Treponemal Disease in the Old World? Integrated Palaeopathological Assessment of a 9Th–11Th Century Skeleton from North-Central Spain

Anthropological Science Advance Publication

Treponemal disease in the Old World? Integrated palaeopathological assessment of a 9th–11th century skeleton from north-central Spain

Belén Lopez1*, José Manuel Lopez-Garcia2, Serafín Costilla3, Eva Garcia-Vazquez4, Eduardo Dopico5, Antonio F. Pardiñas1† 1Departamento de Biología de Organismos y Sistemas, Universidad de Oviedo, C/Catedrático Rodrigo Uría s/n, 33071 Oviedo, Spain 2Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, C/Julián Clavería s/n, 33071 Oviedo, Spain 3Hospital Universitario Central de Asturias, Servicio de Radiodiagnóstico, Calle Carretera de Rubín s/n, 33011 Oviedo, Spain 4Departamento de Biología Funcional, Facultad de Medicina, Universidad de Oviedo, C/Julián Clavería s/n, 33071 Oviedo, Spain 5Departamento de Ciencias de la Educación, Facultad Formación Profesorado y Educación, Universidad de Oviedo, C/Aniceto Sela s/n, 33005 Oviedo, Spain

Received 21 December 2016; accepted 15 May 2017

Abstract The hypothesis of an American origin of acquired (venereal) syphilis and other infectious diseases carried by Columbus’s crew has been considered for a long time as the most plausible one, and has been supported recently by considerable anthropological and molecular evidence. According to this line of thought, it can be argued that Spain would have been the gateway of acquired syphilis, which quickly expanded after the return of Columbus in the late 15th century. However, the hypothesis that syphilis previously existed in Europe but went unrecognized has also been considered by many authors. The present work reports one Pre-Columbian putative case of venereal syphilis found in a Spanish necropolis and radiocarbon dated to the 9th–11th century. The complexities of carrying out accurate diagnoses in remains from archaeological timeframes are addressed by the combined use of palaeopathological, medical, and histological techniques. The results, which point toward third-stage acquired syphilis, spark discussion about the need to revisit or complement the theories and hypothesis on the origin of syphilis in the light of a growing body of cases from European historical populations.

Key words: Palaeohistology, Pre-Columbian populations, Acquired syphilis, Radiology, CT scan

Treponema carateum, does not leave evidence in the bone, Introduction causing only cutaneous disease (Powell and Cook, 2005; Treponematosis is a chronic or subacute infectious dis- Farnsworth and Rosen, 2006; Mandell et al., 2009). The ease whose causative agent is an anaerobic filamentous spi- other three types of treponemal disease are caused by infec- rochete of the genus Treponema (Turner and Hollander, tion of different subspecies of Treponema pallidum: T. 1957). Based on clinical and geographical criteria, four pallidum subsp. endemicum leads to bejel, T. pallidum sub- types of treponemal infection can be distinguished: pinta sp. pertenue causes yaws, and T. pallidum subsp. pallidum is (called mal de pinto or cute), yaws (or frambesia) and bejel the bacteria responsible for both congenital and acquired (also known as non-venereal syphilis or endemic syphilis), syphilis (Antal et al., 2002; Centurion-Lara et al., 2006; which are transmitted by fomites (shared contaminated uten- Mandell et al., 2009). sils) and non-venereal contact (Perine et al., 1984); and ac- Some authors have hinted that syphilis could have acted quired syphilis (or simply syphilis), which might be congen- as a decimating agent in the European continent during the ital or acquired. Of all of these, only pinta, caused by Renaissance and Early Modern Age (Quétel, 1990), although no reliable epidemiological data exists to assess the * Correspondence to: Belén Lopez, Departamento de Biología de real demographic impact of this disease. In the late 15th Organismos y Sistemas, Universidad de Oviedo, C/Catedrático Rod- century, the first outbreak of this epidemic was reported in rigo Uría s/n, 33071 Oviedo, Spain. the Old World, carried and disseminated by mercenaries ac- E-mail: [email protected] companying King Charles VIII of France in the invasion of † Present address: Antonio F. Pardiñas, MRC Centre for Neuropsy- chiatric Genetics and Genomics, School of Medicine, Cardiff Uni- Italy. In the 16th century syphilis became more widespread versity, Hadyn Ellis Building, Maindy Road, Cardiff CF24 4HQ, but reportedly became less virulent (Quétel, 1990; Sefton, UK. 2001; Tognotti, 2009), and a controversy started about Published online 20 July 2017 whether this disease could be a result of the mutual Europe- in J-STAGE (www.jstage.jst.go.jp) DOI: 10.1537/ase.170515 an–American discovery (Berlinguer, 1992). The exchange

