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The Molecular Model: Bacteriemia, Guinea Pig, and Coxiella burnetii

The Molecular Model: Bacteriemia, Guinea Pig, and Coxiella burnetii

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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 [18].

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 [19] 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

[20], and syphilis could be detected starting from Italian samples dating from 16th

century [21]. 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 [22].



5.3. Bones

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 [23]. 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.

5.4. Teeth

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 [24] 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 [25].

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

DNA extraction.



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 [26] 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 [27].

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 [27]. 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 [28]. 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 [29]. 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 [30]. 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



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 [31].

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.




























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Characterization of a Putative

Hemagglutinin Gene in the Caprine Model

for Brucellosis

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.

1. Introduction

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 [4]. 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 [7]. 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 [11].

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




A study done by del C Rocha-Gracia and colleagues [5] 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 [8]. 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 [12]. 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 [13] 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 [12], creating B. abortus 2308-QAE and RB51-QAE.



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 [14]. 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).

2.5. Goats

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 [15]. 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.



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

[16] 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 [17]. 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) [18]. 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 [14]. 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.

2.8. Statistics

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 [19]. Statistical analysis was performed with

Sigma Plot statistical software (Sigma Stat Statistical Software 1.0, Jandel Scientific,

San Rafael, CA).



3. Results

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

7 Days

1 × 104

14 Days

2.7 × 104

21 Days

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.

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