Valentina Giuffra

Recovery of a Medieval Brucella melitensis Genome Using Shotgun Metagenomics

ABSTRACT Shotgun metagenomics provides a powerful assumption-free approach to the recovery of pathogen genomes from contemporary and historical material. We sequenced the metagenome of a calcified nodule from the skeleton of a 14th-century middle-aged male excavated from the medieval Sardinian settlement of Geridu. We obtained 6.5-fold coverage of a Brucella melitensis genome. Sequence reads from this genome showed signatures typical of ancient or aged DNA. Despite the relatively low coverage, we were able to use information from single-nucleotide polymorphisms to place the medieval pathogen genome within a clade of B. melitensis strains that included the well-studied Ether strain and two other recent Italian isolates. We confirmed this placement using information from deletions and IS711 insertions. We conclude that metagenomics stands ready to document past and present infections, shedding light on the emergence, evolution, and spread of microbial pathogens. IMPORTANCE Infectious diseases have shaped human populations and societies throughout history. The recovery of pathogen DNA sequences from human remains provides an opportunity to identify and characterize the causes of individual and epidemic infections. By sequencing DNA extracted from medieval human remains through shotgun metagenomics, without target-specific capture or amplification, we have obtained a draft genome sequence of an ~700-year-old Brucella melitensis strain. Using a variety of bioinformatic approaches, we have shown that this historical strain is most closely related to recent strains isolated from Italy, confirming the continuity of this zoonotic infection, and even a specific lineage, in the Mediterranean region over the centuries. Infectious diseases have shaped human populations and societies throughout history. The recovery of pathogen DNA sequences from human remains provides an opportunity to identify and characterize the causes of individual and epidemic infections. By sequencing DNA extracted from medieval human remains through shotgun metagenomics, without target-specific capture or amplification, we have obtained a draft genome sequence of an ~700-year-old Brucella melitensis strain. Using a variety of bioinformatic approaches, we have shown that this historical strain is most closely related to recent strains isolated from Italy, confirming the continuity of this zoonotic infection, and even a specific lineage, in the Mediterranean region over the centuries.

Diffuse idiopathic skeletal hyperostosis in the Medici, Grand Dukes of Florence (XVI century)

Diffuse idiopathic skeletal hyperostosis (DISH) is a common systemic disorder characterised by the ossification of the anterior longitudinal spinal ligament involving at least three contiguous vertebrae and by diffuse extraspinal enthesopathies. The condition is associated with the male sex and with advanced age; its aetiology is uncertain, but seems to be related to obesity and diabetes. The most recent studies in archaeological series demonstrated a relation between high social status and the incidence of DISH. The present study examines two cases of DISH found amongst the members of the Medici family buried in the Basilica of San Lorenzo in Florence. The skeletons of the Grand Dukes Cosimo I (1519–1574) and his son Ferdinand I (1549–1609) showed the typical features of the condition. This result is related to the obesity of the Grand Dukes, attested by the written and artistic sources, and to the protein-based alimentation demonstrated by a paleonutritional study, thus furnishing further evidence to the significance of DISH as a life style.

Plasmodium falciparum immunodetection in bone remains of members of the Renaissance Medici family (Florence, Italy, sixteenth century).

Medical accounts and ancient autopsy reports imply that tertian malarial fevers caused the death of four members of the Medici family of Florence: Eleonora of Toledo (1522-1562), Cardinal Giovanni (1543-1562), don Garzia (1547-1562) and Grand Duke Francesco I (1531-1587). All members of the Medici family hunted in the endemic malarial areas of Tuscany, such as the marshy areas surrounding their villas and along the swampy Maremma and were, therefore, highly exposed to the risk of being infected by Falciparum malaria. To determine if the original death certificates issued by the court physicians were correct, we carried out immunological investigations and then compared the biological results to the historical sources. Bone samples were examined for the presence of Plasmodium falciparum histidine-rich- protein-2 (PfHRP2) and P. falciparum lactate dehydrogenase (PfLDH) using two different qualitative double-antibody immunoassays. Our findings provide the first modern laboratory evidence of the presence of P. falciparum ancient proteins in the skeletal remains of four members of the Medici family. We confirm the clinical diagnosis of the court physicians, using modern methods. Finally, this study demonstrates that immunodetection can be successfully applied not only to mummified tissues but also to skeletal remains, thus opening new paths of investigation for large archaeological series.