© 2017 The Anthropological Society of Nippon 1 2 B. LOPEZ ET AL. Anthropological Science of infectious diseases in this context, at least from Europe to syphilis, or a closely related treponemal disease, was already America was not rare, and some examples are very well present and widespread both in the New and the Old World known, such as influenza, poliomyelitis, rabies, smallpox, in Pre-Columbian times. In ancient Europe, a possible measles, and mumps. In the opposite direction, from Ameri- source of confounding might have been leprosy, whose ca to Europe, the findings are less clear. For example, it has transmission agent is the Hansen bacillus, Mycobacterium been said that tegumentary leishmaniasis may have originat- leprae (Holcomb, 1934; Hudson, 1961; Meyer et al., 2002; ed in South America and travelled from the New world into Powell and Cook, 2005), and which was only recognized as Europe (Gonçalves et al., 2004; Lukeš et al., 2007). Never- a different entity to syphilis because of a surge of virulence theless, studies from Ancient Egypt and Upper Nubia found (Meyer et al., 2002; Waldron, 2009). In this framework, it is cases of leishmaniasis in humans as early as 2000 BC in thought that recognition of treponemal diseases improved north-east Africa (Zink et al., 2006). The sporadic nature of over time, and references to syphilis became increasingly anthropological findings and the difficulties of phylogenetic more usual. Some authors propose that a reason for the in- dating make these cases of conflicting evidence very hard to crease of awareness of syphilis were important outbreaks resolve (Clark, 2010). (Waldron, 2009) that could have been caused by virulent There is added controversy regarding the origin of syphi- New World strains (El-Najjar, 1979; Kampmeier, 1984). lis. One of the most popular and oldest hypotheses (the ‘Co- While these hypotheses are both supported by palaeo- lumbian hypothesis’) promulgates that the disease came pathological findings, actual strains of treponemal bacteria from America and subsequently spread throughout Europe have been analysed to assess the origin of acquired syphilis (see Naranjo, 1994, for a review). The chronicler Gonzalo using phylogenetic approaches. Hackett (1963) proposed an Fernandez de Oviedo was probably the first person who ar- interesting mutational pattern of the origin and dispersal of gued for the Columbian hypothesis. In 1526, after a decade the four treponemal diseases. He argued that acquired syph- living in Santo Domingo, he published a book (Of the Natu- ilis could have arisen in south-western Asia from a mutation ral History of the Indies), where he described how the na- in the bacteria that causes bejel about 5000 years BP. Later it tives used lignum vitae (Guaiacum sp.) to treat syphilis. would have spread to Europe and the rest of the world. This Nine years later, in his comprehensive study General and theory has been recently tested with phylogenetic methodol- Natural History of the Indies, isles and mainland of the ogy, which showed that pathogenic yaws-causing Trepone- Ocean, he argued for the American origin of syphilis based ma from Africa showed the greatest similarity with simian on his research of indigenous medicine and lignum vitae. strains and were the first to arise in human history (Harper et This followed the common maxim at the time that “wherev- al., 2008). Additional evidence was added by the finding of er God sends punishment or disease, there he also sends the two new strains of T. pallidum in inhabitants from Guyana. cure” (Steinbock, 1976). Modern ethnohistorical and palaeo The Guyana disease, caused by a DNA-related strain of T. anthropological studies from Jamaica have indeed suggested pallidum, seemed like a combination of two diseases, syphi- that the indigenous Taínos (Pre-Columbian inhabitants of lis and yaws, with raspberry-like eruptions and without open the Bahamas and Antilles) suffered endemically from syphi- sores. It has been suggested that this organism could be the lis, and used medicinal plants to treat this (Santos et al., ancestor from which T. pallidum evolved thousands of years 2013). For many years the archaeological evidence support- ago (Harper et al., 2008). ed this American origin (Baker and Armelagos, 1988; Pow- This collection of results excludes the possibility of syph- ell and Cook, 2005), as the vast majority of incontestable ilis being present in Europe before the return of Columbus, syphilis cases were found in New World sites. For example, and points to other treponemal diseases, not acquired syphi- Rothschild et al. (2000) recognized acquired syphilis in lis, as responsible for the above-mentioned Pre-Columbian Pre-Columbian archaeological remains in areas where Co- Old World cases. A recent paper by Harper et al. (2011) lumbus actually had contact with the natives, identifying the showed an extensive review of published syphilis cases be- Dominican Republic as the point of initial contact with longing to the Old World (Africa, Asia, and Australia and syphilis and subsequent spread from the New to the Old Europe). The conclusion of this study was that no single case World. Sailors enrolled in Christopher Columbus’s ships with a convincing diagnosis had a secure Pre-Columbian have long been regarded as the probable first importers of date, though some of its arguments were disputed (Cole and the infection (e.g. Price, 1979), which would have arisen in Waldron, 2012). In any case, the authors of the original pa- the New World before the arrival of the Europeans and per maintained their position and claimed that their intention would have been one of the intercontinental exchanges that was not to rediagnose putative cases but to assess the cer- occurred over time. tainty of both diagnoses and dating, as some of the findings In recent years, some authors have reported lesions com- traditionally held as true by the scientific community had patible with acquired syphilis in Pre-Columbian skeletons in actually been published in journals which lacked a proper regions as distant as Britain (Stirland, 1991; Mays et al., peer-review process (Armelagos et al., 2012). 2003, 2012; von Hunnius et al., 2006; Cole and Waldron, Despite all the methodological and technical advances, 2011), central Europe (Gaul et al., 2015), and the eastern the origin of acquired syphilis remains a controversial mat- Mediterranean (Mitchell, 2003, 2009; Erdal, 2006). These ter. Assuming the Columbian hypothesis and an origin of findings, if their palaeopathological assessments are correct, syphilis in the New World, the Iberian Peninsula would have support a view different to the Columbian hypothesis: the probably been the gateway for the entry of the disease into Pre-Columbian hypothesis. First formulated in the earliest Europe given the large number of sailors who travelled with decades of the 20th century, this argument proposes that Christopher Columbus to the Old World and back (Gould, PUTATIVE PRE-COLUMBIAN SYPHILIS IN SPAIN 3

