EFFECT OF SMOOTH AND ROUGH STRAIN BRUCELLA ABORTUS …

Microsoft Word - Title.1 DOC(LIPOPOLYSACCHARIDES) ON IMMUNE STIMULATION OF
BOVINES
By
ANIMAL SCIENCES, LAHORE- PAKISTAN
2010
DEDICATION Dedicated to my dear parents who devoted their lives to me. My success was made possible by my father who made me learn to fight against failure, and of course my mother as what I am today is all because of her prayers.
DR. RAHEELA AKHTAR
THE COMPASSIONATE,
THE MERCIFUL”.
“Behold! In the creation of the heavens and the earth; in the alternation
of night and day; in the sailing of the ships through the ocean for the
benefit of mankind; in the rain which Allah Sends down from the skies,
and the life which He gives therewith to an earth that is dead; in the
beasts of all kinds that He scatters through the earth; in the change of the
winds, and the clouds which they trail like their slaves between the sky
and the earth -- (Here) indeed are Signs for a people that are wise."
(Surah Al-Baqarah, 2:164)
OF MY RELIGION,
KNOWLEDGE MY WEAPON,
PATIENCE MY DRESS,
FAITH MY DIET,
(LIPOPOLYSACCHAIRDES) ON IMMUNE STIMULATION OF
BOVINES
REQUIREMENT FOR THE DEGREE
2010
To,
The Controller of Examinations, University of Veterinary and Animal Sciences, Lahore.
We, the supervisory Committee, certify that the contents and form of the
thesis, submitted by RAHEELA AKHTAR, have been found satisfactory and
recommend that it be processed for the evaluation by the External Examiner(s) for
Award of the Degree.
ACKNOWLEDGMENT
I owe special thanks to the most Gracious, Merciful, and Almighty
ALLAH who gave me the inspiration, thoughts and opportunity to complete
this task. I bow before my compassionate endowments to HOLY PROPHET
MUHAMMAD (peace be upon him) who is, ever a torch of guidance and
knowledge for humanity as a whole.
I deem it as my utmost pleasure to avail this opportunity to express the
heartiest gratitude and deep sense of obligation to my dedicated Supervisor,
Professor Dr. Zafar Iqbal Chaudhary, Dean Faculty of Veterinary Sciences,
Bhaud Din Zikria University, Multan, Pakistan, for his valuable suggestions,
keen interest, dexterous guidance, enlightened views, constructive criticism,
unfailing patience and inspiring attitude during my studies, research project,
and writing of this manuscript. Infect his day and night pursuance and sincere
efforts made this work to fruitful conclusion.
I gratefully acknowledge invaluable help render by my reverend co-
supervisor and renowned Brucella worker Prof. Dr. Yongqun Oliver HE,
Associate Professor and his research team (Prof. George W Jourdian, Dr.
Fang Chen, Charles B Larson, Halen, Kerthi, Andrew and Thom) University
of Michigan Medical School, USA, for giving me a chance to work with them
at world’s sixth ranked university. They gave me time, energy and offered me
solace, substances and insight during the conduct of this study. They were
always available when I needed them. In fact I do not hesitate to say that
without their untiring efforts, it would not have been possible for this work to
reach its present effective culmination.
I have the honor to express my deep sense of gratitude and profound
indebtedness to Dr. Beatriz Arellano, Department of Microbiology and
Immunology, National University of Mexico, USA for providing me with
moral support and all around help for the fulfillment of my research project.
I am deeply thankful to DR. Mansur ud Din Ahmad, Dr Aftab Anjum,
Dr Azhar Maqbool, Dr Muhammad Younus, UVAS, Lahore and Dr Nasir
Mehmood, School of Biological Sciences, University of Punjab, Lahore,
Pakistan for their personal interest, and valuable advices in my research
project, infect their advices will always serve as a beacon of light throughout
the course of my life. They always shared his extraordinary knowledge with me
that illuminated complex issues and enabled me to grasp their significance.
A big share of thanks goes to Dr Jean Namzek and her lab fellows
(Christ and Dolla), Unit of Laboratory Animal Medicine, University of
Michigan, USA for their technical guidance in research work and
constructive suggestions during my research.
I have no words to thank Dr Carlos Abril, University of Bern,
Swetzerland for his help in completion of this uphill task. It is imperative for
me to thank Dr David O Challghan, France, Dr Commander Nicky,
Veterinary Laboratory Agency, UK for their valuable suggestions and
guidance.
