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The Molecular Model: Bacteriemia, Guinea Pig, and Coxiella burnetii
APPLICATIONS OF PALEOMICROBIOLOGY
in blood and spleens, or the system of detection used (culture) was not sufficiently
sensitive to be able to detect the bacteria after 5 and 10 days. However, because the
tests of detection in blood culture and pulp (PCR) were different, and although they
were concordant with the data in the literature and the laboratory, it was not possible to
draw more conclusions from the difference in the results .
4. Application to the Diagnosis of Bacteriemias
The description of old infectious diseases always was made starting from old texts,
iconography, or anthropological data. These anthropological data were established,
most of the time, starting from observations of the human remains coming from mass
graves discovered fortuitously. However, if the old texts are easier to interpret than the
charts, they raise the problem of their conservation and access to the original texts. It is
very difficult to give an objective interpretation based on such descriptions. Semiology
is not sufficiently reliable to make it possible to make an exact diagnosis and to
establish an analytical classification for ancient old infectious diseases. In addition, it
should be noted that the anthropological and historical analyses allowed an analogical
description of old infectious diseases, generating various etiological and epidemiological
assumptions to account for the same observations, and this often led to controversies.
5. Various Tissues Used for Objective Analysis
5.1. Frozen Tissues
Frozen human tissues represent an ideal situation because these samples allow the
insulation and the culture of the pathogen and its immunological and molecular characterization, but this situation is obviously exceptional. The man discovered in the ice
in the Tyrol gave rise to research  that made it possible to show that his muscles
were rich in bacterial DNA. Nevertheless, taking into account the number of detected
bacterial species and their variety (Sphingomonas, Afipia, Curtobacterium, Microbacterium, Agromyces, and others), the question of external contaminations can be
5.2. Fixed Tissues
Fixed tissues represent samples of good quality for molecular detection but do not
allow the use of a culture medium. These tissues are rarer and more difficult to use if
one takes contamination into account. Nevertheless, Mycobacterium tuberculosis could
be detected starting from soft tissues from a Peruvian mummy dating back 1,000 years
, and syphilis could be detected starting from Italian samples dating from 16th
century . The presence of Carrion’s disease (Bartonella bacilliformis) was detected
in mummified soft tissues from mummies associated with human sacrifice among the
ancient Huaris of southern Peru .
G. ABOUDHARAM, M. DRANCOURT, AND D. RAOULT
Bones are the most abundant remains, but it is difficult to use molecular techniques in
their analysis because of the difficulty in sample preparation, including the washing of
samples and the possibility of their contamination during preparation. DNA must be
extracted from bone before it can be analyzed. This is a difficult requirement, because
the extraction is preceded by a stage of decalcification using EDTA as a chelator.
EDTA must then be removed from the sample by extensive washing, because EDTA is
an inhibitor of DNA polymerases that require magnesium as a co-factor . Once
sample DNA has been extracted, however, PCR amplification allows genomic material
to be obtained in sufficient quantity to enable its characterization, and it eliminates the
problems of the contamination of samples and the specificity of the amplicons obtained.
Teeth constitute a target organ of quality because of their preservation in time, but all
the possibilities that they offer for the detection of bacterial DNA have not been
exploited, whereas, as noted above, the use of dental pulp as sample material in forensic
medicine is well-established. Because blood infections carry bacteria to all parts of the
vasculature in systemic diseases, teeth potentially contain bacterial DNA. This fact was
the motivation for the work that is discussed in detail below.
6. Detection of Yersinia pestis in Dental Pulp
The provision of remains coming from two mass graves identified as containing victims
of the “great plague” gave us the opportunity to apply the techniques developed through
the use of the animal models described above. The origin of the first mass grave located
in the gardens of the monastery of the Observance  did not leave any doubt after
compilation and comparison of the anthropological data with the historical data and the
records of the monastery, which was used as a hospital at that time. The second mass
grave was located close to the site of Fédons. A study of contemporary records, aiming
to specify the statute and the origin of the places better, made it possible to retain the
assumption of an epidemic disease. This archival study led to strong presumptions,
according to which, a cemetery and an infirmary were set up on this site to face an
epidemic of plague .