1925; Díaz-Trechuelo, 1991). In fact, when the first reported outbreak occurred in Naples in 1495, Spanish soldiers garri- Materials and Methods soned there were seen as the transmitters of the disease to the Studied necropolis general population and the mercenaries who later scattered The archaeological intervention was carried out in the throughout Europe (Pearce, 1998). Considering this fact and years 1990 and 1991, at the ‘La Morterona’ necropolis, lo- the existence of one account, albeit of dubious accuracy, of a cated in the municipality of Saldaña, in the present territory syphilis outbreak in Barcelona in 1493 (Meyer et al., 2002), of Castilla-Leon (Figure 1). The necropolis was completely there exists the possibility that many early episodes of the excavated and 56 individuals were recovered—a total of 15 epidemic could have actually taken place in Spain. This subadults and 41 adults of both sexes (López, 2000; López might have been historically unnoticed, given the presuma- Martínez, 2002). Historical and archaeological evidence bly high mobility of sailors in the past and the documented showed that the necropolis was used during the medieval era speed with which the epidemic moved (Tognotti, 2009), (9th–15th centuries) and can be considered of Pre-Columbian which would have made any area of the European continent origin. Osseous material was directly provided to the re- equally affected in a few years. While this is a hypothesis to searchers by the archaeologists in charge of the excavations, consider, it is not currently supported by available data, as, under the responsibility of the Regional Government of to our knowledge, there have been relatively few published Castilla-León. cases of palaeopathological putative acquired syphilis (Reverte, 1979; Lopes and Cardoso, 2000; Codinha, 2002; Dating De Souza et al., 2006; Rissech et al., 2013; Schwarz et al., Due to the importance of knowing exactly the antiquity of 2013) coming from the Iberian Peninsula before the Modern the remains found in this medieval necropolis, radiocarbon Age. dating of a fragment from the basilar region of the occipital To contribute to this currently small body of cases, we bone from the reported individual was performed at the Beta report in this study the analysis of one Pre-Columbian skel- Analytic Inc. laboratory (Miami, FL, USA) in 2011. Accel- eton, which shows lesions that could be attributable to al- erator mass spectrometry was used for dating (Elmore and leged acquired syphilis. Skeletal evidence from Spain such Phillips, 1987), and the laboratory code given was Beta- as this could shed further light on the debate concerning the 311429. The result of the radiocarbon analysis was calibrat- origins of acquired syphilis in Europe. The remains were ed to calendar years using the IntCal09 dataset included in found in an archaeological excavation of a cemetery from the software CALIB v. 6.01 (Reimer et al., 2009; Stuiver et the northern part of the Iberian Peninsula dated to the 9th– al., 2010). 11th century. Those were intense years of military activity in A high dietary intake of marine protein has long been that part of Spain, as a small Christian nucleus, the Kingdom known as a source of bias for radiocarbon dates (Schoe- of Asturias, became established and expanded into the terri- ninger et al., 1983; Bayliss et al., 2004). Thus, the proportion tories of the dominating Umayyad Caliphate (Boyd, 2002). of carbon of marine origin in collagen is usually estimated The territory of this kingdom reached the Douro river in the by analysis of stable isotopes of carbon (δ13C) and nitrogen year 910 and as a consequence King Ordoño II (914–924) (δ15N) (Mays, 1997). Nevertheless, various concerns have moved its political core further south, creating the Kingdom been raised related to accuracy, reliability, and margins of of Leon (O’Callaghan, 2013). Between the 11th and 13th error of this estimation (Harper et al., 2011), and thus we centuries, further expansion resulted in the establishment of used an indirect approach to assess the influence of marine the Kingdom of Castile, the borders of which expanded past intake in our radiocarbon dates. Again, using CALIB v. 6.01, the Spanish central mountains and into the Tagus river basin calibrated radiocarbon ages were calculated by simulating (Zamora, 1973). the presence of up to 80% marine carbon in the sample, at