Last but not least, I am grateful to my family members for their endless
cooperation, assistance and encouragement during this research project.
DR. RAHEELA AKHTAR
BCG: Bacillus Calmette-Guerin
DCs: Dendritic cells
FBS: Fetal bovine serum
Kdo: 2-keto-3-deoxyoctonate
kDa: KilDalton
LPS: Lipopolysaccharide
LPSs: Lipopolysaccharides
PMNs: Polymorph nuclear cells
SG: Specific gravity
SLPS: Smooth lipopolysaccharide
TABLE OF CONTENTS
S. No. Chapters Page no.
1. INTRODUCTION 1
4. RESULTS 46
5. DISCUSSION 89
6. SUMMARY 110
LITERATURE CITED 112
1 Flow Chart of the Study 27
2 Rapid urea positive test for identification of Brucella abortus 47
3 A: SDS-PAGE of Brucella abortus smooth and rough lipopolysaccharides by silver staining.
B: SDS-PAGE of Brucella abortus smooth and rough lipopolysaccharides fractions by Coomassie blue staining.
49
4 4: Standard curve for Rf values of BioRad precision protein marker.
51
6 A: Isolated bovine neutrophils at magnification of 10x
B: Densogram showing isolated bovine neutrophils (a & c) and non neutrophils (b& d).
C: Histograms showing the separation of CH138A positive neutrophils and IgG negative control.
53
54-55
56
7 A: Isolated bovine peripheral blood macrophages are visualized at 10x.
B: Bovine isolated macrophages after seven days of culturing are visualized at 100x. Arrow indicates a mature macrophage.
58
59
9 Visualization of lysozyme release using the agarose plate assay. The LPS samples are (a) rough RB51 LPS, (b) smooth S2308, and (c) combined S2308+RB51 LPSs. The concentration of LPS fractions were 200µg/mL per well.
61
10 10: Standard curve of lysozyme release test 62
11 A: Induction of Lysozyme from Bovine Macrophages Treated with rough, smooth and combined Brucella LPS Fractions.
B: Induction of Lysozyme from Murine Mcrophages Treated with Rough, Smooth and Combined Brucella LPS Fractions.
C: Induction of Lysozyme from Bovine Neutrophils Treated with Rough, Smooth and Combined Brucella LPS Fractions.
63
64
65
12 A: Induction of Reactive Oxygen Species from Bovine Macrophages Treated with Rough, Smooth and Combined Brucella LPS Fractions.
B: Induction of Reactive Oxygen Species (ROS) from Murine Macrophages Treated with Rough, Smooth and Combined Brucella LPS Fractions.
C: Induction of Reactive Oxygen Species (ROS) from Bovine Neutrophils Treated with Rough, Smooth and Combined Brucella LPS Fractions.
67
68
69
13 Nitric Oxide Induction from bovine Macrophages Treated with Rough,
71
14 Sodium nitrite standard curve of for nitric oxide determination
71
15 A: Induction of Nitric Oxide by Bovine Macrophages Treated with Rough, Smooth and Combined Brucella LPS Fractions.
B: Induction of Nitric Oxide from Murine Macrophages Treated with Rough, Smooth and Combined Brucella LPS Fractions.
C: Induction of Nitric Oxide from Bovine Neutrophils Treated with Rough, Smooth and Combined Brucella LPS Fractions.
72
73
74
16 A: TNF-α Induction from Bovine Macrophages Treated with Brucella Rough, Smooth and Combined LPS Fractions.
B: TNF-α Induction from Murine Macrophages Treated with Brucella Rough, Smooth and Combined LPS Fractions.
76
17 A: IL-β Induction from Bovine Macrophages Treated with Brucella Rough, Smooth and Combined LPS Fractions.
B: IL-β Production from Murine Macrophages Treated with Brucella Rough, Smooth and Combined LPS Fractions.
77
78
18 A: IL-6 induction from Bovine Macrophages Treated with Brucella Rough, Smooth and Combined LPS Fractions.
B: IL-6 Induction from Murine Macrophages Treated with Brucella Rough, Smooth and Combined LPS Fractions.
79
19 A: IL-10 Induction from Bovine Macrophages Treated with Brucella Rough, Smooth and Combined LPS Fractions.
81
20 A : IL-12 Induction from Bovine Macrophages Treated with Brucella Rough, Smooth and Combined LPS Fractions.
83
21 CFU Analysis for Determination of Intracllular Survival of Brucella in Bovine Macrophages
85
B. Intracellular Survival of Brucella in Murine Macrophages.
86
23 PCR Amplification Products from Brucella abortus Positive Lymph Node Samples.
88
Sr. No. Title Page No.
