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New Procedures for the Diagnosis of Human
Brucellosis in Mongolia








Inauguraldissertation
zur
Erlangung des Grades eines Doktors der Humanbiologie
des Fachbereichs Medizin
der Justus-Liebig-Universität Giessen













vorgelegt von

Zandraa Jamba

aus Ulaanbaatar, Mongolei






Giessen 2008





I



Aus dem Biochemischen Institut
des Fachbereichs Medizin der Justus-Liebig-Universität Giessen


Geschäftsführender Direktor: Prof. Dr. Klaus T. Preissner





















Gutachter: Prof. Dr. Ewald Beck

Gutachter: Prof. Dr. Rolf Bauerfeind


Tag der Disputation: 24.06.2008

II SUMMARY
The feasibility of developing immunological and molecular diagnostic tools for routine diagnosis
of brucellosis under conditions of a less economically and scientifically advanced country in
general and Mongolia in particular was analysed. Brucellosis is a major healthcare issue in
Mongolia for both, humans as well as lifestock farming, leading to enormous economic losses
every year. Diagnosis of the disease is demanding and insufficiently specific with the available
tests. Using genomic sequences of the most important Brucella strains, new diagnostic
procedures have been developed by means of molecular biotechnology. They include the
production of several recombinant proteins as antigens in immunological assays such as
ELISA. Combining of several of these antigens in a single recombinant fusion protein led to an
unattained highly specific test for infections caused by Brucella. The genus Brucella includes
different species (or biovars) carried by different primary animal hosts and being transmitted by
different routes to humans. Furthermore, the severity of clinical manifestations differs among the
species necessitating specific modalities of treatment and making differential diagnosis on the
species level an important issue. Thus, another part of the study was focused on this topic. By
means of multiplex nested PCR, a highly sensitive and specific differentiation between the
major Brucella strains occurring in Mongolia was obtained. The immunological as well as the
molecular diagnostic principles have been designed to be applicable in laboratory practice
under limited economical conditions, including simple and inexpensive procedures for antigen
production and DNA purification.



III ZUSAMMENFASSUNG
Ziel der Arbeit war zu versuchen, neue immunologische und molekulare Methoden zur
routinemäßigen Diagnose der Brucellose unter wirtschaftlich und wissenschaftlich weniger
privilegierten Bedingung zu entwickeln, wie sie z. B. in der Mongolei vorherrschen. Die
Brucellose ist in der Mongolei ein erhebliches Problem, das nicht nur die Gesundheit der
Bevölkerung betrifft, sondern auch für die Tierzucht relevant ist und dort jedes Jahr zu großen
ökonomischen Einbußen führt. Die Diagnose der Krankheit ist aufwendig und mit den
vorhandenen Methoden wenig spezifisch. In der vorliegenden Arbeit wurden unter
Zuhilfenahme der genomischen Sequenzen der wichtigsten Stämme von Brucella mittels
molekularbiologischer Methoden neue Diagnoseverfahren entwickelt. Auf der einen Seite waren
dies verschiedene rekombinante Antigene, die in immunologischen Tests wie dem ELISA zur
Anwendung kamen. Die Verschmelzung mehrerer solcher rekombinanter Antigene in einem
einzigen rekombinanten Fusionsprotein führte zu einer bislang unerreicht hohen Spezifität der
Diagnose der Brucellose. Das Genus Brucella schließt mehrere Species (oder Biovare) ein,
die bei verschiedenen Tierarten vorkommen und auf unterschiedliche Weisen auf den
Menschen übertragen werden. Die Schwere der klinischen Manifestationen hängt von der Art
des Erregers ab und bedingt unterschiedliche therapeutische Maßnahmen. Daher ist es wichtig,
die Art der Erreger durch eine Differentialdiagnose zu identifizieren. Ein weiterer Teil der Arbeit
befaßt sich daher mit diesem Problem. Mit Hilfe von Multiplex-PCR wurde eine sehr
empfindliche und spezifische Differenzierung zwischen den wichtigsten in der Mongolei
auftretenden Brucella-Stämmem erzielt. Sowohl das immunologische als auch das molekulare
diagnostische Nachweisverfahren wurden so entwickelt, daß sie unter den bezüglich der
Finanzierung und der wissenschaftlichen Laborausstattung limitierten Voraussetzungen der
Mongolei durchführbar sind. Dies betrifft z. B. die Verwendung einfacher und kostengünstiger
Methoden für die Produktion der Antigene für die Immundiagnose und für die Reinigung von
DNA für PCR-Analysen.