Figure 1. Map with the studied necropolis. (1) ‘La Morterona’ (Saldaña, Palencia). 4 B. LOPEZ ET AL. Anthropological Science

10% intervals. Also, to properly take into account the marine the tertiary stage of the disease a reaction may occur in the reservoir effect, a ΔR of 270 ± 40 14C years was considered bone tissue, resulting in either chronic gummatous or as valid for our geographical region and temporal period. non-gummatous inflammation (Steinbock, 1976; Ortner, This quantity was described in a neighbouring region of 2003; Powell and Cook, 2005). These are seen in roughly northern Spain (Galicia) using samples dated at 890 ± 90 BP 10–20% of the cases (Resnick and Niwayama, 1995; (Monge Soares and Alveirinho Dias, 2007). Aufderheide and Rodriguez-Martin, 1998) and are mostly a combination of the two processes (Ortner, 2003). Gumma- Sex determination tous lesions (erosive granulomas) occur due to an alteration Biological sex determination was based on methods that of the blood supply to the tissues, which initiate a reaction relied on classical morphological parameters of sexual di- that creates a granulomatous appearance of nodular foci with morphism in the os coxae (Byers, 2005). The following central liquefying necrosis, called ‘gumma.’ The gumma is morphological features of the pelvis were assessed: presence the classic lesion found in tertiary syphilis (Steinbock, 1976; of a subpubic concavity, ventral arc, and preauricular sulcus; Aufderheide and Rodriguez-Martin, 1998; Ortner, 2003; thickness of the ischiopubic ramus; and a greater sciatic Kinghorn, 2004) and it is found in both the skull and long notch shape in the pelvic bone. In addition, morphological bones. It is widely accepted in palaeopathology that the type cranial characteristics of the skull, such as the robusticity of of lesions that can be attributed to syphilis cannot be distin- the nuchal crest, the size of the mastoid process, the sharp- guished from those caused by bejel and yaws, the other two ness of the supraorbital margin, the prominence of glabella, variants of treponemal disease (Ortner, 2003; Harper et al., and the projection of the mental eminence, were also used 2011). (Buikstra and Ubelaker, 1994). Regarding the skull, lesions tend to start in the frontal bone and usually spread to affect other elements of the cra- Age determination nium. Initial lesions are pits on the surface of the bone which The assessment of age-at-death was based on published may become confluent and deeper (Steinbock, 1976). The standards related to the main macroscopic changes of the boundaries of this lesion usually rise over time, rolling over pelvis with the following criteria: metamorphosis of the pu- the margins of the injuries. As healing progresses, the cavity bic symphysis (Brooks and Suchey, 1990), changes of the starts to be covered by scar tissue until only a low depression auricular surface of the ilium (Lovejoy et al., 1985), and remains, showing radial scarring on the surface. ‘Caries sic- stages of tooth attrition (Hillson, 1996). The general pres- ca’ is the term that refers to the progress in contiguous le- ence of degenerative changes of the skeleton was also used sions that mainly affect the frontal and parietal skull bones (Cox, 2000). (Skinner, 1994; Ortner, 2003; Powell and Cook, 2005). This is a combination of three processes: active lytic lesions, re- Individual SAL 633, female, 30–40 years of age modelling, and scarring (Waldron, 2009). Hackett (1976) il- The state of preservation was poor due the fragmentary lustrated eight stages of this type of lesion, describing the nature of many of its bones. The skull showed an almost sequence of events in the formation of lesions in the skull complete splanchnocranium, left parietal, left temporal, part outer table, and its manifestation in dry bone (Table S1). of the occipital bone, and a complete maxillary bone. The To improve the differential diagnosis and overcome the upper right second and first incisors and both second molars former difficulties to some extent, the lesions of the one in- were lost post-mortem, while the two first molar were lost dividual who was macroscopically catalogued as a ‘putative ante-mortem with full to partial healing of the socket. Only case of treponemal infection’ were inspected in detail by ra- the mandible arch was preserved with all the teeth except diographic and histological techniques. Radiographs were both second molars and the right second incisive, which performed at the Central University Hospital of Oviedo were lost post-mortem. The postcranial skeleton available (Spain) using conventional techniques (40 kv and 2 mAs) includes part of the right humerus, scapulae, and clavicle, and treated in a digital CR Caresteam® system. X-ray com- with some of the ribs of this side, also fragments of sacrum puterized tomography (CT) was performed on the skull re- and both os coxae. The remaining bones are missing. mains of that same individual using an Aquilion 16 scanner (Toshiba Medical System), employing a 0.5 mm slice thick- Identification of palaeopathological indicators ness. Images were visualized in a Vitrea workstation in The bones and teeth of SAL 633 were examined directly which multiplanar and volume-rendered pictures were for detection of macroscopic and radiographic lesions sug- made. Bone samples for light microscopic analysis were re- gestive of treponemal disease (e.g. acquired syphilis) as de- moved using a handheld circular saw and subsequently described by Hackett (1975) and Aufderheide and Rodriguez- hydrated using ascending concentrations of ethanol and Martin (1998). Acquired syphilis manifests in three different embedded in low-viscosity epoxy resin Durkupan-ACM clinical stages, commonly known as primary, secondary, and (Sigma) under vacuum. The embedded samples were ground tertiary syphilis (Tramont, 1995), several of which result in down to a thickness of 15 μm, stained with toluidine blue identifiable skeletal lesions (Ortner, 2003). Soft parts of the and analysed using a Nikon Eclipse E400 microscope body are involved in the disease from the start, and even equipped with phase contrast, following the approach of mild, non-diagnostic lesions may develop in the skeleton Schultz 2001. in the earliest stage (Ehrlich and Kricun, 1976), though the influence of the disease on the skeletal system usually begins from the late second to tertiary phase (Little, 2005). In PUTATIVE PRE-COLUMBIAN SYPHILIS IN SPAIN 5

