Charles L. Greenblatt

Detection and Molecular Characterization of 9000-Year-Old Mycobacterium tuberculosis from a Neolithic Settlement in the Eastern Mediterranean

Background Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis. It has no environmental reservoir and is believed to have co-evolved with its host over millennia. This is supported by skeletal evidence of the disease in early humans, and inferred from M. tuberculosis genomic analysis. Direct examination of ancient human remains for M. tuberculosis biomarkers should aid our understanding of the nature of prehistoric tuberculosis and the host/pathogen relationship. Methodology/Principal Findings We used conventional PCR to examine bone samples with typical tuberculosis lesions from a woman and infant, who were buried together in the now submerged site of Atlit-Yam in the Eastern Mediterranean, dating from 9250-8160 years ago. Rigorous precautions were taken to prevent contamination, and independent centers were used to confirm authenticity of findings. DNA from five M tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex. Conclusions/Significance Human tuberculosis was confirmed by morphological and molecular methods in a population living in one of the first villages with evidence of agriculture and animal domestication. The widespread use of animals was not a source of infection but may have supported a denser human population that facilitated transmission of the tubercle bacillus. The similarity of the M. tuberculosis genetic signature with those of today gives support to the theory of a long-term co-existence of host and pathogen.

Mycobacterium tuberculosis Complex DNA from an Extinct Bison Dated 17,000 Years before the Present

In order to assess the presence of tuberculosis in Pleistocene bison and the origin of tuberculosis in North America, 2 separate DNA extractions were performed by 2 separate laboratories on samples from the metacarpal of an extinct long-horned bison that was radiocarbon dated at 17,870+/-230 years before present and that had pathological changes suggestive of tuberculosis. Polymerase chain reaction amplification isolated fragments of tuberculosis DNA, which were sequenced, and on which spoligotyping was also performed to help determine its relationship to the various members of the Mycobacterium tuberculosis complex. Extensive precautions against contamination with modern M. tuberculosis complex DNA were employed, including analysis of paleontologic and modern specimens in 2 geographically separate laboratories.

Tuberculosis: from prehistory to Robert Koch, as revealed by ancient DNA

During the past 10 years palaeomicrobiology, a new scientific discipline, has developed. The study of ancient pathogens by direct detection of their DNA has answered several historical questions and shown changes to pathogens over time. However, ancient DNA (aDNA) continues to be controversial and great care is needed to provide valid data. Here we review the most successful application of the technology, which is the study of tuberculosis. This has provided direct support for the current theory of Mycobacterium tuberculosis evolution, and suggests areas of investigation for the interaction of M tuberculosis with its host.

Co–infection of Mycobacterium tuberculosis and Mycobacterium leprae in human archaeological samples: a possible explanation for the historical decline of leprosy

Both leprosy and tuberculosis were prevalent in Europe during the first millennium but thereafter leprosy declined. It is not known why this occurred, but one suggestion is that cross–immunity protected tuberculosis patients from leprosy. To investigate any relationship between the two diseases, selected archaeological samples, dating from the Roman period to the thirteenth century, were examined for both Mycobacterium leprae and Mycobacterium tuberculosis DNA, using PCR. The work was carried out and verified in geographically separate and independent laboratories. Several specimens with palaeopathological signs of leprosy were found to contain DNA from both pathogens, indicating that these diseases coexisted in the past. We suggest that the immunological changes found in multi–bacillary leprosy, in association with the socio–economic impact on those suffering from the disease, led to increased mortality from tuberculosis and therefore to the historical decline in leprosy.

