Possible vertebral brucellosis infection in a Neanderthal

The La Chapelle-aux-Saints 1 skeleton of an old (>60-year-old) male Neanderthal is renowned for the advanced osteoarthritis of its spinal column and hip joint, and their implications for posture and lifestyle in these Mid- to Late Pleistocene humans. Reassessment of the pathologic lesions reveals erosions at multiple non-contiguous vertebrae and reactive bone formation extending far beyond the left hip joint, which suggests the additional diagnosis of brucellosis. This implies the earliest secure evidence of this zoonotic disease in hominin evolution. Brucellosis might have been transmitted via butchering or eating raw meat and is well compatible with the range of prey animals documented for Neanderthals. The associated infertility could have represented an important aspect of health in these late archaic humans.

. Preservation of the La Chapelle-aux-Saints 1 Neanderthal skeleton and distribution of the pathological lesions. Possible brucellosis leading to erosions affected the inferior endplates of vertebrae C5 and C6, the right facet joints C4/5 and C7/T1, the left transverse process T1, both facet joints T9/10, and the left hip bone (blue stars). Irregular grooves likely associated with granulomateous processes are found on the laminae of thoracolumbar vertebrae (green stars). Osteoarthritic changes (red) are present in the right temporomandibular joint, the facet joints of the vertebral column, glenohumeral joints, left sternoclavicular joint, elbow joints, right radiocarpal joint, hip joints and right fifth pedal proximal interphalangeal joint. A midthoracic right rib shows a healed fracture (cross). www.nature.com/scientificreports/ 3.2 mm deep lytic lesion with smooth margins (Fig. 4). Irregular grooves with intact cortical bone and smooth margins were also found immediately adjacent to the left superior articular facet and in the middle of the right lamina of vertebra T2, as well as in the laminae of all preserved thoracolumbar vertebrae between T9 and L5 as well in S1 (Fig. 6). The irregular morphology of these grooves suggest that they are caused by nodular, multilobulated granulomateous processes. The depressions measure up to 20 × 9 × 3.5 mm (e.g., in L4) and are thus more extended, deeper and more jagged than imbrication pockets that are associated with hyperextension of the spine and also occur immediately adjacent to the superior articular facets 27 . Another type of depressions represent Schmorl's nodes that can be recognized in the centres of the inferior vertebral body surface of T6 and on both the superior and inferior surfaces of T7-T9 (see Ref. 21 ). Given the relatively low resolution of the medical CT scans of the La Chapelle-aux-Saints 1 skeleton compared to µCT scans, they were not informative save for localizing the pathological lesions (Fig. 7).
The left hip bone is fragmentary (Fig. 8). The articular surface in the weight-bearing posterosuperior portion of the acetabulum is eburnated. The acetabular margin bears large osteophytes and shows reactive new bone formation with erosions extending to the extraarticular portion of the lower ilium and upper ischium. The hip joint is, however, not deformed and shows no indication of hip dysplasia or hip joint luxation. The left proximal femur is not preserved. The right hip joint showed moderate osteoarthritic changes. Eburnation of the subchondral bone was also observed in both proximal humeri.

Discussion
The possibility of osteoarthritis has been considered as an explanation for the vertebral and hip joint lesions observed in the La Chapelle-aux-Saints 1 Neanderthal 2-4 . More precisely, La Chapelle-aux-Saints 1 was thought to have suffered from both osteoarthritis and spondylosis deformans, as the term osteoarthritis is reserved for the disease of diarthrodial joints, while that appellation has been replaced by the term spondylosis deformans for the intervertebral disk articulations [28][29][30] . Both spondylosis deformans and osteoarthritis are recognized by production of osteophytes and subchondral sclerosis [31][32][33][34][35] . In addition, advanced osteoarthritis is characterised by subchondral    www.nature.com/scientificreports/ cyst formation and smoothing or eburnation of joint surfaces 36 . However, eburnation simply identifies sufficient cartilage loss to allow bone to rub on bone. Any pathology that results in cartilage loss, be it mechanical (e.g., osteoarthritis) or inflammatory (e.g., infection or spondyloarthropathy) can produce eburnation 28 . Spondylosis and facet joint osteoarthritis as well as osteoarthritis of the peripheral joints affecting both glenohumeral joints, both hip joints and possibly also the elbow and radiocarpal joints can undoubtedly be observed in La Chapelle-aux-Saints 1, as described earlier [1][2][3][4][5]21 .