Results Only the adult female SAL633 (estimated age 30–40 years) exhibited lesions that could be indicative of acquired syphilis. Radiocarbon analysis provided a date for these remains of 1110 ± 30 BP, and calibration using the IntCal09 dataset provided a corrected date of 879–1001 AD (at 2σ). The 13C/12C ratio measured in the bone sample was of –21.1‰, which has been traditionally considered as indicat- ing no marine contribution to the diet of the living individual (Arneborg et al., 1999). Further evaluation of a possible bias due to marine protein intake by the individual is shown in Figure S1. The maximum calibrated age interval reached by our sample occurs at the value of 80% of marine contribution and is 1313–1439 AD (at 2σ). The skull of this individual had been fragmented during excavation, and was partially reconstructed later in the laboratory. It showed three lesions in the outer table of the frontal bone, of which the bigger one comprised an irregular eroded area of 8 × 5 cm (Figure 2). This area was separated by 30 mm from the glabellum. It strongly affected the outer table, while the inner table presented a slight porosity. The erosion, with the margins rolled inwards towards the center, manifested an important loss of diploe in some points. In an ectocranial view, islands of surviving outer table and confluent pit clusters can be seen (Figure 3). There were other two important lesions of smaller size, situated near to the coronal suture (Figure 4), showing the same erosion and loss of diploe described in the bigger one. There were other two important features in this skull: a smooth porosity (cluster of Figure 2. Photograph of the SAL633 skull. Note the necrotic cavi- pits) over the orbital margin and glabellum, and a widening ties in the frontal bone and the widening of the nasal septum (arrows). of the nasal cavity which appeared empty due the post- mortem destruction of the nasal septum (Figure 2). The maxillary bone also appeared to have a perforation of the Radiographs showed a lytic lesion in the frontal bone, hard palate, but we cannot be sure that this is due to the high with irregular edges without a sclerosing reaction (Figure 5). degree of destruction. CT volume-rendered images show three irregular destruc-

Figure 3. Detail of the area of the SAL633 skull widely destroyed by putative gummas. 6 B. LOPEZ ET AL. Anthropological Science

Figure 4. Detail of the area of the SAL633 skull in which the two minor putatively gummatous lesions are located (arrows, numbers). A tissue sample of 1 × 0.5 × 0.5 cm was taken from lesion 2 and used for histological analysis.

