A 14th century CE Brucella melitensis genome and the recent expansion of the Western Mediterranean clade.

Brucellosis is a disease caused by the bacterium Brucella and typically transmitted through contact with infected ruminants. It is one of the most common chronic zoonotic diseases and of particular interest to public health agencies. Despite its well-known transmission history and characteristic symptoms, we lack a more complete understanding of the evolutionary history of its best-known species-Brucella melitensis. To address this knowledge gap we fortuitously found, sequenced and assembled a high-quality ancient B. melitensis draft genome from the kidney stone of a 14th-century Italian friar. The ancient strain contained fewer core genes than modern B. melitensis isolates, carried a complete complement of virulence genes, and did not contain any indication of significant antimicrobial resistances. The ancient B. melitensis genome fell as a basal sister lineage to a subgroup of B. melitensis strains within the Western Mediterranean phylogenetic group, with a short branch length indicative of its earlier sampling time, along with a similar gene content. By calibrating the molecular clock we suggest that the speciation event between B. melitensis and B. abortus is contemporaneous with the estimated time frame for the domestication of both sheep and goats. These results confirm the existence of the Western Mediterranean clade as a separate group in the 14th CE and suggest that its divergence was due to human and ruminant co-migration.

Correction: The paradox of HBV evolution as revealed from a 16th century mummy.

[This corrects the article DOI: 10.1371/journal.ppat.1006750.].

The paradox of HBV evolution as revealed from a 16th century mummy.

Hepatitis B virus (HBV) is a ubiquitous viral pathogen associated with large-scale morbidity and mortality in humans. However, there is considerable uncertainty over the time-scale of its origin and evolution. Initial shotgun data from a mid-16th century Italian child mummy, that was previously paleopathologically identified as having been infected with Variola virus (VARV, the agent of smallpox), showed no DNA reads for VARV yet did for hepatitis B virus (HBV). Previously, electron microscopy provided evidence for the presence of VARV in this sample, although similar analyses conducted here did not reveal any VARV particles. We attempted to enrich and sequence for both VARV and HBV DNA. Although we did not recover any reads identified as VARV, we were successful in reconstructing an HBV genome at 163.8X coverage. Strikingly, both the HBV sequence and that of the associated host mitochondrial DNA displayed a nearly identical cytosine deamination pattern near the termini of DNA fragments, characteristic of an ancient origin. In contrast, phylogenetic analyses revealed a close relationship between the putative ancient virus and contemporary HBV strains (of genotype D), at first suggesting contamination. In addressing this paradox we demonstrate that HBV evolution is characterized by a marked lack of temporal structure. This confounds attempts to use molecular clock-based methods to date the origin of this virus over the time-frame sampled so far, and means that phylogenetic measures alone cannot yet be used to determine HBV sequence authenticity. If genuine, this phylogenetic pattern indicates that the genotypes of HBV diversified long before the 16th century, and enables comparison of potential pathogenic similarities between modern and ancient HBV. These results have important implications for our understanding of the emergence and evolution of this common viral pathogen.

Metastatic Prostate Carcinoma from Imperial Rome (1st to 2nd Centuries AD).

OBJECTIVE: This study describes and discusses a rare case of metastatic carcinoma that affected the skeleton of an adult male recovered in the necropolis of Casal Bertone in Rome (Italy). The necropolis, which dates back to the Imperial Age (1st to 2nd century AD), is located near some residential structures and a large place identified as a fullery (fullonica). METHODS: Anthropological and paleopathological studies of the skeletal remains were performed via careful macroscopic, microscopic, radiological (X-ray and CT scan), and histological investigation. RESULTS: The skeleton displayed mixed osteoclastic and osteoblastic lesions that mainly involved the axial bones, in particular the sternum, the ribs, the spine, and the scapular and pelvic girdles. The anatomical distribution and the destructive and proliferative nature of the lesions suggested diffuse metastases arising from a soft-tissue primary cancer. The age and sex of the individual, as well as radiographic and histological pictures, allowed diagnosis of an advanced prostate cancer with extensively diffused bone metastases. CONCLUSION: At present, this is the only case of prostate cancer from the Imperial Age recovered in Rome.

