Molecular insights into the history of plague

Michel Drancourt^, and Didier Raoult

Unité des rickettsies, CNRS UPRES-A 6020, faculté de médecine, université de la Méditerranée, 27 boulevard Jean Moulin, 13385  Marseille cedex 5, France

Microbes and Infection
Volume 4, Issue 1 , January 2002, Pages 105-109

Article Outline

1. Historical descriptions of plague

2. The microbiology of plague

3. The limits of historical descriptions of plague and controversial issues regarding the history of the disease

4. Retrospective diagnosis of plague

5. Conclusions

References

1. Historical descriptions of plague

The history of plague is often as confusing as the recorded history of mankind itself. There are numerous references to plagues that may have been due to Yersinia pestis in ancient texts, including the Old Testament. The first recorded outbreak of an epidemic consistent with plague, however, was in Athens in the summer of 430 BC. This occurred at the outbreak of the Peloponnesian War and caused the death of the great statesman, Pericles. It also decimated the general population with an estimated 300,000 deaths (one in every three people) and contributed to the decline and fall of classical Greece. In his documentation of the epidemic, Thucydides, who survived the disease, left us a clear description of the signs of that `plague' including high fever, blistered skin, bilious vomiting, intestinal ulcerations and diarrhea. He noted that animals were also affected. The plague epidemics that occurred between AD 541 and 750 make up the first (Justinian) pandemic. The first epidemic began in Ethiopia and spread quickly from Pelusium, Egypt, through the Middle East to the Mediterranean basin and, to a limited extent, to Mediterranean Europe. The second through eleventh epidemics (AD 558–654) occurred in 8- to 12-year cycles 1 and affected all the `known world', i.e. North Africa, Europe, central and southern Asia and Arabia. Mortality rates of 15–40% were recorded and it has been estimated that 50–60% of the population were lost between AD 541 and 700 2. Clearly, this depopulation was not solely due to plague since other epidemics probably occurred during this period. A number of economic, social and politic consequences, including the weakening of Byzantine Europe, have been attributed in part to this first pandemic.

After an absence of plague for 600 years in Europe, the second pandemic occurred from 1330 to 1346. It probably originated from the steppes of central Asia where there was an epidemic in marmots, and Chwolson, a Russian archeologist, found inscriptions relating to plague on memorial stones in Nestorian graveyards near Issyk Kul Lake dating back to 1338–1339 3. It spread westward along the trade routes entering Europe in 1347 in Sicily and Marseilles, southern France. Trappers collected the fur of the dead animals and sold them to buyers from the West. The first outbreak of bubonic plague occurred along the Volga river, from where it spread west to the Don River and then down to the Black Sea. Gabriel de Mussis, an Italian eyewitness to plague in Caffa (present-day Feodosia, a seaport of South Russia on the east coast of Crimea) reported that Mongolians dying from plague were used as bacteriological weapons and hurled over the city walls of Caffa in 1346 4 and 5. He also reported that plague reached Europe when Genoese vessels docked in Messina, Genoa and Marseilles in November 1347. The first epidemics, later known as the Black Death, then spread again through the `known world' and killed an estimated 17–28 million Europeans, representing approximately 30–40% of the population at the time 6. The French medieval physician Guy de Chaulliac, dean of the oldest French medical university in Montpellier, was appointed by Pope Clement VI in 1347 when the Black Death was sweeping over southern France. He contracted plague and survived and while his original description of the Black Death epidemics was lost, printed copies survived and are still available for historical research 7. His descriptions indicated that the Black Death swept from Marseilles to Avignon (100 km) within 1 month and that the disease presented in both the bubonic and pulmonary forms had an estimated mortality of 42%. Black Death also provided the backdrop for an early Italian Renaissance masterpiece, Boccaccio's Decameron, begun in 1348 in Florence in the grip of the most devastating epidemic of the citys history. Plague then recurred in 2- to 5-year cycles until the beginning of the 18th century. Mortality of up to 40% was described in a detailed study of plague epidemics in 1597–1598 in Penrith, Cumbria 8. In Marseilles, plague reoccurred in May 1720 when a commercial ship named the `Grand Saint-Antoine' arrived from Syria and Lebanon with cases of plague aboard. This epidemic killed 50,000 people in Marseilles and archeological studies of graves of plague victims provided the first evidence that death was verified by driving bronze pins into the toes 9. The third and current pandemic probably started in 1855 in the Chinese province of Yünnan where troop movements during the war in that area caused a rapid spread of the disease to the southern coast of China. It reached Hong Kong and Canton in 1894, Bombay in 1898, and by 1899–1900 steamships had disseminated the disease to Africa, Australia, Europe, Hawaii, India, Japan, the Middle East, the Philippines and North and South America. The etiological agent of plague was discovered at the beginning of this pandemic, opening the door to the modern study of the disease.

