcrowding and unhygienic housing, may have created a favourable environment and probably facilitated the easy dissemination of tuberculosis and other infectious conditions (Hajdu et al., 2012; Jackson, 1988; Lewis, 2011). Acknowledgements We would like to express our gratitude to the Janus Pannonius Museum in Pécs for their permission to conduct this research on the skeletal remains. We would also like to thank the anonymous reviewers for their constructive comments. The authors also wish to thank Katalin Wolff for language editing and Valeria Mattiangeli for assistance with the MiSeq sequencing. This research was supported by the European Research Council Starting Grant of Ron Pinhasi (ERS-2010-StG 263441). Tamás Hajdu assisted in fundamental research in the frame of TÁMOP 4.2.4. 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Int. J. Osteoarchaeol. 21, 208–217. Mays, S., Taylor, G.M., 2003. A first prehistoric case of tuberculosis from Britain. Int. J. Osteoarchaeol. 13, 189–196. ˝ Merczi, M., 2001. Patológiás jelenségek vizsgálata Visegrád-Diós késo˝ római temetojében (Examination of pathological alterations in the Late Roman Age cemetery of Visegrád-Diós). A Wosinsky Mór Múzeum Évkönyve 23, 25–38. Meyer, M., Kircher, M., 2010. Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harb. Protoc., http://dx.doi.org/10.1101/pdb.prot5448. Molnár, E., Maczel, M., Marcsik, A., Pálfi, Gy., Nerlich, G.A., Zink, A., 2005. A csont-ízületi tuberkulózis molekuláris biológiai vizsgálata egy középkori temeto˝ embertani anyagában. Folia Anthropol. 3, 41–51. Ortner, D.J., Putschar, W.G.J., 1981. Identification of Pathological Conditions in Human Skeletal Remains. Smithsonian Institution Press, Washington. Roberts, C.A., Buikstra, J.E., 2003. The Bioarchaeology of Tuberculosis: A Global View on a Reemerging Disease. University Press of Florida, Gainesville. Sánchez-Quinto, F., Schroeder, H., Ramirez, O., Ávila-Arcos, M.C., Pybus, M., Olalde, I., Velazquez, A.M.V., Marcos, M.E.P., Encinas, J.M.V., Bertranpetit, J., Orlando, L.A.A., Gilbert, M.T.P., Lalueza-Fox, C., 2012. Genomic affinities of two 7,000-year-old Iberian hunter–gatherers. Curr. Biol. 22, 1494–1499. Schour, J., Massler, M., 1941. The development of the human dentition. J. Am. Dent. Assoc. 28, 1153–1160. Schubert, M., Ginolhac, A., Lindgreen, S., Thompson, J.F., Al-Rasheid, K.A.S., Willerslev, E., Krogh, A., Orlando, L., 2012. Improving ancient DNA read mapping against modern reference genomes. BMC Genomics 13, 178. Skoglund, P., Northoff, B.H., Shunkov, M.V., Derevianko, A.P., Pääbo, S., Krause, J., Jakobsson, M., 2014. Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal. Proc. Natl. Acad. Sci. U.S.A., http://dx.doi.org/10.1073/pnas.1318934111. Steele, J., Mays, S., 1999. The human bone. In: Niblett, R. (Ed.), The Excavation of a Ceremonial Site at Folly Lane, St. Albans. Brittania Monograph 14. Society for the Promotion of Roman Studies, London, pp. 307–323. Steinbock, R.T., 1976. Paleopathological Diagnosis and Interpretation: Bone Diseases in Ancient Human Populations. Charles C. Thomas, Springfield, IL. Stirland, A., Waldron, T., 1990. The earliest cases of tuberculosis in Britain. J. Archaeol. Sci. 17, 221–230.
