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Using a specialized method to analyze prehistoric disease DNA, researchers have, for the first time, successfully mapped an entire catalogue of infectious diseases – spanning 214 known human pathogens in total – that afflicted prehistoric populations and still circulate today.

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Eske WIllerslev, foto af Jes Larsen

The analyses also provide crucial new insights into the emergence of zoonoses – diseases transmitted from animals to humans, such as louse-borne relapsing fever and plague. The researchers show that many of these diseases first began to appear around 6,500 years ago, which closely coincides with the period when our ancestors started living in close proximity to domesticated animals.

These are the key findings of a research article just published in Nature, one of the world’s leading scientific journals. The study is based on DNA analyses of bones and teeth from 1,313 individuals who lived across Europe and Asia (Eurasia) from the Early Stone Age, around 12,500 years ago, up to about 200 years ago. Remarkably, seven of the samples are even older than the Early Stone Age – the oldest dating back 37,000 years.

The discovery was made by an international team of researchers headed by Eske Willerslev, Professor of Evolutionary Biology at the University of Copenhagen’s Lundbeck Foundation GeoGenetics Centre and at the University of Cambridge.

According to Eske Willerslev, who led the project together with Associate Professor Martin Sikora (University of Copenhagen) and Professor Astrid Iversen (University of Oxford), the mapping of prehistoric infectious diseases offers a range of exciting perspectives – many of which are highly relevant today. 

“In addition to providing information on historical conditions related to infectious diseases, the mapping also provides a deeper understanding of a number of these diseases that can still affect humans today. For example, you can see how some of the pathogenic microorganisms have genetically changed over time,” says Eske Willerslev.

Which, where and when?

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Globe Institute

In 2015, the Danish research team demonstrated that the bones and teeth of prehistoric individuals often contain not only their own genome, but also DNA from infectious diseases they were affected by. In recent years, by studying this type of ancient disease DNA, researchers have been able to reconstruct individual prehistoric outbreaks — for example, of plague or hepatitis.

The ambition to conduct a large-scale mapping of past infectious diseases – to determine which diseases were circulating, when, and where – required an entirely new approach. 

And for every single one of the 1,313 archaeological samples of human teeth or bones included in the Nature study, the researchers left no stone unturned – looking for DNA from pathogenic microorganisms in the form of viruses, bacteria and parasites, which today are known to make humans ill – of which they found 214.

The researchers also looked for ‘other bacteria’ that appeared during this mass search, and found 278 of those. These are bacteria that are not immediately assumed to be pathogenic, including soil bacteria – and bacteria that can be the product of putrefaction processes.

The analysis of the archaeological DNA from the 214 pathogenic microorganisms was carried out using shotgun sequencing. This is a special technique that makes it possible to read the code of DNA that has been broken up into tiny pieces – which would typically be the case when talking about archaeological genomes dating back thousands of years.

When such a piece of ancient DNA has been read, it is compared with registers with current codes of the DNA of pathogenic microorganisms to see if there is a hit.

“The DNA sequences we worked with from the old bones and teeth were really short – typically 50-60 base pairs,” says Martin Sikora, who is Associate Professor in population genetics at the Lundbeck Foundation Geogenetics Centre, UCPH, and who co-led this project. 

“Whether this is enough for identification is hard to say — it depends, among other things, on the type of organism and the number of fragments available,” explains Martin Sikora.

When you compare ancient DNA with modern DNA from the same infectious disease, you can observe how the pathogen has changed over time, says Martin Sikora:

“One example we found in this study is plague, which has evolved – and in its pathogenic form has developed specific genetic characteristics.” 

These changes in Yersinia pestis – the bacterium causing plague – can be traced back 5,500 years in the Nature article, says Assistant Professor Frederik Seersholm from University of Copenhagen, Lundbeck Foundation Geogenetics Centre. He is a specialist in plague analyses of ancient DNA and co-author of the Nature article – and he was very, and pleasantly, surprised when, among the analyses of the 1,313 archaeological tooth and bone samples from prehistoric humans in Eurasia, he saw the old Yersinia pestis hit:

“Because it is simply the oldest example of plague we have seen to date. When you look at this finding, you can really see how plague has evolved over time towards the version we know from ‘The Black Death’ – the plague pandemic that hit Europe in 1346-1353, and in some places killed up to 40 percent of the population,” says Frederik Seersholm.

