Gene detectives place Yersinia pestis at the scene of Plague Pandemics of yore

In the first part, I described the Gram-negative bacillus Yersinia pestis as the etiological agent of human and animal disease; in part deux, I detailed the three major plague pandemics that have devastated human civilization many a times – the modern incarnation of which, complete with medical and epidemiological records, is known to have been caused by Yersinia pestis. In this third and final part, let me tell you about the ancient pandemics.

Based on certain biochemical properties found in modern strains of the bacterium, Yersinia pestis has been traditionally divided into groups called Biovars (namely, Antiqua, Medievalis, and Orientalis), each of which was found to carry specific genetic signatures in terms of mutations. Molecular grouping techniques, based on genetic signatures, are more accurate and help identify specific populations of the microbe. Scientists have begun to identify the family branches (what is known as a ‘phylogenetic tree’) of these populations, using small, isolated changes known as SNPs (pronounced ‘SNiP’; Single Nucleotide Polymorphisms) in their genomes as markers, or points of enumeration. For instance, analysis of such genetic data from 133 genomes of Yersinia pestis strains isolated from various parts of the world has revealed clues about the genealogy of the microbe that could be traced all the way back to the times of  the Justinian Plague and the Black Death pandemics. Genetic analysis has also made it possible to determine that the bacteria – biovar Orientalis – that caused the Third Pandemic belong to the same family branch, or molecular group, as the one that caused the pandemics of 7^th–9^th and 18^th centuries.

However, this determination for the bacteria related to the ancient Justinian Plague and the Black Death pandemics has not been so straightforward. It has been a point of raging controversy carrying on well into recent times, as to whether the plague bacillus, Yersinia pestis, was indeed responsible for causing the first two pandemics that repeatedly ravaged Europe and Asia. Partly responsible for this controversy, of course, is the lamentable – but historically appreciable – lack of accurate medical records during those centuries.

The attribution of the first two pandemics to Yersinia pestis has been hotly debated between epidemiologists and some historians, notably Samuel K Cohn, Jr. – who has argued long and hard that  both these pandemics differed significantly, in epidemiology and symptoms, from the modern plague of 20^th century. Both the Justinian Plague and the Black Death appeared to have spread with great rapidity, much faster than what was observed with Yersinia pestis during the third bubonic plague pandemic of late 19^th century; both were also devastatingly virulent, compared to the third plague which did not kill more than 3% of the populations of the major cities it touched. In addition, while the latter was found to be rarely contagious, the first two, by all contemporary accounts, spread from person to person, decimating households in clusters. The limit of the damage from the third plague may have been, at least in part, due to the rapid institution, and even forced adherence to, public health and sanitation measures in many countries, but Cohn disputes that, speculating that acquisition of immunity to Yersinia pestis by humans was a likelier reason.

Again, from historical evidence, the waves of the Justinian Plague, as well as the European outbreaks during late medieval and early modern periods, were preceded by famines, whereas during the third pandemic, bumper crops – according to Cohn – appeared to have encouraged the number of infected rats and fleas, leading to higher numbers of human infections. (Note: Although at least on one occasion, there was an exception to this: right before the third pandemic hit India in 1897, India was in the throes of the great famine of 1896.)

Cohn also points out that contrary to expectations, no scholar has demonstrated archaeological or historical evidence for any mass death of rodents during the first or second pandemic; in fact, Cohn argues in his 2008 paper that the flea vectors (Xenopsyllis cheopsis) associated with bubonic plague transmission would have found inhospitable the hot and dry summers of the southern Mediterranean and the Near East (time of year when both first and second pandemics were around), and that these fleas were rare or absent in Europe during the Black Death.

Male Xenopsylla cheopis (oriental rat flea) engorged with blood. This flea is the primary vector of plague in most large plague epidemics in Asia, Africa, and South America. Both male and female fleas can transmit the infection. Image file courtesy Wikimedia commons.

It is an unfortunate fact that, because of the stagnation of scientific inquiry during the Middle Ages, modern epidemiologists and historians are often forced to rely on contemporary accounts, missives, and religious texts, for gaining knowledge about these events. One such contemporary account, sent from the papal court at Avignon, recounts a tale of mortality and pestilence in September 1347 occurring in Eastern India; apparently, this event was accompanied by a rain of frogs, snakes, lizards, scorpions, hailstones of great sizes, and fire that consumed all. Giovanni Boccaccio’s (1313-75) Decameron details his brush with plague in Florence, and appears to describe buboes, or swellings in the axillary and inguinal regions. The plague in Padua was described in florid terms as God’s punishment on unbelievers. Because plague patients showed respiratory distress, ‘infected air’ became a common term to describe the scourge. In parts of England, accounts described a human monster, with two bodies navel upwards – a male and a female – but joined at the legs. A scientific explanation, attempted in a report by the Paris Medical faculty in 1348, indicated that the plague was caused by a ‘major conjunction of three planets in Aquarius’ which caused a ‘deadly corruption of air’. I am sure you, dear reader, get the point by now: in absence of good data, good conclusions are impossible to reach.

Cohn has been at pains to point out the collective opinion of some physicians of the early 20^th century, who considered that the typical symptoms, such as lymph node swellings, may not be unique to Yersinia pestis; additionally, he has painstakingly combed through various historical records of 14^th through 19^th century, to discover descriptions of ancillary, variant symptoms which didn’t appear to fit the traditional plague model. Some scientists have questioned the currently-accepted rat/flea/human transmission model, pointing out other possibilities.

As the controversy carries on, modern molecular methods have been able to shed some light on some of the aspects.