The first part of this work consisted in developing a technique for the recovery of
dental pulp and the extraction of the pulpar DNA, which permitted a second phase
to amplify specific gene fragments of Yersinia pestis. The selection of teeth is a
significant stage. The teeth on which research is carried out must answer criteria of
selection: young teeth having belonged to a child or an adolescent and, preferably,
single-root teeth with the apex almost closed; unerupted teeth are preferred for their
absence of contact with any external elements. The technique for the removal of pulp
consisted in the preparation of a preliminary fracture line and the fracture itself along
the largest axis of the tooth, followed by the recovery of the powdery organic remains
that line the pulp cavity using an excavator. Pulpar DNA extraction using the classical
gave the best results; we chose this method after testing several other protocols for
APPLICATIONS OF PALEOMICROBIOLOGY
The second part of the work was the search for the DNA of Y. pestis. The selected
molecular targets were twofold. First, various fragments of small sizes of the plasmidborne pla gene were tested (250–300 nt). It is specific to Y. pestis, and it is present in
multiple copies per genome. The fact that it is present in several copies per bacterium
represented a significant advantage, taking into account the possible degradation of the
DNA. The second gene tested was a fragment of 133 bp of the rpoB gene  specific
to Y. pestis. This second gene is present as a single copy per bacterial chromosome and
is used in routine diagnosis in the laboratory. The results confirmed the cause of death
of the individuals because the various fragments of genes tested were found. The results
also confirmed the colonization of dental pulp via the hematogen ducts in a bacteriemia
and that the tooth represented a choice tool for this type of investigation .
7. Development of a New Protocol of Amplification to Prevent the Risk
of Contamination: “Suicide PCR”
In preceding work, for the first time, we established a paleomicrobiological diagnosis
of plague in human remains dating from the 16th and 18th centuries, thus establishing
the diagnosis of an old, septicemic disease . In addition to the difficulties relating to
the fact that old DNA is fragile and fragmented and that one finds only a small number
of copies of the required molecular target, other technical difficulties, primarily related
to problems of contamination of old DNA, appeared. Only the most stringent laboratory
hygiene made it possible to avoid these contaminations; the more sensitive the techniques
of detection, the greater the risk of contamination . These problems of contamination
can be related to the handling by the operator or the organization of the laboratory.
To mitigate such disadvantages, the laboratory is organized so as to avoid cross
contaminations and false-positive detections . The circulation of the samples and
the amplicons should never cross, taking into account the ease of spread of the latter
and the number of copies at the end of a reaction of PCR. DNA extractions and PCR
reactions are carried out in different, physically separated sites. Exposure of working
surfaces to ultraviolet irradiation also makes it possible to reduce the risk of contamination.
To take into account the difficulties encountered in preceding work and to establish
a diagnosis that can in no case be debatable, samples of human material dated according to
anthropological and historical criteria from the 14th century were analyzed in a novel
way. Moreover, to make a diagnosis could be of considerable historical interest because
it is reported that, from 1,347 to 1,351, the Black Death killed nearly 30 million
Europeans. Although for a long time historians put forth the assumption that it was
indeed the plague, this assumption had not been objectively confirms with experimental
evidence. Moreover, taking into account the spread of the epidemic, doubts remained as
to the responsibility of Y. pestis. Given the symptoms described in historical accounts,
a diagnosis of hemorrhagic fever could also reasonably be made. Only a paleomicrobiological diagnosis could eliminate or confirm Y. pestis as the etiological agent responsible for the Black Death. In this work, we showed, thanks to the strategy of “suicide
amplification,” that the Black Death of 1,348 was indeed caused by Y. pestis . This
procedure was performed using sample preparation methods and protocols that were
developed specifically for this project. The absence of contamination being able to be
G. ABOUDHARAM, M. DRANCOURT, AND D. RAOULT
related to the presence of DNA in an aerosol provided confidence in the accuracy of
our results. PCR was carried out in the absence of any positive controls, which themselves could have been the source of contaminating DNA. This original approach
demanded a theoretical calculation of the conditions of amplification. However, while
the results generated are indisputably unaffected by contamination from positive control
DNA, the correct amplification of target sequences can be confirmed only by the
sequencing the resulting amplicons. In “suicide PCR”,, a single primer set is used in
only one PCR reaction ever in the analytical laboratory. This single-use strategy avoids
the possibility that previously amplified DNA will contaminate future PCR reactions.
