This document is the summary of my thesis work carried out at Indian Veterinary Research Institute, Izatnagar, India (2001) I am sorry, I cant provide a download option for this

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3. INDIAN VETERINARY RESEARCH INSTITUTE
IZATNAGAR - 243 122, U.P.
Dr. D.K. Singh
M.V.Sc., Ph.D. BRUCELLA LABORATORY
SCIENTIST (SR. SCALE) FAO/WHO COLLABORATING CENTRE FOR RESEARCH
AND TRAINING IN VETERINARY PUBLIC HEALTH
DIVISION OF VETERINARY PUBLIC HEALTH
I.V.R.I., IZATNAGAR, U.P., INDIA
DATE : 15TH JUNE 2001
Certificate
Certified that the research work embodied
in this thesis entitled “Studies on in vitro effects of
Brucella melitensis cytosolic antigen on ovine neutrophils” submitted
by Dr. V.I. Bishor, Roll No. 3925, for the award of Master of
Veterinary Science degree in Veterinary Public Health at Indian
Veterinary Research Institute, Izatnagar, is
the original work carried out by the candidate
himself under my supervision and guidance.
It is further certified that Dr. V.I. Bishor, has
worked for about 21 months in this Institute
and has put in more than 150 days attendance
under me from the date of registration for the
M.V.Sc. degree of this Deemed University, as
required under the relevant ordinance.
(D.K. Singh)
3
Chairman