Sex identification of archaeological human remains based on amplification of the X and Y amelogenin alleles

Sex identification of archaeological human remains is essential for the exploration of gender differences in past populations. Traditional morphometric analyses fail to identify the gender of incomplete skeletal remains and that of immature individuals. In the present work, we have established a sensitive and reliable method, based on amplification of the single-copy amelogenin-encoding gene (AMG). The Y allele carries a small deletion in the first intron, facilitating the design of distinct X- and Y-specific polymerase chain reactions. Amplification with three primers, two of which are allele-specific, allows unambiguous identification of both X and Y chromosome signals in a single reaction, providing an internal control. For added confidence, the reaction may be performed in separate tubes for each allele. Using this method, the sex was determined from the skeletal remains of 18 individuals, including young children, out of 22 examined from periods ranging from 200 to around 8000 years ago. The state of skeletal preservation ranged from poor to good. Cortical and cranial bones, as well as teeth, were found to provide sufficiently preserved DNA. The success of retrieval of amplifiable DNA was not related either to the period or to the burial site. On the other hand, the method of DNA purification was critical. In our hands, direct DNA purification by Chelex from minute samples of bone/tooth powder gave the best results. This study demonstrates the applicability of the method for gender determination in skeletal remains from different periods.

Micrococcus luteus - Survival in Amber

A growing body of evidence now supports the isolation of microorganisms from ancient materials. However, questions about the stringency of extraction methods and the genetic relatedness of isolated organisms to their closest living relatives continue to challenge the authenticity of these ancient life forms. Previous studies have successfully isolated a number of spore-forming bacteria from organic and inorganic deposits of considerable age whose survival is explained by their ability to enter suspended animation for extended periods of time. However, despite a number of putative reports, the isolation of non-spore-forming bacteria and an explanation for their survival have remained enigmatic. Here we describe the isolation of non-spore-forming cocci from a 120-million-year-old block of amber, which by genetic, morphological, and biochemical analyses are identified as belonging to the bacterial species Micrococcus luteus. Although comparison of 16S rRNA sequences from the ancient isolates with their modern counterparts is unable to confirm the precise age of these bacteria, we demonstrate, using complementary molecular and cell biological techniques, evidence supporting the view that these (and related modern members of the genus) have numerous adaptations for survival in extreme, nutrient-poor environments, traits that will assist in this bacteria’s persistence and dispersal in the environment. The bacteria’s ability to utilize succinic acid and process terpine-related compounds, both major components of natural amber, support its survival in this oligotrophic environment.

Evolutionary Genomics and Conservation of the Endangered Przewalski’s Horse

Przewalskis horses (PHs, Equus ferus ssp. przewalskii) were discovered in the Asian steppes in the 1870s and represent the last remaining true wild horses. PHs became extinct in the wild in the 1960s but survived in captivity, thanks to major conservation efforts. The current population is still endangered, with just 2,109 individuals, one-quarter of which are in Chinese and Mongolian reintroduction reserves [1]. These horses descend from a founding population of 12 wild-caught PHs and possibly up to four domesticated individuals [2-4]. With a stocky build, an erect mane, and stripped and short legs, they are phenotypically and behaviorally distinct from domesticated horses (DHs, Equus caballus). Here, we sequenced the complete genomes of 11 PHs, representing all founding lineages, and five historical specimens dated to 1878-1929 CE, including the Holotype. These were compared to the hitherto-most-extensive genome dataset characterized for horses, comprising 21 new genomes. We found that loci showing the most genetic differentiation with DHs were enriched in genes involved in metabolism, cardiac disorders, muscle contraction, reproduction, behavior, and signaling pathways. We also show that DH and PH populations split ∼45,000 years ago and have remained connected by gene-flow thereafter. Finally, we monitor the genomic impact of ∼110 years of captivity, revealing reduced heterozygosity, increased inbreeding, and variable introgression of domestic alleles, ranging from non-detectable to as much as 31.1%. This, together with the identification of ancestry informative markers and corrections to the International Studbook, establishes a framework for evaluating the persistence of genetic variation in future reintroduced populations.