The success of this strategy has implications beyond the characterization of Y. pestis
in ancient human samples. “Suicide PCR” now is applied routinely for the molecular
diagnosis of contemporary infectious diseases.
8. Genotyping of Yersinia pestis
Based on the conventionally determined geographical origins of Y. pestis and the
historical writings that indicate the geographical origin of plague pandemics, it was
suggested that each pandemic came from a different biovar: the biovar Antiqua of East
Africa caused the first pandemic, and the biovar Medievalis of Central Asia caused the
second. The bacteria related to the third pandemic are biovar Orientalis. In this study,
we evaluated this assumption for the first time by detecting biovars in old human
Starting from the two available genomes of Y. pestis – strain CO92 and Kim –
bioinformatic analysis made it possible to locate eight intergenic spacers, enabling the
differentiation of the three biotypes of Y. pestis. This multispacer typing then was
applied to 35 contemporary strains from different geographical origins to establish
the genotyping of these strains. With the use of this multispacer typing, three groups
were determined, representing the three biovars. This multispacer typing was used to
test the dental pulp from the remains of several individuals. These remains dated from
the Justinian plague and the Black Death. The analysis showed that the samples
attributed to the Justinian plague indeed were contaminated by Y. pestis and that the
first two pandemics, attributed historically to the Antiqua and Medievalis biotypes,
could in fact be attributed to the Orientalis biovar .
9. Conclusions and Prospects
This study showed that DNA signatures of pathogens can be found in ancient dental
pulp. Because of its exceptional ability to preserve DNA, dental pulp constitutes an
extremely interesting sample matrix for the research of old pathogens. Dental pulp and
the tools we have developed for its analysis therefore comprise an important new set of
research tools in microbiology and paleomicrobiology.
Future courses of research include the use of protocols of repair of DNA to collect
more data on ancient pathogens and the use of universal molecular targets to be able to
answer several etiological questions.
APPLICATIONS OF PALEOMICROBIOLOGY
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Characterization of a Putative
Hemagglutinin Gene in the Caprine Model
Quinesha L. PERRY1, Sue D. HAGIUS2, Joel V. WALKER2, Lauren DUHON1,
and Philip H. ELZER1,2
Department of Pathobiological Sciences, School of Veterinary Medicine,
Louisiana State University, Baton Rouge, Louisiana
Department of Veterinary Science, Louisiana State University Agricultural Center,
Baton Rouge, Louisiana
Abstract. With the completion of the genomic sequences of Brucella melitensis
16M and B. abortus 2308 and the vaccine strain RB51, a putative hemagglutinin
gene was identified that is present in 16M and absent in B. abortus. The possibility
of this hemagglutinin being a potential host specificity factor was evaluated via
expression in trans in B. abortus 2308-QAE and RB51-QAE. Using the caprine
brucellosis model, colonization and pathogenesis studies were performed to
evaluate the strains.
Brucella species are short, nonmotile, nonsporulating, nonencapsulated, gram-negative
aerobic rods. They are facultative intracellular pathogens of animals and humans [1, 33,
34, 39, 41]. The Brucella genus is highly homogeneous, with all members showing
greater than 90% homology in DNA–DNA pairing studies [2, 3], and little is known about
Brucella virulence. The genus Brucella consists of six species, each with a preference for a
primary host and varying degrees of pathogenicity. B. melitensis primarily infects
goats and is the most pathogenic for humans; B. abortus infects cattle.