4. 4

5. Acknowledgement
I consider it a great privilege to express my deepest sense of gratitude to
Dr. D.K. Singh, my guide and chairman of the advisory committee for suggesting
this problem, his constructive councel, critical appreciation and his relentless efforts
during the entire research work and preparation of this manuscript.
I express my sincere thanks to Dr. V.N. Bachhil, Head, VPH division for his
timly help.
I owe my deep gratitude to Dr. T.K. Goswami, Scientist, Sr. Scientist,
Immunology Section for his valuable suggestions, constant interest, unstinted
encouragement and more than that his charming company.
I am highly thankful to the Director, Joint Director (Acad) and Scientific
Coordinator, IVRI, for the financial assistance in the form of the IVRI-JRF and
providing necessary facilities to carry out this work.
It gives me immense pleasure to aknowledge the encouragement and valuable
help received from Dr. D.K. Sinha, Scientist, division of Epidemiology without
which the work might not have been compleated so smoothly.
I owe special gratitude and feel highly esteemed to thank Dr. K.N. Kapoor,
Dr. R.K. Agarwal, Dr. S.V.S. Malik, Dr. K.N. Bhilagaonkar and Dr. R.S. Rathore
of the VPH division for their valuable suggestions and altruistic help in my research
work.
I acknowledge my cordial thanks to Dr. G.C. Ram, i/c immunology section,
for providing me the necessary laboratory facilities.
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6. A special note of appreciation and thanks are due to Dr. G.L. Koul, Head,
Animal Genetics division and Dr. S.M. Deb, Scientist, Animal Genetics division
for their help in the flourescent microscopic studies.
It gives me pleasure to acknowledge the valuable help received from Dr.
J.C. Verma, Dr. V.P. Singh (B&M division), Dr. K.P. Singh, Dr. Rajendra Singh
(CADRAD) and Dr. Pallab Chaudhary (NBC).
I always cherish the happy moments, shared with Jacob sir and his family.
I shall forever remain indebted to my collegues Dr. Elezebeth and Dr. Ajith
for helping me out at odd times with their inspiring and comforting words.
My sincere thanks to my senior Dr. Sandeep and to my collegues Dr.
Chatlod, Dr. Rajkumar and Mr. Purushottam for their valuable help during my
research work.
It is my pleasure to acknowledge the technical assistance and heelp received
from Jhaji, Jayanthi Ji, Mohan and all the VPH staff members.
I express my heartful gratitude to Mr. Harpreet and all the staff of CIF,
MLB for their help.
I can’t fail to mention the cheerful company given by my friends Jaison,
Eyas, Archana, Unni, John, Suman, Sabari, Sriram, Anish, Abi, David, Reghu,
Manoj, Mohan, Pramod, Jayakumar, Salim, Pankaj, Sanjay, Sathu, Jajati, ....
I feel immense pleasure to thank Sethu Chechi, Dr. Anamika and Dr. Anjali
for their scholarly advises, affection and care they always showed to me.
I am thankful for the remarkable encouragement and valuable suggestions
extended to me by my seniors Drs. Ghatak, Banerjee, Siddharth, Saravanan,
Hazarika, Neelima, Chitra, Goutam, Banasure, Suresh, Amith, Munjal and
my junior collegues Deepa, Balamurugan and Vijaykumar.
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7. Mr. Anirudh deserves a special thanks for his inexplicable photographic
work.
I also wish to express my thanks to the staff of National Library of Veterinary
Sciences and University Office for their valuable help.
I sincerely thank all my senior and Juniors at IVRI who made the stay in IVRI
a memorable experiance.
Thanks are also due to Mr. Dharmendra and Mr. Narender for the neat
typing of this manuscript.
My heartiest thanks are due to all the members of south indian mess for the
homly atmosphere they provided me.
I cant forget those innocent animals who served themselves for my
experiment. I will always owe them for the study.
I feel immense pleasure in expressing my gratitude to my father, mother and
my brothers, whose love, support and encouragement has brought me to this stage.
At last, I remember the Almighty who gave me strength, courage and
perseverance to achieve this goal.
Bishor V.I.
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8. Contents
CHAPTER PAGE NO.
1. INTRODUCTION 1-4
2. REVIEW OF LITERATURE 5-13
3. MATERIALS AND METHODS 14-29
4. RESULTS 30-34
5. DISCUSSION 35-43
6. SUMMARY 44-45
7. MINI ABSTRACT 46
8. HINDI ABSTRACT 47
9. REFERENCES 48-60
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9. Abbreviations
µg : Microgram
µl : Microlitre
µM : Micromoles
APS : Ammonium persulphate
Bis : N,N’-methylene bisacrylamide
bp : Base pair
CFU : Colony forming unit
CO2 : Carbon dioxide
DAB : 3,3'-diaminobenzidine dihydrochloride
DMSO : Dimethyl sulphoxide
DNA : Deoxy ribonucleic acid
DTH : Delayed type hypersensitivity
EDTA : Ethylenediamine tetra-acetic acid
ELISA : Enzyme linked immunosorbent assay
FAO : Food and Agriculture Organization
Fig. : Figure
FITC : Fluorescin isothiocyanate
g : Gram
h : Hour
H2O2 : Hydrogen peroxide
HEPES : N-2-hydroxyethylpiperazine -N'-2-ethanesulphonic
acid
HRPO : Horseradish peroxidase
IU : International units
kDa : Kilodalton
lbs : Pounds
LPS : Lipopolysaccharide
M : Molar
mA : Milli ampere
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10. mg : Milligram
min : Minute
ml : Millilitre
mM : Millimolar
MTT : 3-[4, 5-dimethylthiazole-zyl]-2,5-diphenyl
tetrazolium bromide
MW : Molecular weight
N : Normal
NBCS : Newborn calf serum
NCM : Nitrocellulose membrane
nm : Nanometer
nM : Nanomoles
NO : Nitric oxide
NO–2 : Nitrite
o
C : Degree centigrade
OPD : O-phenylene diamine dihydrochloride
PBMC : Peripheral blood mononuclear cells
PBST : Phosphate buffered saline-Tween-20
PMNs : Polymorphonuclear cells
RNI : Reactive nitrogen intermediates
ROI : Reactive oxygen intermediates
SDS-PAGE : Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis
TEMED : N,N,N,’N’-tetramethyl ethylene diamine
Tris : Tris [Hydroxymethyl] aminomethane
UV : Ultraviolet
V : Volt
v/v : Volume/volume
VPH : Veterinary Public Health
w/v : Weight/volume
WHO : World Health Organization
xg : Centrifugal force equal to gravitational acceleration
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11. INTRODUCTION
Members of the genus Brucella are facultative intracellular gram negative
bacteria capable of causing brucellosis in man and animals. The disease is prevalent
worldwide and endemic in many countries especially underdeveloped and developing
countries. Brucellosis is still considered to be a serious public health problem and
an ever increasing concern in many developing countries. It is estimated that there
are more than 500,000 new cases of brucellosis in man every year.
During the course of infection phagocytes are the first to encounter the
Brucella and, thus, play an important role in the defence against the invading micro-
organisms. These phagocytes constitute innate immune system of the host as against
the adoptive immune system mediated by the lymphocytes. There are two types of
phagocytes, viz., polymorphonuclear (PMN) cells which are short lived and the
long lived mononuclear leukocytes. Polymorphonuclear cells such as the neutrophils
are supposed to be the first line of defence. Thus, the PMNs play an important role
in establishment of the infection by Brucella.
The ability of PMNs to phagocytize and then kill the ingested bacteria is
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12. critical for resistance to pathogenic bacteria such as Brucella. Inability of
these cells to efficiently destroy virulent Brucella at the primary site of infection
is a key factor in establishment of the organism in the regional lymphnodes and
eventually to spread and localization in the reticuloendothelial system (RES) leading
to, in most cases, establishment of chronic brucellosis.
For the bacteria to survive within the phagocytes, they must be able to resist
the antibacterial activity of phagolysosome, escape from the phagosome or prevent
the metabolic burst associated with phagocytosis. B. abortus can survive the
oxidizing activity in macrophages and neutrophils, inhibit the fusion of phagosome
with lysosome by releasing guanosine monophosphate (GMP) and inhibits the
migration of neutrophils from clots, and circumvent phagocytosis by cleaving Fc
portion of IgG in whey.
The survival of pathogenic intracellular bacteria inside cell, especially in
phagocytes, is essential for establishment of disease. There are many mechanisms
for entry into the phagocytes used by intracellular pathogens.Once inside the cell,
different bacteria utiilize different mechanism to circumvent the immune response
thereby ensuring their survival and multiplication. Precise mechanism to evade
killing used by many pathogenic bacteria including brucellae are yet not clearly
explained. Similar is the situation with respect to the host immune mechanism
leading to reduction in number of invading microorganism and elimination from
the system.
It has been established that brucellae are readily phagocytosed by the PMNs
and macrophages and the brucellae have the ability not only to survive inside these
phagocytes but they also grow and multiply. Many of the virulence factors have
been described which can alter the phagocytic cell function.
Role of some surface molecules of Brucella has been emphasised. However,
it is not clear which surface component play role in determining virulence of
facultative intracellular pathogen such as Brucella. The fact that these are readily
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13. ingested by the phagocytes points to the possibility that surface components of
brucellae do play some role in survival of organisms inside the cell. These
components help enhance adhesion and facilitate ingestion of bacteria thereby
protecting them from bactericidal factors and even antibacterial agents such as
antibiotics present in the plasma. Lipopolysaccharides (LPS) of Brucella has been
hypothesised to be one such cell surface component. Different species and strains
of brucellae have been reported to differ in their ability to resist bactericidal
activities of the phagocytes. This ability is also dependent on the smoothness and
roughness of the strain. B. abortus contains a nucleotide like substance with
molecular weight <1000, later identified as 5' GMP which is responsible for the
resistance to killing by PMNs. The virulent B. abortus produces 2,3-dihydroxy-
benzoic acid which acts as siderophore and help in the uptake of iron from medium.
B. abortus seems to have adapted and developed multiple mechanism to
avoid intracellular destruction. The 5'-GMP and adenine released inhibit the MPO-
H2O2-halide antibacterial system of bovine neutrophils by inhibiting degranulation.
The viable cells (but not the cell wall fractions) of virulent B. abortus inhibit primary
metabolic burst associated with phagocytosis in bovine neutrophils. Water extracts
of virulent strain prevent the fusion of phagosome with lysosome in macrophage,
thereby, enabling B. abortus to survive. However, it is also suggested that LPS
from B. abortus doesn’t contribute to the ability of the extracts to inhibit fusion.
Of the PMNs, the neutrophils are supposed to be the major cells responsible
for defences of host by eliminating the invading brucellae. They phagocytize and
digest brucellae to get rid of them. This ability of neutrophils to kill and digest the
invading microorganisms is mediated primarily through two mechanism.: a) oxidative
and b) non-oxidative. While the non-oxidative mechanism is dependent on
antimicrobial proteins and peptides, the oxidative mechanism operates through
respiratory burst to generate toxic microbicidal oxidants. The respiratory burst is
mediated through the enzymes that are present in the phagocytes and activated
following the ingestion of pathogen. These oxidants known as reactive oxygen
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14. intermediates (ROI) are very powerful microbicidal agents and include superoxide
anions (O–2), hydrogen peroxide (H2O2), hydroxyl radicals (OH–), hypochlorous
acid (HOCl) and chloramine.
In addition to these oxidants, a reactive nitrogen intermediate (RNI), nitric
oxide (NO) has also been reported to possess strong microbicidal activity. It is
also produced by neutrophils of many species.
It is an established fact that the phagocytes ,viz., neutrophils and macrophages,
do produce varying amount of these reactive molecules on stimulation. The stimulus
could be the phagocytosis of the pathogen or through production of cytokines in
response to infection by the microorganisms. Though, there has been reports dealing
with the phagocytosis of brucellae by the neutrophils and their distruction within
them, these are few and far between. Also, most of the studies have been performed
with bovine neutrophils and few with caprine and human neutrophils. There have
been no studies regarding the response of the ovine neutrophils with Brucella.
Apart from this, the cell surface component or antigen(s) that play role in
modulating the neutrophil response of the host, so as to circumvent their
brucellicidal activity leading to their survival has not been clearly identified.
Thus, it is clear that even though there is agreement that the brucellae are
capable to evade the phagocyte's brucellicidal activities with consequent survival,
the underlying mechanism is far from elucidated. Also, the cell component involved
is still to be identified. Accordingly, the present study has been designed to be
undertaken with the objectives as follows.
1. Identification and purification of immunodominant fractions from
Brucella cytosolic antigen.
2. Immunochemical characterization of isolated antigen
3. To study the interaction of antigens with ovine neutophils.
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15. Review
of
Literature
Brucellosis is an acute and chronic infectious disease of a wide range of
animals readily transmissible to man. It is caused by members of the genus Brucella
which are Gram-negative, facultative intracellular organism capable to survive in a
variety of cells including the phagocytes (Bounous et al., 1993). Brucellosis
remains a disease of considerable importance for man and domesticated animals
worldwide. In the last meeting of FAO/WHO expert committee on brucellosis, it
has been described as an ever increasing cause of concern to public health (Anon,
1986).
Brucella antigens
Brucellae are Gram-negative organisms with lipopolysaccharide (LPS) as
the major immunodominant antigen ( Nielson et al., 1989; Berman et al., 1980).
However, it also has a wide array of antigenic components that directly or indirectly
influence the overall immune response of the host leading to either elimination of
brucellae or establishment of infection. Identification and characterization of
antigen(s) that modulate the host immune effector system is primary to the
development of a diagnostic test or vaccine.
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16. Among the various antigens, LPS is the most important. The O-polysaccharide
(O-PS) associated with lipid A is the major component of LPS. The cross reactivity
of Brucella with other bacteria like Yersinia, Vibrio cholerae, Salmonella,
Escherichia coli and Pseudomonas has essentially been found due to structural
similarity in the O-PS (Hurvell and Lindberg, 1973).
Besides LPS, various protein antigens extracted from Brucella have also
been found to be immunogenic. These protein antigens can be cell-wall antigen or
cytosolic antigen. The major fraction of cell-wall protein antigen have been shown
to be outer membrane protein (OMP). The Brucella OMP contains three groups
of protein with molecular weights of 88-94 kDa (group 1), 35-40 kDa (group II)
and 25-30 kDa (group III) (Verstreate and Winter, 1984). Of these, the group II
proteins were later identified as porins (Douglas et al., 1984). These porins were
also found to induce lymphocyte proliferation and strong delayed type-
hypersensitivity (DTH) reaction in infected cattle (Winter 1987). The cell wall of
B. abortus has been described as a complex structure composed of at least 40
proteins (Sowa et al., 1991) which are strongly bound to peptidoglycan (Dubray,
1973).
Cytosolic antigens
Brucella contain a variety of soluble proteins inside the cells as cytosolic
antigen. One such protein, brucellin, contained atleast 20 protein fractions with no
detectable LPS (Denoel et al., 1997b). Bhongbhibhat et al. (1970) prepared this
antigen by cold hypertonic saline (2.7%) extraction followed by ethanol
precipitation. Later Jones et al. (1973) reported that ultracentrifugation at 100,000
x g for 6 hours was effective in removing the high molecular weight membrane
parts. Brucellin also contained significant amount of nucleic acid material along
with its proteins (Alton et al., 1975).
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17. On SDS-PAGE analysis Fensterbank and Dubray (1980) showed 20 bands
with major bands at 70-72 kDa , 35-40 kDa and 16-18 kDa. Blasco et al. (1994)
detected 40 kDa and 35 kDa bands by SDS-PAGE which represented 90% of the
protein detected in cytosol. Brooks-Worrell and Splitter (1992) could separate
SDS extracted B. abortus S19 antigen to several bands ranging from molecular
weights of 6 kDa to 87 kDa by SDS-PAGE and two dimensional electrophoresis.
The carbohydrate contamination of this antigen was also reported by
Bhongbhibhat et al. (1970) and Cherwonogrodzky et al. (1990). However, Blasco
et al. (1994) could not detect any rough LPS in the brucellin prepared by them.
They reported that the preparation contained 45.3% protein distributed in over 25
polypeptides of molecular weight 72 kDa to 14 kDa while the cold saline extract
(CSA) contained 53.5% protein with 15 polypeptides in the same molecular weight
range.
Immunoblotting with sheep sera showed a 20 kDa immunodominant band
in the cytosolic antigen prepared from B. melitensis B115 cells (Zygmunt et al.,
1992). But earlier workers (Fensterbank and Pardon, 1977; Fensterbank and Dubray,
1980) reported that these allergens when injected, did not elicit the formation of
either agglutinins or complement fixing antibodies in cow and rabbits. Denoel et
al. (1997a) purified a 39 kDa protein from brucellergen by anion exchange
chromatography on mono Q sepharose fast flow column HR5/5. The antigen was
immunoreactive and did not cross react with any other bacteria.
Humoral immune response to Brucella infection
Immune respone of host to Brucella infection is mediated through both
humoral as well as cellular compartment. The humoral response mediated through
generation of immunoglobulins(Ig), has been shown to provide a low degree of
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18. protection usually limited to minor reduction in the count of Brucella during the
initial stages of infection.
The primary humoral response to Brucella infection has been shown to be
an early production of IgM which is overtaken by IgG with disease progression
(Herbert, 1970). Subsequent to vaccination , too, IgM is produced initially which
remains predominant isotype. IgG, though produced to a low titre, does not persist
(Rice et al., 1966). Bossery and Plomet (1980) reported that the cytoplasmic
proteins that elicit DTH reaction was not immunogenic and did not protect mouse
against infection. In the Western blotting using infected cattle sera, a common
group of proteins with molecular weights ranging from 31-45 kDa and 66- 71 kDa
have been found to be reactive (Belzer et al., 1991).
Cellular immune response to Brucella infection
Brucella is a facultative intracellular pathogen capable of surviving within
the autophagic vesicles of the host cells. Eventhough the mechanism of survival of
Brucella species remains unresolved, many factors are supposed to contribute to
the survival of Brucella inside the phagocytic cells.
One of the major function of PMNs and macrophage is to phagocytize, to
eliminate parasites and to present the processed antigens to the lymphocytes. For
most pathogenic intracellular bacteria, this process is led astray by the parasite that
survive and multiplies within the phagocytic cells. This intracellular survival is due
to specific factor, produced by bacteria, that interfere with macrophage / PMN
physiology (Caron et al., 1994b). These factors are responsible for the virulence
of the pathogen.
The internalization of the bacterial pathogen is the initial morphologic event
of host- bacteria interaction. Ocon et al. (1994) reported that some factors
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19. associated with the Brucella are capable of inhibiting the migration of phagocytes.
The phagocytosis of these bacilli are facilitated by opsonins (Gallego et al ., 1989).
After the phagocytosis Brucella are reported to be able to escape the host
bactericidal mechanism (Price et al., 1990, Smith and Ficht, 1990). Many factors
contribute to this intracellular survival. In the macrophages, Brucella have been
shown to inhibit the primary metabolic burst following phagocytosis and prevent
fusion of phagosome with lysosome (Frenchick et al., 1985; Arena et al., 2000).
They are also reported to induce the synthesis of some stress proteins, viz., HSP,
GroEL, following phagocytosis by macrophage (Lin and Ficht, 1995). A siderophore
(2,3-dihydroxybenzoic acid) produced by B. abortus prevent its intracellular
killing (Leonard et al., 1997).
Brucella after invasion of the cells of reticuloendothelial system
(RES),develop within the mononuclear phagocytes and the infected monocytes play
an important role in the dissemination of the bacteria. Brucella multiplies in the
membrane bound compartments of phagocytic and nonprofessional phagocytic cells
(Baldwin and Winter 1994; Detilleux et al., 1990) with endoplasmic reticulum as
replication site (Anderson and Cheville, 1986). Following phagocytosis, Brucella
interact first with the early endosomes and by pass the late endosomal compartment
to localize into the endoplasmic reticulum where it multiplies (Pizarro- Cerda et
al., 1998).
The intracellular environment of the phagocytic cells are inhospitable for
most bacteria. The acidic environment of phagosomes as well as phagolysosomes
are unsuitable for bacterial survival (Lin and Ficht, 1995). Porte et al. (1999)
reported that this vacuole acidificaion of pH between 4 and 4.5 in the phagocytic
cells has been shown to essential for intracellular survival of Brucella. It is possible
that expression of virulence genes is coordinately regulated in response to
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20. environmental changes, which include genes responsible for the change in the pH
of environment (Miller et al., 1989). Such type of gene expression has been
demonstrated in case of S. Typhimurium (Aranda et al., 1992).
Recently, modulation of apoptotic mechanism by the invading intracellular
bacteria to evade the host cell killing mechanism has been proposed . Many bacteria
viz., Shigella flexneri, Legionella pneumophila, Yersinia enterocolitica,
Bordetella pertussis, L. monocytogenus and S. Typhimurium have been found to
induce apoptosis in infected cells (Gao and Kwaik, 2000). In the PMNs
Streptococcus pneumoniae induce cell death, depending on the intensity of
stimulus (Zysk et al., 2000). The human granulocytic ehrlichiosis (HGE) agent
replicating in PMNs is reported to be capable of inhibiting the apoptosis of human
neutrophils (Yoshiie et al., 2000). Baran et al. (1996) observed that granulocytes
and monocytes reacted differently to phagocytosis of bacteria where the conditions
that induced apoptosis in monocytes prolonged the survival of granulocytes. Some
organisms like mycobacteria and rickettsia have been found to be able to induce as
well as inhibit apoptosis (Gao and Kwaik, 2000). It was, thus, postulated that
inhibition of host cell apoptosis protects the intracellular pathogen by shielding
the immune attacks from outside. Recent reports suggests that Brucella has also
got such a mechanism, where the survival of Brucella inhibit the programmed cell
death of human monocytes (Gross et al., 2000). This inhibition of apoptosis did
not involve LPS and requires Brucella survival within the host cells.
Oxidative Mechanism in Intracellular Killing of Brucella
The destruction of pathogens within the PMNs and mononuclear phagocytes
is accomplished through mainly two mechanisms : oxidative and non-oxidative.
The oxygen could act as a toxicant was first reported by Gerschman (1959) who
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21. demonstrated that the toxicity of oxygen is due to the generation of reactive oxygen
species (ROS) which include superoxide anion (O–2), hydroxyl radical (OH-), peroxyl
(ROO-), alkoxyl (RO–) radicals and radicals of nitric oxide (NO), nitrogen dioxide
(NO2), peroxy nitrite (ONOO–) and possibly singlet oxygen (1O2). Besides these,
hydrogen peroxide (H2O2) and lipid peroxide are not free radicals. They act as
reservoirs for the highly reactive OH–, ROO– and RO– radicals. The ROS are oxidants
and highly toxic to all types of biological molecules. Most of their activities are
mediated by hydroxyl radicals (OH–).
Generation of ROS in cells could be deliberate under certain circumstances
by the activated phagocytic cells as part of their bactericidal role (Datta et al.,
2000). These ROS produced by phagocytic cells such as neutrophils and
macrophages could be broadly catagorised as ROI and RNI.
The phagocytic cells upon proper stimulation increase their utilization of
oxygen (respiratory burst) and convert oxygen to metabolic ROI such as O-2, H2O2,
OH- and 1O2 (Robinson and Badwey, 1994). In addition to this, they can respond
through induction of nitric oxide synthase (iNOS) and the enzymatic conversion of
L-arginine to citrulline, releasing RNI and NO, which is quickly oxidised to nitrite
(NO2-) or nitrate (NO3-) (Moncada et al., 1991). These toxic products are supposed
to contribute significantly to the destruction of extracellular as well as intracellular
pathogens (Nare et al., 1990).
Role of Reactive Oxygen Intermediates (ROI)
The ROI production by the phagocytes is a key process in the defence of the
host against various microorganisms including Brucella (Babior, 1987). Inside the
PMNs, brucellae inhibit myeloperoxide (MPO)-H2O2 - halide system. The major
antigens involved are probably LPS and a low molecular weight necleotide like
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22. material (Bertram et al., 1986), later identified as 5'-guanosine monophosphate
(GMP) and adenine (Canning et al., 1986). A reduced superoxide and lysozyme
production by Brucella LPS compared to Salmonella LPS has been reported (Rasool
et al., 1992) which might contribute to the intracellular survival of Brucella.
A partial or total inhibition of degranulation of primary granules to release
MPO has been observed in contrast to adequate generation of respiratory burst in
patients with active brucellosis (Ocon et al., 1994). Earlier, IFN- mediated increase
in the production of O2– and MPO-H2O2 - halide activity of neutrophil in the presence
of Brucella has been recorded (Canning and Roth, 1989). Recently, Iyankan (1998)
reported LPS mediated decrease in H2O2 production, in contrast to OMP and killed
Brucella cells in a dose-dependant manner.
Role of Reactive Nitrogen Intermediates (RNI)
Nitric oxide (NO) is considered to be one of the most important mediators
of the host defence against microbial infection (Nathan and Hibbs, 1991). The
production of NO is catalysed by nitric oxide synthase (NOS) leading to the
formation of L-citrulline and NO from L-arginine. Two distinct NOS isoenzymes
are known. The constitutive NOS exist in various host cells and accounts for basal
NO synthesis whereas the inducible NOS (iNOS) is primarily found in professional
phagocytes and responsible for microbial killing (McCall and Vallance, 1992). The
iNOS expression is induced by proinflammatory cytokines such as IFN- , tumor
necrosis factor (TNF- ), IL-1 as well as microbial products such as LPS and
lipoteichoic acid (Fang, 1997). The mechanism of this activity is little understood
(Gross et al., 1998), but one possibility is that during infection NO could combine
with superoxide anion to generate the deleterious ON OO– anion (Zhu et al., 1992).
22

