It seems I may have spoken too soon. Quoting myself:
One historical event that has been the subject of much speculation over the decades has been the Plague of Athens, a mysterious outbreak that is thought to have changed the direction of the Peloponnesian War, and for which the cause still remains uncertain.
This plague has been attributed to bubonic plague, toxic shock syndrome and/or necrotizing fasciitis due to Streptococcus pyogenes or Staphylococcus aureus, Salmonella, yellow fever, malaria, Ebola, influenza, and smallpox, to name just a few. Typhus seems to fit the description best, but it’s likely that a cause will never be known with certainty.
Little did I know when I posted that on my old blog (just last month!) that a study had already been accepted to the International Journal of Infectious Diseases suggesting that it’s not typhus (caused by Rickettsia prowazekii), but typhoid fever (Salmonella enterica serovar Typhi) that appears to be the cause of the plague.
As I mentioned at the old site, the plague of Athens refers to an outbreak lasting from ~430-426 BC, reported by the Greek historian Thucydides. While his description of the plague was extensive, it didn’t seem to quite fit the clinical spectrum of a number of infectious diseases. Since that was the only thing we had to go by, it appeared that the cause of the plague would go down in history unresolved, but with mountains of speculation and competing hypotheses.
But as any good forensic scientist will tell you, a body can tell a good story–and it seems that’s what they found in 1994. A mass burial site containing at least 150 bodies was unearthed in Athens, and dated to ~430 BC. For a number of reasons (such as the crudeness of the pit, the placement of the bodies, and the rarity of mass graves in Greece except in times of epidemic), the researchers concluded the dead were likely victims of the infamous plague.
So, what good does this do us? The bodies are almost 2500 years old; surely they can’t provide a testable sample. Right? Luckily, people cleverer than I have figured out a way to search for microbial DNA in ancient samples: via dental pulp. This is used because it’s well-protected, and generally considered to be free of contamination (until you break open the tooth, that is). That’s what they did in this study as well, using the polymerase chain reaction (PCR) to search for DNA from Yersinia pestis, typhus, anthrax, tuberculosis, cowpox, and cat-scratch disease (Bartonella henselae) in addition to S. enterica. But, there are a number of problems with the study.
First, they didn’t use any positive controls. Their explanation is reasonable: in these types of studies, contamination is your worst enemy, and not having any positive controls to run side-by-side dramatically decreases the likelihood of contamination. Problem is, they went through these primer sets one-by-one until they got a positive result (in other words, they did PCR for the six organisms listed above and got all negatives; when they got a positive with S. enterica, they stopped.) But since no positive controls were used, they can’t be certain that the negative results they got for the first six were real, or a result of bad primers, enzymes, incorrect cycling conditions, etc. (The PCR reactions for the other genes had been previously optimized so they *should* work, but still…)
Second, the DNA sequence had only 93% similarity to the same gene (narG, which encodes a part of an enzyme involved in anaerobic respiration) in modern-day S. enterica. This isn’t necessarily bad–after all, there’s been around 2500 years’ worth of potential changes–but is it enough to call it S. enterica instead of another species? It’s suggestive, but IMO more genes should have been checked before going to publication. (They do note in their discussion that it could be S. enterica serovar Typhi “or a bacterial species very closely related to it,” but that could have been strengthened by just doing a bit more PCR).
Third, why stop at S. enterica? They even mention in their discussion that not all the symptoms described fit with modern-day typhoid fever. This could mean that it simply presented differently back then (due to changes in the bacterium and/or the host population), or it could mean that the plague wasn’t caused by a single agent. It’s likely that this S. enterica (or S. enterica-related) organism played some role; it was found in all three of the teeth examined (taken from 3 different individuals). Suggestive, but not conclusive that this was the only cause. In their Table 1, they list 31 different “theories on the cause of the Plague of Athens.” Some–such as scurvy–aren’t infectious agents. Others, such as sweating sickness, don’t have a known organism associated with them–they’re also mysteries. Still, many of the remaining hypothesized causes could have been tested in the same manner they tested the other 6 agents. If they’d all been negative (and again, positive controls would be a good thing), that would be more convincing.
Finally, limited number of samples. Even with ~150 individuals in the grave, though, this isn’t as easy as it sounds. The teeth need to be in good shape so as to avoid contamination with contemporary bacteria (including bacteria from the soil they were buried in). They do conclude that further investigation of DNA material from the grave is needed, so I’d assume they’re planning to do this.
All in all, a very intriguing study. It’s amazing what can be done with a little bit of tissue, a thermocycler, and a DNA sequence. As far as me being wrong, don’t expect me to own up to that very often. But I’m in good company, at least. In America’s Forgotten Pandemic by Alfred Crosby (discussing the 1918 influenza pandemic), he states:
It has been the dream of scientists working on influenza for over a half century to somehow obtain specimens of the virus of Spanish influenza, but only something as unlikely as a time capsule could provide them.
Jeffery Taubenberger et al. found that unlikely “time capsule” in the form of archived tissue samples and frozen lungs, just waiting for the right techniques to allow them to share their viral gold mine. Likewise, these samples from Athens are a wonderful time capsule that permit us to reach back into the past, and investigate an epidemic that I thought we’d never have access to just a month ago. Amazing stuff.