Diversity of Microorganisms Isolated from Amber

A bstractClaims that organisms can be cultured from amber, if substantiated, would be significant contributions to our understanding of the evolution, tenacity, and potential spread of life. Three reports on the isolation of organisms from amber have been published. Cano and Borucki recently reported the isolation of Bacillus sphaericus and Lambert et al. have described a new species designated Staphylococcus succinus from 25–40 million year old Dominican amber. These characterized organisms were phylogenetically distant from extant relatives and the Staphylococcus sp. sufficiently far removed from other extant staphylococci to be considered a new species. Here we report the culture of bacteria from Dominican and previously untested 120 million year old Israeli (Lebanese lode) amber. Twenty-seven isolates from the amber matrix have been characterized by fatty-acid profiles (FAME) and/or 16S rRNA sequencing. We also performed a terminal restriction fragment pattern (TRF) analysis of the original amber before prolonged culture by consensus primer amplification of the 16S rRNA followed by restriction enzyme digestion of the amplicons. Sample TRFs were consistent with a sparse bacterial assemblage and included at least five of the isolated organisms. Finally, we microscopically mapped the internal topography of an amber slice.

Leishmaniasis in ancient Egypt and upper Nubia

To the Editor: Leishmaniasis is a disease caused by parasites of the genus Leishmania. The infection is transmitted to humans through the bites of female sandflies and manifests mainly in 3 forms: visceral, cutaneous, and mucocutaneous. Visceral leishmaniasis or kala-azar, the often fatal form of the disease, is caused by species of the Leishmania donovani complex. These parasites were responsible for severe recent outbreaks in Sudan and other countries and are thought to originate in East Africa (1–4). In this report, we describe the successful amplification of L. donovani DNA in ancient Egyptian and Christian Nubian mummies dating back 4,000 years. Besides the first proof for visceral leishmaniasis in paleopathology, we provide evidence that leishmaniasis was present in Nubia in the early Christian period and that the organism also infected ancient Egyptians, probably because of close trading contacts to Nubia, during the Middle Kingdom. We analyzed 91 bone tissue samples from ancient Egyptian mummies and skeletons and 70 bone marrow samples from naturally mummified human remains from Upper Nubia. The Egyptian material derived from the Pre- to Early Dynastic site of Abydos (n = 7; 3500–2800 BC), a Middle Kingdom tomb in Thebes West (42; 2050–1650 BC), and different tomb complexes in Thebes West, which were built and used between the Middle and New Kingdom until the Late Period (42; c. 2050–500 BC). The Nubian samples were taken before the flooding caused by the Aswan Dam from 2 early Christian burial sites at Kulubnarti, between the second and third cataracts of the Nile River in northern Sudan. One site was on an island in the Nile and dated from 550 to 750 AD. The other was on the western bank of the Nile and was in use from c.750 to 1500 AD. All samples were tested for Leishmania spp. DNA and further characterized by direct sequencing. In 4 of the 91 Egyptian and 9 of the 70 Nubian samples, a 120-bp fragment of a conserved region of the minicircle molecule of kinetoplastid mitochondrial DNA of the parasite (5,6) could be successfully amplified and, with the first primer pair, unambiguously related to L. donovani species after sequencing (Figure). The positive samples from ancient Egypt exclusively originated from the Middle Kingdom tomb, while no molecular evidence for ancient Leishmania DNA was found in the Pre- to Early Dynastic and the New Kingdom to Late Period specimens. Figure PCR amplification of a 120-bp fragment of kinetoplastid mitochondrial DNA of Leishmania spp. in Egyptian and Nubian mummies. Lane 1, 50-bp ladder lanes 2–8, mummy samples; lanes 9,10, extraction controls; lane 11, PCR controls. Lane 6 provides ... In the Middle Kingdom, the Egyptians extended trade relationships and military expeditions to Nubia, the modern Sudan, with particular interest in the gold resources of the country and in obtaining slaves to serve as servants or soldiers in the pharaoh’s army. Today, the Sudan is one of the highly endemic countries for visceral leishmaniasis or kala-azar, which is thought to have originated in East Africa and later spread to the Indian subcontinent and the New World (4). Therefore, the high incidence of Leishmania DNA in the Middle Kingdom samples (4 [9.5%] of 42) and the lack of findings in earlier or later time periods, may indicate that leishmaniasis was introduced into Egypt at this time. Leishmaniasis did not likely become endemic in the Egyptian Nile Valley because the disease is closely linked to its vector, the phlebotomine sandfly, and the distribution of Acacia-Balanites woodland (7). That ancient Egyptians became infected because of close trade contacts and associated travel with Nubia during the Middle Kingdom seems more plausible. The high frequency of Leishmania DNA–positive samples in the Nubian mummies (12.9%) suggests that leishmaniasis was endemic in Nubia during the Early Christian period and, in light of the data on the ancient Egyptian mummies, probably already several thousand years before. Taken together, our results support the theory that Sudan could have been indeed the original focus of visceral leishmaniasis (4). Our study shows a completely new aspect of molecular paleopathology. The detection of ancient pathogen DNA is not only used to identify a certain disease and gain information on its frequency and evolutionary origin but also to trace back cultural contacts and their role in the transmission and spread of infectious diseases.