23. In murine macrophages, inducible NO has been shown to kill or inhibit
tumour cells, Leishmania major, Trypansoma cruzi, Schistosoma, certain viruses,
mycobacteria and Legionella (Zhao et al., 1996). Gross et al. (1998) reported
that NO is one component in antibrucella activity but only in IFN- treated
macrophages infected with opsonized Brucella. Iyankan (1998) found that NO2–
production by Brucella LPS is much greater than that of OMP and killed Brucella
cells and it is lesser than that of Pasteurella and E. coli LPS. This decrease or
increase on NO2– production has been shown to be dose-dependent and not affected
by the vaccination status of the animals. Recently, Lopez-Urrutia et al. (2000)
reported that B. abortus and B. melitensis S-LPS and lipid A induce NO production
in rat peritoneal macrophage by a mechanism involving transcriptional up-regulation
of the iNOS gene.
23

24. Materials
and
methods
Materials and Methods
Organisms
Brucella melitensis 16M, Rev 1 and B115 maintained at the FAO/WHO
collaborating center for research and training in Veterinary Public Health, IVRI,
India, were used in the present study. The cultures were tested for purity and
biochemical characters before use.
Experimental animals
Apparentely healthy adult female cross bred sheeps, serologically negative
for brucellosis, procured from sheep and goat farm, IVRI, were used in the study.
They were maintained on standard diet consisting of concentrate and fodder. Rabbits
used in the study were adult male rabbits of Newzealand white breed obtained from
the Laboratory Animal Resource Section, IVRI. They were maintained hygienically
on a standard rabbit diet. The animals were provided ad libitum water.
Media, Buffers and Reagents
The composition of media, buffers and reagents used in the present study is
given in Appendix.
24