IV
CONTENTS
ABBREVIATIONS III
1 INTRODUCTION 1
1.1. General facts about brucellosis 1
1.1.2 Brucella species: taxonomy, structure and biochemical characteristics 1
1.1.3 Virulence and pathogenesis 4
1.1.4 Immune response 6
1.1.5 Clinical spectrum of Brucella infection 8
1.1.6 Treatment 9
1.1.7 Control and prevention 10
1.1.8 Epidemiology of brucellosis worldwide 12
1.1.9 Brucellosis in Mongolia 15
1.2. Diagnostic methods for brucellosis 18
1.2.1 Clinical diagnosis 18
1.2.2 Laboratory diagnosis 19
1.2.2.1 Bacterial culture 20
1.2.2.2 Immunological methods 21
1.2.2.3 Molecular methods 23
1.3 Goals and objectives of the study 25
2 MATERIALS AND METHODS 27
2.1 Instruments 27
2.2 Materials 28
2.2.1 Chemicals 28
2.2.2 Bacterial strains 30
2.2.3 Bacterial DNAs 30
2.2.4 Antisera 31
2.2.5 Enzymes 31
2.3 Buffers and solutions 32
2.3.1 Buffers and solutions for protein gel electrophoresis 32
2.3.2 Buffers and solutions for DNA gel electrophoresis 32
2.3.3 Buffers and solutions for methods of molecular biology 33
2.3.4 Buffers for total DNA extraction 34
2.3.5 Buffers for alkaline lysis/silica method for plasmid preparation 34
® 2.3.6 Buffers for purification of His-tagged proteins with TALON 35
2.3.7 Buffers for immunoassays 35
I 2.4 Methods 37
2.4.1 DNA purification 37
2.4.2 Purification of recombinant Taq DNA polymerase 39
2.4.3 Standard cleavage assay 40
2.4.4 Standard ligation assay 40
2.4.5 Transformation of E. coli cells by electroporation 40
2.4.6 Expression and purification of His-tagged proteins 41
2.4.7 Line blot 42
2.4.8 ELISA 43
2.4.9 PCR procedures 44
2.4.10 Gel electrophoresis 45
2.4.11 Serological tests for brucellosis 46
3 RESULTS 47
3.1 Clinical specimen 48
3.2 Molecular methods: PCR 49
3.3 Establishing immunodiagnosis 55
3.3.1 Selection of diagnostic antigens 55
3.3.2 Synthesis of recombinant antigens 57
3.3.3 Cloning in pQE-30 vector and protein expression 60
3.3.4 Specificity and sensitivity of the antigens 62
3.3.5 Fusion of selected antigens 66
3.3.6 Example of fusion: P15-bp26-P39 67
3.3.7 Tests of the antigens with human sera samples in ELISA 69
3.3.8 Comparison of antigens with existing serological methods 70
4 DISCUSSION 72
4.1 Patient samples 73
4.2 Recombinant antigens 74
4.3 Specificity 75
4.4 Antibodies during the course of Brucella infection 76
4.5 ELISA 76
4.6 PCR 77
4.7 Specimens and DNA extraction 77
4.8 Primers. Design, PCR conditions, reaction mix, ready-to-use mixes 79
4.9 Brucellosis in Mongolia 81
5 CONCLUSION 83
6 REFERENCES 84
7 APPENDIX 93
II ABBREVIATIONS
Ab Antibody
Amp Ampicillin
AMOS Abortus Melitensis Ovis Suis
AP Alkaline Phosphatase
APS Ammonium persulphate
BCIP 5-Bromo-4-Chloro-3-Indolylphosphate
BCV Brucella containing vacuole
BSA Bovine serum albumin
CDC Center for disease control
CSF Cerebrospinal fluid
CNS Central nervous system
dNTP Deoxyribonucleoside triphosphate
DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
ELISA Enzyme-Linked ImmunoSorbent Assay
ER Endoplasmic reticulum
ERIC Enterobacterial Repetitive Intergenic Consensus
ERES exit site
FPA Fluorescence polarization assay
HRP Horseradish peroxydase
HOOF Hypervariable Octameric Oligonucleotide Fingerprints
IFA Indirect immunofluorescence assay
IFN Interferon
Ig Immunoglobulin
IL Interleukin
IPTG Isopropyl-beta-D-thiogalactopyranoside
IRS Interspersed Repetitive Sequence
IS Insertion sequence
LB-Medium Luria-Broth-Medium
LPS Lipopolysaccharide
MLVA Multi Locus VNTR Analysis
NBT Nitro blue tetrazolium
NCCD National Center for Contagious Diseases
NK Natural Killer
OD Optical density
III OMP Outer membrane protein
PAGE Polyacrylamide gel electrophoresis
PCR Polymerase Chain Reaction
PO Per os
RBC Red blood cell
RBPT Rose Bengal Plate Agglutination Test
RBT Rose Bengal Agglutination Test
REP Repetitive extragenic palindromic sequence
RFLP Restriction Fragment Length Polymorphism
RNase A Ribonuclease A
RNI Reactive nitrogen intermediate
ROI Reactive oxygen intermediate
rpm Rotations per minute
RT Room temperature
RTD Routine test dilution
S Svedberg Unit
SARS Severe Acute Respiratory Syndrome
SAT Serum agglutination test
SDS Sodium dodecylsulphate
SMZ Sulfamethoxazole
S-LPS Smooth-lipopolysaccharide
Taq Thermus aquaticus
Th1 T helper cell 1
TMP trimethoprim
T&S Test and slaughter
U Units
UV Ultraviolet
VNTR Variable Number Tandem Repeats
WHO World Health Organisation








