Co-occurrence of Yersinia pestis and other zoonoses during European prehistory

This study presents the earliest evidence of *Yersinia pestis* in Southern Europe from a 4,950-year-old Italian individual who was co-infected with *Erysipelothrix rhusiopathiae* and Hepatitis B virus, while also expanding the ancient genomic record of *Erysipelothrix* species to better understand prehistoric zoonotic disease dynamics and transmission.

The Gist

Imagine you are a time traveler visiting a dusty, forgotten cave in Tuscany, Italy, roughly 5,000 years ago. You find a pile of ancient bones, jumbled together like a messy deck of cards. This paper is the story of what happens when scientists take those bones, dust them off, and look at them through a high-tech microscope to see what invisible "ghosts" (diseases) were haunting these people.

Here is the story of the paper, broken down into simple parts:

1. The Mystery of the Mixed-Up Bones

The researchers went to a site called Grotta della Spinosa. It was a Copper Age burial site where the bones of about eight people had been mixed up over thousands of years. It was like trying to solve a puzzle where the pieces from eight different people were all in the same box.

Using advanced DNA technology, they managed to sort the pieces out. They found that most of these people were local Italians from that time period. But one teenager (about 16 or 17 years old), whom they named GSP013, had a very special and scary secret.

2. The "Triple Threat" Infection

When they looked at the DNA of teenager GSP013, they found something shocking: this young person wasn't just fighting one illness; they were fighting a three-way battle.

• The Big Bad: They had Plague (Yersinia pestis). This is the same germ that caused the Black Death centuries later. But this was a "prehistoric" version, older than any plague found in Italy before.
• The Silent Partner: They also had Erysipelothrix, a bacteria usually found in pigs and other farm animals. Think of this as a "farm bug" that jumped from an animal to the human.
• The Viral Guest: Finally, they had Hepatitis B, a liver virus.

It's like the teenager's body was a crowded bus where three different dangerous passengers were sitting next to each other. The fact that they found Hepatitis B in four different people at the site suggests this virus was actually quite common in their community, like a cold that everyone caught.

3. The "Missing Link" Plague

The plague bacteria found in GSP013 is a big deal for history.

• Time Travel: It is about 200 years older than the next oldest plague found in Southern Europe. It pushes the timeline of the plague's arrival in Italy back significantly.
• The "Unfinished" Plague: Modern plague is terrifying because it spreads easily via fleas on rats. But this ancient version was "unfinished." It was missing a specific genetic tool (a gene called ymt) that acts like a shield, allowing the bacteria to survive inside a flea's stomach.
• The Analogy: Imagine a modern plague is a fully equipped soldier with a bulletproof vest and a rifle. This ancient plague was like a soldier who had the rifle but forgot the vest. It could still kill, but it probably couldn't spread as easily from rat-to-flea-to-human. It likely spread through direct contact with sick animals or their waste, rather than a swarm of fleas.

4. The "Farm Bug" Detective Work

The paper also did something amazing with the second bacteria, Erysipelothrix.

• The Hunt: The researchers didn't just look at the Italian cave. They went on a digital treasure hunt through hundreds of other ancient DNA studies from all over Europe.
• The Discovery: They found 60 new cases of this bacteria in ancient humans and animals, dating back 8,300 years!
• The Connection: They found that this "farm bug" often showed up in people who also had plague. This suggests that in prehistoric times, humans were getting sick from a "zoo" of diseases. As people started farming and living closer to pigs, dogs, and cows, they opened the door for these animal diseases to jump into humans.

5. Why This Matters

Think of human history as a long movie. Before this paper, we knew the plague arrived in Europe, but we didn't know exactly when or how it got to Italy.

• The Map: This study draws a new dot on the map, showing the plague was in Southern Europe much earlier than we thought.
• The Warning: It shows that when humans started farming and living in tight communities with animals, they created a "perfect storm" for diseases to mix and match. The teenager in the cave was a victim of this new, dangerous lifestyle.
• The Lesson: It reminds us that diseases don't just appear out of nowhere; they often travel with our pets and livestock. The "farm bug" (Erysipelothrix) was likely a constant companion to these ancient people, a reminder that their close bond with animals came with a hidden cost.

In a nutshell: This paper is a time-traveling detective story. It proves that a teenager in ancient Italy died from a rare mix of plague, a pig-bacteria, and a virus. It tells us that the plague arrived in Italy earlier than we knew, and that the rise of farming brought a whole new world of dangerous germs to our ancestors.

Technical Summary

1. Problem Statement

The study addresses significant gaps in the understanding of prehistoric plague (Yersinia pestis) epidemiology and zoonotic disease dynamics in Europe. Specifically:

• Geographical and Temporal Gaps: While Y. pestis has been detected in the Late Neolithic–Bronze Age (LNBA), its spread into Southern Europe (specifically the Italian peninsula) and its precise timing relative to other European strains remain unclear.
• Transmission Mechanisms: The transmission routes of early Y. pestis strains (lacking the ymt gene required for flea-borne transmission) are debated. It is unclear how these strains infected humans without the classic rodent-flea cycle.
• Co-infection Dynamics: The role of co-infection with other zoonotic pathogens (e.g., Erysipelothrix rhusiopathiae) in prehistoric communities is poorly understood, particularly regarding how animal husbandry and environmental factors facilitated spillover events.
• Data Scarcity: Ancient genomic data for Erysipelothrix spp. is extremely limited, hindering the reconstruction of its evolutionary history and host range.

2. Methodology

The researchers employed a multi-faceted paleogenomic approach combining ancient DNA (aDNA) extraction, metagenomic screening, and phylogenetic analysis.