25. Conjugates
Anti-caprine, anti-rabbit, anti-bovine and anti-human HRPO and anti-rabbit
FITC conjugates were procured from National Institute of Immunology, New Delhi,
India.
Chemicals
All chemicals used in the present study were purchased from Difco, Sigma
(USA), BDH, Glaxo, Merck, SRL, Sd.fine and Genei Banglore and were of analytical
reagent or molecular biology grade.
Glasswares and plastic wares
All glasswares used were purchased from Borosil (India) or Corning (India).
All plastic wares (96 well flat bottom plate, 24 well plate, ELISA plates and
petridishes) were from Greiner/Millipore/Corning.
Newborn calf serum
Day old, colostrum deprived male calf was obtained from dairy of IVRI,
Izatnagar. Blood was collected and serum separated under aseptic conditions. Serum
was inactivated by heating at 56oC for half an hour. It was then filter sterilized
through seitz filter and stored at –20oC.
Equipments
The following equipments were used in the study : Centrifuge [Sovall RT-
6000, Sorvall Ultra PRV 80, high speed centrifuge REMI-R8C), Monopan balance
(Aldair dutta, India], U-V spectrophotometer [UV-1201 Shimadzu, Japan], Microscan
ELISA reader [ECIL, India], Modulo freeze dryer [Edwards, England], MES-
Soniprep : 150 sonicator [Sanyo, Japan], Vertical electrophoresis apparatus and
25

26. powerpack [Atto, Japan], Semidry western blotting apparatus [Atto, Japan], Inverted
microscope [Olympus, Japan], Fluorescent microscope (Nikon TS100, Japan),
Millipore water purification system [Millipore, USA], Gel doc [UVP-White/UV
transilluminator, UK] and Gel dryer [Drygel Sr. Hoefer Scientific instruments,
Sanfrancisco].
Propagation and harvesting of Brucella organisms
B. melitensis B115 was propagated in potato infusion agar in Roux flask as
described by Alton et al. (1975). The seed suspension was prepared by harvesting
and suspending the growth from potato infusion slant in 500 ml of sterile phosphate
buffered saline [PBS : 0.01M, pH 7.2]. The suspension was checked for purity.
Each Roux flask was inoculated with 5-10 ml of seed suspension, spread uniformly
and left for 30-45 minutes with agar side towards bottom. It was then incubated for
72 h at 37oC with agar side upwards. At the end of incubation, the liquid contained
in the flask was discarded into a disinfectant solution and 10 ml of normal saline
solution (NSS) or PBS added to each flask. Flasks were kept for 30 min, and gently
agitated to detach the cells and the cultures in each Roux flask were examined for
purity by gram staining. The bacterial suspension was aspirated, pooled in one
flask, filtered through sterile absorbent cotton and centrifuged at 6000 x g for 30
minutes to settledown the cells. The cells were washed twice with PBS and
resuspended in PBS.
Preparation of acetone dried cells
The method described by Alton et al. (1975) was followed. The bacteria (1
x 109) suspended in PBS was added to two volumes of acetone at –20oC and allowed
to stand at 4oC for 18 hours. Then, the sterility was checked by plating on a trypticase
soya agar (TSA) plate. The bacteria were sedimented by centrifugation at 6000 x g
for 30 min at 4oC, washed three times with cold acetone and freeze dried.
26

27. Preparation of cytosolic antigen
The method by Bhongbhibhat et al. (1970) as modified by Jones et al. (1973)
was followed with little modifications. Briefly, the acetone dried bacterial cells
were suspended in 2.5% sodium chloride solution at 4oC to give a 5% (w/v)
suspension. It was agitated for three days at 4oC and then centrifuged at 6000 x g
for 30 minute at 4oC. The supernatant was collected and treated with three volumes
of cold ethanol with constant stirring. The mixture was held for 24 hours and
centrifuged at 13000 x g for 30 min at 4oC to collect the precipitate. The precipitate
was dissolved in distilled water and extensively dialysed against distilled water at
4oC. The solution was ultracentrifuged at 100,000 x g for 2 hours to remove high
molecular weight material and the resultant supernatant fluid was collected and
stored at –20oC.
Other antigens
Heat killed B. melitensis 16M were prepared by heating the cells to 65oC
for 1 h in a waterbath. B. abortus LPS was obtained from the Brucella laboratory,
Division of VPH, IVRI.
Raising of Brucella anti-serum in rabbit
Three adult healthy rabbits were inoculated with sonicated B. melitensis
16M cells in Freund’s incompleate adjuvants. A booster was given on day 14. The
sera tested one week after booster for Brucella antibody by standard tube
agglutination test (STAT) using B. abortus plain antigen obtained from Division of
Biological Products, IVRI. At the desired antibody titre, rabbits were bled, serum
collected, pooled and stored at –20oC in aliquotes.
27

28. Chemical analysis of antigen
The protein was determined by the modified Lowry method (Lowry et al.,
1951, Peterson, 1979) using bovine serum albumin (Sigma) as standard.
Carbohydrate content was estimated by phenol sulphuric acid method (Dubois et
al., 1956) using D-glucose as standard. The nucleic acid content was assessed by
comparing the optical densities at 260 and 280 nm.
SDS-PAGE analysis
The cytosolic antigen prepared from B. melitensis B115 cells was analysed
by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) either
on a 12.5% gel or on a gradient gel of 5 to 15% acrylamide [Appendix] as per the
method of Laemmli (1970). The samples were mixed in a ratio of 4:1 with sample
buffer (Appendix) and kept on a boiling waterbath for 3 minutes. The gels were run
at 100 V until the tracking dye reached the bottom and stained with silver stain
(Blum et al., 1987). The molecular weights (MW) of peptides were determined by
comparing their relative mobilities with that of standard molecular weight markers
[Genie, Banglore] plotted on a graph as described by Rosenberg, (1996).
Immunoblotting
The immunoblotting was done to asses the immunoreactivity of various
peptide fractions. The procedure of Towbin et al. (1979) was followed with little
modifications. After the SDS-PAGE of cytosolic antigen in a vertical slab gel
apparatus the proteins were transferred onto the nitrocellulose membrane [Sartorius,
Germany] using a semidry electroblotting apparatus (Atto, Japan) at a constant
current of 2mA/cm2 for 70 minutes. After blotting, the membrane was air dried
briefly, and then transferred to 5% skim milk powder in PBS containing 0.05%
Tween-20 for blocking the nonspecific sites overnight at 4oC. The nitrocellulose
membrane was then washed three times with PBS-T and incubated for 2h at 37oC in
28

29. 1:100 dilution of the B.melitensis hyperimmune sera,anti-B.abortus bovine sera
or clinical sera from human in PBS-T. The membrane was again washed throughly
with PBS-T and incubated with the HRPO conjugated anti-rabbit, anti-bovine or
anti-human antibody [1:1000] for 2h at 37oC. It was then washed three times with
PBS-T and developed in substrate solution containing 0.6 mg/ml diaminobenzidine
[Sigma] and 2 µl/ml of 30% hydrogen peroxide in PBS. The colour development
was stopped by immersing membranes in the distilled water.
Fractionation of cytosolic antigen by anion exchange chromatography
Fractionation was done by the method of Denoel et al. (1997a) with suitable
modifications. Briefly, cytosolic antigen [10 mg] was applied to a DEAE Sepharose
(Sigma) column equillibrated with 10mM Tris-HCl [pH 8.5] buffer and having a
bed volume of 15 ml. The column was washed with 60 ml of equillibrating buffer
and then eluted with 120 ml of linear gradient of 0 to 1M NaCl in the same buffer
at a flow rate of 0.5 ml/minute. The absorbence of elutes was monitered at 280 nm
and fractions of interest were pooled.
Analysis of fractions
Protein content of each fraction was determined from their absorbence at
260 and 280 nm. Carbohydrate content was estimated by phenol-sulphuric acid
method.
SDS-PAGE and Immunoblotting
SDS-PAGE and western blotting of the fractions were done as described
previously.
ELISA
Immunoreactivity of each fraction was tested in ELISA by method described
by Riezu-Boj et al. (1988). Briefly, 1 µg protein from each fraction was coated
29

30. overnight at 4oC on a 96 well flat bottom ELISA plate (Greiner, Germany) in
carbonate-bicarbonate buffer. The coated plates were washed with PBS-T and
blocked overnight with 5% skim milk in PBS-T at 4oC. After washing the plates
1:100 dilution of serum was added to the wells and incubated at 37oC for 2 h. The
plates were washed thrice with PBS-T and HRPO conjugated anti-caprine antibody
was added to each well [1:5000]. The plates were incubated for 2 h at 37oC. Washed
three times as earlier with PBST and 100 µl of O-phenylene diamine (OPD, Sigma)
0.6 mg/ml in substrate buffer containing H2O2 (0.01%) was added to each well.
Plates were incubated for 10 min in dark and reaction was stopped by 100µl/well
of 2N H2SO4. The OD was measured at 492nm by ELISA reader and OD value 2
times that of negative was considered positive.
dot-ELISA
the dot-ELISA was done by the method of Chand et al.(1989). Briefly, the
nitrocellulose dip-sticks were coated with 1µl of antigen. The coated strips were
air dried and blocked by dipping in 5% skim milk in PBS-T for 2 h at 37oC. The
dipsticks were then washed with PBS-T and incubated with test sera (1:100 ) for 2
h at 37oC. The strips were washed throughly and allowed to react with HRPO
conjugated anti-bovine or anti-caprine antibody (1:1000) for 2 h at 37oC. The
dipsticks were again washed with PBS-T and reaction was visualized by dipping the
strips in substrate solution (DAB : 0.6 mg/ml in PBS containing 0.01% H2O2). The
colour development was stopped by washing the dipsticks in distilled water.
Isolation of neutrophils from peripheral blood
The PMNs from the peripheral blood of sheep were separated by method of
Eggleton et al. (1989).
Briefly, 20 ml blood from jugular vein of sheep was drawn into 2 ml of
2.7% EDTA in PBS (0.01 M, pH 7.2). The blood was added to ice cold (4oC) isotonic
30