1 Primer Sequences used for the Detection of Brucella abortus 43
2 A: Rf Values and Log Molecular Weight of Marker Bands.
B: Rf Values and Log Molecular Weight of Sample Bands.
50
3
A: Induction of Lysozyme from Bovine Macrophages Treated with Rough, Smooth and Combined Brucella LPS Fractions.
B: Induction of Lysozyme from Murine Macrophages Treated with Rough, Smooth and Combined Brucella LPS Fractions.
C: Induction of Lysozyme from Bovine Neutrophils Treated with Rough, Smooth and Combined Brucella LPS Fractions.
62
63
64
4
A: Induction of Reactive Oxygen Species (ROS) from Bovine Macrophages Treated with Rough, Smooth and Combined Brucella LPS Fractions.
B: Induction of Reactive Oxygen Species (ROS) from Murine Macrophages Treated with Rough, Smooth and Combined Brucella LPS Fractions.
C: Induction of Reactive Oxygen Species (ROS) from Bovine Neutrophils Treated with Rough, Smooth and Combined Brucella LPS Fractions.
66
67
68
5
A: Induction of Nitric oxide from Bovine Macrophages Treated with Rough, Smooth and Combined Brucella LPS Fractions.
B: Induction of Nitric Oxide from Murine Macrophages Treated with Rough, Smooth and Combined Brucella LPS Fractions.
C: Induction of Nitric Oxide from Bovine Neutrophils Treated with Rough, Smooth and Combined Brucella LPSs Fractions.
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73
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Introduction
1
Chapter-1
INTRODUCTION
Brucellosis is a spreading disease transmitted to animals and humans mainly
by direct contact between infected hosts or through oral, respiratory, cutaneous,
ocular and sexual routes (Neta et al., 2009). The six Brucella species exhibit variation
in their host specificities and pathogenicities. Frequency of this disease varies from
country to country, but is higher in agrarian countries including the Middle East and
South West Asia (Pappas and Memish, 2007). Several studies demonstrated that
brucellosis is also endemic in Pakistan and its incidence is increasing (Akhtar et al.,
1990; Ahmad and Munir, 1995; Ramazan, 1996; Nasir et al., 2004; Nasir et al., 2005;
Hussain et al., 2008). Brucellosis has a major impact in terms of economic losses due
to abortion of calves, reduced milk yield, infertility in the male, and potential
infection of humans (Greiner et al., 2009).
The etiological agent of brucellosis is a non-motile, non sporforming and
facultative intracellular bacteria of genus Brucella (Jean, 2005). It is a potential agent
for biological warfare. This has sparked a growing interest in its biology (Department
of Army, USA, 1977).
Brucella LPS is a biologically active component present in the cell
membrane and comprised of three domains:
Polysaccharide or O-side chains or O-antigen (non toxic portion)
Core polysaccharide
Lipid A (toxic portion)
O-antigen is a homopolymer consisting of 96-100 units of α 1-2 linked
perosamine. It is considered the immunodominant subunit of LPS (Caroff et al.,
1984; Ugalde et al., 2003). Structurally, the O-antigen resembles with that of Vibrio
cholera LPS, but is totally different from other enterobacteriaceae LPS (Duenas et al.,
Introduction
2
2004). The Middle portion of the LPS, the core polysaccharide, consists of the
trisaccharide 2-ketodeoxy 3-octonate (Kdo). The embedded part of lipid A is
composed of long chain saturated fatty acids and small amounts of hydroxylated fatty
acids, but lack β-OH mystric acid-linked fatty acids (Aragon et al., 1996). Wild type
and attenuated Brucella strains present smooth or rough types of LPS based on the
presence or absence of an O-chain. The virulence of Brucella species has been linked
with the type of LPS present (Zygmunt et al., 2009).
Brucella LPS is released into body fluids where it is readily ingested by
phagocytic cells through pinocytosis or receptor-mediated endocytosis (Poussin et al.,
1998). Two days after ingestion, Brucella LPS molecules are found inside
macrophages in small vacuoles that coalesce together. The lipid A moiety of LPS is
deacylated and dephosphorylated (Wuorela et al., 1993; Leyva-Cobian et al., 1997).
