Brief communication Co-detection of Bartonella quintana and Yersinia pestis in an 11thЦ15th burial site in Bondy France.код для вставкиСкачать
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 145:489–494 (2011) Brief Communication: Co-Detection of Bartonella quintana and Yersinia pestis in an 11th–15th Burial Site in Bondy, France Thi-Nguyen-Ny Tran,1 Cyrille Le Forestier,2 Michel Drancourt,1* Didier Raoult,1 and Gérard Aboudharam1 1 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR CNRS 6236, IRD198, IFR48, Faculté de médecine, Université de la Méditerranée, 13005 Marseille, France 2 Institut National de Recherches Archéologiques Préventives UMR 6130, Centre d’Etudes Préhistoire, Antiquité, Moyen Âge, Direction interrégionale Centre, Ile de France, France KEY WORDS dental pulp; plague; Bondy; ‘‘suicide PCR’’ ABSTRACT Historical and anthropological data suggest that skeletons excavated from an 11th to 15th century mass grave in Bondy, France, may be those of victims of the Great Plague. Using high-throughput realtime PCR investigation of the dental pulp collected from 14 teeth from ﬁve such skeletons, we detected Bartonella quintana DNA in three individuals and Yersinia pestis DNA in two individuals. DNA from ﬁve other deadly pathogens was not found. Suicide PCR genotyping conﬁrmed Y. pestis DNA belonging to the Orientalis biotype. One individual had co-infection. These data suggest a plague epidemic in a population already infected by the body louse-transmitted B. quintana or a body lousedriven transmission of the plague that drove a medieval epidemic in inland Europe. Am J Phys Anthropol 145:489–494, 2011. V 2011 Wiley-Liss, Inc. Historical texts and works of art have related two massive, deadly epidemics that erupted in medieval Mediterranean countries and swept over inland European countries during the 6th–8th centuries and the middle of the 14th century (Perry and Fetherston, 1997; Gage and Kosoy, 2005). Later epidemics, known collectively as the Black Death, entered Mediterranean ports in 1347 and reached as far as Scandinavia and Russia (Perry and Fetherston, 1997; Byrne, 2004). The Black Death was estimated to have killed up to one-third of the European population at that time and to have profoundly inﬂuenced the history of major medieval states (Biraben, 1975; Cantor, 2002; Byrne, 2004). The Black Death presented as a deadly epidemic in which victims were afﬂicted with painful, inﬂamed lymph nodes. These inﬂamed lymph nodes are highly indicative of the plague bubo, which is caused by the bacterium Yersinia pestis (Yersin, 1894). The large number of people affected within short periods of time, the length of the epidemics, the clustering of cases (e.g., household cases), and the patterns of spatial and temporal spread, however, have raised controversies regarding the etiology of the Black Death. Indeed, these particular epidemiological features of the Black Death were clearly incompatible with the rat-ﬂea transmission model commonly suggested for the plague (Wren, 2003). Because of these inconsistencies, some scholars have denied the potential role of Y. pestis as the causative agent of the Black Death, and several alternative hypotheses have been proposed, including anthrax (Twigg, 1985; Cantor, 2002), inﬂuenza (Teh et al., 1923), and hemorrhagic fever (Duncan and Scott, 2005). None of these hypotheses, however, has received additional support from any experimental data. One paleomicrobiological study failed to detect Y. pestis DNA in human remains collected from ﬁve different burial sites dated from the 13th to 17th centuries in Northern Europe (Gilbert et al., 2004). Further studies performed by several research teams, however, have detected the presence of Y. pestis-speciﬁc DNA sequences and the pathogen’s F1 antigen at other burial sites in France, Italy, Germany, the Netherlands, and England (Drancourt et al., 1998, 2004; Raoult et al., 2000; Pusch et al., 2004; Wiechmann and Grupe, 2005; Bianucci et al., 2007, 2008, 2009; Cerutti et al., 2007; Drancourt et al., 2007; Donat et al., 2008; Hadjouis et al., 2008; Haensch et al., 2010; Wieschmann et al., 2010). These paleomicrobiological data, while conﬁrming that some burial sites in medieval Europe were plague burial sites, do not help