Alicia K. Wilbur

Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis

Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-old mycobacterial genomes from Peruvian human skeletons, revealing that a member of the M. tuberculosis complex caused human disease before contact. The ancient strains are distinct from known human-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean.

Tuberculosis and Leprosy in Perspective

Two of humankinds most socially and psychologically devastating diseases, tuberculosis and leprosy, have been the subject of intensive paleopathological research due to their antiquity, a presumed association with human settlement and subsistence patterns, and their propensity to leave characteristic lesions on skeletal and mummified remains. Despite a long history of medical research and the development of effective chemotherapy, these diseases remain global health threats even in the 21st century, and as such, their causative agents Mycobacterium tuberculosis and M. leprae, respectively, have recently been the subject of molecular genetics research. The new genome-level data for several mycobacterial species have informed extensive phylogenetic analyses that call into question previously accepted theories concerning the origins and antiquity of these diseases. Of special note is the fact that all new models are in broad agreement that human TB predated that in other animals, including cattle and other domesticates, and that this disease originated at least 35,000 years ago and probably closer to 2.6 million years ago. In this work, we review current phylogenetic and biogeographic models derived from molecular biology and explore their implications for the global development of TB and leprosy, past and present. In so doing, we also briefly review the skeletal evidence for TB and leprosy, explore the current status of these pathogens, critically consider current methods for identifying ancient mycobacterial DNA, and evaluate coevolutionary models.

Diet, Tuberculosis, and the Paleopathological Record

Osseous manifestation of infectious disease is of paramount importance to paleopathologists seeking to interpret ancient health, but the relationships among infectious agent exposure, development of disease, and skeletal involvement are complex. The outcome of an exposure strongly depends on multiple factors, including ecology, diet, nutrition, immune function, and the genetics of pathogen and host. Mycobacterial diseases are often studied in ancient remains but also are especially influenced by these factors; individual and population differences in severity and course are apparent following onset of active disease. The osteological record for these diseases represents the complex interplay of host and pathogen characteristics influencing within‐ and among‐individual skeletal lesion prevalence and distribution. However, many of these characteristics may be assessed independently through the archaeological record. Here, we explore the contributions of dietary protein and iron to immune function, particularly the course and outcome of infection with Mycobacterium tuberculosis. We emphasize how nutrition may influence the dissemination of bacilli to the skeleton and subsequent formation of diagnostic lesions. We then generate models and hypotheses informed by this interplay and apply them to four prehistoric New World areas. Finally, discrepancies between our expectations and the observed record are explored as a basis for new hypotheses.

From the mouths of monkeys: detection of Mycobacterium tuberculosis complex DNA from buccal swabs of synanthropic macaques.

Although the Mycobacterium tuberculosis complex (MTBC) infects a third of all humans, little is known regarding the prevalence of mycobacterial infection in nonhuman primates (NHP). For more than a century, tuberculosis has been regarded as a serious infectious threat to NHP species. Advances in the detection of MTBC open new possibilities for investigating the effects of this poorly understood pathogen in diverse populations of NHP. Here, we report results of a cross‐sectional study using well‐described molecular methods to detect a nucleic acid sequence (IS6110) unique to the MTBC. Sample collection was focused on the oral cavity, the presumed route of transmission of MTBC. Buccal swabs were collected from 263 macaques representing 11 species in four Asian countries and Gibraltar. Contexts of contact with humans included free ranging, pets, performing monkeys, zoos, and monkey temples. Following DNA isolation from buccal swabs, the polymerase chain reaction (PCR) amplified IS6110 from 84 (31.9%) of the macaques. In general, prevalence of MTBC DNA was higher among NHP in countries where the World Health Organization reports higher prevalence of humans infected with MTBC. This is the first demonstration of MTBC DNA in the mouths of macaques. Further research is needed to establish the significance of this finding at both the individual and population levels. PCR of buccal samples holds promise as a method to elucidate the mycobacterial landscape among NHP, particularly macaques that thrive in areas of high human MTBC prevalence. Am. J. Primatol. 74:676–686, 2012.

Patterns of tuberculosis in the Americas: how can modern biomedicine inform the ancient past?

Tuberculosis (TB) is an infectious disease that continues to take its toll on human lives. Paleopathological research indicates that it has been a significant cause of death among humans for at least five thousand years. Because of the devastating consequences to human health, social systems, and endangered primate species, TB has been the subject of many and varied research efforts throughout the world, efforts that are amassing an enormous amount of data concerning the causative agent Mycobacterium tuberculosis. Despite sequencing of the M. tuberculosis genome and numerous molecular epidemiological studies, many questions remain regarding the origin, evolution, and future co-evolutionary trajectory of M. tuberculosis and humans. Indeed, the origin of pre-Columbian New World TB has been and remains hotly debated, and resolution of this controversy will likely only come with integration of data and theory from multiple disciplines. In this paper, we discuss the pre-Columbian TB controversy, and then use research from biological and biomedical sciences to help inform paleopathological and archaeological studies of this ubiquitous disease that plagued our ancient forbears.