A historical case of amelogenesis imperfecta: Giovanna of Austria, Grand Duchess of Tuscany (1547-1578).

The skeletal remains of Giovanna of Austria (1547-1578), daughter of the Emperor Ferdinand I of Habsburg (1503-1564) and first wife of the Grand Duke of Tuscany, Francesco I (1541-1587), exhumed from the Basilica of San Lorenzo in Florence, were submitted to paleopathological study. Examination of the dentition, which was in a good state of preservation, showed maxillary retrognathism, together with a caries lesion, moderate periodontal disease, malposition of the upper second premolars and tooth wear. Furthermore, several horizontal grooves were observed in both the buccal and the lingual crown surfaces of almost all teeth, especially the anterior ones. The orthopantomogram showed hypomineralized enamel and alveolar bone loss. Two third-molar teeth were investigated using micro-computed tomography (micro-CT) analysis, revealing highly irregular enamel caps with reduced average thickness. The observed features suggest a diagnosis of hypoplastic amelogenesis imperfecta, a developmental condition affecting enamel formation.

A great enigma of the Italian Renaissance: paleopathological study on the death of Giovanni dalle Bande Nere (1498-1526) and historical relevance of a leg amputation.

BACKGROUND: The Medici project consisted in archeological and paleopathological researches on some members of the great dynasty of the Italian Renaissance. The remains of Giovanni de' Medici, so-called "dalle Bande Nere" (Forlì 1498- Mantua 1526) have not been investigated yet. The enigma of the fatal injury and leg amputation of the famous Captain excited curiosity of paleopathologists, medical scientists and Italian Society of Orthopedic and Traumatology which contributed to realize the project of exhumation and study of his skeletal remains. The aim of the study is to report the first anthropological and paleopathological results. CASE PRESENTATION: The tomb of Giovanni and his wife Maria Salviati was explored and the skeletal remains were investigated. Anthropological and paleopathological examination defined: age at death, physical constitution and activity, skeletal diseases. The bones of the leg were studied macroscopically, under stereoscopic microscope, at X-ray and CT scans to detect type of injury and level of amputation. CONCLUSIONS: The skeleton and muscular insertions of Giovanni revealed a young-adult and vigorous man, subjected to stresses of military activity since adolescence. Right tibia was amputated below the proximal half of diaphysis leaving long tibio-fibular stumps with a horizontal cut only at the lateral portion. Thus, the surgeon limited to complete the traumatic hemi-amputation. Amputation in the Sixteenth Century technically consisted in guillotine incisions below the knee using crescent shaped knife and bony saw, usually leaving a quite long tibial stump. Amputations in the Sixteenth Century were contaminated and grossly performed not providing vascular binding nor wound closure. The surgeon performed the procedure in conformity with surgical knowledge of that period.

Quinto Tiberio Angelerio and new measures for controlling plague in 16th-century Alghero, Sardinia.

Plague, a zoonotic disease caused by the bacterium Yersinia pestis, has been responsible for at least 3 pandemics. During 1582-1583, a plague outbreak devastated the seaport of Alghero in Sardinia. By analyzing contemporary medical texts and local documentation, we uncovered the pivotal role played by the Protomedicus of Alghero, Quinto Tiberio Angelerio (1532-1617), in controlling the epidemic. Angelerio imposed rules and antiepidemic measures new to the 16th-century sanitary system of Sardinia. Those measures undoubtedly spared the surrounding districts from the spread of the contagion. Angelerio seems to have been an extremely successful public health officer in the history of plague epidemics in Sardinia.

Osteobiography of a 19th century elderly woman with pertrochanteric fracture and osteoporosis: a multidisciplinary approach.