2. The microbiology of plague

During the Hong Kong epidemic in June 1894, Alexandre Yersin 10 and Shibasaburo Kitasato independently announced within a few days of one another the isolation of the plague organism, which was named Yersinia pestis in 1944 11 and 12. Yersin gave us a clear description of bubonic plague in Hong Kong and noted that buboes occurred in 75% of cases. While Yersin described a connection between rats and plague, it was Paul Louis Simon who discovered the role of the rat flea in the transmission of the disease during the Indian epidemics in 1897 13. Mollaret and collaborators finally showed that Y. pestis survives in the litter and the soil in the burrows of infected animals for years 14 and 15 leading to infection of any subsequent occupiers of the burrows 16. Although the mortality rates and dissemination of the sporadic plague outbreaks that still occur today are greatly reduced, stable enzootic foci are still to be found on every continent except Australia. Plague, then, remains a significant public health concern in some countries and is now classified as a re-emerging infectious disease because of an increase in the number of cases reported to the World Health Organization 17 and 18. It reappeared in 1994 in epidemic form in countries including Malawi, Mozambique and India where the disease had been silent for 15–30 years. There has also been a gradual extension of existing foci of the disease as is the case in the United States, for example 19. We would note, however, that controversial data from the 1994 epidemic in India have now raised suspicion that the epidemic may not have been plague 20. In Madagascar, new genotypes of Y. pestis are emerging 21 as well as plasmid-mediated resistance to antibiotics 22 and 23. Y. pestis has now been identified as a subspecies of Yersinia pseudotuberculosis on the basis of DNA/DNA hybridization studies and 16S rDNA sequence analysis 24 and 25. The name Y. pestis has, however, been retained because of the special epidemiological and clinical features of this bacterium. Recent studies have shown that Y. pestis emerged as a separate clone within Y. pseudotuberculosis 1,500–20,000 years ago, shortly before the first known pandemics 26. Three biotypes named Antiqua, Medievalis and Orientalis have been distinguished among Y. pestis strains collected during the 20th century on the basis of their ability to reduce nitrate and to ferment glycerol and mellobiose 27. Molecular analyses of a collection of 70 strains belonging to the three biotypes collected over a 72-year period established correlations between the ribotypes and the biotypes of strains 28. In particular, two ribotypes, designated B and O, were found to comprise 65.7% of strains under study; ribotype B comprised all the biotype Orientalis isolates while ribotype O comprised biotypes Antiqua and Medievalis isolates 28. Clear-cut differences were found in the current geographical distribution of biotypes and ribotypes on the five continents 27 and 28: ribotype B (biotype Orientalis) was found on all five continents, while ribotype O (biotypes Antiqua and Medievalis) was restricted to Central Africa and Central Asia. These geographical patterns have been proposed to result from the past spread of biotypes during each of the three pandemics 27. It has been stated that plague originated in the Central Asiatic plateau 29 and that biotypes Antiqua and Medievalis originated from this focus 27. Ribotyping enabled this hypothesis to be refined and led to the proposal that a main clone of Y. pestis ribotype O (biovar Antiqua) spread from Central Asia to Central Africa and caused Justinian's plague. Later, a second clone from the same pulsotype (biovar Medievalis), which lost the ability to reduce nitrate, spread from Central Asia to Crimea and was responsible for the Black Death. Still later, a third clone of pulsotype B (biovar Orientalis) spread from Asia to cause the third pandemic. Recently, sequence analysis of five housekeeping genes in Y. pestis has provided support for the above hypothesis 26. Further support using molecular typing of ancient strains is, however, yet to be provided.