The use of mercury against pediculosis in the Renaissance: the case of Ferdinand II of Aragon, King of Naples, 1467-96.

The hair samples of Ferdinand II of Aragon (1467–1496), King of Naples, whose mummy is preserved in the Basilica of San Domenico Maggiore in Naples, showed a high content of mercury, with a value of 827ppm. Furthermore, examination using a stereomicroscope and a scanning electron microscope (SEM) of head and pubic hairs of Ferdinand II, revealed a lice infestation. The reasons for the massive presence of the mercury in the kings hair are discussed and contemporary literature regarding the use of this metal in medical therapies and in cosmetic practices is analysed. As a result, the high value of mercury in the hair of Ferdinand II can be attributed to antipediculosis therapy, applied as a topic medicament. This case represents an important finding for the history of medicine, because demonstrates that in the Renaissance mercury was applied locally not only to treat syphilis, as well attested by direct and indirect sources, but also to prevent or eliminate lice infestation.

Royal pediculosis in Renaissance Italy: lice in the mummy of the King of Naples Ferdinand II of Aragon (1467-1496)

Pediculosis seems to have afflicted humans since the most ancient times and lice have been found in several ancient human remains. Examination of the head hair and pubic hair of the artificial mummy of Ferdinand II of Aragon (1467-1496), King of Naples, revealed a double infestation with two different species of lice, Pediculus capitis, the head louse, and Pthirus pubis, the pubic louse. The hair samples were also positive for the presence of mercury, probably applied as an anti-pediculosis therapy. This is the first time that these parasites have been found in the hair of a king, demonstrating that even members of the wealthy classes in the Renaissance were subject to louse infestation.

Malaria was the killer of Francesco I de' Medici (1531-1587)

alaria Was “the Killer” of Francesco I de’ Medici 1531-1587) ino Fornaciari, MD, Valentina Giuffra, PhD, Ezio Ferroglio, DVM, PhD, Raffaella Bianucci, PhD Division of Paleopathology, History of Medicine and Bioethics, Department of Oncology, Transplants and Advanced Technologies in edicine, University of Pisa, Italy; Laboratory of Parasitology and Parasitic Diseases, Department of Animal Production, pidemiology and Ecology, University of Turin, Grugliasco Torino, Italy; Laboratory of Anthropology, Department of Animal and uman Biology, University of Turin, Torino, Italy; UMR 6578 CNRS-EFS (Biocultural Anthropology), University of Marseille, rance.

An investigation of Etruscan cremations by Computed Tomography (CT)

The cremation urn is a tiny archaeological site of its own, with finds, features, stratification and structure. The old prescription was to take the pot apart, or slice it, and micro-excavate with inevitable damage and loss. Here is a new methodology – the application of a CT scan as used in medicine. The authors evaluate the results on 35 Etruscan cremations, finding that CT not only provides an excellent guide for micro-excavation, but allows the degree of fragmentation to be appreciated inside the pot and maps those metal objects that have corroded to a crust and do not survive excavation. They emphasise the value of the method in making a ‘first resort’ primary record especially in commercial archaeology.

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.

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.

Visceral Leishmaniasis during Italian Renaissance, 1522–1562

To the Editor: Leishmaniasis, an infectious disease caused by parasites of the genus Leishmania, is transmitted to humans through the bite of a female sandfly. The 3 forms of leishmaniasis are visceral (VL) and cutaneous (CL), which are typical of the Old World, and mucocutaneous leishmaniasis, which occurs primarily in Central and South America. VL (also called kala-azar) is caused by species of the L. donovani complex (including L. infantum), and CL is mainly caused by L. major or L. tropica (1). In Italy, VL and CL are caused by L. infantum. The origin and spread of leishmaniasis are a matter of debate. Widespread in antiquity, visceral leishmaniasis has been identified only in mummies from ancient Egypt and upper Nubia (2). Similarly, only 4 cases of mucocutaneous leishmaniasis have been identified in skulls from northern Chile (3).