In this paper the osteobiography of an elderly woman recovered from a cemetery tomb where she was buried in 1850, affected by hip fracture and osteoporosis, is described. The overall anthropological characteristics of the individual have been investigated. Macroscopic, radiographic, tomographic, microscopic, and chemical and structural examinations have been performed to give a detailed account of the condition of the skeleton. A non-union pertrochanteric fracture not surgically treated and probably due to senile osteoporosis was diagnosed. The consequences of the fracture to the bones show that this individual likely survived several years following the injury. The osseous features we describe (remodelled bone at the fracture site, asymmetry of entheseal changes likely related to the particular walking pattern of the individual) may be useful in personal identification of skeletons of legal interest. Regarding the recognition of osteoporosis in unearthed skeletons, our study underlines that the cortical thickness, microscopic features, degree of crystallinity and Ca/P ratio represent more useful elements than the mean bone density, mineral/matrix ratio and mineral maturity, which are more sensitive to diagenetic changes that affect the mineral phase post-mortem.

The privileged burial of the Pava Pieve (Siena, 8th Century AD).

RATIONALE: The 6(th) archaeological excavation campaign performed at the 'Pieve di Pava' (San Giovanni d'Asso, Siena, Italy) unearthed a privileged stone burial of a single individual (US 2378), covered by a monolithic slab and placed in front of an altar. The skeletal remains of a young male (18-20 years old), not in anatomical connection, were found at the bottom floor of a tomb (160 cm long, 40 cm large and over 70 cm deep). METHODS: A multidisciplinary study has been carried out concerning that privileged bone burial. The study combines paleopathology studies, stable isotope palaeodietary reconstruction, radiocarbon dating and archaeological analyses. RESULTS: (14) C dating of the skeleton revealed a date between 650 and 688 AD. Stable isotope analysis (δ(18) O, δ(13) C, δ(15) N) attested that he was probably a member of the local population, whose diet was rather rich in animal proteins. The paleopathological study diagnosed a case of acromesomelic dysplasia, a congenital anomaly with disproportion of the limbs. Archaeological evidence regarding the circular delimitation of the bones suggested that the skeleton was a secondary deposition, transported to the church in a sack. CONCLUSIONS: We argue that the relic was used for the re-consecration of the church, following the restoration work in the 8(th) century. We conclude that the skeleton belonged to an eminent personage (e.g., either the member of a local elite family or a saint).

Soft tissue tumors in palaeopathology: a review.

OBJECTIVE: To perform a review of soft tissue tumors in palaeopathology. METHODS: Examination of palaeopathological literature. RESULTS: Up to now, observations of neoplastic soft tissue lesions in mummified remains are limited to 15 cases, and only 4 of them present malignancies; 14 cases of calcified benign tumors, including 13 neoplasms of the female reproductive system and 1 teratoma, are also documented. CONCLUSIONS: The main reasons for the rarity of soft tissue tumors in palaeopathology, especially of malignancies, are apparently the short life span of past populations, the scarcity of mummified remains available in comparison with skeletal remains, and technical difficulties in the detection of neoplastic lesions in mummified tissues.

Palaeopathology of human remains from the Roman Imperial Age.

The increasing attention of archaeological and anthropological research towards palaeopathological studies has allowed to focus the examination of many skeletal samples on this aspect and to evaluate the presence of many diseases afflicting ancient populations. This paper describes the most interesting diseases observed in skeletal samples from five necropolises found in urban and suburban areas of Rome during archaeological excavations in the last decades, and dating back to the Imperial Age. The diseases observed were grouped into the following categories: articular diseases, traumas, infections, metabolic or nutritional diseases, congenital diseases and tumors, and some examples are reported for each group. Although extensive epidemiological investigation in ancient skeletal records is impossible, palaeopathology allowed highlighting the spread of numerous illnesses, many of which can be related to the life and health conditions of the Roman population.

A 16th century Escherichia coli draft genome associated with an opportunistic bile infection.