Brucella LPS is designated as a non-classical endotoxin that plays a pivotal role in
pathogenesis and modifies phagocytosis, phagolysosome fusion, cytokine secretion,
and apoptosis (Beninati et al., 2009). In contrast to most endotoxins, it is
nonpyrogenic, does not induce a localized Shwartzman reaction, does not increase the
susceptibility to histamine and does not activate complement to any significant level.
Despite these properties, it is reported as the major antibody-inducing antigen present
in Brucella infections (Zaitseva et al., 1996).
Invading Brucellae are mainly found in and metabolized by short-lived
neutrophils and long-lived macrophages. Two mechanisms are used, non-oxidative or
oxidative pathways. Under non-oxidative conditions, antimicrobial proteins such as
lysozyme (LZ) and peptides are released, while under oxidative conditions,
brucellicidal oxidants are formed. These oxidants are recognized as reactive oxygen
species (ROS), including superoxide anions, hydrogen peroxide, chloramines,
hydroxyl radicals and hydrochlorous acid (Liautard et al., 1996) and reactive nitrogen
intermediates (RNI) (Jinkyung and Splitter, 2003).
Introduction
3
Killing Brucella requires enhanced macrophage and neutrophil stimulation
that augment the release of LZ, ROS, and RNI and ultimately cell mediated
immunity. Lysozyme has the ability to degrade Brucella cell walls by hydrolyzing
constituent peptidoglycan molecule and cleaving the glycosidic bonds between NAG
and NAM (Mc-Ghee et al., 1970; Maria et al., 2003), ROS damages fatty acid side
chains of the Brucella cell wall (Jinkyung and Splitter, 2003). RNI inhibits cellular
respiration (Gross et al., 1998). Therefore, taken together these metabolic functions
(LZ, ROS, RNI) are of importance for antibrucella activity. Additional support for
this statement comes from the observation that treatment of macrophages with
methylene blue (an electron carrier) enhances killing of intracellular Brucellae,
indicating their susceptibility to ROS. Similarly, inhibition of RNI by NG-
monomethyl L-arginine resulted in blocking of macrophage anti-Brucella activity
(Jiang et al., 1993b).
Activation of immune cells (macrophages and neutrophils) can be achieved
with number of stimulators including bacterial cell-wall components,
lipopolysaccharides (LPS), cytokines, interferon-gamma (IFN-γ) and tumor necrosis
factor (TNF-α). Each of these factors acts independently or in combination to elicit
various states of activation (Connelly et al., 2003). Evidence (Goldstein et al., 1992;
Rasool et al., 1992) exists that of the immune cells stimulators listed, Brucella LPS is
of particularly important to study, because it exhibits minimal endotoxic activity
compared to other stimulators (10,000 times less toxic than E. coli LPS and 1000
times less toxic than Salmonella typhimurium LPS). This property may contribute to
its potential use for immune cell stimulation studies and as an adjuvant in Brucella
vaccine (Goldstein et al., 1992). Attention has been focused on the fact that Brucella
LPS can serve as potential cell stimulator without adverse effect.
Previous studies on Brucella smooth and rough LPS have emphasized
extraction procedures (Alina et al., 2007), biological properties (Schurig et al., 1991;
Aragon et al., 1996), anti-LPS antibodies detection (Khatun et al., 2009), vaccine
Introduction
4
design (Apurba et al., 2002), immunogenic mimicking of LPS epitopes (Benninatic et
al., 2009), macrophage activation in an artificial metastasis model (Schultz et al.,
1978) and comparison of the LPS properties of Brucella with the LPSs of other
species (Escherichia coli and Salmonella) (Jarvis et al., 2002). However, the precise
role of LPS in induction of anti-Brucella immunity remains unresolved (Billard et al.,
2007). To better understand the differential immunological roles of smooth and
rough Brucella LPS, it is critical to study their differential stimulatory activities in
treated macrophages and neutrophils.
The present study focuses on the protective immunological properties of
rough Brucella LPS resulting from enhanced production of LZ, ROS, RNI and pro-
inflammatory cytokines. It is hypothesized that smooth and rough Brucella LPSs
utilize differential stimulatory activities on lysozyme resulting in oxidative burst, and
nitric oxide production which in turn could lead to different brucellicidal reactions of
infected cells (macrophages and neutrophils). In this study the rough and smooth
LPSs were used for the first time to study the reaction of immune cells and to
evaluate the differences in stimulatory activities of individual and combined LPSs.
Background of study
The unique properties of low pyrogenicity, minimal endotoxic shock and good
stimulation of immune cells suggests that Brucella abortus LPS has potential as a
candidate for vaccine preparation or as a vaccine carrier (Ayman et al., 2001).