Zoonoses

Approximately 70 percent of all new infectious diseases discovered in recent years are zoonotic – that is diseases that can be transmitted from animals to humans. Known zoonoses include diseases such as salmonella, listeriosis, Yersinia enterocolitica (which causes gastrointestinal infection), Borrelia recurrentis (which causes louse-borne relapsing fever), rabies and MRSA. As well as many others, because the list of zoonoses is long – there are a total of about 200.

Science has long been trying to find out when and under what circumstances zoonotic diseases first really made their appearance. The researchers behind the Nature article can provide an answer to this: We have to go back around 6,500 years to see DNA from zoonotic diseases increase in the archaeological tooth and bone samples from the 1,313 prehistoric humans who have been examined.

“Before 6,500 years ago, we only found DNA from one pathogenic microorganism in the samples from Eurasia, which we could classify as a zoonosis. After that time, zoonoses, to some extent, start causing people to die, and about 5,000 years ago, zoonoses really took off, according to our analyses of ancient human remains ,” says Astrid Iversen. She is professor of virology and immunology at University of Oxford and one of the co-authors of the Nature article.

The significant increase in the incidence of zoonoses around 5,000 years ago coincides with a migration to north-western Europe from the Pontic Steppe – that is from parts of present-day Ukraine, south-western Russia and western Kazakhstan.

The people embarking on this migration – and who to a large extent passed on the genetic profile found among people in north-western Europe today – belonged to the Yamnaya herders. Their primary source of food was meat and dairy products – and in present-day Denmark, among other places, the Yamnaya displaced and replaced the peasant population that lived here at the time and had relatively few livestock.

“The Yamnayas, on the other hand, lived really close to their animals – they had cows, sheep, and bulls – and therefore they have also been highly exposed to diseases from their animals,” says Kristian Kristiansen, an archaeologist affiliated with the Lundbeck Foundation GeoGenetics Centre at University of Copenhagen. 

It is well known that a wide range of pathogenic bacteria, viruses and parasites can be transmitted from animals to humans, including domesticated animals such as cows, sheep and goats; for example, leptospirosis, bovine tuberculosis, brucellosis and listeriosis – the last three often via milk, says Professor Astrid Iversen:

“Zoonoses first became a major problem for humans when we started keeping animals together in large herds and living close to these animals – eating their meat and drinking their milk. This meant that the animals could more easily infect each other, and that the risk of them infecting humans increased.”

The rise of zoonoses 5,000 years ago also shows that the way we live has a major impact on which pathogenic microorganisms we are exposed to. It is also a reminder that zoonoses are not a static phenomenon, says Astrid Iversen:

“Pathogens are constantly changing as they evolve and adapt to their host(s), which can affect both their prevalence, the risk of transmission, and the outcome of a human infection – and our way of life can significantly increase or reduce our risk of infection with various pathogens. In addition, over generations, some resistance to certain pathogens can also develop. There is something very fascinating about examining ancient DNA for evidence of infectious diseases in humans – and observing the relationship between the composition and burden of infectious challenges and lifestyle. This interplay between infection risk and lifestyle continues to this da��.”

New studies

The 214 different infectious diseases identified by the researchers in the old bone and tooth samples are all blood-borne – and the infection has at some point passed into the blood of these prehistoric humans. The infectious diseases also had in common that their genome is DNA.

Once the two conditions are met – and when a certain amount of pathogens are present – the researchers can use the shotgun method to identify infectious diseases in archaeological samples. And this is an opportunity that can and should be exploited to carry out new studies, says Associate Professor Martin Sikora from the Lundbeck Foundation Geogenetics Centre:

“We’ll try to expand the datasets we’re working with – and besides, it would be really cool if we could conduct similar studies elsewhere in the world, for example in Asia and on the American continent.”

According to Professor Eske Willerslev, studies of DNA from infections that affected prehistoric humans could also be used for testing and the development of vaccines:

“Using shotgun analyses, it is possible to follow mutations in the genome behind an infection through time – right up to today. For example, you can see when a virus and bacterium has mutated, how it has mutated, how quickly the genome has changed – and which mutations appear to have been particularly significant. And the mutations that have been successful in the past are likely to reappear.”

This provides information that gives you both a picture of the efficacy of vaccines in the future, and an idea of what tomorrow’s vaccines should be able to do, says Eske Willerslev:

“There is also another area that may hold a number of interesting perspectives: the possibility of finding new correlations between infectious diseases and changes in the human genome over time. I'm convinced that this will enable us to study and in this way, among other things, gain new knowledge about genetic conditions that play a role in connection with the development of resistance.”