resolve the lingering questions concerning the epidemiology of the Black Death. Using multiple molecular approaches, recent investigations of human remains dated from the 11th to 15th centuries in Bondy, France, have revealed co-infection of victims by Y. pestis and Bartonella quintana, a human liceborne organism (Raoult and Roux, 1999). Together with other recently gathered experimental data, our observations pave the way toward a renewed understanding of the transmission scenarios for the Black Death, including lice-borne and human-to-human transmission. These ﬁndings will help resolve previous epidemiological inconsistencies. C 2011 V WILEY-LISS, INC. C *Correspondence to: Michel Drancourt, Unité des Rickettsies, Faculté de Médecine, 27, Boulevard Jean Moulin-cedex 5, France. E-mail: email@example.com Received 22 November 2010; accepted 17 January 2011 DOI 10.1002/ajpa.21510 Published online 3 May 2011 in Wiley Online Library (wileyonlinelibrary.com). 490 T.-N.-N. TRAN ET AL. Fig. 1. Skeletons from the 11th to 15th centuries in Bondy, France yielded evidence for Yersinia pestis/Bartonella quintana coinfection. Copyright: Nicolas Latsanopoulos (Bureau de l’Archéologie, Service du patrimoine du Conseil général de Seine-SaintDenis, France). [Color ﬁgure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] MATERIALS AND METHODS Archaeological samples In 2007, during piping work in Bondy, a small city located 13 km east from Paris, France, three multiple burial sites dated from the 11th to the 15th century were discovered. The burial sites contained a total of 11 individuals of both genders and various ages (see Fig. 1). Initially, stratigraphy and the analysis of the burial remains were used to date the sites. C14-based datation further yielded 1297–1373 with 70% probability and 1377–1414 with 30% probability. Individuals at the site were covered with earth and not buried in cofﬁns. Simultaneous deposition of the bodies was evidenced by the direct contact between bones, the mechanical stress between individuals, and the migration of some bones to the contacts of the underlying skeletons (Duday, 2008). All the buried corpses were laid on their backs in the same east–west orientation with their heads placed at the western position. In four individuals, the forearms were bent 908 at the humeral axis (see Fig. 2). The upper limbs of individuals 072 and 075 were not bent and were found to be positioned alongside the body. The bodies did not appear to have been thrown into the pit but appeared to have been successively deposited next to each other, except for subjects 071 and 073, which were deposited on top of other bodies. Individuals 069, 072, and 073 were placed ﬁrst, followed by individuals 070 and 075, and ﬁnally individual 071. The multiple depositions involved individuals of both genders and various ages. The skeletons were handled according to the principles of anthropological ﬁeldwork, and the teeth were placed into plastic bags. In the laboratory, the teeth were washed with water and dried gradually. Teeth American Journal of Physical Anthropology belonging to different individuals were never in physical contact from the time of their excavation to the time of molecular biological analysis. Molecular detection of multiple pathogens Fourteen teeth were collected from ﬁve skeletons excavated from two Bondy burial sites. These skeletons did not show any macroscopic signs of infectious disease. Total DNA was extracted from the dental pulp collected from teeth as previously described (Drancourt et al., 1998). Using total DNA as a template, molecular detection of seven pathogens, including Bacillus anthracis, Borrelia recurrentis, Bartonella quintana, Rickettsia prowazekii, Salmonella enterica Typhi, Poxvirus, and Y. pestis, was performed using multiplex high-throughput PCR and the 7900 HT Fast Real-Time PCR System (Applied Biosystem, Courtaboeuf, France) as previously described (Nguyen-Hieu et al., 2010; Table 1). Each plate included two wells containing sterile water as negative controls and two wells containing DNA extracted from dental pulp collected from the skeletons of individuals lacking macroscopic and anthropological evidence of infection. Y. pestis genotyping Y. pestis genotyping was performed