3. The limits of historical descriptions of plague and controversial issues regarding the history of the disease

Historical descriptions of plague are found in paintings and in old texts which provide epidemiological and clinical descriptions of the disease. The interpretation of historical texts is often limited by the absence of the original text itself, problems with the translation of ancient words into contemporary language and a lack of precise medical terms in old languages. Also, until the second part of 19th century, confusion existed between diseases presenting with similar signs and symptoms, for example typhus and typhoid fever 30. In historical documents, a description of an epidemic associated with high mortality rates that occur within a few days and the presence of buboes probably has the highest predictive value for plague. Although encountered in other contemporary infectious diseases, buboes––large, painful, engorged lymph nodes––are a useful sign to distinguish plague. None of the above three characteristics alone, however, is sufficient for a diagnosis of plague, and this has caused controversies regarding the etiology and the epidemiology of the disease 31. In particular, for suspected plague epidemics that occurred before 1894, only degrees of probability can be offered as to whether these were in fact caused by Y. pestis. Descriptions of the plague of Athens for example 32 are not consistent with any disease we know and bubonic plague, typhus, smallpox 33, staphylococcal toxic shock syndrome-complicated influenza 34, Ebola fever 32, melioidosis 35 and other diseases 36 have been regarded as the diseases probably involved. Also, as the high levels of mortality and transmissibility associated with the Black Death were not observed during the third plague pandemic, controversies arose regarding the etiology of the Black Death, and some historians believed it was not plague. The alternatives have included anthrax, typhus, tuberculosis and hemorrhagic fever 8; 37 and 38. As we show below, modern molecular methods have enabled us to demonstrate clearly that the second pandemic was in fact due to Y. pestis 39 and 40 Controversies have also arisen regarding the reservoirs in epidemics of plague. Yersin established that the black rat, Rattus rattus was the reservoir of Y. pestis during the third pandemic 10. Observation that some plague epidemics lacked an obvious murine reservoir, however, led to the discovery of telluric resistance Y. pestis 14. Changes in the numbers and geographical distribution of R. rattus populations have been found to correlate with plague epidemics during the first and second pandemics, supporting the role of this species as a reservoir for Y. pestis during these periods. Several workers described the presence of R. rattus on the southern coasts of the Mediterranean sea in the ancient world and the rat was confirmed to be present in Corsica, the Baleares Islands and Pompei in the 4th–2nd century BC 41. R. rattus developed a close association with humans during the Roman expansion and the early Middle Ages but remained localized in small territories in urban areas until the 11th century. Archeological data from Antiquity indicate that black rats migrated from Mediterranean harbors along the routes and their geographical distribution matched that of the Justinian plague 42. During the second millenium, economic prosperity resulted in a rapid expansion of trade between European and Eastern countries, and between southern and northern European countries. The R. rattus population grew very rapidly and this species has now been found in almost all the archeological sites in Europe, reaching the highest density in the 14th century. When plague entered southern Europe in 1347 R. rattus was present at high density everywhere in Europe. Rattus norvegicus appears to have entered Europe during the 16th century and expanded its range during the 18th century. The role of rats, however, has been disputed because, except in a few instances, historical records have not mentioned rat deaths preceding plague epidemics 43. The nature of the vector of plague has also been disputed. Plague was observed to be seasonal in Switzerland and Great Britain during the second pandemic. This is compatible with an arthropod-borne disease but offers no indication as to whether rodent or human ectoparasites were the dominant carriers 44. The Eastern rat flea Xenopsylla cheopis has been recognized as the main vector of Y. pestis during the third pandemic 13. X. cheopis, however, was unlikely to have been present in medieval Europe; instead it was most likely Nosopsyllus fasciatus, a species that rarely feeds on humans. Also, a notable clustering of cases in family and household groups seems to have been a constant feature of past plague epidemics, a phenomenon that has not occurred in modern Asian epidemics. Historical texts contain numerous references to the role of clothes and bedding in the transmission of plague. For example, in the introduction of the Decameron, burning the clothing of plague victims is described as an effective measure to prevent the transmission of the disease. Also, the measures taken to prevent transmission of plague amongst people, such as quarantine and cordons sanitaires 45, remain controversial. In fact, some of the contemporary precautions used were unlikely to be effective if plague epidemics had only an epizootic base. Pulex irritans, a human flea has therefore been proposed as a major vector of past plague outbreaks 1 and 46 by directly transmitting Y. pestis from person to person. P. irritans was an endemic human ectoparasite during the second pandemic. Finally, the reasons for the disappearance of plague from western Europe during the 17th century remain controversial.