However, most studies have emphasized the interaction of Brucella abortus LPS with
human and murine macrophages. Limited studies have focused on bovine
macrophages and neutrophils. Studies on the immune response of Brucella abortus in
bovines are of utmost importance since they are the major reservoir of brucellosis and
are responsible for transmitting the disease to humans. Moreover, there is a need for
insight into the differential activities of smooth and rough Brucella abortus LPSs, i.e.
which exhibits greater stimulatory activity. The present study also addresses possible
differential interaction of bovine and murine macrophages with Brucella abortus
smooth, rough, and with combined (rough + smooth) LPSs.
Introduction
5
Mice are not the natural host of Brucella and display a certain resistance to infection
(Zhan and Cheers, 1998). Much of the work has been performed in the mouse model
due to its convenient use as compared to other lab animals. In the present study, the
murine macrophages were compared with bovine macrophages after stimulation with
Brucella abortus LPSs.
The present study was designed to achieve the following objectives:
1- To extract, purify and characterize Brucella abortus smooth and rough LPSs.
2- To explore the mechanism of stimulation of bovine macrophages, neutrophils
and murine macrophages by Brucella rough and smooth LPS (separately and
in combination)
3- To determine (a) the outcome of infection by observing the interaction
between the pathogen (Brucella) and (b) LPS stimulated immune cells and to
find out the type of LPS (smooth, rough or combined) and optimal
concentration (00, 0.02, 0.2, 2, 20, 200 µg/ml) of Brucella LPS that yields
maximum stimulatory activity.
4- To compare the susceptibility of murine and bovine macrophages to Brucella
LPS stimulus in order to determine differences in species susceptibility.
5- To determine the major cellular organelles involved in Brucella death by
comparing LPS mediated stimulation of bovine macrophages and neutrophils.
Review of Literature
REVIEW OF LITERATURE
Brucellosis or Malta fever is a serious public health problem caused by a
Gram-negative bacterium of the genus Brucella. The genus contains six species.
These are Brucella abortus, Brucella melitensis, Brucella suis, Brucella neotomae,
Brucella ovis and Brucella canis. Brucellosis has been an emerging disease of interest
since 79 A.D when skeletons of Romans buried with carbonized cheese revealed
brucellosis lesions on examination by scanning electron microscope (Capasso, 2002)
and later the discovery of Brucella melitensis by Bruce in 1887 until the identification
of marine reservoirs (Godfroid et al., 2005). Bovine brucellosis is characterized by
abortion in last trimester of gestation, stillbirth and weak calves. The commonly
observed clinical signs are pyrexia, anorexia, polyarthritis, meningitis, pneumonia
and endocarditis. The postmortem lesions include in placentitis in dam and interstitial
pneumonia in aborted foetus (Sauret and Vilissova, 2002). Human infection is due to
consumption of contaminated and/or unpasteurized milk, and milk products (cheese),
or laboratory acquired infection. (Young, 1983). Brucellosis is currently ranked at
number fifth on the list of important diseases of the world (Corbel, 1997). In some
geographical areas, Brucella melitensis has emerged as a cause of brucellosis
infection in non-bovine species including ovines and caprines. Brucellosis has been
an important animal and health issue in Pakistan from many years (Sheikh et al.,
1967). The incidence of diseases is very high in different districts of Punjab province
(Akhtar et al., 2010c). It not only affects humans zoonotically, but also exerts an
adverse influence on the Pakastani economy by perturbing the livestock sector. This
sector comprises 49.6% of Pakastan’s agriculture income and shares 10.4% of its
national gross domestic product (GDP) (Economy survey 2008-09). Consumption of
contaminated foods and occupational contact with animals remain the major sources
of infection. Human-to-human transmission by tissue transplantation or sexual
contact has been rarely reported. Brucellosis can be diagnosed by culturing and
isolating the pathogen and is standardized by the rose Bengal plate test (RBPT), milk
ring test (MRT), serum agglutination (SAT), complement fixation (CFT) and PCR
assay. Prevention of human brucellosis depends on the control of this disease in
animals.
Extraction and Purification of Brucella abortus Rough and Smooth Strains LPS The presence of LPS as round bodies embedded in Brucella cell wall was
reported by Bobo and Foster, (1964). With the aid of electron microscopy they
proposed that LPS was a subunit of cell wall. They used several sequential treatments
for lysis of Brucella cell wall including trypsin, pronase and lysozyme. These
reagents were more effective for the extraction of LPS than ribonuclease, pepsin,
lipase and non-enzymatic agents. The properties of Brucella LPS were studied by
Nicolas et al., (2006) who demonstrated by biochemical techniques and electron
microscopy that Brucella abortus LPS was recirculated through macrophages.