Yet, not all damage can be attributed to osteoarthritis. Features that are incompatible with the diagnosis of osteoarthritis and spondylosis deformans include the presence of actual subchondral and marginal erosions at the inferior endplates of vertebrae C5 and C6, erosions in the right facet joints C4/5 and C7/T1 as well as both facet joints T9/10, lytic lesions in the extra-articular areas of T1 to S1, and erosions and reactive bone formation extending far beyond the left hip joint 28,30 . Rather, the mixed picture of destructive and proliferative processes is typical of an inflammatory aetiology. Although the term erosion has also been applied to the cartilage damage that osteoarthritis produces, this damage is characterized by mechanical abrasion and fissures and not by the inflammatory process that produce erosions in rheumatologic disorders or chronic infection 28,30,35,37 . Rheumatologic disorders that mainly affect the spine are classified as spondylarthropathies. They include ankylosing spondylitis, reactive arthritis (previously referred to as Reiter's syndrome), psoriatic arthritis and enteropathic arthritis associated with inflammatory bowel diseases. The scattered distribution of the erosive lesions along the axial skeleton of La Chapelle-aux-Saints 1 and the presence of extensive granulomateous processes suggest, however, a chronic infectious or tumorous aetiology. Osteolytic tumours typically grow from the vertebral body into the appendicular regions and preferentially destroy the anterior and posterior edges of the vertebral bodies rather than the endplates 38 . Chronic infections producing spondylitis and spondylodiscitis, on the other hand, mostly include tuberculosis and brucellosis. Other infections like actinomycosis, blastomycosis, coccidiodomycosis, and echinococcosis are extremely rare and mainly lead to spondylitis and spondylodiscitis in immunodeficient patients 38 . Tuberculosis, on the other hand, typically causes a wedge-shaped destruction of one or two adjacent  www.nature.com/scientificreports/ vertebral bodies leading to the pathognomonic gibbus deformity, which is most often located in the mid-thoracic region. In the La Chapelle-aux-Saints 1 skeleton, vertebra L1 is slightly anteriorly wedge-shaped with an angle of 12° between the superior and inferior vertebral surface, but there are no indications of destructive processes in that vertebra. Rather, the wedge-shaped vertebral body of L1 might have resulted from a healed vertebral fracture or from Scheuermann's disease (for diagnostic criteria see Refs. 39,40 ). The pattern of the erosive defects observed in the La Chapelle-aux-Saints 1 vertebrae and the reactive new bone formation at the left hipbone is, however, characteristic of brucellosis infection. Thus, vertebrae are involved in 2-53% of brucellosis infections 17,41,42 . Typically, the disease initially affects a single vertebral endplate, mostly in the lumbar spine, but it can also be observed at multiple levels, affecting non-contiguous endplates and facet joints also in the thoracic and cervical spine 17,42 . In addition, monoarthritis or oligoarthritis may occur, with the knee and hip joints being most commonly affected 12 . The lesions have a specific erosive character and a location that is seemingly pathognomonic for the disease, initially manifesting as an osteolysis/groove on the anterior aspect of superior vertebral endplates 17,[43][44][45][46] . Specifically referred to as Pedro i Pons' sign 47,48 , such grooves are not found with pyogenic (e.g., Staphylococcus) vertebral infections 49 or tuberculosis 28 . Unfortunately, this characteristic destruction of the anterosuperior margin of the lumbar vertebrae cannot be observed in La Chapelle-aux-Saints 1 due to taphonomic damage of the anterior portion of all thoraco-lumbar vertebrae.
However, the well-preserved vertebrae C4 to T2 of La Chapelle-aux-Saints 1 show several pathological lesions suggestive of brucellosis. Examination of vertebrae C5 and C6 revealed erosive changes to the inferior vertebral surface (Fig. 2) that do not occur in spondyloarthropathy 28,30,50 . The smooth character of the base of the groove in C6 evidences reactive new bone formation and not simply taphonomic bone loss (which would have exposed trabecular bone). Confluent erosion with new bone formation is quite different from the edge (Romanus) lesions noted with spondyloarthropathy 28,51 . That appellation refers to apophyseal ring erosion, producing loss of the edge of the endplate in spondyloarthropathy, in contrast to production of a groove on its subchondral (diskfacing) surface. Such vertebral endplate grooves, however, are quite characteristic of the damage caused by brucellosis 17,[43][44][45][46][47][48] . In later stages of the infection, the erosive changes gradually expand over the entire vertebral endplate, producing the condition seen in vertebra C5.