31. ammonium chloride solution (Appendix) in 1:4 ratio (blood : ammon chloride
solution), mixed well and kept for 15 minutes. The cell suspension was then
centrifuged at 160 x g for 10 min in plastic conical bottom tubes (Tarsons, India).
The supernatant was discarded, the cell pellet was gently suspended in 5 ml PBS
(pH 7.2) and recentrifuged at 55 x g for 10 min. The washing was repeated twice at
same speed. The resultant PMNs were suspended in appropriate media and kept at
4oC until used. The viability of cells was determined by Trypan blue dye exclusion
test and differential count was done by Giemsa staining.
Estimation of nitric oxide
The nitrite (NO2–) production by neutrophils after stimulation with heat killed
B. melitensis 16M cells, LPS and cytosolic antigen was assessed using a
colorimetric assay for nitrite based on Griess reaction (Green et al., 1982).
Effect of whole cells
The test was done in 96 well tissue culture plates. PBS (300 µl) containing
2 x 107 PMNs/ml was dispensed in the wells. Heat killed B. melitensis 16 M cells
were added at different concentrations (0, 1.5, 3, 6 x 107/ml) in triplicate wells and
incubated for 60 min at 37oC. The plate was then centrifuged at 600 x g for 10 min
and the supernatant was collected. To the 100 µl of supernatant, equal amount of
Griess reagent (Appendix) was added and incubated for 10 min at room temperature
and absorbence was taken at 540 nm in an ELISA reader. The results were expressed
as µM of NO2–/2 x 107 PMNs/hour calculated from a pre-calibrated standard curve
using sodium nitrite as standard.
Effect of B. abortus LPS
B. abortus LPS at concentrations of 0, 12.5, 25, 50, 75, 150 and 300 µg/ml
was added for the activation of PMNs and NO2– was estimated and results expressed
as above.
31

32. Effect of cytosolic antigen
The PMNs were stimulated with cytosolic antigen at concentrations of 0,
12.5, 25, 50, 75, 150 and 300 µg/ml and NO2– level was determined and results
expressed as described previously.
Production of hydrogen peroxide by neutrophils
The method described by Pick and Keisari (1980) was followed with little
modifications.
Effect of whole cells on H2O2 production
Neutrophils (2 x 107 cells/ml) in buffered phenol red solution (PRS) was
added to the wells of a 96 well tissue culture plate. Heat killed B. melitensis 16M
cells at various concentrations (0, 1.5, 3 and 6 x 107/ml) were added to respective
wells in triplicate. The plate was incubated for 60 min at 37oC and centrifuged at
600 x g for 10 min. The supernatant was transferred to a microtitre plate and 10 µl
of 1N NaOH was added to each well to bring the pH to 12. The plate was shaken
gently and absorbence at 610 nm was taken in an ELISA reader. The H2O2 production
was determined from a standard curve of H2O2 prepared for each experiment and
results were expressed in nM/2 x 107 PMNs/minute.
Effect of B. abortus LPS
The PMNs were stimulated with B. abortus LPS at concentrations 0, 12.5,
25, 50, 75, 150, 300 µg/ml and H2O2 produced was estimated and results expressed
as above.
Effect of cytosolic antigen
The PMNs were stimulated with cytosolic antigen at concentrations of 0,
25, 50, 75, 150, 300 µg/ml and H2O2 production was estimated and results expressed
as above
32

33. Colorimetric assay for oxidative metabolism of PMNs
Method as described by Bogdan et al. (1997) was followed. After harvesting
supernatant for NO2– estimation, 20 µl of MTT [3,[4, 5-dimethylthiazol-zyl]2, 5-
diphenyltelrazolium bromide, 5 mg/ml in PBS] was added to each well. The plate
was incbuated for 1 hr at 37oC. At the end of incubation formazan crystals were
dissolved by adding 150µl of dimethyl sulfoxide (DMSO) and mixed properely.
The optical density was determined at 595 nm. The results were expressed as the
percentage of OD comparing test wells and control wells as follows.
Mean OD in test well
––––––––––––––––––––––––– x 100
Mean OD in control well
Phagocytosis and intracellular killing index
The assay was performed as per the method of Gallego et al. (1989) with
modifications from Hampton and Winterbourn, (1994).
Preparation of bacteria
B. melitensis 16M was cultured on trypticase soya agar (TSA) slant. After
48 h of incubation, bacteria were washed with Hank’s balanced salt solution (HBSS
pH 7.4, Appendix), centrifuged at 1000 rpm for 10 min to settled down the agar
particle. Colony count was adjusted to 1 x 107 cells/ml turbidimetrically.
Opsonization
Bacteria (1 x 107/ml) was suspended in HBSS with 10% heat inactivated
sheep serum from Brucella free animals. The tubes were incubated in shaker water
bath at 6 rpm for 20 min at 37oC and used immediately.
33

34. Treatment of neutrophils with antigen
Neutrophils in glass tubes were incubated with 50 µg/ml of cytosolic antigen
or B. abortus LPS for 1 h at 37oC.
Phagocytic index
To 1 ml of PMN suspension (1 x 107/ml), 1 ml bacteria (1 x 107/ml) and 0.2
ml serum were mixed and incubated at 37oC under continuous rotation. At different
time intervals (t15, t30, t60), a 0.5 ml aliquot of cell suspension was removed and
added to 1.5 ml ice cold HBSS. The tubes were then centrifuged at 110 x g for 5
min to pellet the PMNs. The supernatant (2 ml) was seperated and serially diluted.
The plate count was determined by the method of Miles and Misra (1938) after
plating onto TSA plates and incubating for 3 days.
The phagocytosis after t min was expressed as phagocytic index (PI) and
was calculated as,
PI (t) = Log NO – Log Nt
Where,
NO : initial number of viable bacteria at time 0.
Nt : number of viable extracellular bacteria at time t.
Intracellular killing index
Here 1 ml of bacteria (1 x 107/ml), 1ml of neutrophils (1 x 107/ml) and 0.2
ml serum were mixed and incubated with continuous rotation. At each time point
(t15, t30, t60) , 0.5ml of suspension was removed and added to 1.5 ml ice cold HBSS.
The suspension was then centrifuged at 110 x g for 10 min at 4oC. The supernatant
was discarded and pellet of neutrophil resuspended in 2 ml HBSS. To it, 20 µl of
saponin (0.05% w/v in PBS) was added to lyse the neutrophils. The serial dilution
34

35. of this was made in sterile PBS and 10µl of each dilution was inoculated on TSA
plate. The colony count was determined after incubating for 3 days as described
earlier.
The index of intracellular killing expressed as bactericidal index (BI) after t
min was calculated as
BI (t) = Log NO– Log Nt.
NO = initial number of viable intracellular bacteria
Nt = number of viable intracellular bacteria after t min.
Studies on apoptosis
Preparation of bacteria and antigens
B. melitensis 16 M and Rev 1 strains were grown on glycerol dextrose agar
(GDA) for 48 hours at 37oC, harvested in PBS (0.01 M, pH 7.2) and centrifuged
briefly at 1000 rpm to settle down the agar particles. The bacterial suspension was
then washed three times in PBS and resuspended in RPMI-1640 containing 10%
new born calf serum (NBCS) and 2mM L-glutamine without any antibiotic, after
adjusting the cell count to 3 x 108 cells/ml. In some experiments, the bacteria were
heat killed at 65oC for 1 h in a waterbath and suspended in RPMI-1640.
Isolation of PMNs
The PMNs were isolated by method of Eggleton et al. (1989) as described
previously. The cell count was adjusted to 1 x 106 to 2 x 106 cells/ml in RPMI-1640
containing 10% NBCS.
Isolation of monocytes
The peripheral blood mononuclear cels (PBMC) were seperated from sheep
blood by method of Boyum (1968).
35

36. Briefly, 40 ml blood was collected in sterile syringes containing 4 ml of
2.7% EDTA in PBS and centrifuged at 1000 x g for 40 min. The buffy coat was
pipetted out, mixed with equal volume of PBS and layered over histopaque (1.077)
and centrifuged at 400 x g for 45 min. The interface ring was collected and washed
with PBS three times, including one low speed centrifugation to eliminate the plate
lets. The washed cells were suspended in RPMI-1640 containing 10% NBCS, 25mM
HEPES ,50µg/ml gentamycin and 100 µg/ml of penicillin and streptomycin. The
cell count was adjusted to 2 x 107 cells/ml and viability was checked by trypan blue
dye exclusion test.
To seperate the monocytes, the PBMC (2 x 107 cells/ml) was dispensed to
sterile glass petridishes (Borosil) or plastic petridishes (Millipore, USA) or 24
well tissue culture plates with 12 mm cover slips. The cells were allowed to adhere
for 2 hours and the nonadherent cells were extensively washed out with PBS. The
adherent cells were reincubated with RPMI-1640 for further 4-5 days for maturation.
Infection of neutrophils and monocytes
The PMNs and monocytes were infected in vitro with B. melitensis 16M or
Rev. 1. The infection was performed usually with a multiplicity of infection (MOI)
of 20 for 1 h at 37oC in antibiotic free media. The non-phagocytosed bacteria were
extensively washed out using PBS. The cells were then reincubated with RPMI-
1640 containing 50 µg/ml of gentamycin. At this concentration, only extracellular
bacteria were killed while intracellular bacteria survived (Gross et al., 2000). The
neutrophils were cultured for 24 h and monocytes for 48 hours. In some experiment,
neutrophils were treated with 50 µg/ml of cytosolic antigen or LPS and incubated
for 24 hours.
36