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Strouhal, E., 1989. Palaeopathology of the Christian population at Sayala (Egyptian Nubia, 5th–11th cent. A.D.). In: Capasso, L. (Ed.), Advances in Paleopathology. Journal of Paleopathology Monographic Publications 1. Chieti, pp. 191–196. Suzuki, T., Inoue, T., 2007. Earliest evidence of spinal tuberculosis from the Aneolithic Yayoi Period in Japan. Int. J. Osteoarchaeol. 17, 392–402. Waldron, T., 2009. Palaeopathology. Cambridge University Press, Cambridge. Wiltschke-Schrotta, K., Berner, M., 1999. Distribution of tuberculosis in the skeletal material of Eastern Austrian sites. In: Pálfi, G., Dutour, O., Deák, J., Hutás, I. (Eds.), Tuberculosis: Past and Present. Golden Book Publishers and Tuberculosis Foundation, Budapest/Szeged, pp. 543–548. Wiltschke-Schrotta, K., Teschler-Nicola, M., 1991. Das spätantike Gräberfeld von Lentia/Linz. Linzer Archäologische Forschungen 19. Yang, D.Y., Eng, B., Waye, J.S., Dudar, J.C., Saunders, S.R., 1998. Technical note: improved DNA extraction from ancient bones using silica-based spin columns. Am. J. Phys. Anthropol. 105, 539–543.
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Childhood bone tuberculosis from Roman Pécs, Hungary L. Hlavenková a,∗, M.D. Teasdale b, O. Gábor c, G. Nagy d, ˇ s e, A. Marcsik f, R. Pinhasi g, T. Hajdu h,i R. Benuˇ a Institute for History of Medicine and Foreign Languages, Charles University in Prague, U Nemocnice 4, 121 08 Prague 2, Czech Republic b Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland c Archaeological Museum, Janus Pannonius Museum, Széchenyi tér 12, H-7621 Pécs, Hungary d Lánc Street Outpatient Polyclinics, Lánc utca 2, H-7626 Pécs, Hungary e Comenius University in Bratislava, Faculty of Natural Sciences, Department of Anthropology, Mlynská dolina B2, 842 15 Bratislava 4, Slovakia f Retired associate professor, University of Szeged, Mályva utca 23, H-6771 Szeged, Hungary g Earth Institute and School of Archaeology, Belfield, University College Dublin, Dublin 4, Ireland h Eötvös Loránd University, Faculty of Science, Institute of Biology, Department of Biological Anthropology, Pázmány Péter sétány 1/c, H-1117 Budapest, Hungary i Hungarian Natural History Museum, Department of Anthropology, Ludovika tér 2, H-1083 Budapest, Hungary
a r t i c l e
i n f o
Article history: Received 21 March 2014 Accepted 6 October 2014
a b s t r a c t A child from a Roman necropolis in Pécs, Hungary (4th century CE) was initially diagnosed with severe spinal osteomyelitis. The postcranial skeleton displayed bone alterations in the lower thoracic and upper lumbar segments, including vertebral body destruction, collapse and sharp kyphosis, and additional multiple rib lesions, suggesting a most likely diagnosis of pulmonary and spinal tuberculosis. This study discusses a number of selected diagnoses in the context of our pathological findings, complementing the macroscopic examination with radiological and biomolecular analyses. © 2014 Elsevier GmbH. All rights reserved.
∗ Corresponding author. Tel.: +421 903961993. E-mail address: [email protected] (L. Hlavenková). http://dx.doi.org/10.1016/j.jchb.2014.10.001 0018-442X/© 2014 Elsevier GmbH. All rights reserved.