Escherichia coli - one of the most characterized bacteria and a major public health concern - remains invisible across the temporal landscape. Here, we present the meticulous reconstruction of the first ancient E. coli genome from a 16th century gallstone from an Italian mummy with chronic cholecystitis. We isolated ancient DNA and reconstructed the ancient E. coli genome. It consisted of one chromosome of 4446 genes and two putative plasmids with 52 genes. The E. coli strain belonged to the phylogroup A and an exceptionally rare sequence type 4995. The type VI secretion system component genes appears to be horizontally acquired from Klebsiella aerogenes, however we could not identify any pathovar specific genes nor any acquired antibiotic resistances. A sepsis mouse assay showed that a closely related contemporary E. coli strain was avirulent. Our reconstruction of this ancient E. coli helps paint a more complete picture of the burden of opportunistic infections of the past.

Examining pathogen DNA recovery across the remains of a 14th century Italian friar (Blessed Sante) infected with Brucella melitensis.

OBJECTIVE: To investigate variation in ancient DNA recovery of Brucella melitensis, the causative agent of brucellosis, from multiple tissues belonging to one individual MATERIALS: 14 samples were analyzed from the mummified remains of the Blessed Sante, a 14 th century Franciscan friar from central Italy, with macroscopic diagnosis of probable brucellosis. METHODS: Shotgun sequencing data from was examined to determine the presence of Brucella DNA. RESULTS: Three of the 14 samples contained authentic ancient DNA, identified as belonging to B. melitensis. A genome (23.81X depth coverage, 0.98 breadth coverage) was recovered from a kidney stone. Nine of the samples contained reads classified as B. melitensis (7-169), but for many the data quality was insufficient to withstand our identification and authentication criteria. CONCLUSIONS: We identified significant variation in the preservation and abundance of B. melitensis DNA present across multiple tissues, with calcified nodules yielding the highest number of authenticated reads. This shows how greatly sample selection can impact pathogen identification. SIGNIFICANCE: Our results demonstrate variation in the preservation and recovery of pathogen DNA across tissues. This study highlights the importance of sample selection in the reconstruction of infectious disease burden and highlights the importance of a holistic approach to identifying disease. LIMITATIONS: Study focuses on pathogen recovery in a single individual. SUGGESTIONS FOR FURTHER RESEARCH: Further analysis of how sampling impacts aDNA recovery will improve pathogen aDNA recovery and advance our understanding of disease in past peoples.

Hyperostosis frontalis interna (HFI) and castration: the case of the famous singer Farinelli (1705-1782).

The famous castrato singer Farinelli (1705-1782) was exhumed by our research group in July 2006 for the purpose of gaining some insight into his biological profile through a study of his skeletal remains. Farinelli was castrated before puberty to preserve the treble pitch of the boy's voice into adult life. His powerful and sweet voice became legendary. In spite of its bad preservation state, the skeleton displayed some interesting characteristics that are probably related to the effects of castration, including long limb-bones, persistence of epiphyseal lines and osteoporosis. In particular, the frontal bone was affected by severe hyperostosis frontalis interna (HFI). This condition consists in a symmetrical thickening of the inner table of the bone. The epidemiology of HFI shows that it is relatively common in postmenopausal women but very rare in men. Men affected by this pathology suffer from diseases, syndromes or treatments causing androgen deficiency. In the case of Farinelli, castration was probably responsible for the onset and development of this lesion.

Syndromic craniosynostosis in a modern-age skeleton from Siena, Italy.

The skeleton of a female adult found in archaeological excavations carried out in Siena (central Italy) and dated back to the modern age showed a severe skull malformation due to the premature bilateral closure of the coronal suture, which determined a deformed brachycephalic skull. This craniosynostosis was associated with other malformations, such as shallow orbits, hypertelorism, mandibular prognathism, and consequent malocclusion, but there was absence of anomalies in the remaining bones of the extremities. These features did not seem to be related to an isolated condition but to a more complex genetic syndrome, suggesting a possible case of Crouzon syndrome. Besides representing a rare finding in archaeological material, the present case provides the opportunity to observe in an adult subject lesions typical of this congenital disorder, which is at present surgically corrected in infantile age.