4. Retrospective diagnosis of plague

Molecular biology tools enable the detection of microbial genome fragments in ancient human remains and thus the possibility of making retrospective diagnoses of ancient diseases. By polymerase chain reaction (PCR) sections of microbial DNA may be amplified enzymatically and then sequenced to assess the percentage of similarity between parts of the genomes of ancient microorganisms and those of their modern-day counterparts deposited in electronic databases. DNA is known to persist for long periods after the death of an organism 47. There are, however, chemical modifications and fragmentation of the ancient DNA 48 that may occur and limit the application of the above techniques. Also, uncharacterized inhibitors of the PCR may be present in ancient DNA samples 49. Molecular tools were first applied to detect mycobacterial DNA in ancient human corpses that had macroscopic signs of either tuberculosis 50 or leprosy 51. Mummified tissues 51 and 52 or, more frequently, remnants of bones have been used to detect microorganisms that might have infected the person 48; 53 and 54. While mummified tissues are rare, bones can be found more readily but require time-consuming decalcification processing before DNA extraction can be carried out. During these procedures the samples may be contaminated with microorganisms present in the environment, a particular problem when trying to detect Y. pestis, which may be present in soil. Also, bone samples are best used to detect microorganisms that cause lesions within the bones themselves. For example in syphilis or tuberculosis, where the causative bacteria may multiply in the bones and therefore multiple DNA copies are available for amplification. Where septicemia is a feature of the disease, such as plague, bone may not be a good sample as there may only be very few bacteria present.

In humans with plague, death usually occurs during the septicemic phase of the disease and virtually all well-vascularized tissues are contaminated by Y. pestis, including the dental pulp. We therefore postulated that dental pulp, by virtue of its good vascularization, durability and natural sterility, would be a suitable sample on which to attempt the demonstration of Y. pestis by molecular biology techniques. We have tested our hypothesis on other organisms causing septicemia and have detected Coxiella burnetii DNA in dental pulp extracted from experimentally infected mice 55 and Rickettsia rickettsii DNA in the dental pulp of experimentally infected guinea-pigs (unpublished data). To determine if this was also possible for Y. pestis, we extracted DNA from the dental pulp of teeth extracted from skeletal remains of people suspected to have died of plague 39. Molecular targets that we attempted to amplify included the plasmid-encoded pla gene, encoding a virulence factor of Y. pestis, and the rpoB gene, encoding the beta-subunit of the bacterial RNA polymerase, which is a molecular target used for the identification of enteric bacteria 56. In our first set of experiments, dental pulp was extracted from the teeth of three skeletal remains of people suspected of having died of plague in Marseilles in 1722 and from two corpses buried in 1590 in Lambesc, a southern French village where there had also been a plague outbreak 39. As negative controls, dental pulp was extracted from skeletal remains in medieval tombs in Toulon, France where there had been no history of plague. The specific pla sequence was amplified from six of the twelve dental pulps collected from suspected plague victims, confirming the diagnosis in each individual, but from none of the seven dental pulps used as negative controls. The main problem with the PCR technique is the high risk of cross-contamination, especially when several experiments are carried out in the same laboratory using the same amplified sequence. As amplicons obtained from this first set of experiments could contaminate further specimens resulting in false positive results, we designed a new protocol which we named a `suicide PCR' and applied it to the detection of Y. pestis DNA in the skeletal remains of two adults and a child excavated from a 1348 plague grave in Montpellier, southern France 40. In this protocol, the primers are used only once in a laboratory and there are no positive controls, so that any amplicons obtained must result from the on-going experiment and cannot be due to contamination. We again demonstrated the presence of Y. pestis DNA in the three individuals, demonstrating for the first time that the Black Death was indeed synonymous with plague 40. We also found one mutation in the amplified pla gene fragment, the significance of which remains to be studied.

5. Conclusions

Combining the use of dental pulp as a source of DNA and our `suicide PCR' protocol may facilitate the detection of the DNA of ancient pathogens responsible for septicemic diseases in the past. Cremation, as practiced by Greeks during the Athens plague, prevents workers from obtaining genetic information on ancient microorganisms and there will, then, be long-term controversy over the etiology of this first documented human epidemic. The protocols we designed, however, do open up new opportunities for retrospective diagnoses to be made on ancient epidemics due to septicemia, including those due to plague, typhus or viral diseases such as smallpox. They could also be applied to the remains of rodents and ectoparasites to enable us to better understand their roles as reservoirs and vectors of Y. pestis during past plague epidemics. Finally, when the complete chromosome sequences of Y. pestis are announced this will enable comparisons of ancient Y. pestis sequences with those of the modern day organism to assess the evolution of the pathogen. This will also allow the hypothesis that each one of the three Y. pestis genotypes/biotypes was responsible for the three plague pandemics to be further evaluated.

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