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Introduction Tuberculosis is a chronic infectious disease caused by bacteria from the Mycobacterium tuberculosis complex. Human tuberculosis is caused by the Mycobacterium tuberculosis and is transmitted from person to person through the inhalation of infectious aerosols produced by persons with active pulmonary disease. Mycobacterium bovis is the causative agent for bovine tuberculosis and its transmission from animals to humans occurs through direct exposure or consumption of contaminated meat and dairy products (Évinger et al., 2011; Waldron, 2009). Tuberculosis is an infection known to primarily affect the soft tissues of the body, spreading into the lungs, intestines, skin or lymph nodes. In limited cases, the disease can also involve the skeleton, producing lesions that may develop in any portion of the body and may differ in nature in both adults and sub-adults (Köhler et al., 2012; Lewis, 2011; Roberts and Buikstra, 2003; Steinbock, 1976). The spine is the most commonly affected site in all age groups. In children, involvement of the joints (especially the hip, knee and ankle), the short bones of the hands and the feet, the inner surfaces of the ribs and the endocranial surfaces is common and can be regarded as possible indicators of tuberculosis (Évinger et al., 2011; Lewis, 2011). Herein, we reassess an old case of a young individual from late Roman Pécs (4th century CE) showing signs consistent with tuberculous infection; a diagnosis that was not considered in the initial osteological investigation in the 1970s (Éry, 1973). Archaeological background The Roman necropolis in Pécs, situated in the south-western part of Hungary, was established outside the ancient city of Sopianae and was in use during the 2nd–5th century CE. It occupied a large territory with the oldest burials discovered in its eastern part. These cremations, dated to the 2nd–4th centuries CE, belonged presumably to the pagan inhabitants. Some 3rd–5th century skeletal inhumations were also sparsely located among them. The north-western burial area, which was founded in the 3rd or 4th century CE, contained mainly simple pit graves or tombs constructed of bricks that grouped around more elaborate familial monumental buildings with funerary chapels or relic shrines (Hudák and Nagy, 2009). Their owners were the highest ranking citizens of Sopianae with burial chambers that were decorated with early Christian symbols and frescoes (Kárpáti and Gábor, 2004). During the 1958 and 1970 excavations a larger Christian grave group was excavated at the northwestern part of the necropolis, chronologically dated between 320 and 350 CE (Fülep, 1984). Based on the archaeological evaluation of the artefacts we may presume that these 104 single, double and even multiple inhumations were of craftsmen, merchants or freedmen. Materials and methods The skeletal remains under study were recovered from grave L/74 from the north-western area of the necropolis, and are now in the collection of the Janus Pannonius Museum in Pécs (inventory number: 74.1.95). The skeleton is of a child that was buried in a simple earth grave with a W-E (head–feet) orientation, reflecting the Christian burial customs here. No grave goods were found, but signs of grave robbing were recorded in the area of lower extremities (Fülep, 1984). The bones were moderately preserved with some vertebrae, the sacrum, the sternum, the right clavicle, and several small bones of the hand and feet missing. The age estimation was based on dental eruption (Schour and Massler, 1941) and long bone diaphyseal lengths (Johnston, 1962), which were originally used by Éry (1973) in her study. The age at the time of death was around 9.5–10 years. Traditional macroscopic examination was carried out with an emphasis to determine skeletal features of tuberculosis and any other anomalies indicating the health status of the child. The bone lesions described in the original study (Éry, 1973) were diagnosed as severe osteomyelitis in the spine with destruction and fusion of the vertebrae. However, these observations did not associate the pathological changes with tuberculosis and some additional inflammatory changes and lesions that were present on the ribs have not been documented. We applied radiological and molecular methods to further test our observations.
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Fig. 1. Grave L/74: lateral view of the thoraco-lumbar spine showing sharp angular kyphosis due to vertebral destruction in T12. Fusion of zygapophyseal joints between T11 and T12 can be seen.