tive lesions of worm-eaten appearance, which involve the outer table. Multiplanar slices show an irregular destruction on the outer table and some portions of the diploe (Figure 6). Microscopic analysis of bone samples from this lesion demonstrated the presence of many characteristic features of diagenetically modified bone originated by the post-mortem destruction of bone tissue by taphonomic processes. Howev- er, it is remarkable that the fundamental histological pattern of the bone, characterized by concentric bone layers encir- cling Haversian canals, was clearly recognizable (Fig- ure 7A). Furthermore, some regions showed osteons with a modified lamellar layering pattern consisting of irregular accumulations of stacks of non-concentric lamellae (Fig- ure 7B). This structure is consistent with partial healing after necrosis by formation of less organized bony tissue which would be subsequently remodelled and implies the occur- rence of bone formation, a distinctive feature of caries sicca. Most of the other skeletal material from SAL633 was lost during the excavations and before the archaeological team arrived. The remains recovered included the mandibular arc, with partial dentition but without the ramus mandibulae; the left humerus without the distal third; the left scapula and some ribs (Figure S2). None of these elements showed any signs that could be pathognomic for syphilis or other diseases.

Discussion Radiocarbon dating showed that SAL633 lived during the War of Reconquista period (9th–11th century), in the Pre-Columbian era. Even though these dates could be biased by an undetected marine intake in the diet of the individual, the antiquity of the remains does not encompass Post- Figure 5. Apical plane radiograph of SAL633 skull. The arrow Columbian times in any way. Simulation analysis of marine marks the biggest lytic lesion in the frontal bone of the skull, which carbon in our sample showed that the calendar date of the affects the outer table and diploe. return of the first Columbian expedition (1493 AD) is never PUTATIVE PRE-COLUMBIAN SYPHILIS IN SPAIN 7

Figure 6. CT images of the frontal calvaria of SAL633 in different planes (A, axial; B, coronal; C, saggital). All the images show an irregular bone destruction affecting the calvaria and in which the outer table and diploe are involved. (D) 3D volume-rendered image reconstructed using sections of 0.5 mm thickness. The irregular worm-eaten nature of the destructive lesions can be appreciated (arrows) which has been reported as a characteristic of osteomyelitis associated with syphilis. Parameters used to generate this image were: 120 kVP (peak kilovoltage); 250 mA; 500 msec; 125 mA/s; 0.5 mm thick. reached, even when marine uptake is raised up to 80%. It new bone tissue formation. This is consistent with the hy- should be noted that such a high proportion of marine-based pothesis that the living bone could have suffered an inflam- carbon would be at best improbable in an inhabitant from an matory process induced by bacterial infection, which would inland peasant population such as the one we studied, with have caused a diminishing of blood supply to the centre of no direct access to marine resources other than importing the lesion and, over time, necrosis. them from neighbouring regions (Polet and Katzenberg, 2003; Müldner and Richards, 2005). Differential diagnosis Palaeopathological results are based on direct inspection It is important to consider that these bone lesions, albeit of morphological osseous lesions, as well as radiographic similar to others which had been identified as the result of and histologic study. While full certainty can hardly be acquired syphilis, could also have been caused by pathologic achieved even combining all these methods, a detailed anal- processes other than treponematosis. The first circumstance ysis of skull and bone lesions is still considered the most that we must consider is the possibility that post-mortem reliable method for identification of acquired syphilis in past taphonomic processes could have caused them, since the al- populations (Ortner, 2009). In this case, the CT appearance teration of the characteristic morphology of the outer or in- of our specimen (Figure 6) is very similar to images which ner table of bones due to the action of natural factors such as have been reported by Gurland et al. (2001) and Huang et al. animals, plants, water and soil features is possible. Post- (2007) in HIV-infected patients with acquired syphilis; and mortem damage might sometimes be difficult to differentiate also by Kang et al. (2010) in a patient with confirmed T. from certain pathological conditions that affect the skeletal pallidum infection. This morphological appearance has been structure, but some processes can hardly be mimicked by previously reported as characteristic of acquired treponemal natural post-mortem damage. This is the case of the altera- infection on radiographic and palaeopathological assess- tions in the SAL633 skull, which exhibits both bone osteol- ments (Thompson and Preston, 1952; Ortner, 2003; Shang ysis and neoformation, as these cannot be caused by taphon- and Trinkaus, 2008; Boone et al., 2009; Reinus, 2010). As omy. for the microscopic structure, it is relatively well preserved Regarding the specificity of lesions to acquired syphilis, it and scar tissue can be unmistakably recognized. The occur- has to be said that osseous reactions, although reproducible rence of this scar tissue implies a healing process including for each variety of treponemal disease (except pinta, which 8 B. LOPEZ ET AL. Anthropological Science

