Clinical Microbiology and Infection 22 (2016) 911e915

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Clinical Microbiology and Infection

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Review

Molecular history of plague

M. Drancourt, D. Raoult*
Aix Marseille Universit
e, INSERM, CNRS, IRD, URMITE, Marseille, France

a r t i c l e i n f o a b s t r a c t

Article history: Plague, a deadly zoonose caused by the bacterium Yersinia pestis, has been firmly documented in 39
Received 19 July 2016 historical burial sites in Eurasia that date from the Bronze Age to two historical pandemics spanning the
Received in revised form 6th to 18th centuries. Palaeomicrobiologic data, including gene and spacer sequences, whole genome
30 August 2016
sequences and protein data, confirmed that two historical pandemics swept over Europe from probable
Accepted 30 August 2016
Available online 8 September 2016
Asian sources and possible two-way-ticket journeys back from Europe to Asia. These investigations made
it possible to address questions regarding the potential sources and routes of transmission by completing
Editor: L. Leibovici the standard rodent and rodenteflea transmission scheme. This suggested that plague was transmissible
by human ectoparasites such as lice, and that Y. pestis was able to persist for months in the soil, which is a
Keywords: source of reinfection for burrowing mammals. The analyses of seven complete genome sequences from
History the Bronze Age indicated that Y. pestis was probably not an ectoparasite-borne pathogen in these pop-
Palaeomicrobiology ulations. Further analyses of 14 genomes indicated that the Justinian pandemic strains may have formed
Pandemia a clade distinct from the one responsible for the second pandemic, spanning in Y. pestis branch 1, which
Plague
also comprises the third pandemic strains. Further palaeomicrobiologic studies must tightly connect
Yersinia pestis
with historical and anthropologic studies to resolve questions regarding the actual sources of plague in
ancient populations, alternative routes of transmission and resistance traits. Answering these questions
will broaden our understanding of plague epidemiology so we may better face the actuality of this deadly
infection in countries where it remains epidemic. M. Drancourt, CMI 2016;22:911
© 2016 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious
Diseases.

Introduction: A Brief Plague History
Plague, a deadly zoonotic disease caused by the bacterium Yer-
sinia pestis [1], is an epidemic infectious disease. It is a disease well
described in medical historical texts and in the general literature
[2], as well as by pictorial and sculptural representations, and it a
disease for which the infectious aetiology has been indisputably
proven by numerous palaeomicrobiologic studies [1,3]. The study of
plague has been complicated by the fact that two kinds of teams
worked in this field using different approaches: specialists in
diagnosing infectious diseases, who have extensive experience of
PCR-based diagnosis, which is currently used worldwide to make
tens of millions of diagnoses annually; and scientists working on
ancient human DNA, who at the beginning of their studies noted
cases of contamination due to human DNA. This is understandable;
it is indeed much more likely that samples will be contaminated
, INSERM, CNRS, IRD,
* Corresponding author. D. Raoult, Aix Marseille Universite
URMITE, Marseille 13005, France.
E-mail address: didier.raoult@gmail.com (D. Raoult).
with human DNA than with plague bacteria. This story of plague
shows the profound differences of two scientific fields with little in
common. However, PCR is currently still the most modern tool of
diagnosis in infectious diseases.
Ancient plague epidemics greatly reduced the European popu-
lation. The mortality rate was estimated at 50% over 6 months in
urban communities such as Marseille [4]. However, the aetiology of
these outbreaks remained unknown until the end of the 19th
century, when Alexandre Yersin, a Franco-Swiss researcher from
the Institut Pasteur, discovered for the first time the bacterium by
investigating the plague epidemic in Hong Kong in 1894 [5].
Therefore, various assumptions regarding the aetiology of this
disease have been proposed [6]. Much documentary and pictorial
historic data available which essentially retrace the ancient epi-
demics in Europe, North Africa and the Near and Middle East, while
there is very little information in the international literature con-
cerning the rest of the African continent and Asia before the 19th
century. Plague entered the United States in the late 1890s with the
first cases in San Francisco, followed by a gradual spreading over a
part of the North American continent, where it is still present [7].