according to the principles of ‘‘suicide PCR’’ as follows: (1) no positive control was used at any step of the processing of ancient materials; (2) ampliﬁcation was performed using a previously used glpD-PCR system (Drancourt et al., 2007) in a building and in a laboratory where Y. pestis and its DNA have never been previously worked on using PLAGUE IN MEDIEVAL FRANCE 491 Fig. 2. Site plan featuring the positions of the studied individuals. Copyright: Nicolas Latsanopoulos (Bureau de l’Archéologie, Service du patrimoine du Conseil général de Seine-Saint-Denis, France). disposable instruments (Raoult et al., 2000); (3) each step was conducted in a separate room and in a negative air pressure hood; (4) three negative controls of DNA dental pulp were placed between samples, and negative control teeth (collected from skeletons of individuals lacking anthropologic and macroscopic evidence of infection) were from a mass grave dating from the 5th to 7th centuries. A total of 4 ll (1 ll for nested PCR) of DNA were ampliﬁed in a 25-ll mixture containing 10 pmol of each primer, 200 lmol/l each deoxyribonucleotide triphosphate (Invitrogen, Cergy-Pontoise, France), 1.5 U HotStarTaq DNA Polymerase (Qiagen, Courtaboeuf, France), and 2.5 lL MgCl2 (50-mmol/l) in 13 Taq buffer. Suicide-nested PCR targeting the glpD gene was performed as previously described (Drancourt et al., 2007) using a T3 thermocycler (Biometra, Archamps, France) under the following conditions: an initial 2 min of denaturation at 958C was followed by 43 cycles (38 cycles for nested PCR) of denaturation for 30 s at 948C, annealing for 30 s at 588C, and extension for 90 s at 688C. The ampliﬁcation was completed by holding the reaction mixture for 7 min at 688C. PCR products were puriﬁed using a QIAquick PCR puriﬁcation kit (Qiagen, Courtaboeuf, France). PCR products were separated by electrophoresis for 25 min at 135 V in a 2% agarose gel in 0.53 TBE. Marker VI was used as a DNA ladder when determining the size of the ampliﬁed fragments. Puriﬁed positive products were sequenced using ABI Prism1 Big Dye1 Terminator V1 and a 1 Cycle sequencing Kit (Applied Biosystem, Courtaboeuf, France) in a 3130xl Genetic Analyzer (Applied Biosystem). The sequences were compared to the sequences deposited in the GenBank database (www.ncbi.nlm.nih.gov/GenBank). RESULTS Molecular detection of multiple pathogens A total of 14 dental pulp specimens were analyzed by multiple molecular detection methods. All the negative controls had negative results, and real-time PCR detected B. quintana DNA in 3 of 14 dental pulp specimens (identiﬁed tooth C33.JUG104.3 of individual 041, tooth C33.JUG180.1 of individual 070 and tooth C33.JUG183.2 of individual 073) and Y. pestis DNA in 4 of 14 dental pulp specimens (identiﬁed tooth American Journal of Physical Anthropology 492 T.-N.-N. TRAN ET AL. TABLE 1. Sequences of primers and probes used for the molecular detection of seven pathogens Target gene Speciﬁc pathogen Bacillus anthracis (anthrax) pag Borrelia recurrentis (louseborne relapsing fever) Bartonella quintana (Trench fever) ITS Rickettsia prowazekii (typhus) ompB Salmonella Typhi (typhoid fever) Poxvirus (smallpox) HA Yersinia pestis (plague) pla Primer Sequence Lengh (bp) Bant_pag_P Bant_pag_F Bant_pag_R Brec_P Brec_F Brec_R Barto ITS_P Barto ITS_F Barto ITS_R Rpr_ompB_P Rpr_ompB_F Rpr_ompB_R Styp_put_P Styp_put_F Styp_put_R Var_HA_P Var_HA_F Var_HA_R Yper_PLA_P Yper_PLA_F Yper_PLA_R 6 FAM-TAC CGC AAA TTC AAG AAA CAA CTG 50 -AGG CTC GAA CTG GAG TGA A-30 50 -CCG CCT TTC TAC CAG ATT T-30 6 FAM-CTG CTG CTC CTT TAA CCA CAG GAG 50 -TCA ACT GTT TTT CTT ATT GCC ACA-30 50 -TCC TTA TGT TGG TTA TGG GAT TGA-30 6 FAM-GCG CGC GCT TGA TAA GCG TG 50 -GAT GCC GGG GAA GGT TTT C-30 50 -GCC TGG GAG GAC TTG AAC CT-30 6 FAM-CGG TGG TGT TAA TGC TGC GTT ACA 50 -AAT GCT CTT GCA GCT GGT TCT-30 50 -TCG AGT GCT AAT ATT TTT GAA GCA-30 6 FAM-GCT TTT TGT GAA GCA ACG CTG GCA 50 -CTC CAT GCT GCG ACC TCA AA-30 50 -TTC ATC CTG GTC CGG TGT CT-30 6 FAM-AAG ATC ATA CAG TCA CAG ACA CTG 50 -GAC KTC SGG ACC AAT TAC TA-30 50 -TTG ATT TAG TAG TGA CAA TTT CA-30 6 FAM-TCC CGA AAG GAG TGC GGG TAA 50 -ATG GAG CTT ATA CCG GAA AC-30 50 -GCG ATA CTG GCC TGC AAG-30 24 19 19 24 24 24 20 19 20 24 21 24 24 20 20 24 20 23 21 20 18 