Biomolecular analysis DNA was extracted from two skeletal elements from this burial: a tooth root (Grave L/74 – mandibular left first molar; Hpath1) and rib bone (Grave L/74 – rib; Hpath2) following previously published ancient DNA (aDNA) extraction protocols (MacHugh et al., 2000; Yang et al., 1998), in a specialised aDNA laboratory in Trinity College Dublin. Illumina sequencing libraries were then prepared from the aDNA extracts following the protocol of Meyer and Kircher (2010) with modifications by SánchezQuinto et al. (2012), further the end-repair step was replaced with the NEBNext End Repair Module (E6050). Seventy base pair single-end Illumina DNA sequencing of the libraries was completed on an Illumina MiSeq at TrinSeq, St James’s Hospital Dublin. The sequencing reads produced from this analysis were aligned to the human (UCSC – hg19), M. tuberculosis (NC 000962.3) and Brucella melitensis (NC 003317.1, NC 003318.1) genomes using standard aDNA alignment settings (Schubert et al., 2012). MapDamage2 and PMDtools were used to assess the authenticity of the aligned DNA from both samples using standard settings (Jónsson et al., 2013; Skoglund et al., 2014). Results Pathological alterations were noticeable predominantly in the axial skeleton, where they affected the ribs and in the spine characteristic changes were registered in the lower thoracic and upper lumbar regions. A complete destruction of the vertebral body occurred in T12 and caused a vertebral collapse between T9 and L1 segments, which bodies were atrophied. This collapse resulted in a gibbus deformity, forming a sharp angular kyphosis of about 90◦ (Fig. 1). Anterior and lateral portions of the affected vertebrae were too damaged to display any traces of remodelling, new bone formation or ankylosis, but on the posterior side, the zygapophyseal joints between T11 and T12 were fused. On radiographs, no destructive and osteolytic lesions, sclerosis or other hidden changes were illustrated in the preserved vertebral bodies (Fig. 2). New bone formation was observed on twelve ribs from both sides of the chest (Fig. 3). Diffused periostitis appeared in slight to moderate form and was mostly located on the costal groove and
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Fig. 2. Radiographs showing the affected vertebrae from anterior and right lateral views.
Fig. 3. Rib lesions in the form of diffused new bone formation were located on the visceral surfaces and costal grooves of several ribs, shown by arrows.
the visceral surfaces of the upper/middle portions of the ribs. More destructive changes, that might be suggestive of abscess formations, were detected on one left rib (Fig. 4). A smaller oval lesion was present on the upper rim that became thickened and enlarged in this area and another smooth erosive lesion was observable on the inner surface of the rib. One out of the examined ribs showed abnormalities of curvature (Fig. 4). Less pronounced changes were noted on lower limbs. Slight traces of periosteal reactions could be observed on the linea aspera of the right femur and in the two thirds of both tibiae (Fig. 5), affecting their posterior side. There was a slight enthesopathy in the left radial tuberosity, a very slight dental calculus and two carious lesions in deciduous molars in the mandible of the child. Apart from these findings, the skeletal remains produced no distinctive evidence of stress or malnutrition. Results of molecular examination A preliminary Illumina shotgun-sequencing screen of the aDNA extracts from Hpath1 and Hapth2 produced 4,124,047 raw reads (Hpath1 = 3,303,529, Hpath2 = 820,518) of which 0.46% aligned uniquely to the human genome. However, following a stringent PMDtools analysis (PMD score = 3) to discern authenticable endogenous ancient DNA (Skoglund et al., 2014) only 0.03% of the reads
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Fig. 4. Two large lytic lesions on the left rib, suggestive of the presence of paravertebral abscess formation, and the rib with abnormal curvature.
Fig. 5. Right tibia (middle portion): remains of periosteal new bone formation.