http://dx.doi.org/10.1016/j.cmi.2016.08.031
1198-743X/© 2016 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.
912 M. Drancourt, D. Raoult / Clinical Microbiology and Infection 22 (2016) 911e915
These historical data allowed us to recognize three plague pan-
demics. The first pandemic of the 6th to 8th centuries, which
affected mainly the periphery of the Mediterranean Sea, is called
the Justinian plague [8]. The second pandemic, which affected the
Mediterranean region and Continental Europe spreading as far as
Moscow and Scandinavia, is called the Black Death by Anglo-Saxon
writers. The entry of the second pandemic had been precisely dated
at 1347. Finally, the third pandemic broke out in Hong Kong in 1894
and spread over all the continents except the poles.
Long before the palaeomicrobiology data confirmed the aeti-
ology of these pandemics and epidemics, the European populations
distinguished the plague epidemics from other epidemic infectious
diseases present at that time. Indeed, the major bubonic form was
characterized by one or more superficial inflammatory and painful
lymph nodes, the epidemic nature of which was so novel that
people quickly named it the bubonic plague.
There is virtually no other epidemic infectious disease that
causes outbreaks of inflammatory lymphadenopathies of this na-
ture. Unsurprisingly, this disease was perfectly well recognized by
laypeople as well as members of the medical profession. Precise
descriptions were made at the beginning of the second pan-
demicdfor example, by Guy de Chauliac, the founder of the
Montpellier Faculty of Medicine in the 13th century and the per-
sonal doctor of the pope, who lived in Avignon at that time. Written
documents such as those of Guy de Chauliac help describe the
important features of ancient plague as well as its high mortality
rate, which exceeded 80%. Once it broke out, the plague spread at
an average speed of 3 to 5 km per day, which corresponds to the
average walking speed at the time.
The classic epidemiologic path for plague was established in the
work of the Indian Plague Commission. In the first half of the 20th
century, the commission imposed a dogma that the plague was a
zoonotic disease circulating in wild small mammals with a trans-
mission by ectoparasites, including fleas, followed by flea-borne
transmission to anthropophilic mammals and humans. Obviously
this standard pattern is not consistent with the historical data on
old plagues, and these inconsistencies have raised much contro-
versy about the aetiology of ancient epidemics until palae-
omicrobiologic studies confirmed that it was plague. Several
controversial issues regarding the aetiology of past epidemics have
therefore arisen in the literature that our major palae-
omicrobiologic studies [9,10], subsequently confirmed by others
[11e15], challenged. However, controversy raised by a single group
of researchers who claimed that the Black Death was probably not
caused by Y. pestis and that teeth were not suitable for DNA re-
covery for palaeomicrobiologic analysis delayed any general
agreement on this topic [16].
The Questions
Several questions were raised from historical data on large Eu-
ropean epidemics. The first issue was the question of whether it
was plague epidemics or other deadly epidemic infectious diseases.
The lack of microbiologic evidence has left room for a number of
various assumptions, including the hypothesis of epidemics of viral
haemorrhagic fever [17], a Spanish flu pandemic for Justinian pla-
gue [18] and even an unknown microorganism [19,20] (proposed
without any evidence). Also, the question was issued of whether
the strains of Y. pestis responsible for historical epidemics differed
from recent strains responsible for quite different epidemiology,
from sporadic cases to limited outbreaks. The hypothesis was put
forward that three biochemical variants, known as Antiqua, Medi-
evalis and Orientalis, have each been responsible for one of the
three main European pandemics [21]. The epidemiologic cycle
observed by the Indian Plague Commission, and in particular the
transmission of plague from anthropophilic mammals such as rats
via blocked fleas, which can first transmit Y. pestis approximately
30 days after a blood meal, has been established as a dogma starting
from the work of Simond et al. in the 1920s [22].
Palaeomicrobiology of Plague
We applied techniques commonly used for the routine diagnosis
of modern infections to ancient clinical specimens [9,10]. Plague is
not known to leave any skeletal lesions, so we used dental pulp as a
suitable clinical specimen on which to base a diagnosis of plague.
We understood that dental pulp, being a rich vascularized tissue,
may indeed harbor Y. pestis remnants in the case of septicaemia and
deadly plague. We devised a specific protocol to recover the pul-
verulent ancient dental pulp, and this choice of specimen is now
adopted in all the studies dealing with ancient plague, even if some
authors do use the entire tooth instead of specifically using the
dental pulp, as we still advocate in order to limit any risk of
contamination [11,15]. There is a higher amplifiable Y. pestis DNA
concentration in teeth (37%) than in bones (5.7%) [23], and bones
consistently failed to yield the diagnosis of plague based on DNA
even though the specific F1 antigen could be detected [24].
Intriguingly, the F1 antigen was more often detectable from bones
than from the dental pulp of plague victims, probably because later
specimens yielded more proteins [25]. These data validated our
postulate that dental pulp, not bones, should be used to detect DNA
of ancient blood-borne pathogens that lack specific bone involve-
ment [9,10]. Even though we have used PCR protocol for routine
diagnosis in the absence of any positive control, we were careful to
amplify at least two different genetic targets. Because the routine
pla gene is not specific for Y. pestis [26,27], and because we had
extensive experience with PCR-based diagnoses, our initial work
was challenged by English colleagues as resulting from mere lab-
oratory contamination [16,28]. This controversy, created by col-
leagues inexperienced in molecular diagnosis, threw the field into
confusion and contributed to the delay of further work. It is now
ended after the recovery of 11 complete Y. pestis genomes recovered
from Justinian [11] (two individuals) and Black Death burial sites in
Europe [12,13,15] (nine individuals) in addition to seven Y. pestis
genomes recovered from several burial sites in Eurasia dating back
to 3000 BC [14,29]. The laboratory techniques evolved from our
pioneer PCR sequencing works [9,10,24] to further detection of
specific DNA sequences by immuno-PCR [30], and now next-
generation sequencing after enrichment of the extracted DNA by
using a Y. pestis microarray [11] or a Yersinia pseudotuberculosis
microarray [15]. In parallel, pioneering detection of the Y. pestis
antigenic F1 protein [25,31e33] is now surpassed by the palae-
ometaproteomic approach that we recently used for the neutral
detection of Y. pestisespecific proteins in ancient dental pulp (M.
Drancourt, unpublished data). On the basis of genomic data, the
genotyping of ancient Y. pestis strains evolved from our pioneering
multispacer sequence typing (MST) [34] to the current analysis of
selected single nucleotide polymorphisms located in either coding
or spacer regions [11e13,15,35,36].
Palaeomicrobiology Answers Questions
The first contribution of palaeomicrobiology to the study of
ancient plague is the confirmation of Y. pestis as the cause of death
in plague burial sites. After our first two publications, numerous
research teams provided undisputed pieces of evidence proving
that plague is indeed plague, and that most of the massive burial
sites in Europe are plague burial sites. Altogether, 39 sites in Eurasia
have now been confirmed by the detection of specific DNA se-
quences and the immunochromatographic detection of the Y. pestis
 M. Drancourt, D. Raoult / Clinical Microbiology and Infection 22 (2016) 911e915 913