Brucella LPS has important cell surface properties, yet there is no evidence
showing its role in invasion . Other outer membrane proteins also may play a role in
the organisms’ virulence [5, 6, 40]. An organism’s ability to adhere to a mucosal surface is
a crucial first step in the pathogenesis of many pathogens . Initial attachment of
the brucellae to epithelial cells is mostly unknown. With the completion of Brucella
genomes, specifically B. melitensis 16M and B. abortus 2308, studies have been done and
are currently underway to detect and characterize novel genes that may be involved in
Brucella pathogenicity [8, 9]. Of particular note is a putative hemagglutinin gene found
within the B. melitensis 16M genome that is absent in B. abortus [9, 10]. The gene is
present in B. suis and B. canis but with minor nucleotide substitutions. There are two
copies of the gene in B. ovis .
K.P. O’Connell et al. (eds.), Emerging and Endemic Pathogens,
DOI 10.1007/978-90-481-9637-1_9, © Springer Science + Business Media B.V. 2010
Q.L. PERRY ET AL.
A study done by del C Rocha-Gracia and colleagues  explored the possibility of
hemagglutinins on the cell surface of brucellae serving as adhesins to eukaryotic cells
through the ability of B. abortus and B. melitensis to hemagglutinate human and animal
(rabbit, hamster, guinea pig, rat, mouse, sheep, and dog) erythrocytes and attempted to
identify a receptor moiety involved in that reaction. All Brucella strains (B. abortus 2308,
B. abortus S19, B. abortus 02, and B. melitensis 03) tested showed hemagglutination with
the red blood cells from the various sources, with B. melitensis 03 showing the highest
hemagglutination titers against all red blood cells and B. abortus 2308 the lowest titer.
This study evaluated the host specificity of Region E, a putative ~2.0-kilobase
hemagglutinin gene using the completed genome of B. melitensis 16M . Experiments
using variants of B. abortus 2308 and RB51 expressing Region E in trans were carried
out in the caprine brucellosis model to provide insight into possible vaccine development.
2. Materials and Methods
2.1. Bacterial Strains
Virulent B. abortus strain 2308, vaccine strain RB51, and B. melitensis strain 16M were
used in these studies to create B. abortus 2308-QAE, RB51-QAE. B. abortus 2308,
RB51 and B. melitensis 16M were grown on Schaedler Brucella Agar (SBA) (Difco
Laboratories, Detroit, MI) and B. abortus 2308-QAE, RB51-QAE were grown on SBA
containing 100 μg/mL ampicillin or 45 μg/mL kanamycin, respectively. Plates were
incubated at 37°C in a 5% CO2 atmosphere for 2–3 days.
Inoculation doses of B. abortus 2308, RB51, B. melitensis 16M, and B. abortus
2308-QAE, RB51-QAE were made as previously described . Viability counts on
SBA plates, SBA plates with ampicillin (100 μg/mL), and SBA plates with kanamycin
(45 μg/mL) using serial dilutions were done to validate the concentration of the
inoculation doses the day of use.
2.2. Creation of B. abortus 2308 and RB51 Variants
A 4,950-bp plasmid called pBBR1MCS-4  was digested using EcoR V (New
England Biolabs, Beverly, MA). Region E, an ~2.0-kilobase PCR-amplified putative
hemagglutinin gene, was generated from B. melitensis 16M genomic DNA using the
primers ORF-944F (5'-GAATTGGCGACCTGACTGAGGA-3') and ORF-944R (5'CTCACGGCTGTTCTCCTTTAACA-3') (the Institute of Molecular Biology and
Medicine at the University of Scranton, Scranton, PA). PCR-amplified Region E was
ligated into the EcoR V-linearized, gel-purified pBBR1MCS-4 plasmid using the FastLink™ DNA Ligation Kit for Blunt End Ligation (Epicentre Biotechnologies, Madison,
WI) to create pQAE. The ligation mixture then was used to transform One Shot®
Chemically Competent Cells (Invitrogen Corporation, Carlsbad, CA) according to the
manufacturer’s directions. Successful transformants were cultured and their plasmids
isolated using the Qiagen Buffer System (Qiagen, Inc., Valencia, CA). The isolated
plasmid DNA was electroporated into B. abortus 2308 and RB51 as previously
described , creating B. abortus 2308-QAE and RB51-QAE.