remained. This lack of authenticable endogenous data prevented any further human genomic analyses in this study. The raw reads were further aligned to the Mycobacterium tuberculosis and B. melitensis genomes. Only 0.001% of the reads aligned to the Mycobacterium tuberculosis genome post PMDtools filtering (PMD score = 3) precluding the testing of the osteological diagnosis. A similar number also aligned to the B. melitensis genome (0.001%). In both these cases the recovered reads are however, most likely
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to be sequences of environmental bacterium origin, which have aligned to the target genomes due to sequence homology. Discussion Alternative diagnostic options for the pathological changes discussed in this paper include brucellosis, pyogenic osteomyelitis, Scheuermann’s disease and fungal infections. Brucellosis may result in destruction of vertebral bodies, preferably affecting the lumbar and lumbosacral segments, but unlike in tuberculosis, vertebral collapse in brucellar spondylitis is rare, bony repair is often prominent and usually accompanied by osteophyte formation. Gibbus deformity is not usual (Aufderheide and Rodríguez-Martín, 1998; Matos et al., 2011; Ortner and Putschar, 1981). Moreover, while in children there is a tendency for the joints, especially the hip and knee, to be more involved than the spine (Lewis, 2011), no such changes were noticed. Although pyogenic osteomyelitis can lead to vertebral collapse and kyphosis, the infection usually affects a single vertebra and often involves neural arches and spinous process. Usually prominent new bone formation occurs (Mays and Taylor, 2003; Ortner and Putschar, 1981). Scheuermann’s kyphosis is more usual in juveniles and causes vertebral body destruction with possible fusion of the anterior vertebral aspects and kyphosis. However, the spinal curving is not angular and multiple anterior wedging of the vertebrae is a significant feature (Aufderheide and Rodríguez-Martín, 1998; Lewis, 2011). Various fungal infections such as blastomycosis, cryptococcosis or aspergillosis may eventually result in vertebral collapse, but mostly the posterior elements are involved and this contrasts to the pattern seen in our presented case (Aufderheide and Rodríguez-Martín, 1998; Canci et al., 2005; Suzuki and Inoue, 2007). On the multiple rib involvement, a specific diagnosis is difficult to establish. Rib lesions are not reliable indicators and several pleural diseases, including pulmonary tuberculosis, bacterial pneumonia or actinomycosis, may all be regarded as plausible causes resulting in the development of the similar type of rib lesions (Lambert, 2002; Masson et al., 2013; Waldron, 2009). According to the observations, the thoraco-lumbar pathologies suggest that tuberculosis is the most probable cause of death of the 9.5–10-year old child. The features described here bear a strong resemblance to the typical vertebral deformity, recognised in cases of spinal tuberculosis commonly known as Pott’s disease. The collapse and atrophy of vertebral bodies, marked kyphosis and slight ankylosis of the posterior parts in association with pulmonary manifestations support this diagnosis in the present dry bone specimen (Holloway et al., 2011, 2013; Ortner and Putschar, 1981). A preliminary shotgun aDNA screen of a tooth and rib bone from this burial using next generation sequencing, failed to provide sufficient data to test the osteological diagnosis. Given the technical difficulties of detecting pathogens in skeletal remains via this method (Campana et al., 2014), this result was not unexpected. Further analyses using genomic capture which has been shown to be successful in retrieving pathogen genomes from archaeological samples, could be conducted in the future (Bos et al., 2011; Bouwman et al., 2012). However these methods were outside the limits of this preliminary study. Tuberculosis is mainly a disease of childhood (Dawson and Robson Brown, 2012), when children usually contract the illness from adults suffering from chronic pulmonary disease. Its presence is also an indication of a reverse transmission within a population (Lewis, 2011). Thus, it is reasonable to suppose that the presented child case may not be a single occurrence of this infectious condition in Sopianae. Of the approximately 69 sub-adults, only one 7-year-old child from grave L/99 had