F1 antigen (Table 1). Such confirmation culminated with the re-
covery of 18 complete Y. pestis genomes from six burial sites scat-
tered in France, Spain, England, Germany and Russia for the two last
pandemics [11e13,15] and in Eurasia for human strains from the
Bronze Age [14] (Fig. 1).
A second contribution of plague palaeomicrobiology is to pro-
vide insights into Y. pestis variants that were potentially responsible
for ancient plague epidemics. We first aimed to answer the ques-
tion by developing MST by sequencing intergenic spacers [34]. MST
analysis yielded interpretable sequences in three individuals of the
Justinian site of Sens and in five individuals from the early second
pandemic sites of Dreux. Montpellier and BLAST (Basic Local
Alignment Search Tool) analyses consistently yielded better scores
for the Orientalis-type sequences [34]. Further, the Medievalis
biovar is characterized by a stop codon in the napA gene (G to T
mutation) [47] and the Orientalis biovar by a 93 bp deletion in the
glpD gene [47e49]. Both genetic events were absent in the Antiqua
biovar [21]. We detected a 93 bp deletion in the glpD gene in two
individuals in the Justinian site of Vienne and three individuals in
the second pandemic sites of Martigues and Marseille [38]; in one
plague individual in Venice, 14th to 16th centuries [50], and one
further individual in the second pandemia site of Bondy [43]. These
data suggested the existence of Orientalis biotype strains recovered
from the Justinian and medieval pandemics.
These conclusions have been further challenged by the observa-
tion of a 3-T homopolymeric tract which differed from the 5-T ho-
mopolymeric tract of the Orientalis Y. pestis CO92 type strain in four
individuals in the second pandemia site of Manching-Pichl [44]. This