ohistological diagnosis: Grenzstreifen (border stripes), pol- sters (padding) and ‘sinous lacunae.’ Other authors, such as Weston (2009), have suggested that these microscopic features cannot be used to diagnose specific pathological conditions at all, as they were found to occur in pathological bone specimens with different disease aetiologies. The above reasons indicate that, if a pathological explanation is considered, some alternative causes should also be considered. One is osteomyelitis, but there is no evidence of the sequestra or involucra typical of such disease (Rogers and Waldron, 1989; Aufderheide and Rodriguez-Martin, 1998; Waldron, 2009). A possible metastatic neoplasm can also be discarded because it implies multiple perforating skull holes, which are not present in our case (Aufderheide and Rodriguez-Martin, 1998; Shang and Trinkaus, 2008). Furthermore, the bony changes and alterations exhibited by these remains do not correspond with other infectious diseases that can be confounded with acquired syphilis. Tuber- culosis, for example, typically produces a lone lesion that destroys both tables, the destruction being much more evi- dent in the inner table where the disease first manifests in the skull (Mitchell, 2003; Ortner, 2003; Shang and Trinkaus, 2008). Leprosy also destroys bone, but its pathological changes are not frequently found in the skull vault as in our case (Møller-Christensen, 1965; Ortner, 2003). Paget’s disease, of unknown aetiology, can also be ruled out, as the engrossing of the cranial vault is very different to our case (Steinbock, 1976; Aufderheide and Rodriguez-Martin, 1998). Finally, it should be said that the different types of treponematosis are nearly impossible to differentiate by the lesions they leave on archaeological specimens (Hackett, Figure 7. Histological analysis of the largest putative gummatous 1976; Webb, 1995; Ortner, 2003; Harper et al., 2011). Nev- lesion of SAL633. (A) Low-power micrograph showing two recogniz- ertheless, the geographical location in which our remains able osteons (arrows) with a central empty canal (harvesian) encircled were found makes it unlikely for yaws to be the causative by concentric layers, 110×. (B) Micrograph of an osteon having an ir- agent of our possible case of treponematosis, as this is a regular accumulation of lamellae with a non-concentric disposition tropical disease (Farnsworth and Rosen, 2006). The possibil- (arrows), 250×. The inset shows a detail of the arrangement of the layers, 500×. ity of bejel cannot be completely discarded and has to re- main open, as even though this disease is uncommon in Spain (Berco, 2011) and typical of hot desert areas, it has does not manifest in bone), are not uniform between skele- also been described as endemic in some regions of temperate tons. Thus, there is no histological, biochemical, immuno- Europe (Baker, 2005). Of course, apart from this last remark, logical, or microbiological technique that can be used to one must consider the possibility that our specimen could distinguish them (Noordhoek et al., 1990; Rothschild and have contracted one of these diseases during a voyage to Rothschild; 1995, Waldron, 2009). This led syphilis long another country, or even that the remains belong to an immi- being called “the great imitator” (Ehrlich and Kricun, 1976; grant who was buried in the cemetery of Saldaña. Sefton, 2001), as both its clinical pathogenesis and skeletal As for other kind of analyses which can identify acquired alterations are very similar to other diseases such as leprosy, syphilis it should be said that they exist, but their use in ar- tuberculosis, Paget’s disease, or haematogenous osteomyeli- chaeological remains is unclear. Some years ago, T. pallidum tis (von Hunnius et al., 2006). Thus, in archaeological spec- subsp. pallidum, the causative agent of syphilis, was detect- imens it is very difficult to achieve a differential diagnosis, ed in a 200-year-old skeletal specimen from Easter Island by especially for an incomplete skeleton. The great majority of DNA analysis (Kolman et al., 1999), and sensitive molecular lesions commonly attributed to syphilis, such as periostitis markers for detection of this bacteria in living tissues have and tibial bowing, are non-specific and can be caused by long been known (e.g. Burstain et al., 1991). However, fur- different diseases, both infectious and no-infectious, so it is ther assays failed to detect treponemal DNA in bones other- not unusual that bone alterations of putatively syphilitic in- wise suitable for ancient DNA analysis, even when using dividuals mimic several diseases. Von Hunnius et al. (2006) highly sensitive PCR protocols, suggesting that treponemal recommended a microscopic approach to differentiate dis- DNA is not well preserved in human bone and that ancient ease processes, but also commented on the difficulties in DNA analysis is not adequate, at present, to study ancient using the features reccomended by Schultz (2001) for palae- cases of putative acquired syphilis on a routine basis (Bouw- PUTATIVE PRE-COLUMBIAN SYPHILIS IN SPAIN 9 man and Brown, 2005; Barnes and Thomas, 2006). Trepone- tian archaeological case of treponematosis from the first mal bacteria (and, consequently, its