37. Detection of apoptosis
DNA fragmentation
Method described by Hayashi et al. (1997) was followed for isolating the
fragmented low molecular weight DNA. The PMN or monocytes, (1-2 x 106) were
washed out using chilled PBS after the end of incubation and pelletted by
centrifugation at 200 x g for 10 min in microcentrifuge tube. The supernatant was
discarded and to the pellet 0.5 ml of Tris-triton-X-EDTA (TTE) solution (Appendix)
was added and vortexed vigorously. The tubes were then centrifuged at 13000 x g
for 10 min at 4oC. The supernatant containing fragmented DNA was collected and
0.1 ml of ice cold NaCl (5M) was added. The tube was vortexed and 0.7 ml ice cold
isopropanol was added. It was again vortexed and the tubes were kept at –20oC
overnight to precipitate the DNA. The tubes were then centrifuged at 13000 x g for
10 min at 4oC and the supernatant was removed carefully without disturbing the
DNA pellet. Then the tubes were half-filled with ice-cold 70% ethanol and again
centrifuged at 13000 x g for 10 min at 4oC. Supernatant was removed and DNA
pellet was air dried for 4 h. The DNA pellet was incubated for 24 to 72 h in 30 µl of
TE buffer (Appendix) to dissolve it.
Agarose gel electrophoresis of DNA
The fragmentation of DNA was assessed by agarose gel electrophoresis
using 0.8% agarose gel in Tris-borate-EDTA (TBE) buffer (Appendix) containing
0.5 µg/ml of ethidium bromide in a horizontal gel electrophoresis apparatus. Sample
DNA (6 µl) was loaded into wells after diluting it with 5 x loading dye (Appendix)
and the gel was run for 2 hour at 70V. The DNA was visualized and photographed
under U-V gel documentation system (UVP-white/UV-trasilluminator, UK).
37

38. Morphological assessment of apoptosis by fluorescent microscopy
The method described by Duke and Cohen (1992) was followed with little
modifications. At the end of incubation, 10 µl of dye mixture (100 µg/ml acridine
orange + 100 µg/ml ethidium bromide in PBS) was added to the monocytes in the
petridish, mixed well and incubated for 5 minutes. The cells were then examined
under a 40 X dry objective of epifluorescent microscope (Nikon TS 100). The
apoptotic cells were identified from their nuclear morphology (bright chromatin,
highly condensed or fragmented nuclei) and atleast 200 cells were counted. The
apoptotic index was calculated as follows :
Total number of cells with apoptotic nuclei
% of apoptotic cells = ––––––––––––––––––––––––––––––––––––––x100
Total number of cells counted
Intracellular localization of brucellae in the infected PMNs and
monocytes
To locate the bacteria in the infected cells and to assess their role in the
apoptosis, the bacteria were stained by the method of Heesemann and Laufs (1985)
with slight modifications. The cells on coverslips or in 24 well plates were washed
throughly with PBS containing 5% bovine serum albumin (PBS-B). It was then
fixed with methanol at –15oC for 5 minutes. The smears were air dried and then
overlayed with anti Brucella serum raised in rabbits and incubated for 1 h at 370C.
The smears were then washed with PBS-B and goat anti-rabbit FITC conjugate
(1:200) in PBS was applied for one hour at 370C. The smears were washed with
PBS and treated with ethidium bromide (100µg/ml in PBS) for 5 min at 370C. The
smears were again washed with PBS and mounted on 90% glycerine in PBS and
observed in an epifluorescent microscope (Nikon, TS 100, Japan) using 40 X dry
objective. By this method brucellae have an intense green- yellow fluorescence
while nucleas of the cells have red fluorescence.
38

39. Simultaniously, the coverslips were fixed in methanol, stained with Giemsa
stain for 20 min and observed under the oil immersion objectives and the infected
cells were counted
Statistical Analysis
Statistical analysis was done by using students two tailed t-test for
independent means.
39

40. Results
Results
Chemical Analysis of antigen
The cytosolic antigen contained 6.1 mg/ml of protein and 0.198 mg/ml of
carbohydrate.It contained significant amount of nucleic acid as detected from its
absorbence at 260 and 280 nm.
Ion exchange chromatography
The ion exchange chromatographic profile of cytosolic antigen is given in
Fig. 1. There were four distinct peaks which eluted with the linear gradient of 0 to
1M NaCl. The protein and carbohydrate content of each fraction is given in Fig. 2.It
is apparent that proteins could not be seperated from the carbohydrate.
SDS-PAGE of cytosolic antigen
A total of 16 peptide bands with different molecular weights from 62kDa to
8kDa were visible when the cytosolic antigen was put to SDS-PAGE on a 12.5%
acrylamide gel(Fig.3).The major peptides were appeared to have MWs of
62,45,39,14,11 and 8kDa.The antigen was ultracentrifuged at 100,000xg and the
SDS-PAGE profile is shown in a gradient gel of 5-15% acrylamide in Fig.4.
40

41. Western blotting of cytosolic antigen
Western blot of cytosolic antigen using anti- B. melitensis 16 M sera raised
in rabbit, clinical sera from human and anti- B. abortus bovine sera is shown in Fig.
5. A common group of proteins (62kDa, 42kDa and 39 kDa) were found to be
reactive with all sera. The anti- B. abortus serum reacted weakly in comparison to
the anti- B. melitensis serum.
SDS-PAGE and Western blot analysis of fractions after ion exchange
chromatography
When SDS-PAGE was performed with different fractions of antigen after
ion exchange chromatography, peptides with MWs of 62 to 8kDa were visible in all
fractions.However ,in the first peak a prominent band of 39kDa was seen which
beecame faint subsequently(Fig.6).At the beginning of the first peak ,peptides with
MWs 34,29,23,11and 8kDa were found to be prominent.Interestingly,one 8kDa
band was prominantly visible in peak 1(Fig.6). In western blot (Fig.7) many bands
were visible with 39 and 8kDa being the prominent.
ELISA and dot-ELISA
The ELISA OD of fractions is given in the Fig. 8. Most of the fractions
showed reactivity with positive goat sera in ELISA as well as in dot ELISA. In dot
ELISA cattle serum was also used which reacted weakly in comparison to goat
serum .
Nitrite production by ovine PMNs in response to different stimulants
The effect of cytosolic antigen and LPS on the NO2– production by ovine
PMNs is shown in table 1 and Fig 9. There was slight increase in the production of
nitrite at lower lower concentration which became significant at a higher
41

42. concentration (300µg/ml) of LPS (P<0.05). However, stimulation with cytosolic
antigen did not elicit significant production or suppression of nitrite.Also the two
antigens showed no significant difference in the stimulation of nitrite production.On
stimulation of PMNs with heat killed B.melitensis 16M cells ,a dose dependent
suppression was noticed which was non significant.(Table 2,Fig.10).
Hydrogen peroxide production by ovine PMNs in response to different
stimulants
Table 3 and Fig. 11 shows the effect of cytosolic antigen and LPS on H2O2
production by ovine PMNs. Stimulation of PMNs with LPS showed
significant(P<0.05) suppression of H2O2 production in a dose-dependent manner.
Contrary to this the cytosolic antigen caused a dose-dependent increase in the H2O2
production which was significant . Heat killed B. melitensis 16M cells, too, showed
a dose-dependant elevation of H2O2 production (Table. 2,Fig.12) which was
significant at higher concentration (P <0.05).
MTT metabolism of PMNs on stimulation with different antigens
The MTT metabolism of PMNs after treating with LPS or cytosolic antigen
is given in Table 4 and Fig. 13. At the given concentration, the antigens did not
affect the MTT metabolism of PMNs. However, MTT metabolism increased with
increase in cell number when heat killed B. melitensis 16M cells was used as stimulant
(Table 2,Fig.14).
Phagocytic indices of B. melitensis 16M by ovine PMNs on stimulation
with LPS and cytosolic antigen
The rate of phagocytosis in all groups increased significantly with progression
of time (Table 5 and Fig. 15). Between the antigen no significant difference in
phagocytic index was noticed.
42

43. Intracellular killing indices of B. melitensis 16 M by ovine PMNs on
stimulation with LPS and cytosolic antigen
The intracellular killing indices have been given in Table 6 and Fig. 16 There
was significant (P<0.05) increase in the rate of killing in all group with increase in
incubation period. A mild suppression in killing index was noticed in LPS and
cytosolic antigen treated groups, which was non significant.
Apoptosis of neutrophils
In an attempt to see whether apoptosis in neutrophil was affected by B.
melitensis and cytosolic antigen, the agarose gel electrophoresis of DNA isolated
from control and stimulated PMNs were performed and the pattern is shown in
Fig. 17. None of the stimuli viz., live B. melitensis 16M, B. melitensis Rev 1,
heat killed B. melitensis 16M cells or cytosolic antigen appear to modulate
apoptosis as laddering of DNA was noticed in both control as well as in infected
group at 24 h. However, the invasion of PMNs with Brucella varied in different
groups. In the B. melitensis 16M treated group, 70% of the cells were infected.
But the B. melitensis Rev 1 could invade only 50% cells. The killed cells showed
the lowest invasiveness (30%) in PMNs. The invading bacteria could be located in
the PMNs by fluorescent antibody technique (FAT) (Fig. 18) as well as by Giemsa
staining.
Apoptosis in monocytes
As in case of neutrophils, role of B. melitensis if any, in modulation of
apoptosis in monocytes was also examined and the DNA analysis of treated
monocytes are given in Fig. 19. There was no DNA laddering in the B. melitensis
16M treated monocytes after 48 h indicating that Brucella probably prolonged the
monocyte’s life span suggesting prevention of apoptosis. In the non-infected cells,
43

44. there was evidence of spontanious cell death as indicated by the laddering of DNA.
Apoptotic index was also low (12%) in the infected monocytes while the non-
infected monocytes showed a high apoptotic index (32%). The phase contrast
microscopic appearance of monocytes is shown in Fig. 20. In the non-infected
group, the monocytes started detaching from the adhered surface as against the
infected monocytes which remained adhered to the surface.
44