Leishmania donovani: physicochemical, immunological, and biological characterization of excreted factor from promastigotes

Abstract Leishmanial excreted factor (EF) from promastigote cultures was enriched from the crude product by differential precipitation with ammonium sulfate and perchloric acid, followed by column chromatography; and by boiling EF-antibody complex. Boiling destroyed the antibody, releasing the EF, which retained its ability to precipitate antibody. Enriched EF from Leishmania donovani promastigotes was found to be a highly negatively charged, carbohydrate-like material with a molecular weight approximating to 33,000, when monitored against a series of protein markers by gel filtration. Its ability to precipitate with antibody was unimpaired by boiling, lyophilization, pH changes from 1 to 11, treatment with high concentrations of NaCl, 10% phosphotungstic acid in 10% HCl, 0.6 M perchloric acid, 5% H 2 SO 4 , acetone, or dioxan. It did not absorb at wavelengths between 220 and 750 nm. Treatment with trypsin, Pronase, neuraminidase, and hyaluronidase did not affect its activity. Biochemical analysis showed that enriched EF contains carbohydrates but, at our level of detection, no protein, lipid, triglycerides, fatty acids, DNA, RNA, pentoses, amino sugars, sialic or uronic acid. Precipitation of EF by antibody was studied and the optimal molecular proportions for complete precipitation determined. EF-antibody complex, prepared at optimal proportions, and EF complexed with methylated bovine serum albumin, like EF alone, did not elicit antibody production in rabbits. EF in 0.5% phenol-saline elicited a delayed skin response of induration and erythema in guinea pigs cured of L. enriettii . Elevated temperature increased the release of EF from promastigotes, while the presence of trypsin acting at 37 C seemed to inhibit this effect slightly. Fractionation of mechanically broken promastigotes, by differential centrifugation and stepwise sucrose gradients, revealed a factor that precipitated rabbit antibody against whole promastigotes. This factor was associated with the soluble, organelle-free fraction and resembled EF when monitored by gel diffusion. This factor did not migrate when the complete extract from the broken promastigotes was run in immunoelectrophoresis. Boiling the extract for 5 min released a factor, which migrated to the anode. This factor appeared to be associated with another component in the promastigote, from which it dissociated on boiling. Boiling hamster tissues infected with leishmanial amastigotes, i.e., spleens containing L. donovani and epididymides containing L. tropica , also released factors similar to EF. These precipitated antibody in the same way, producing precipitation arcs that were continuous with those formed by EF from the homologous promastigotes. EF acted as a conditioner for culture promastigotes. Conditioned cultures showed maximal growth before similar, unconditioned cultures. However, both types of culture produced equal numbers of promastigotes per unit volume by the end of exponential growth.