CHARACTERIZATION OF A PUTATIVE HEMAGGLUTININ GENE
2.3. Confirmation of B. abortus 2308 and RB51 Variants
Expression of pQAE in trans in B. abortus 2308 and RB51 was achieved by the
introduction and maintenance of the low copy number plasmid in the cell. The new
plasmid containing Region E from B. melitensis (pQAE) was electroporated into
B. abortus 2308 or RB51 and screened for successful transformation using SBA plates
supplemented with 100 µg/mL ampicillin. The new variant of B. abortus 2308 was
named B. abortus 2308-QAE, and RB51 was named RB51-QAE. Presence of the gene
was confirmed via PCR amplification of the putative hemagglutinin using the Region E
primers and restriction enzyme digestion of pQAE.
2.4. Standard Identification Tests
Potential variant/mutant colonies were isolated for Brucella typing using techniques
commonly performed to differentiate Brucella species from other gram-negative
organisms. Standard biochemical tests were performed, including urease, oxidase, and
catalase, along with observing colony morphology and growth rate . Suspected
variants/mutants along with their parental strains also were tested for sensitivity to
dyes: azure A, basic fuchsine, crystal violet, pyronin, safranin, and thionin, according to
the manufacturer’s protocol (Key Scientific Products, Round Rock, TX).
For all animal studies, male or female Angora or Spanish goats were obtained from
commercial herds or from the Louisiana State University (LSU) herd (LSUniversity
Agricultural Center, Baton Rouge, LA). All animals were housed throughout the
study at the Ben Hur Large Animal Isolation Facility, a restricted-access United States
Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS)
Veterinary Services – and Centers for Disease Control and Prevention (CDC) – approved
facility. All animals were cared for in accordance with the LSU AgCenter Animal Care
and Use Committee guidelines.
For the colonization studies, 30 male or nonpregnant female goats divided into five
equal groups were inoculated conjunctivally with either 1 × 109 colony forming units
(cfu) of B. abortus 2308, RB51, B. abortus 2308-QAE, RB51-QAE, or B. melitensis
16M . At predetermined time points, the goats were euthanized by captive-bolt and
exsanguination. Two animals from each group were sacrificed on days 7, 14, and 21.
The following tissues were collected and examined bacteriologically: parotid, prescapular,
internal iliac, inguinal, and supramammary lymph nodes; liver; and spleen. Results
were recorded as colony-forming-units per gram (cfu/g) of tissue.
For the pathogenesis studies, dams were bred with Brucella-negative billies, and
their pregnancies later were confirmed via ultrasound examination. Goats in late gestation
were exposed conjunctivally to either the virulent parental strains B. melitensis 16M or
B. abortus 2308 or the variant B. abortus 2308-QAE with 1 × 107 cfu. Pregnancies
were monitored until delivery, and kids were recorded as aborted/weak or live/healthy.
Q.L. PERRY ET AL.
Live kids were euthanized by CO2 asphyxiation, and lung tissue and abomasal fluid
were collected on all kids born or aborted. A month following the last birth or abortion,
all dams were euthanized by captive-bolt and exsanguination. The following tissues
were collected: parotid, prescapular, internal iliac, and supramammary lymph nodes;
liver; spleen; and mammary gland. All tissues collected were stored at –20°C until
cultured for bacteriological analysis.
2.6. Serological Analysis
All animal sera samples were brucellosis card tested and evaluated by Western blot
 before any experimentation to confirm the absence of Brucella-specific antibodies.
Necropsy samples were tested similarly. For Western immunoblot analysis, cell lysates
of B. abortus 2308, RB51, B. melitensis 16M, B. abortus 2308-QAE, and RB51-QAE
were prepared by sonication and dilution in Laemmli sample buffer . Cell lysates
were separated by polyacrylamide gel electrophoresis (SDS-PAGE) using 12% TrisHCl Ready Gels (BioRad Laboratories, Inc., Hercules, CA) and transferred to a nitrocellulose membrane (Osmotics, Livermore, CA). After blocking with 1% skim milk,
individual blots were incubated in a 1:40 dilution of test serum on a shaker at room
temperature overnight. After incubation, blots were washed with tris buffered saline
(TBS)-Tween and TBS and incubated on a shaker for 45 min at room temperature in a
1:800 dilution of rabbit anti-goat IgG peroxidase conjugate (Sigma-Aldrich Co.,
St. Louis, MO). Blots were developed using 4-chloro-1 napthol tablets (Sigma-Aldrich
Co.) in a TBS-methanol-3% hydrogen peroxide solution. Reactions were stopped by
the addition of dH2O.