C33.JUG181.1 and tooth C33.JUG181.2 of individual 072 and tooth C33.JUG183.1 and tooth C33.JUG183.2 of individual 073). Tooth C33.JUG83.2 of individual 073 yielded DNA from both pathogens. No pathogens other than B. quintana and Y. pestis were detected in these specimens. Suicide-nested PCR detection and genotyping of Y. pestis In the presence of negative controls, suicide-nested PCR conﬁrmed the presence of Y. pestis in 2 of 14 dental pulp specimens (tooth C33.JUG181.2 of individual 072 and tooth C33.JUG183.1 of individual 073). Sequencing of the pathogen DNA from tooth C33.JUG181.2 of individual 072 yielded 100% sequence similarity with the reference sequence of Y. pestis CO92 (GenBank accession number AL590842). The partial glpD sequence of the pathogen DNA extracted from tooth C33.JUG181.2 exhibited a 96-bp pair deletion indicative of the Orientalis biotype (Motin et al., 2002) when compared with homologous Y. pestis reference sequences in GenBank. Combining the results of the molecular detection methods and suicide PCR, Y. pestis DNA was detected in four teeth collected from two different individuals buried in Bondy. Y. pestis was not detected in any of the three negative controls. DISCUSSION In this study, all ancient specimens were manipulated according to the agreed upon paleomicrobiological protocols to ensure the authenticity of the data (Drancourt and Raoult, 2005). In particular, direct contact between individual remains was avoided at all times, both in the ﬁeld and in the laboratory. In addition, the teeth used for examination were selected on the basis of a closed apex, the absence of dental caries, and a lack of traumatic lesions to minimize any risk of external contamination of the dental pulp. Genotyping experiments were performed in a laboratory where the targeted pathogens had not been previously worked on, using the principles American Journal of Physical Anthropology Amplicon Tm 94 bp 608C 111 bp 608C 102 bp 608C 134 bp 608C 138 bp 608C 100 bp 608C 98 bp 608C of the suicide PCR protocol (Raoult et al., 2000). In all experiments, negative controls were composed of dental pulp collected from individuals without historical or anthropological evidence of epidemic disease, and PCR mixes without DNA were used. In addition, the detection of Y. pestis DNA was performed in two independent experiments by different operators and conﬁrmed by sequencing. The detection of Y. pestis conﬁrms the presence of plague in the 11th–15th centuries in Northern France. A previous study of bones and teeth collected from ﬁve sites in Northern Europe failed to detect Y. pestis DNA. The study, however, yielded 16S rDNA sequences indicative of the genus Yersinia, including 16S rDNA sequences closely related to Y. pestis (Gilbert et al., 2004). Recovery of Y. pestis DNA in Bondy, currently located in the suburbs of Paris at a latitude of 4889 north, along with a previous report of Y. pestis DNA in skeletons exhumed in Dreux, another French city located at 4884 north (Drancourt et al., 2004) and the recent detection of Y. pestis DNA in Black Death skeletons in England and in the Netherlands (Haensch et al., 2010), conﬁrmed Black Death in inland medieval Europe. These locations depict the geographical extension of the Black Death, starting from the Mediterranean sea ports where DNA sequencing studies and immunological studies have previously demonstrated its presence (Drancourt et al., 1998; Raoult et al., 2000; Cerutti et al., 2007) toward Northern and Western inland France, where its presence has been conﬁrmed by DNA sequencing studies (Drancourt et al., 2004, 2007) and the detection of the F1 antigen known to be indicative of Y. pestis (Pusch et al., 2004; Bianucci et al., 2008, 2009). In Bondy, we conﬁrmed an Orientalis biotype of Y. pestis, the same biotype that has been found to date in the majority of historical European plague victims (Drancourt et al., 2004, 2007). A recent study based on the analysis of single-nucleotide polymorphisms found two non-Orientalis genotypes of Y. pestis in 10 individuals in three archeological sites: Bergen op Zoom, the Netherlands (middle 14th century); Hereford, England (1335), and Saint-Laurent-de-Cabrerisse, France (1348–1374) (Haensch et al., 493 PLAGUE IN MEDIEVAL FRANCE 