Genotyping, Orientalis-like Yersinia pestis, and
Plague Pandemics
M. Drancourt, et al. 2004. Emerging Infectious Diseases. 10(9):1585-1592
“Plague Village”
Eyam, England 1665-1666
Boundary stone outside Eyam
Site of George Viccars’
Church Street cottage
Eyam
St. Stephens Church services moved to
outdoor locations
“The Great Pestilance”
Xenopsylla cheopis
Pneumonic
Septicemic
Bubonic - refers to swollen lymph nodes
Plague pandemics
Justinian plague (541-750 A.D.) – Africa, Middle East
Black Death (8th – 14th centuries) – Asia, followed trade routes to Europe
Modern Plague (1800 onward) – China, followed shipping routes to Africa,
North & South Americas, Australia
Plague ……not just a medieval scourge
• • • Modern outbreaks:
• 1924-25; Los Angeles; currently 10-15
cases reported annually
• 1994; western India; 6,300 suspected
cases and 54 deaths
• Ongoing: 1000 – 2000 cases/year
reported wordwide
Examples of virulence factors:
• pla – plasminogen activator : activates
mammalian plasma proenzyme into
plasmin (which degrades fibrin),
cleaves complement component C3,
mediates adhesion to eukaryotic cells
• lcr - low calcium response
• F-1 antigen – anti-phagocytic
envelope protein expressed at
37ºC
Fatality and Treatment:
• UT bubonic 40% -60%; UT septicemic
& pneumonic 100%
• Streptomycin and tetracycline
“Even if all suspected cases were
confirmed and resulted in death, these
numbers would be dwarfed by those
due to other infectious diseases
occurring yearly in India” Perry &
Fetherston, 1997. Clinical Microbiology Reviews
(10)1:35-66.
Detection of 400-year-old Yersinia pestis DNA in human dental pulp: An
approach to the diagnosis of ancient septicemia
by Michel Drancourt et al. 1998. PNAS (95):12637-12640
 Excavated mass graves containing
victims from plague quarantine hospitals
in France:
 Lambesc (1590) – 4 teeth
 Marseille (1722) – 8 teeth
 Controls:
 Toulon (medieval)- 7 teeth
Unerupted
teeth
 Extracted DNA from dental pulp
 PCR targeted β-globulin, and Y. pestis
specific genes rpoB & pla
 rpoB = RNA polymerase β subunit
 pla = plasminogen activator, associated
with virulence
http://www.sfendo.net/
endo1.htm
Detection of 400-year-old Yersinia pestis DNA in human dental
pulp: An approach to the diagnosis of ancient septicemia
by Michel Drancourt et al. 1998. PNAS (95):12637-12640
pla:
(+)
(-)
S61 S61 S122 S122
(1 PCR)
rpoB:
(2 PCRs)
(-) (+) (+)
(-) S19 S19 S87 S87
“Molecular identification by “suicide PCR” of Yersinia pestis as
the agent of medieval black death” by Raoult et al. 2000 PNAS
97:12800
Others could not repeat results & were not convinced…..
“Absence of Yersinia pestis-specific DNA in human teeth from
five European excavations of putative plague victims” by
Gilbert et al. 2004. Microbiology. 150:341.
Drancourt and Raoult persist…..
“Molecular detection of Yersinia
pestis in dental pulp” by
Drancourt & Raoult. 2004.
Microbiology. 150:263.
Who’s right???
Y. pestis biovars and their relationships
to historic plague pandemics
Antiqua
Medievalis
Orientalis
Glycerol fermentation
+
+
–
Reduces NO3¯ to NO2¯
+
–
+
First pandemic
Second pandemic
(Justinian plague)
(Black Death)
Third pandemic
(Modern plague)
Hypothesis:
The 3 plague pandemics were caused by Y. pestis serovars Antiqua,
Medievalis, and Orientalis respectively.
Yersinia pestis genomes: CO92 and
KIM
CO92 (Orientalis)
KIM (Medievalis)
1. Identify intergenic spacer regions in CO92
2. Use flanking gene sequences to find (via
BLASTN) homologous spacers in KIM
3. Align these sequences and use for primer
design (Table 2)
4. Test 35 isolates to see if spacer sequences can
be used to distinguish biovars (Multiple Spacer
Typing –MST method)
5. Test ancient DNA
Table 1: Alleles of eight spacers in three Y. pestis
biovars
Figure 1: Phylogenetic tree of 35 Y. pestis
isolates
Medievalis
Consistent trees using several
different methods:
• Unweighted pair group
method
• Parsimony
• Neighbor-joining
Orientali
s
Antiqua
Unique Antiqua strain
• Maximum likelihood
Sources of Ancient DNA
• • Justinian era DNA:
– 1989 excavation in Sens, France
– Mass graves
– Radiocarbon dating showed skeletons
were from 5th – 6th century
Black Death era DNA:
– 1990 excavations in Dreux, France
• 2-22 skeletons per grave
• Ceramic fragments dated graves
from 12th – 14th centuries
• Burial patterns, absence of bone
fractures, indications of age & sex
used as supporting evidence for
death by epidemic
– Montpellier France
• 13th century remblai (small burial
mound)
• 14C dating of ceramic fragments
dated graves to 14th century
Dreux, France cemetary excavated in
1990
Teeth collected for analysis:
Sens
10
Dreux
4
Montpellier
5
Marseille
17 (-) controls
Amplification of Ancient DNA
Multiple precautions to avoid
contamination:
– use of several Y.pestis
free buildings
– various technicians
– chronological processing
of teeth with negative
controls done last
– Use of new reagents,
new forceps
Results and Conclusion
Results:
o 46 PCRs found 10 Y. pestis
sequences
• YP1 → 1 positive
• YP8 → 6 positives
• YP3 → 3 positives
• All 17 (-) controls were negative
o GenBank BLAST results found most
homology with CO92, an Orientalis
biovar
o MST database BLAST results found
YP3 matches to Orientalis
sequences
Conclusion:
o All three pandemics
were caused by Yersinia
pestis genotype
Orientalis
Questions and Comments
Are you convinced?
Venice “Quarantine” Island
15th-16th century mass plague graves
discovered in 2004 on Lazzaretto Vecchio
Source: National Geographic