• Sample Collection:
  • Primary Site: Grotta della Spinosa (Tuscany, Italy), a Copper Age site (~4950 cal BP).
  • Samples: 9 skeletal samples (teeth, petrous bones, auditory ossicles) from disarticulated, commingled remains.
  • Identification: Genetic screening (READv2) identified 8 unique individuals, merging two samples (GSP005 and GSP013) from the same individual.
• Sequencing and Library Preparation:
  • Double-stranded DNA (dsDNA) libraries were constructed.
  • Shotgun Sequencing: Performed on all samples using Illumina platforms (NextSeq500/2000).
  • Target Enrichment: For the positive sample (GSP013), custom myBaits enrichment was used for Y. pestis and Y. pseudotuberculosis genomes to increase coverage.
• Metagenomic Screening:
  • Tools: KrakenUniq for initial taxonomic classification, followed by mapping validation.
  • Broad Screening: 2,495 published ancient datasets (humans, animals, sediments) were screened for Erysipelothrix spp. (E. rhusiopathiae and E. tonsillarum).
• Bioinformatic Analysis:
  • Authentication: Assessment of cytosine deamination (C>T) patterns and fragment length to confirm ancient origin.
  • Phylogenetics:
    • Y. pestis: Maximum Likelihood (ML) trees built using RAxML-NG on 1,042 variant sites.
    • Erysipelothrix: Recombination filtering (ClonalFrameML) followed by ML tree construction (6,809 SNPs) and projection of low-coverage genomes (epa-ng).
  • Virulence Profiling: Coverage analysis of specific virulence genes (e.g., ymt, pla, ureD, pde-2) and antibiotic resistance markers.
  • Population Genetics: Principal Component Analysis (PCA) of human host ancestry using the 1240k SNP panel.

3. Key Results

A. Discovery of Early Yersinia pestis in Southern Europe

• Individual GSP013: An adolescent female (16–18 years) from Grotta della Spinosa was found to be co-infected with Y. pestis, Erysipelothrix rhusiopathiae, and Hepatitis B virus (HBV).
• Dating: The sample dates to ~4950 cal BP (3100–2920 BCE), making it the earliest evidence of Y. pestis in Italy and Southern Europe, predating previous Central/Eastern European findings by ~200 years.
• Phylogeny: The strain belongs to the LNBA- clade (ymt-negative). It is basal to later European diversity but slightly derived from Northern Caucasus (RK1001) and Altai (RISE509) strains.
• Virulence Profile:
  • Missing: ymt (flea survival), YPMT1.66c (innate immunity resistance), and the filamentous prophage.
  • Ancestral pla: Possesses the ancestral I259 allele (less efficient in mammalian tissue colonization) rather than the derived T259 allele.
  • Implication: The lack of ymt and YPMT1.66c suggests bubonic plague (flea-borne) was unlikely. The ancestral pla suggests potential for pneumonic transmission or less severe systemic infection, though the strain was still virulent enough to cause death.

B. Expansion of Ancient Erysipelothrix Genomics

• New Genomes: The study retrieved 60 new ancient genomes of Erysipelothrix spp. (45 E. rhusiopathiae, 15 E. tonsillarum) from humans, animals (dogs, aurochs, cattle), and sediments, dating from 8,300 BP to 100 BP.
• Co-occurrence: Erysipelothrix was found in 15 individuals previously known to be infected with Y. pestis, supporting a model of shared zoonotic reservoirs.
• Phylogeny: Ancient genomes cluster at the base of Clade 2, showing no clear host or geographic specificity, indicating a generalist pathogen in prehistory.
• Gene Content Differences:
  • Ancient-Specific Genes: Ancient human genomes retained genes involved in cell wall synthesis (wcaJ, dapDC, rmlB), adhesion (inlJ), and immune evasion (EEP), which are largely absent in modern strains.
  • Modern-Specific Genes: Modern strains possess antibiotic resistance genes (macB, drrA, ykoD), absent in ancient samples, reflecting the lack of selective pressure from antibiotics in prehistory.

C. Hepatitis B Virus (HBV)

• HBV was detected in 4 of the 8 individuals from Grotta della Spinosa, suggesting endemic circulation of the virus in this Copper Age community.

4. Significance and Implications

• Revised Epidemiological Model: The findings challenge the assumption that early Y. pestis relied on the rodent-flea-human cycle. The presence of ymt-negative strains in Southern Europe, combined with co-infection by Erysipelothrix (a pathogen associated with livestock contact), supports a "bridge host" hypothesis. This suggests transmission occurred via direct contact with infected domestic animals (e.g., pigs, dogs) or their waste, rather than via fleas.
• Zoonotic Landscape: The study highlights the Neolithic transition (sedentism, animal husbandry) as a critical period for increased human-animal contact, facilitating the spillover of multiple pathogens simultaneously.
• Evolutionary Trajectory: The genomic data reveals that E. rhusiopathiae has undergone significant gene loss (specifically in virulence factors like inlJ and cell wall synthesis genes) and gained antibiotic resistance genes only in the modern era. This suggests ancient strains may have had different infectivity profiles compared to modern clinical isolates.
• Geographical Expansion: The discovery pushes the known boundary of LNBA Y. pestis significantly south and west, indicating a more widespread and established presence of the bacterium in prehistoric Europe than previously documented.

Conclusion

This study provides the earliest evidence of Y. pestis in Southern Europe and significantly expands the ancient genomic record of Erysipelothrix. It demonstrates that prehistoric plague likely spread through complex zoonotic networks involving domestic animals, rather than solely through the classic flea-borne mechanism. The co-occurrence of multiple pathogens in a single individual underscores the high burden of infectious disease in Copper Age communities and the critical role of animal husbandry in shaping human pathogen evolution.