Notation of erosions of the right facet joint of C4/C5 and C7/T1 and both facet joints of T9/T10 (Figs. 3, 4, 5) suggested differential diagnostic consideration of spondyloarthropathy or granulomatous infection. Facet joint involvement is found in up to one-third of individuals with brucellosis 6,44,52,53 . However, such erosions of the facet joints and vertebral endplate are not anticipated with spondyloarthropathy 28,30,50 . In addition, the transverse process of T1 and the laminae of the lower thoraco-lumbar vertebrae show large erosions characteristic of spaceoccupying granulomatous processes (see Figs. 4, 5 and 6) 28,54-56 . The latter are caused by infectious processes that may be attributed to tuberculosis, fungal disease and brucellosis 28,30 . Fungal disease produces erosions that do not respect variation in bone density, preserving a spheroid shape 54 . Tuberculosis is characterized by zones of bone resorption 30,56 . Neither pattern is observed in the La Chapelle-aux-Saints 1 Neanderthal.
The severe arthritic changes at the left hip joint have previously been attributed to trauma 3 , but there is otherwise no evidence for trauma. The presence of erosions and new bone formation suggest that on top of the age-related primary osteoarthritis that might initially have been present (and which can also be observed in the right hip joint and both humeri), a secondary phenomenon, i.e., hip joint infection by brucellosis occurred. In fact, osteoarthritis-related changes would be confined to the joint region itself and do not extend to the extraarticular region as it is the case of La Chapelle-aux-Saints 1. Inflammatory processes that extend beyond the joint region are, however, not unusual in brucellosis, analogous to the paraspinal granulomas probably present in vertebrae T1 and T10 (see also Ref. 12 ).
Neanderthals likely were infected by brucellosis during butchering of prey animals, not unlike the abattoir experience today 13 , or by eating raw meat. The main causative organisms of brucellosis have been found in a wide range of wildlife. Particularly, Brucella melitensis has been reported in ibex and chamois, while B. abortus and B. suis have a preference for most other bovines and suids, including wild sheep, goats, wild cattle, European bison, reindeer, roe and red deer, wild boar, but also horses, hares and marmots [57][58][59][60][61] . All these animals were important components of the Neanderthal diet [62][63][64] . Only the two largest Neanderthal prey animals, mammoths and woolly rhinoceros, might not have been reservoirs for Brucella as inferred from their extant relatives [65][66][67] . The host preference of the different Brucella species might suggest that La Chapelle-aux-Saints 1 was infected by B. abortus rather than B. melitensis. While the latter species often leads to acute pain and immobilization, B. abortus is known to cause a milder course of the disease. This might therefore explain the advanced stage of the arthritis in La Chapelle-aux-Saints 1 and his survival to the advanced age of perhaps well over 60 years 5 .
Symptoms of brucellosis not only include fever, arthritis, endocarditis, neurologic defects, spinal deformity, reduced milk production, but also epididymitis/orchitis, infertility, still births and abortions 68 . This could have represented an important aspect of health in Neanderthals, or more generally in Palaeolithic humans.

Materials and methods
The La Chapelle-aux-Saints 1 burial of an old male Neanderthal was discovered in 1908 in a pit in the Bouffia Bonneval cave near the village of La Chapelle-aux-Saints in south-western France 1 , together with dental remains of four additional individuals. Electron spin resonance (ESR) dating suggested a geological age of 47 ka ± 3 ka BP (early U-uptake model) or 56 ka ± 4 ka BP (linear U-uptake model) 69 . The auricular surface of the ilium suggests an individual age of 62 ± 13 years 5 . The partial skeleton preserves an almost complete skull with extensive post-canine tooth loss, a scapular fragment, portions of all long bones and both hipbones, as well as 19 variously complete vertebrae and a partial sacrum. The vertebrae include C1 and C2, C4 to T2, two fragmentary upper thoracic vertebrae (possibly T3 or T4 and T6 or T7), T8 to T12, L1, and L3 to S2 5,70 (Fig. 1).