IV 1 INTRODUCTION
1.1 General facts about brucellosis
Brucellosis is a group of closely related diseases caused by the members of the genus
Brucella in animals and humans. It is a zoonosis transmittable to humans with a high degree of
morbidity. More than 500,000 new cases of brucellosis are reported each year, and according to
World Health Organization, this figure underestimates the magnitude of the problem. There
were historically a number of synonyms for this infection: Malta fever, Mediterranean fever,
Gibraltar fever, Cyprus fever, and undulant fever, but in the meantime all infections by species
of Brucella are referred to as brucellosis. The organism was first isolated in 1887 by Sir David
Bruce, who recovered a suspect organism from the spleens of British soldiers dying of Malta
fever, hence the name for this illness. From this point on, it has progressively become clear that
closely related bacteria caused all of these diseases. Thus, Meyer and Shaw created the genus
Brucella in 1920 to accommodate these microorganisms.
Brucellosis is a complex disease and the range of primary hosts of Brucella includes several
domestic or semi-domestic animals, cetaceans, pinnipeds and some wild rodents. In domestic
animals the disease manifests by abortion and infertility. Humans usually acquire brucellosis
from domestic animals through direct contact or consumption of their products and are not
themselves source of contagion. The disease in humans is rarely deadly, but debilitating with
possible severe consequences. The high degree of morbidity, for both animals and humans, is
an important cause of financial loss and represents a serious public health problem in many
developing countries (Corbel, 1997).
1.1.2 Brucella species: taxonomy, structure, biochemical characteristics and
polymorphisms
Genus Brucella belongs to class I Alpha-2 Proteobacteria of phylum Proteobacteria.
Brucellae are Gram-negative, facultative intracellular pleomorphic bacteria that can infect
humans and many species of animals (Figure 1.1).
1
Figure 1.1 Electron microscopy of B.abortus (Dennis Kunkel Microscopy, Inc., 2004).
Six classical species were formerly recognized within the genus Brucellae: B. melitensis,
B. abortus, B. suis, B. neotomae, B. ovis and B. canis. Two new species were isolated from
marine animals B. pinnipedialis and B. ceti in mid 1990s (Foster et al., 2007), while recently, a
novel species, Brucella microti has been detected and isolated from common vole (Scholz et al.,
2008). This classification is mainly based on differences in pathogenicity and host preference.
Although the six classical species can be differentiated by conventional phenotypic tests, they
show a high degree of homology in their DNA-DNA hybridization assays (>90 % identity)
suggesting that the Brucella genus should comprise a single species, B. melitensis, with the
remaining species considered as biovars. However, molecular genotyping revealed that
Brucella species display significant DNA inter-specific polymorphisms, justifying the current
classification. Since the epidemiology and the severity of the diseases in humans is influenced
by the Brucella type and its source (Corbel et al, 2000), the practical approach in classification
is also of great importance. In general, B. abortus is associated with cattle, B. melitensis with
sheep and goats, B. suis with swine, B. ovis causes infections specific for sheep and has not
been implicated in human diseases, B. canis is usually associated with diseases in dogs but
occasionally causes human brucellosis, and B. neotomae has been isolated on few occasions
and has never been implicated in human diseases. The pathogenicity for humans of other
Brucella species (B. pinnipedialis, B. ceti, and B. microti) still has to be clearly established.
The most common Brucella species to affect humans is B. melitensis, the most
pathogenic species producing the most intense symptoms, the greatest tissue damage, and the
most frequent incidence of localization in body organs, systems and tissue.
2