A group of French biologists, seeking to unravel the mystery of plague etiology, started out with the idea that remnants of microbial DNA may be present in relatively good condition, enclosed within the dental pulp (marrow of the teeth) of young teeth still within the jawbone (a.k.a. unerupted teeth). In the late 1990s, they got access to a great many skeletons from two mass graves which were once used to ancient bubonic plague victims; the grave in Lambesc had 133 skeletons from later half of 1590 CE, and one in Marseille, 200 skeletons from middle of 1722 CE. The group focused on a few skeletons from both places, carefully extracted unerupted teeth, and extracted DNA from the dental tissue. Using a sensitive technique called the Polymerase Chain Reaction (PCR), they looked for two genetic signatures of Yersinia pestis (known as rpoB and pla) while maintaining strict experimental controls to rule out inadvertent mixing (or ‘contamination’) of materials. They confirmed that Yersinia pestis DNA was indeed present in the teeth of these victims of the Black Death.

Within two years, in 2000, the same group – led by Didier Raoult and Michel Drancourt of the University of the Mediterranean at Marseilles – made another stunning discovery. Working with skeletons unearthed at 13^th-14^th century graves in Montpellier, Southern France, they isolated DNA from 23 teeth from the remains of a male, a female, and a child of approximately 8-10 years. Using a modification of their previous PCR technique and maintaining strict conditions, they again looked for the signature pla gene, which is highly specific for Yersinia pestis, and confirmed the presence of the plague bacterium. As an alternative hypothesis, they looked for signatures of two other microbes, the possibility of whose involvement had been raised by historians and scientists opposed to the Yersinia pestis theory of plague; however, these could not be found.

Image © CDC. Source: Drancourt et al., 2007 Photograph by: S Tzortzis. Skeletons from a mass grave in Martigues (France), 1720–1721.

These opponents were not sitting idle, though. Molecular biologists in this group continued to cast doubts on these observations, citing variously laboratory contamination, misused methods, and so forth, while historians pointed out the absence of similar results in well-preserved plague pits in Britain, Denmark, France, and Italy. In 2004, a British team lead by Thomas Gilbert and Michael Prentice of the Oxford University reported their inability to find Yersinia pestis-specific rpoB and pla DNA in any adult tooth sample recovered from the plague victim graves, dated late 13^th to 17^th centuries, found at five northern European sites (Copenhagen, Denmark; Royal Mint and Spitalfields in London; Verdun and Angers in France). They attributed this to a technique they used to minimize any kind of DNA contamination.

In rebuttal, Drancourt and Raoult pointed out that their choice of dental pulp, a well-vascularized soft tissue, was superior to bone or dentine (used by Gilbert’s team), and was more suitable for molecular detection of microbial DNA. Additionally, they took issue with Gilbert’s procedure for recovery of the dental pulp, which they considered not as effective as theirs, and criticized the design of the PCR. In reply, Gilbert and his team dismissed these claims, but both teams stressed on the need for independent verifications of the data.

In any event, while for some this controversy has not been laid to rest, other investigators (including some working with Drancourt and Raoult’s team in France) have made significant progress in discovering Yersinia pestis DNA in dental pulp specimens from Black Death-ravaged parts of Europe, such as Venice (Italy), Bavaria (Germany), and Bondy (France).

In 2010, a multi-nation group of European investigators combined the genetic analysis of ancient DNA from dental pulp samples, with analysis of a specific Yersinia pestis protein, called F1 antigen, detected on the skeletons recovered from mass graves found in five plague-burial-related archeological sites in England, France, Germany, Italy, and the Netherlands. In a fascinating study with rigorous controls, they demonstrated the presence of Yersinia pestis in these Black Death-associated mass graves. What is even more interesting is that by testing for specific genetic signatures, the authors were able to raise the possibility that different waves of the second pandemic may be attributed to at least two related, but genetically distinct, bacterial populations that have since gone extinct.

Since even small genetic differences may translate to distinct phenotypic characters, it is not unwarranted to speculate that these distinct genotypes – among other factors – may have been responsible for the differences that modern historians have noted in epidemiological parameters, such as transmission rates. Chinese researchers, studying SNPs, have recently determined that most Yersinia pestis lineages are virulent enough to cause human pandemics, and raised the intriguing possibility that genetically, this property has been present in this microbe ever since it evolved from the Yersinia pseudotuberculosis ancestor approximately 1,500–6,400 years ago.

In 2011, using dental pulp DNA from Black Death victims found at the East Smithfield burial site near London, Kirsten Bos and colleagues were able to reconstruct a draft genome for the ancient Yersinia pestis by means of DNA enrichment techniques and targeted high-throughput DNA sequencing; they were also able to confirm that the ancestral microbial strains may no longer exist in modern times.

In 2013, a group of European and American researchers in Anthropology and Genetics confirmed the presence of Yersinia pestis DNA in human teeth from an early medieval cemetery (name Aschheim, in Bavaria, Germany), that dates back to the of the 5^th–8^th centuries CE (contemporaneous with the Justinian Plague); they also traced the genealogy of the microbial strain to Asia, confirming Asian origin of the first pandemic.

Some of the ancillary points raised by Cohn and other opponents of the Yersinia pestis hypothesis remain unanswered – such as the absence of evidence for mass death of rodents during Black Death. Given the unreliability of the contemporary accounts, we may never fully assuage all the vexatious queries. What we need now is additional investigations into the contributions made by non-microbial factors, such as environment, vector dynamics, and host susceptibility; we also need a body of molecular evidence for ancient plague deaths in continents other than Europe. However, from the data already in evidence, the presence of Yersinia pestis during these ancient plague pandemics is unmistakable. Perhaps this would finally put to rest the controversy over the etiology of these recurrent, devastating events.