2.7. Bacteriological Analysis
Tissue samples were thawed, weighed, homogenized in sterile phosphate buffered
saline, and plated on SBA plates supplemented with 5% bovine blood and Brucella
Selective Supplement (Oxoid Ltd., Basingstoke, Hampshire, England) . After a 14day incubation period at 37°C in a 5% CO2 atmosphere, the total number of colonies
present on each plate was counted and cfu/g of tissue calculated. The limit of detection
for our laboratory using this system is 13 cfu/g or mL. Brucella species were identified
by colony morphology, growth rate, and biochemical tests . B. abortus 2308-QAE
and RB51-QAE were differentiated from B. abortus 2308 and RB51 based on their
ability to grow on SBA plates containing 100 µg/mL ampicillin.
Numbers of colonized dams, colonized kids, and abortions in the pathogenesis study
were compared between two groups at a time using a Fisher exact probability test, with
P < 0.05 being considered significant . Statistical analysis was performed with
Sigma Plot statistical software (Sigma Stat Statistical Software 1.0, Jandel Scientific,
San Rafael, CA).
CHARACTERIZATION OF A PUTATIVE HEMAGGLUTININ GENE
3.1. Dye-Sensitivity Analyses
Dye-sensitivity analysis of B. abortus 2308-QAE and RB51-QAE was typical of that
usually seen with B. abortus 2308 and RB51.
3.2. Colonization of B. abortus 2308-QAE and RB51-QAE
A short-term colonization study was performed to see whether B. abortus 2308-QAE or
RB51-QAE could colonize nonpregnant goats. At predetermined time points, two
animals from each group were sacrificed; and the following tissues were collected for
bacterial culture: parotid, prescapular, internal iliac, inguinal, and supramammary lymph
nodes; liver; and spleen. Results were recorded as cfu/g of tissue (Table 1). There were
no significant differences between the experimental groups and the controls. Both
modified strains were capable of colonizing the caprine hosts at levels comparable with
the parental strains.
TABLE 1. Colonization of Nonpregnant Goats Inoculated with Brucella abortus 2308, B. abortus
2308-QAE, Brucella melitensis 16M, RB51, or RB51-QAE
Brucella abortus 2308
1 × 104
2.7 × 104
1.4 × 104
B. abortus 2308-QAE
Brucella melitensis 16M
3 × 104
5.4 × 104
1.5 × 102
1.0 × 103
2.3 × 105
8.6 × 104
1.0 × 101
3.2 × 103
6.8 × 104
3.2 × 104
1.2 × 102
1.2 × 103
Calculated in mean cfu/g of tissue.
All resulting colonies were evaluated to verify their Brucella origin via oxidase,
catalase, and urease tests. All colonies were confirmed to be Brucella and grew on the
appropriate antibiotic-supplemented media.
Serological analysis of the colonization goats on days 7, 14, and 21 via Brucella
card test and Western immunoblot analysis revealed that all animals given 2308 or 16M
on days 14 and 21 were seropositive. RB51 animals were seronegative on the card test.
3.3. Pathogenesis of B. abortus 2308-QAE
To assess the pathogenicity of the experimental strains in the ruminant host, pregnant
goats in late gestation were exposed to conjunctivally one of the three strains of
Brucella. Study results are presented in Table 2. Goats inoculated with B. abortus 2308
displayed a 27% abortion rate as compared with goats infected with B. abortus 2308QAE, which exhibited a 67% abortion rate (P < 0.05). Additionally, 78% of the animals
inoculated with B. melitensis 16M aborted.