Table 1
Review of ancient plague studies

Site, country Datation Technique Method Data Study

Marseille; Lambesc, France 1722; 1590 PCR sequencing First detection of ancient plague Drancourt, 1998 [9]
Montpellier, France 14th PCR sequencing Suicide PCR Raoult, 2000 [10]
Sens; Dreux; Montpellier, France 5the6th; 12the14th; MST Drancourt, 2004 [34]
13the14th
Stuttgart, Germany 17th PCR Southern blot F1 First immunologic detection Pusch, 2004 [31]
antigen detection of ancient plague
Aschheim, Germany 6th PCR sequencing First detection of Justinian plague Wiechmann, 2005 [37]
Vienna, Austria; Martigues, 7the9th; 1720e1721; Suicide nested PCR Orientalis-like strain Drancourt, 2007 [38]
France; Marseille, France 1722
Biançon, France 17th F1 antigen detection Cerutti, 2007 [39]
Venice, Italy 14e16th
Genoa, Italy 14th
Marseille; Martigues; 1720e1722; 1720; F1 antigen detection Bianucci, 2007 [25]
Draguignan; Lambesc, France 17th; 1590
Lambesc, France 1590 F1 antigen detection Bianucci, 2008 [32]
Saint-Maurice, France 1636 PCR sequencing First identified plague Hadjouis, 2008 [40]
victim, Sir T. Craven
Puy Saint-Pierre, France 1628e1632 F1 antigen detection Bianucci, 2009 [33]
Draguignan, France 1649e1650
Martigues, France 1720e1721

Berre l’Etang, France 1720e1721
Marseille, France 1722
La Chaize-le-Vicomte, France 17the18th F1 antigen detection Bianucci, 2009 [33]
Poitier, France 17th F1 antigen detection
Parma, Italy 1629e1630? PCR sequencing undeleted glpD Prebranch 1 and 2 variants Haensch, 2010 [24]
St-Laurent de la Cabrerisse, France 1348 or 1374? F1 antigen detection coding napA
Augsburg, Germany 1500e1699
Hereford, England 1355 ± 54
Bergen op Zoom, Netherlands 1349e1350?
Manching-Pichl, Germany 14th Suicide PCR Non-Orientalis-like strain? Wiechmann, 2010 [41]
Bondy, France 11the15th PCR sequencing deleted glpD Bartonella quitana coinfection Tran, 2011 [42]
Venice, Italy 14the 16th High-throughput glpD-deleted B. quintana coinfection Tran, 2011 [42]
multiplex PCR
Sens, France 5the6th ELISA; Immuno-PCR Malou, 2012 [30]
Venice, Italy 14the16th
Puy-Saint-Vincent, France 1628e1632
Bourges, France
Barletta, Italy 1656e1658 RT-PCR Scasciamacchia, 2012 [44]
Aschheim, Germany 431e631 q PCR SNP analysis Harbeck, 2013 [8]
Manching-Pichl, Germany 1300e1399
Brandenburg, Germany 1640 PCR sequencing Seifert, 2013 [45]
Basel, Switzerland 1300e1490
Aschheim, Germany 541e543 NGS 2 genomes Justinian plague branch Wagner, 2014 [11]
Bateni, Russia 2909e2677 BC NGS 2 genomes Rasmussen, 2015 [14]
Sope, Estonia 2575e2349 BC NGS 1 genome Rasmussen, 2015 [14]
Bulavovo, Russia 2280e2047 BC NGS 1 genome Rasmussen, 2015 [14]
Chociwel, Poland 2135e1923 BC NGS 1 genome Rasmussen, 2015 [14]
Kytmanovo, Russia 1746e1626 BC NGS 1 genome Rasmussen, 2015 [14]
Kapan, Turkey 1048e855 BC NGS 1 genome Rasmussen, 2015 [14]
Barcelona, Spain 1300e1420 NGS 1 genome Spyrou, 2016 [15]
Bolgar City, Russia 1362e1400 NGS 1 genome Spyrou, 2016 [15]
Ellwangen, Germany 1485e1627 NGS 1 genome Spyrou, 2016 [15]
Brandenburg, Germany 1618e1648 PCR sequencing SNP analysis Seifert, 2016 [46]