DNA) likely dissemi- millennium AD (Ortner, 2003), its dating methodology was nates to bone early during the first stages of infection but is never provided (Harper et al., 2011). Apart from that, there not present in the same location at later stages of the disease, are no other clear syphilis cases coming from other African making ancient DNA techniques unreliable for studies on archaeological sites. This could be due to the high degree of acquired syphilis in past human populations (von Hunnius et deterioration that skeletal material quickly suffers in arid al., 2007). environments, both after and prior to burial (Maat, 1993; Trueman et al., 2004), which may erase identifiable traces of Conclusions treponematosis. Even so, it is worth mentioning that if an African origin of syphilis were proposed, the Iberian Penin- Some conclusions can be obtained from our results. The sula would again appear as a probable gateway for the entry first is that acquired syphilis may have occurred in Spain of treponemal infections into Europe, as the Strait of Gibral- before the European–American interchange, as early as in tar is thought to have been a place of frequent contact be- the 9th century. This supports other studies reporting tween European and African human populations (Escacena, Pre-Columbian syphilis in Europe and adds to a recently 2000; Pardiñas et al., 2014). growing field of evidence that does not agree with the Co- As for the Columbian hypothesis, one of its last main sup- lumbian hypothesis of an American origin of syphilis (e.g. porters is the phylogenetic approach of Harper et al. (2008), Mitchell, 2009; Mays et al., 2012; Cole and Waldron, 2011). which suggested an American origin of trepanematoses. Nevertheless, all these cases and many others have been re- Harper et al. compared modern DNA sequences of isolates cently evaluated by Harper et al. (2011), in a study that from different countries, including American, Asian (India) raised numerous critiques of their diagnostic and dating cri- and African (South Africa) strains of T. pallidum subsp. pal- teria, eventually concluding that the only incontestable re- lidum as well as strains of other pathogenic subspecies (per- ported cases of syphilis from Pre-Columbian times have tenue, endemicus) and even different Treponema species. their origin in the New World. Even in light of this recent The resulting phylogeny supported an American origin of review, the pathologic evidence shown in our remains, cou- acquired syphilis, but no Spanish strains of T. pallidum sub- pled with their antiquity, forces us to propose that the Euro- sp. pallidum were represented, nor were strains from other pean Pre-Columbian existence of acquired syphilis is, at parts of Europe, such as Britain or the Mediterranean, re- least, possible. Thus, we also have to seek an explanation for ported. A more recent analysis of whole-genome data from the origin of syphilis in the Old World. European contacts T. pallidum strains from worldwide patients showed all the with the New World in the form of Viking expeditions from samples shared a common ancestor around the time of the Greenland have been proposed as an explanation, but this is Columbian voyages, and while this supports that population not supported by existing historical or skeletal evidence dynamics after this date favoured the emergence of a syphi- (Mays et al., 2003). Another explanation could be the intro- lis pandemic, it does not resolve where the ultimate origin of duction of syphilis in Europe by the pilgrims and crusaders the pathogen lies (Arora et al., 2016). It might be the case that marched to the Near East during the Crusades (Hudson, that genetics, or indeed any of the individual techniques 1963). These took place during the 11th–13th centuries, outlined in this article, might not be able to resolve such a greatly enhancing human and trade flow between Europe persisting question on its own, and multidisciplinary ap- and south-west Asia (Lewis, 1991). proaches on current and future palaeopathological findings A possible earlier origin has been defended using the cas- will surely be needed. es of congenital syphilis found in the Greek colony of Meta- ponto (southern Italy), dated between the 1st and 4th centu- Acknowledgments ries BC (Henneberg and Henneberg, 1994). Greek colonies in this period were scattered throughout Europe and western This study has been supported by the research Contract Asia (Cerchiai et al., 2004), and syphilis could have arrived EL01/98, funded by the Consejería de Educación y Cultura at this particular location from some other place in the Med- de la Junta de Castilla y León. iterranean. 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Figure S1. Probability distributions of the dating of the SAL633 individual. The vertical axis shows a supposed percentage of marine contribution to the diet, while the horizontal axis shows 2σ calibrated calendar dates (AD). Curves were smoothed using an estimated age-at-death from the individual of 40 years. The 13C/12C ratio measured in the SAL633 bone sample was of –21.1‰, which indicates a 0% marine contribution to the diet. 14 B. LOPEZ ET AL. Anthropological Science

Figure S2. Conserved skeletal remains (shaded) of the SAL633 individual.