45. Discussion
Discussion
Host defence and pathogenic mechanism of bacteria are continuously in-
teracting and constantly evolving. As such, the microorganisms have evolved mecha-
nisms to survive and replicate within complex environment inside the cells. These
pathogen use a wide variety of extracellular and/or intracellular components to
modulate the host cell environment in order to ensure their survival.
Neutrophils are a prominent component of host defence armamentarium
against invading microbial pathogens. These cells have the innate capacity to ingest
and kill a wide range of pathogens and are the first to arrive at the site of invasion,
because of which they are considered as the first line of defence. The neutrophils
accomplish this through production and release of a variety of toxic agents capable
of killing the invading pathogens. The two such agents are reactive oxygen interme-
diates (ROI) and reactive nitrogen intermediates (RNI), the production of which is
triggered by phagocytosis or exposure to certain inflammatory mediators includ-
ing cytokines (Robinson and Badwey, 1994, Fang et al., 1997).
Brucella is a facultative intracellular bacteria which use the phagocytic cells
such as PMNs and monocytes for their survival and multiplication. This intracellu-
lar survival is due to specific factors produced by the bacteria, that interfere with
45

46. the host cell physiology. It is not clear what roles the surface structure and cytosolic
components play in the virulence of these intracellular pathogens. Thus, the present
study was aimed to investigate the role of surface as well as cytoplasmic compo-
nents in the pathogenesis of Brucella infection in sheep. Their interaction with and
response of the ovine neutrophils has been studied.
In the present study method by Bhongbhibhat et al. (1970) was followed
after adopting some modifications from Jones et al. (1973) for extraction of
cytosolic antigen (brucellin). As observed by Jones et al. (1973) ultracentrifugation
was found to remove some high molecular weight components from the cytosolic
antigen. The antigen prepared in the present study contained significant amount of
nucleic acid material. Similar observations were also made by Alton et al. (1975).
A little amount of carbohydrate could also detected in this antigen as has been
previously reported (Bhongbhibhat et al., 1970; Cherwonogrodzky et al., 1990).
On SDS-PAGE analysis, more than 16 peptide bands with MWs ranging from
8 kDa to 62 kDa was noticed.Previous work by Fensterbank and Dubray (1980)
could detect 20 bands with major bands at 70-72 kDa, 35-45 kDa and 16-18 kDa.
Bachrach et al. (1994) and Blasco et al. (1994) noticed 20-30 proteins in this
antigen. Subsequently, Denoel et al. (1997a) reported variation in peptide banding
pattern on SDS-PAGE even in different batches of same antigen. Thus, the observed
difference in the number of peptide bands in the present study was some what ex-
pected and could be because of factors such as strain used, modifications in the
method and other variations in the experiment.
In immunoblotting, the antigen was found to be weakly reactive to clinical
serum from cattle and man. The cytosolic nature of this antigen may be the reason
for this weak reaction. However, it was reactive to hyperimmune sera raised against
B. melitensis 16M sonicate in rabbit as was also reported by Belzer et al. (1991).
46

47. The antigen being a cytosolic preparation, probably these are not at all or inad-
equately presented to the immune system in case of natural infection for genera-
tion of antibodies to the level that could be detected by the serological tests used.
Another reason could be the time elapsed from infection till the drawel of serum
used in the blotting experiment. Explanation of this requires further study using
serum at different point of time for a longer duration. Previous studies, too, has
shown that this antigen did not elicit the formation of agglutinating and comple-
ment fixing antibodies in cows and rabbit (Fensterbank and Dubray, 1980;
Fensterbank and Pardon, 1977).
Fractionation of cytosolic antigen has been attempted by many workers
(Bhongbhibhat et al., 1970; Denoel et al., 1997a). Bhongbhibhat et al. (1970)
fractionated this antigen on a Sephadex-G100 column and obtained 3 peaks having
MWs of 120 kDa, 30 kDa and 8-6 kDa. In the present study cytosolic antigen was
fractionated on a DEAE-sepharose column according to the method of Denoel et
al. (1997a). The separation could yield similar peaks with a 39 kDa protein as the
major protein. Besides this some other proteins with MWs of 34, 29, 23, 11 and 8
kDa were also noticed on SDS-PAGE analysis of the various peaks. However, the
procedure failed to separate the protein and carbohydrate, indicating strong asso-
ciation of lipopolysaccharide with the protein in Brucella. As the protein antigen
has been speculated to be the choice antigen for overcoming many of the associ-
ated problems in diagnosis and vaccination (Anon, 1986), some other methods
needs to be explored to achieve this separation. In the western blotting, too, the 39
kDa protein was found immunoreactive indicating that, this protein is one of the
major immunogens in cytosolic antigen as has been shown by Denoel et al., 1997b,
1997a).
In containing the infection and elimination of intracellular pathogens like
Brucella, cell mediated immunity (CMI) plays pivotal role. Of the two arms of the
47

48. CMI, the PMNs, i.e. neutrophils and mononuclear cells, viz. monocytes and
macrophages, the neutrophils are the first to encounter the pathogens at the site of
invasion (Clark, 1990). In the ensuing encounter, if the neutrophil fail to eliminate
the pathogen, brucellae spread and get localized in the regional lymphnodes drain-
ing the area. The neutrophils produce ROI and RNI in response to infection follow-
ing phagocytosis, so as to kill and destroy the brucellae. Thus, the neutrophils from
ovines, were separated from peripheral blood and its response to stimulation with
cytosolic antigen, LPS and killed B. melitensis 16 M cells were studied, with re-
spect to ROI and RNI release. Besides this, the phagocytosis and killing of B.
melitensis 16M cells by neutrophils were also examined.
For the isolation of PMNs from blood many methods have been described
(Ganz, 1987; Selested et al., 1984; Eggleton et al., 1989). In the present study a
method described by Eggleton et al. (1989) was followed as the PMNs isolated
from fresh blood by this method has been reported to be minimally exposed to
chemical stimuli. The neutrophils isolated in the present study had >90% purity as
determined by Giemsa staining and with >92% viability as determined by Trypan
blue dye exclusion test. The results were according to the findings of Eggleton et
al. (1989).
Recent researches on the free radicals suggest that they are very important
in the antibacterial activity of phagocytic cells. But limited work has been done on
the nitric oxide expression in the ovine neutrophils. The NO mediated killing by
Brucella has been reported in murine macrophages (Gross et al., 1998). Iyankan
(1998) also reported that the Brucella species are sensitive to NO in vitro. But
neutrophils are considered as poor source of NO. In the present study, too, no
significant production or suppression of NO by any stimuli was noticed which is
required for bactericidal action. Goff et al. (1996) reported that bovine neutrophils
were unable to produce NO under the stimulation with LPS, IFN- or TNF- , which
48

49. are otherwise considered as strong stimuli for inducing NO in macrophages. Stud-
ies by Padgett and Pruett (1995) also suggest that rat, mouse and human neutrophils
donot produce detectable amount of nitrite. They were of the opinion that the RNI
production by PMNs are insufficient to provide antimicrobial activity.
Alternatively, this could be because of a reaction of superoxide with RNI
produced leading to production of end products other than nitrite as has been re-
ported by Schmidt et al. (1989) and McCall et al. (1989). However, the role of
iNOS derived production of NO by neutrophils need to be investigated. Overall, the
RNI doesnot appear to the agent for antimicrobial effect in case of ovine neutrophils
as has been in case of rat, mouse and human neutrophils (Padgett and Pruett, 1995)
as opposed by the macrophages (Keller et al., 1990; Takema et al., 1991).
The production of hydrogen peroxide is a measure of ROI production by the
phagocytic cells. The respiratory burst produces superoxide anion leading to the
formation of various toxic agents such as H2O2, HOCl and possibly hydroxyl radi-
cals and singlet oxygen, catalysed by the membrane associated enzyme NADPH-
oxidase (Robinson and Badwey, 1994). In the present study Brucella LPS has been
found to suppress the H2O2 production by ovine PMNs, while cytosolic antigen and
heat killed Brucella cells elevated its production.
A LPS mediated inhibition of respiratory burst in PMNs by Brucella has
been proposed by Bertram et al. (1986) and Kreutzer et al. (1979). Iyankan (1998)
also reported a suppression of H2O2 production from bovine neutrophils on stimu-
lation with LPS. Rasool et al. (1992) reported Brucella LPS to be 100 to 1000
times less stimulatory than Salmonella LPS. The biological acitivity of Brucella
LPS differs considerably to a classical endotoxin activity of many LPS viz. Salmo-
nella and E. coli (Leong et al., 1970). Similar findings were also observed in the
present study where the MTT metabolism by the LPS and cytosolic antigen treated
PMNs were very less in line with the findings of Gallego Ruiz and Lapena Honso
49

50. (1989), who reported that LPS of Brucella was less reductive for NBT in caprine
neutrophils.
The ability of LPS to suppress the oxidative metabolism in PMNs, sup-
ported by the observations in the study of low H2O2 production and low killing
index after stimulation might support the contention that LPS has some role in the
intracellular survival. Earlier too, the LPS has been suggested to play role in the
survival of brucellae inside the phagocytic as well as nonphagocytic cells (Bertram
et al., 1986). However, the mechanism that involves the role of LPS in protection
of brucellae is not yet known.
The intracellular survival of Brucella are modulated by many factors. In the
present investigation, attempt was made to study whether preactivation of neutrophils
with Brucella cell components will alter their phagocytic and intracellular killing
ability. This preactivation is not a natural situation. But it was attempted because of
the poor penetration of the active components of the extract into the PMNs as
reported earier by Frenchick et al. (1985). The results in the present investigation
suggest that the stimulation of PMNs with LPS and cytosolic antigen did elevate
the phagocytic response. But the killing ability of PMNs were negatively affected
leading to some degree of suppression by LPS and cytosolic antigen. The suppres-
sion was little more marked with LPS, though non-significant statistically. Similar
observations were also made by Caron et al. (1994a), where they have showed that
stimulation of monocytes with LPS enhances the phagocytosis of B. suis. Freevert
et al. (1998) reported that in vivo stimulation of rats with LPS was found to asso-
ciated with increased phagocytosis in alveolar macrophages. Frenchick et al. (1985)
could observe an inhibition of phagosome-lysosome fusion on macrophages after
treating with water soluble extracts of Brucella. This may help the brucellae from
the intracellular killing. The suppression of H2O2 production from PMNs by LPS
may also contribute to this reduced killing ability of PMNs.
50