2010). Altogether, these data indicate that several Y. pestis genotypes circulated in medieval Europe. In this study, B. quintana DNA was found to have a prevalence of 21%, which is consistent with previous observations (Raoult and Roux, 1999). Several mass graves have yielded evidence for the B. quintana infection, suggesting that this pathogen was widely distributed with a high prevalence in historic populations in Europe. Such evidence is consistent with the estimated high prevalence of the body louse vector of B. quintana and with the pathogen’s relatively low mortality rate (Brouqui et al., 1999). B. quintana has even been detected in 4,000-year-old remains discovered in France (Drancourt et al., 2005). Paleomicrobiology techniques have previously detected B. quintana associated with the epidemic typhus pathogen Rickettsia prowazekii found in the remains of soldiers from Napoleon’s 1812 Great Army buried in Vilnius (Raoult et al., 2006) and in dental pulp specimens dated to 1710–1712 in Douai, France (Nguyen-Hieu et al., 2010). The association was interpreted as being indicative of body louse infestation in the dead individuals, because both pathogens are known to be transmitted by this human ectoparasite (Raoult and Roux, 1999). To our knowledge, this is the ﬁrst report of a historical co-infection of B. quintana and Y. pestis, and we are not aware of any such association in modern times. In Bondy, the fact that B. quintana and Y. pestis were detected in the same mass grave and even in the same individuals may be interpreted as coincidental plague in B. quintana-infected individuals. Alternatively, other observations raise questions about the mechanism of transmission for the Black Death in Bondy. Plague can be transmitted by rodent ectoparasites and is primarily transmitted by rat ﬂeas (Perry and Fetherston, 1997; Wren, 2003; Gage and Kosoy, 2005); however, several inconsistencies argue against the role of rat ﬂeas in the transmission of the Black Death. In medieval Europe, plague-carrying rats were present in port areas and not in inland areas; the plague epidemics were not associated with the expected rodent die-offs; and plague epidemics were active in the winter, which is a season when rodent ﬂeas would not have been active (McLean and Fall, 2010). In Morocco, in 1941, the body louse, which transmits B. quintana (Raoult and Roux, 1999), was also found to be a plague vector in familial cases (Blanc and Balthazard, 1941). In this situation, familial cases of bubonic plague occurred in the absence of dead or alive rats. Body lice were observed, but no ﬂeas in the vicinity of the infected cases were noted (Blanc and Baltazard, 1941, 1942, 1945). Blanc and Baltazard demonstrated the transmission of Y. pestis in mammals and rats by inoculating them subcutaneously with infected human body lice or their feces (Blanc and Baltazard, 1941; Blanc and Baltazard, 1942; Blanc and Balthazard, 1945). The potential role of lice in the transmission of plague and the possibility that they may constitute a vector for the plague has been further demonstrated experimentally in rabbits (Houhamdi et al., 2006), and it was recently shown that such body louse transmission is restricted to the Orientalis biotype of Y. pestis (Ayyadurai et al., 2010). This Orientalis biotype was found in Bondy and also in several other ancient plague burials. These observations suggest that body lice was the vector for interhuman transmission of plague in Bondy and other inland medieval plague epidemics after the plague had been introduced in Europe by infected rats and their ﬂeas through Mediterranean ports (Drancourt and Raoult, in press). CONCLUSIONS Plague should be added to the list of deadly pathogens that can be transmitted to humans by human body lice. The body louse remains prevalent in some developing countries where plague may persist and should be examined as a potential vector of plague in these populations. Additional studies may help to elucidate the role of body lice in plague transmission and aid in efforts to prevent of future plague epidemics. ACKNOWLEDGMENTS The authors acknowledge Annick Bernard (URMITE, Marseille, France) for her assistance with multiplex high-throughput PCR. 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