nonspecific stress conditions. Some active new bone formation was evident on the anterior aspects of the third and fourth lumbar bodies, while remodelling was noted on the inner surface of three ribs. Yet, without a MTB DNA analysis, the aetiology of the spinal changes remains uncertain. Respiratory diseases seem to be a common health concern in this urban community, with at least fifteen adults and one juvenile displaying pulmonary lesions of unspecified origin. Other skeletal evidence pathognomonic of tuberculosis was not recognised in adult or non-adult remains, although it should be noted that typical skeletal features are usually present in only around 3–5% of all infected individuals (Roberts and Buikstra, 2003). Concerning the sub-adult archaeological skeletal record, cases of child tuberculosis from the Roman period are rarely discussed (e.g., Baxarias Tibau, 1997). Most recently, Lewis (2011) described seven
L. Hlavenková et al. / HOMO - Journal of Comparative Human Biology 66 (2015) 27–37 Table 1 Distribution of tuberculosis in Roman period samples from the Old World. Country Austria
Egypt and Nubia
Location
Period (CE)
Halbturn Linz
2nd–4th century Late Antique
Age (years)
40–60
Osteomyelitis tuberculosa in left carpal bones
Sayala (Nubia)
4th century
22–24
Spinal involvement
Strouhal (1989), Roberts and Buikstra (2003)
Alington Ave., Dorchester, Dorset
2nd–4th century
Early 20s
Destruction of seven thoracic vertebral bodies (T2–T8) with collapse in T2, kyphosis Spinal involvement
Stirland and Waldron (1990)
Mature Ancaster, Lincolnshire
2nd–4th century
Mature Young adult
England
Ashton, Northamptonshire
2nd–4th century
Early 20s 20s
Cirencester, Gloucestershire
2nd–4th century
17–25 Adult Adult
Poundbury, Dorset
1st–3rd century
10.6–14.5
10.6–14.5
10.6–14.5
2.6–6.5
10.6–14.5
2.6–6.5 1.0–2.5
Description
References
Lytic lesion L1
Wiltschke-Schrotta and Berner (1999) Wiltschke-Schrotta and Teschler-Nicola (1991)
Roberts and Buikstra (2003) Collapse of T9 and kyphosis Cox (1989), Anderson (2001) Multiple joint involvement Cox (1989), Anderson (2001) Cavitations in T11–T12, Stirland and Waldron lesions T5–L5 (1990) Collapse of T10 and partial Stirland and Waldron collapse of adjacent (1990) vertebrae Spinal and rib involvement Roberts and Buikstra (2003) Pott’s disease Farwell and Molleson (1993), Lewis (2011) Pott’s disease Farwell and Molleson (1993), Lewis (2011) Lytic lesions and active Lewis (2011) new bone formation on thoratic vertebrae, sacrum, ilium and ribs, dactylitis on metacarpals, periostitis on ulna and radius. New bone formation and lytic lesions on postcranium Active new bone formation Lewis (2011) on body of L4–L5 and S1. Lytic lesion S1, active new bone formation on one rib Profuse active new bone on Lewis (2011) long bones, possible osteomyelitis on right scapula Osteomyelitis of the Lewis (2011) mandible, active new bone formation on innominates and ribs Dactylitis on metatarsals Lewis (2011) and metacarpals, active new bone formation on all long bones, scapulae, pelvis and four ribs Active rib periostitis on Lewis (2011) visceral aspects Active new bone formation Lewis (2011) on visceral aspects of ribs, femora and tibiae
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Table 1 (Continued) Country
France
Hungary
Location
Period (CE)
Age (years)
Description
References
Queensford Mill, Dorset Tolpuddle Hall, Dorset Towcester, Water Lane, Northamptonshire Victoria Rd, Winchester, Hampshire West Hall
2nd–4th century 2nd–4th century 2nd–4th century
Adult
Spinal involvement
Adult
Spinal involvement
30–40
Pott’s disease T2–T9
Roberts and Buikstra (2003) Roberts and Buikstra (2003) Anderson (2001)
2nd–4th century
Adult
Spinal and rib involvement
Holloway et al. (2011)
4th century
Northern France (site not specified)
4th–5th century
˝ Gyor
3rd–4th century
25–30
Visegrad-Diós
4th–5th century
56–60
Herculaneum
1st century
35–40
Italy
30–35
Mitza Salida (Sardinia) Rome, Nomentana way
1st–3rd century 1st–2nd century
Rome
2nd–3rd century
Adult
Marvelè
2nd–3rd century
30–35
Lithuania
Verˇsvai
Spain
Prat de la Riba, Tarragona
Adult 25–35
3rd–4th century
3rd century
Roberts and Buikstra (2003)
15–17
4 individuals, details not available
Roberts and Buikstra (2003)
Tuberculous spondylitis in upper thoracic vertebrae (T2–T5) with slight anterior kyphosis Vertebral lesions T9–T11
Hajdu et al. (2012)