NGS, next-generation sequencing; qPCR, quantitative PCR; SNP, single nucleotide polymorphism.
914 M. Drancourt, D. Raoult / Clinical Microbiology and Infection 22 (2016) 911e915
Fig. 1. Locations of plague burial sites which yielded 18 ancient complete Yersinia pestis genome sequences from the second pandemics (as of June 2016). Compiled from:
[11,12,14,15].
was later shown to be CO92 strain specific and not Orientalis biotype
specific [42]. More contradictory was the observation of an undeleted
glpD gene and a coding napA gene in individuals from 14th-century
Netherlands, France and England [24]. At that point, these data sug-
gested that at least two different strains had been responsible for the
second pandemic. Further analyses were all based on single nucleo-
tide polymorphism (SNP) determination derived from a phylogenetic
tree distinguishing five branches: 0 (containing modern representa-
tives of pestoids and Antiqua), 1 (containing Antiqua and Orientalis
modern representatives), 2 (containing Medievalis and Antiqua
modern representatives), 3 and 4 (containing modern representa-
tives of Antiqua) [36]. SNPs derived from PCR sequencing analyses
found the Justinian plague strains to be intermediate between branch
0 (pestoids) and the four other branches [8], whereas the most recent
analysis of the second pandemic strains indicated that they belong to
branch 1, which also comprises modern representatives of the third
pandemic [15]. Altogether, these data indicate one Y. pestis clade for
the first pandemic and another Y. pestis clade for the second and third
pandemics. The analysis of the oldest Bronze Age genomes further
indicated that at the time, plague was not an ectoparasite-borne
disease, as Y. pestis then lacked the gene considered critical for
arthropod transmission [14].
However, the analysis of the ancient Y. pestis genomes did not
reveal any potential specific “virulence” trait [15]. Genome
sequenceebased typing is now fueling contradictory hypotheses
regarding the existence of stable plague foci in medieval Europe
and routes of plague in medieval Europe. The prevalent “one-way
ticket” hypothesis is that Asian or Eurasian plague foci were the
sources for westward plague waves possibly driven by climatic
factors [51]. More recently, an investigation of three skeletons in
18th-century Marseille [12] and a single skeleton in Germany (and
finally a unique SNP) resulted in evidence interpreted as indicating
an eastward route of medieval plague back to its sources in Russia
and China and as evidence for elusive plague foci in medieval
Europe [15]. Whether this is an overinterpretation of data or a
verified alternative will depend on additional data from further
plague sites. In any case, the precise nature of the ancient plague
sources remains to be determined.
Also, the nature of the transmission of Y. pestis from sources to
populations remains unproven. It is admitted that the classical
scheme of rats and their fleas does not explain the situation in the two
historic pandemics [22]. Indeed, this scheme does not fit with the
large continental extension of medieval plague or with the season-
ality of medieval plague and its high attack rate. We alternatively
proposed that ancient plague epidemics were vectorized from one
patient to another by human ectoparasites, including human lice [52].
This hypothesis was supported by the demonstration of coinfection
with Y. pestis and the acknowledged human liceevectorized Barto-
nella quintana in one 11th- to 15th-century burial site in France [42],
by the recent observation of Y. pestis in human lice collected in
modern Congo [53] and by the experimental demonstration that
human lice are competent to vectorize Y. pestis [54]. Detection in
ancient lice would definitely prove that this human-to-human
transmission played a role in ancient bubonic plague epidemics [52].
Conclusions
Within 20 years, starting with our seminal observations [9,10],
the palaeomicrobiology of plague has made great achievements in
the detection and genome-based characterization of ancient
Y. pestis strains infecting past populations from the Bronze Age [14]
to the late 18th century [12]. These data left partially resolved
questions regarding the exact sources of contamination, including
the hypothesis of vanished plague foci in Europe and the modes of
transmission besides mammal and human ectoparasite-borne,
majority bubonic cases. Also, the potential role of plague-
protection factors in ancient populations, such as the CCR5 D 32
deletion [55], as well as the converse role of plague in selecting
some crucial immunologic traits, such as the Toll-like receptors
[56], remains in its infancy. The areas of uncertainty have to be
filled by further research. We suggest combining new laboratory
approaches with interdisciplinary collaborations among re-
searchers in the fields of microbiology, anthropology and history to
more quickly resolve these unanswered questions A lack of
communication between microbiologists and specialists in human
ancient DNA has “plagued” the field in the past. It matters to
 M. Drancourt, D. Raoult / Clinical Microbiology and Infection 22 (2016) 911e915
modern-day plague, still affecting populations in Africa and Asia,
because a thorough understanding of plague transmission,
including the role of lice, may help limit the number of cases by
innovations in plague foci surveillance and plague transmission.
Transparency Declaration
Financial support was received from URMITE, Marseille, France.
Both authors report no conflicts of interest relevant to this article.
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