51. The intracellular fate of brucellae may depend on the bacterial species or
the type of phagocyte ingesting them. Opsonization with complement in vitro leads
to uptake and killing of B. abortus by human PMNs where as the more virulent B.
melitensis survives under these conditions (Young et al., 1985). Recently,
Kusumawati et al. (2000) observed no effect on opsonization as the nonopsonized
bacteria entered and differentiated inside the human monocyte as efficiently as the
opsonized bacteria.
In the present investigation phagocytosis experiments were performed af-
ter opsonizing the brucellae with normal heat inactivated sheep serum. It was ob-
served that the strain affected the phagocytosis by PMNs as the B. melitensis 16M
infected the highest percentage of neutrophils followed by Rev 1 and B. melitensis
16M killed cells. This indicated some relationship between the infectivity and viru-
lence of Brucella strains as the B. melitensis 16M is the virulent strain ,while Rev
1 is an attenuated vaccine strain (Montaraz and Winter, 1986). But no such refer-
ences could be found in the available literature. However, relationship between the
survival of brucellae inside the phagocytes as well as nonphagocytic cells with
their virulence has been demonstrated (Detilleux et al. 1990; Pizarro-Cerda et al.,
1998), with virulent Brucella strain surviving the phagocytes while the nonvirulent
or attenuated strains were killed and digested (Jones and Winter, 1992). The prac-
tical implication of this relationships could be increased survivability of the brucellae
inside the cell culminating in the establishment of infection and consequently dis-
ease in the host.
Many intracellular pathogens have been found to modulate apoptosis in
phagocytic cells (Gao and Kwaik, 2000). In the present study, none of the stimuli
viz. B. melitensis 16M live cells, killed cells, B. melitensis Rev 1 live cells or
cytosolic antigen were found to modulate the PMN apoptosis. On the other hands,
in monocyte, the B. melitensis 16M appeared to inhibit the apoptosis (fig. 19). The
51

52. finding derives support from observations of Gross et al. (2000), who showed that
B. suis infection in human monocyte modulated its apoptotic response to the ad-
vantage of the pathogen preventing the host cell elimination. This might well repre-
sent as one strategy of many known as well as unknown of Brucella for develop-
ment in infected host cell as has been proposed for some other intracellular patho-
gens (Gross et al. 2000). However, no reference could be traced on the apoptotic
response of PMNs with respect to Brucella infection. The bacteria like
Streptococcus pneumoniae induce PMN cell death, while pneumolysin induce
necrosis in PMNs (Zysk, 2000). The work by Baran et al. (1996) also says that
PMNs and monocytes can behave differently to phagocytosis of bacteria. They
observed that infection with S. aureus, E. coli, P. aereginosa or S. enterica pro-
mote monocyte apoptosis, but prolong the PMN life span. Such prolongation of
phagocytic cell life span with brucellae inside, protect the pathogen from the
microbicidal factors including antibodies in the external environment of the
phagocytes. It has been observed in case of many other bacteria (Gao and Kwaik,
2000). However, the exact role played by some cell components as well as the
whole brucellae in affecting or modulating the apoptosis of phagocytic cells need
to be throughly investigated.
The inhibition of apoptosis in phagocytic cells have also been noticed in
Chlamydia infected cells (Fan et al., 1998), M. bovis infection (Kremer et al.,
1997) and Leishmania infection (Moore and Matlashewski, 1994). There are many
mechanisms by which the bacteria modulate apoptosis. It was found that TNF-
partially mediates antiapoptotic effect of M. tuberculosis. Caron et al. (1994b)
reported that the capacity of Brucella spp. to use pathways avoiding TNF- pro-
duction during infection may be considered a major attribute of virulence Gross et
al. (2000) indicated that brucellae trigger a cell signalling which inturrupt the IFN-
apoptotic pathway, blocking a central step of apoptosis in invaded cells like the
52

53. suppression of mitochondrial cytochrome C release necessary for caspase activa-
tion in cytoplasm. This inhibit several apoptotic pathways. Modulation of host cell
apoptosis could eliminate key defence cells that are necessary to eliminate patho-
gen, inhibit or allow bacterial replication, facilitate the release of intracellular bac-
teria after termination of intracellular replication or promote inflamation, which
aids clearance or prevents further spread of pathogen within tissues (Gao and Kwaik,
2000). Further investigation is necessary to resolve the molecular events occuring
following Brucella infection in the phagocytic cells, which modulate the host cell
apoptosis. Such studies could thus, help understanding the disease pathogenesis.
thus on the basis of the present study following conclusions can be made.
1. The cytosolic fraction of Brucella elicit poor humoral immune response.
2. A 39 kDa protein is the major component of the cytosolic antigen, besides
some other minor peptides at 14 kDa and 11 kDa.
3. Chromatographic separation appears to be of little aid in separating the an-
tigen of choice. Other methods need to be explored.
4. Ovine neutrophils appear to be poor producers of NO, while Brucella LPS
inhibit H2O2 production from PMNs.
5. The LPS and cytosolic antigen of brucellae enhances the phagocytic rate
but reduce the killing ability of PMNs.
6. B. melitensis 16M inhibit the programmed cell death of ovine monocytes,
but donot appear to have any effect on neutrophils so far as apoptosis is
concerned. However this requires further studies.
53

54. Summary
abstract
Brucellosis still remains as an important zoonotic disease worldwide. The
organism can survive and multiply inside the phagocytic cells. The mechanisms and
the factors of Brucella spp. that modulate its survival is still unclear. In the present
investigation an attempt has been made to identify the antigen responsible for this.
Interaction of the neutrophils with Brucella has also been analysed.
The cytosolic antigen of B. melitensis B115 was prepared by cold-hypertonic
saline-ethanol extraction method and was found to contain high protein with little
carbohydrate. This antigen produced > 16 peptide bands in the SDS-PAGE with
molecular weights ranging from 8 kDa to 62 kDa. In the western blot, the antigen
reacted poorly with clinical sera from human and cattle. But in hyperimmune sera a
common group of proteins with molecular weights 62 kDa, 42 kDa and 39 kDa
were found to be immunogenic.
The antigen was fractionated by anion exchange chromatography, which pro-
duced four peaks. A 39 kDa protein was highly reactive to antisera in western blot-
ting besides some minor bands at MWs of 34, 29, 23, 11 and 8 kDa. However, all
the fractions from the peaks showed immuno-reactivity in ELISA. In dot-ELISA
the bovine serum reacted weakly in comparison to caprine serum.
54

55. Studies on the interaction of the cytosolic antigen with ovine PMNs was
carried out and compared with heat killed B. melitensis 16M cells and Brucella
LPS.
The PMNs were separated using isotonic ammonium chloride and was of
>90% purity and >92% viability. These antigens were not found to be stimulatory
for nitrite production from PMNs. But H2O2 production showed a dose dependent
elevation after the stimulation with heat killed cells and cytosolic antigen. On the
other hand, Brucella LPS caused a dose dependent suppression of H2O2 produc-
tion. The MTT metabolism by PMNs was not affected by the LPS and cytosolic
antigen, but the heat killed Brucella cells elevated the MTT metabolism by PMNs.
The prestimulation of PMNs with LPS and cytosolic antigen enhanced the
phagocytosis of B. melitensis 16M. But the intracellular killing was lowered non-
significantly, suggesting a minor role of these antigens on the intracellular survival
of Brucella. The invasiveness of the various Brucella strains varied, with B.
melitensis 16M, a virulent strain invading 70% of PMNs, followed by Rev. 1, a
vaccine strain (50%). The killed cells were the least invasive (30%).
To investigate the molecular mechanism of intracellular survival of Brucella,
modulation of apoptosis by these bacteria in PMNs and monocytes was studied.
After 48 h of infection with B. melitensis 16M, the ovine monocyte appeared healthy,
while the noninfected monocytes were detaching from the adhered surface. The
DNA analysis of monocyte showed typical ladder pattern in the noninfected
monocytes, but was absent in infected monocytes. The apoptotic index was also
low in infected monocytes (12%) as compared to noninfected monocytes (32%).
Such inhibition of apoptosis was not evident in the PMNs treated with various stimuli
like B. melitensis 16M, Rev 1, heat killed B. melitensis 16M or cytosolic antigen.
55

56. Mini Abstract
Mini Abstract
The cytosolic fraction brucellin from Brucella melitensis B115 on SDS-
PAGE separated into 16 peptide bands with molecular weights ranging from 8 kDa
to 62 kDa. Common groups of proteins with molecular weights 62 kDa, 42 kDa and
39 kDa were found to be immunogenic in Western blotting. On fractionation on a
DEAE sepharose column, the various fractions of man antigen showed poor reac-
tivity to bovine sera in comparison to goat sera on dot-ELISA. None of the stimuli
produce significant change in the nitrite production by ovine neutrophils. But H2O2
production was suppressed by Brucella LPS, while it was enhanced by killed cells
and cytosolic antigen. The preactivation of PMNs with LPS or cytosolic antigen
elevated the phagocytic response of PMNs but the intracellular killing activity was
nonsignificantly suppressed. In the ovine monocytes the B. melitensis 16M was
found to inhibit the spontaniously occuring apoptosis. But no such inhibition of
apoptosis was noticed in PMNs treated with B. melitensis 16M, B. melitensis Rev
1, heat killed cells or cytosolic antigen.
56

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57

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