Detection of Mycobacterium tuberculosis DNA on the oral mucosa of tuberculosis patients

Diagnosis of pulmonary tuberculosis (TB) usually includes laboratory analysis of sputum, a viscous material derived from deep in the airways of patients with active disease. As a diagnostic sample matrix, sputum can be difficult to collect and analyze by microbiological and molecular techniques. An alternative, less invasive sample matrix could greatly simplify TB diagnosis. We hypothesized that Mycobacterium tuberculosis cells or DNA accumulate on the oral epithelia of pulmonary TB patients, and can be collected and detected by using oral (buccal) swabs. To test this hypothesis, 3 swabs each were collected from 20 subjects with active pulmonary TB and from 20 healthy controls. Samples were tested by using a polymerase chain reaction (PCR) specific to the M. tuberculosis IS6110 insertion element. Eighteen out of 20 confirmed case subjects (90%) yielded at least 2 positive swabs. Healthy control samples were 100% negative. This case-control study supports past reports of M. tuberculosis DNA detection in oral swabs. Oral swab samples are non-invasive, non-viscous, and easy to collect with or without active TB symptoms. These characteristics may enable simpler and more active TB case finding strategies.

Naturally acquired Mycobacterium tuberculosis complex in laboratory pig-tailed macaques

Here we present a case series from a primate research facility. The index case, a 4-year-old pig-tailed macaque (Macaca nemestrina) experimentally infected with chimeric simian-human immunodeficiency virus (SHIVSF162 P4), developed weight loss and was euthanized. Based on necropsy results the animal was diagnosed with opportunistic atypical mycobacteriosis associated with simian AIDS (SAIDS). Subsequently, tissues from the index animal, as well as tissues and oral mucosal swabs from six SHIV-infected contacts, were analyzed using molecular methods and found to contain nucleic acid sequences characteristic of Mycobacterium tuberculosis complex (MTBC). These data suggest that existing protocols fail to reliably detect MTBC infection in laboratory primates used as experimental models.

TB infection in the nonhuman primate biomedical model: tip of the iceberg?

Biomedical research in the 21st century increasingly relies on pathogen-free nonhuman primates (NHPs) to model human pathophysiology. Despite adherence to protocols designed to maintain pathogen-free colonies, reports of tuberculosis regularly appear. We hypothesize that, undetected by standard screening protocols, mycobacteria of the Mycobacterium tuberculosis complex (MTBC) continue to circulate in established NHP colonies and may, in addition, be periodically reintroduced with newly imported animals. The tuberculin skin test (TST), the accepted standard screening test for tuberculosis, relies on the hosts immune response to detect infection, but empirical data suggest that TST lacks both specificity and, particularly in certain NHP species and in immune compromised animals, sensitivity. In order to improve the detection of MTBC infection in NHP colonies we propose new screening protocols that incorporate molecular methods to detect mycobacteria. These new tests do not rely on the hosts immune response and may allow for strain typing of the pathogens - enhancing our ability to elucidate patterns of disease transmission. Moreover, the ability to rapidly and noninvasively collect specimens could lead to an improved appreciation of the burden of MTBC circulating in populations of NHPs and humans, including drug-resistant strains, data that are invaluable to public health efforts.

Pre-Columbian tuberculosis in Tierra del Fuego? Discussion of the paleopathological and molecular evidence

This work contributes to ongoing discussions about the nature of tuberculosis in the Western Hemisphere prior to the time of European contact. Our example, from the extreme south of South America was, at the time of our study, without firm temporal association or molecular characterization. In Tierra del Fuego, Constantinescu (1999) briefly described vertebral bone lesions compatible with TB in an undated skeleton from Myren 1 site (Chile). The remains of Myren are estimated to represent a man between 18 and 23 years old at the time of death. The objectives of this research are to extend this description, to present molecular results, to establish a radiocarbon date, and to report stable isotopic values for the remains. We provide further description of the remains, including tuberculosis-like skeletal pathology. Radiocarbon dating of 640±20 years BP attributes this individual to the precontact fourteenth-fifteenth centuries. Isotopic ratios for nitrogen and carbon from bone collagen suggest a mixed diet. Molecular results were positive for the rpoB quantitative PCR (qPCR) assays but negative for two independent IS6110 and IS1081 qPCR assays. Further testing using genomic methods to target any mycobacteria for specific identification are needed.

Detection of Mycobacterium tuberculosis Complex in New World Monkeys in Peru

The Mycobacterium tuberculosis complex causes tuberculosis in humans and nonhuman primates and is a global public health concern. Standard diagnostics rely upon host immune responses to detect infection in nonhuman primates and lack sensitivity and specificity across the spectrum of mycobacterial infection in these species. We have previously shown that the Oral Swab PCR (OSP) assay, a direct pathogen detection method, can identify the presence of M. tuberculosis complex in laboratory and free-ranging Old World monkeys. Addressing the current limitations in tuberculosis diagnostics in primates, including sample acquisition and pathogen detection, this paper furthers our understanding of the presence of the tuberculosis-causing bacteria among New World monkeys in close contact with humans. Here we use the minimally invasive OSP assay, which includes buccal swab collection followed by amplification of the IS6110 repetitive nucleic acid sequence specific to M. tuberculosis complex subspecies, to detect the bacteria in the mouths of Peruvian New World monkeys. A total of 220 buccal swabs from 16 species were obtained and positive amplification of the IS6110 sequence was observed in 30 (13.6%) of the samples. To our knowledge, this is the first documentation of M. tuberculosis complex DNA in a diverse sample of Peruvian Neotropical primates.