Lytic cavitation T11 and active new bone formation T12–L1, lytic lesions on ribs and eroded sternal xiphoid apophysis Collapse of L2 and active new bone formation L5, lytic lesions on ribs Pott’s disease
Capasso and Di Tota (1999)
Anterior scalloping T6–T9, fusion of T7–T8, resorptive lesion T10 Pulmonary calcification
Merczi (2001)
Capasso and Di Tota (1999) Manos and Floris (2005) Canci et al. (2005)
Cucina et al. (1999)
Pott’s disease, destruction in the head of one rib, thickening of the metatarsal bones Elbow and wrist joint lesions Tuberculous spondylitis in lower thoracic vertebrae with gibbus formation
Jankauskas (1999)
Cervical and thoracic involvement
Baxarias Tibau (1997)
Jankauskas (1999) Jankauskas (1999)
probable cases of tuberculosis found at the Roman Poundbury Camp in England, involving active new bone formation and lytic lesions in the spine, rib periostitis, diffuse active new bone formation on long and flat bones, osteomyelitis in the mandible or dactylitis on metacarpals. Such a wide range of lesions was not detected in Sopianae, in which the diagnosis of tuberculosis shows certain similarities with some cases documented in Hungarian or Portuguese skeletal series. A case of a child, 10–11 years old, with tuberculous spondylitis has been reported from a cemetery in Bácsalmás Homokbánya, dated to the 16th–17th century CE (Molnár et al., 2005). A second case of a 12-year-old child, displaying tuberculous spondylitis, was discovered at the cemetery of the São Miguel Church, Portugal, which was in use from 13th/14th to 19th centuries CE. The pulmonary form of tuberculosis was also noted in the same individual (Matos et al., 2011).
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Contemporary osteological and written evidence from the Old World suggests that tuberculosis was a relatively widespread and common disease in ancient Roman populations. The majority of cases currently reported in the paleopathological literature (Table 1) are from Roman Britain (also see: Conheeney, 2000; Steele and Mays, 1999), though the disease is known to had been present in other parts of Europe as well, emerging in France, Spain, Italy, Austria and Lithuania. In Hungary, two cases of bone tuberculosis, dated to the Roman period, have been reported previously. Hajdu et al. ˝ a small 3rd–4th century settlement located near a military facility (2012) described a case from Gyor, in Arrabona, where hypertrophic and reactive bone formation and slight angulation were seen in the upper thoracic spine of a young adult woman. Another female individual from Visegrad-Diós, dated between 340 and 430 CE, displays vertebral lesions possibly of tuberculous origin (Merczi, 2001). The presented case from Pécs is the first case of childhood tuberculosis identified in Roman Hungary. It also yields new evidence on the distribution of tuberculosis in the Roman world and among different social groups living in urban areas. For the studied population of Sopianae, osteological findings and chemical results revealed that part of the urban community, particularly women and children, had experienced inadequate nourishment (Éry, 1973). Such stress markers were absent in the skeletal remains of this child, indicating that he was very likely well provided for by his family and largely spared periods of nutritional and physical stress. However, little information is available about the living conditions and occupation of this population. Given the status of the city as an administrative centre of the Valeria province, it is likely that a number of factors, including increased urbanisation, overcrowding and unhygienic housing, may have created a favourable environment and probably facilitated the easy dissemination of tuberculosis and other infectious conditions (Hajdu et al., 2012; Jackson, 1988; Lewis, 2011). Acknowledgements We would like to express our gratitude to the Janus Pannonius Museum in Pécs for their permission to conduct this research on the skeletal remains. We would also like to thank the anonymous reviewers for their constructive comments. The authors also wish to thank Katalin Wolff for language editing and Valeria Mattiangeli for assistance with the MiSeq sequencing. This research was supported by the European Research Council Starting Grant of Ron Pinhasi (ERS-2010-StG 263441). Tamás Hajdu assisted in fundamental research in the frame of TÁMOP 4.2.4. 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