Furthermore, LPS was shown to act as detergent and was able to remain intact for
many months in macrophages by resisting destruction by methyl β-cyclodextrin
through the development of rigid surface membrane complexes. Many Brucella
workers developed different methods of LPS extraction to observe the relationship
between the extraction procedure and the biological properties of Brucella LPS.
Westphal and Tann, (1965) showed that rough Brucella LPS could isolated from an
aqueous phase whereas smooth LPS could be purified only from the phenol phase.
This was confirmed by Redfearn (1960) who showed that Brucella rough and smooth
LPSs were not only different from each other in their mode of isolation, but also with
respect to their biological properties. Baker and Wilson (1965) compared the
chemical composition and biological properties of B. abortus LPS and E. coli LPS
(based upon nitrogen, phosphorus, fatty acid amides, ester, hexose, hexosamine and
9
total fatty acids). They found that LPS preparations from E. coli possessed much
greater biological activity of hypoferremia and lethality in mice than B. abortus LPS.
Smooth and rough Brucella LPS preparations were also compared by Moreno et al.,
(1979), using two different modifications of phenol-water extraction method
(Westphal and Tann, 1969). They used chemical, immunological and SDS-PAGE
analysis to determine the difference in properties of Brucella rough and smooth LPS.
They not only revealed differences in the protein contents of two fractions (S-LPS
and R-LPS) but also in their isolation characteristics. The smooth LPS was obtained
from the phenol phase and the rough LPS was obtained from the aqueous phase. The
differences noted in biological activity of LPS were on per mass basis. The LPS
associated proteins were not the part of either LPS structure; rather they were firmly
attached to S-LPS and were not removed during purification.
Differences in the biological properties of smooth and rough Brucella LPSs
found by various laboratories prompted development for more effectual isolation
methods of each LPS. Jones et al., (1976b) demonstrated that both S and R LPS could
not be consistently extracted by previously developed methods as that the aqueous
phase was totally deficient in LPS while phenol phase had chief fraction of LPS.
Darveau and Hancock, (1983) developed an improved method for the extraction of
both smooth and rough LPS with high yields (51 to 81%) and purity. They used a
combination of cell breakage and nucleic acid digestion along with ethanol water
extraction method. The dried bacterial cells were preferred over wet cells for isolation
of large quantity of LPS. The contamination with protein (0.1%), nucleic acids (1%),
lipids (2 to 5%), and other bacterial products was much less than that obtained using
previously used methods. Subsequently, it was affirmed by the studies of Kreutzer et
al., (1979 a) that smooth intermediate (45/0) LPS was present in the aqueous and
phenol phases. Rough LPS (45/20) was only present in the aqueous phase. It was
proposed that in addition to being toxic, the phenol-soluble S-LPS could be a major
virulence factor in intracellular survival of B. abortus. The authors also proposed that
10
in addition to LPS, the rest of the components of aqueous and phenolic phase also
differed. LPS in the phenolic phase contained nine to 16 times less heptose and lower
amounts of dideoxyaldoses than did the aqueous phase. The major neutral sugars
found were glucose, galactose, and mannose. fβ-hydroxymyristic acid which is a
fatty acid and a common marker of enteric LPS, was absent. The only fatty acids
present, hydroxylated and nonhydroxylated with chain lengths of 16, 18, and 20
carbons respectively (Kreutzer et al., 1979 a), were present in the higher amounts.
Detailed characterization of the LPS from rough B. abortus, B. melitensis, B. ovis and
B. canis LPSs was made by Moreno et al., (1984). These authors also compared the
structural properties of Brucella rough and smooth LPSs side by side and
demonstrated a granular pattern on electron micrograph of Brucella R-LPS. This was
in contrast to characteristic lamellar structures found for S-LPS. The chemical,
physical and serological characteristics of Brucella rough and smooth LPS were
studied and monospecific mouse sera were developed against B. ovis R-LPS. Another
advanced, modified and specific method for Brucella rough LPS extraction was
described by Galanos et al., (1969). The extraction mixture was monophasic,
containing aqueous phenol, chloroform and petroleum ether. This method offers the
advantage that it can be carried out below 10°C and is easy to perform and yields
higher amounts of LPS than phenol-water extraction procedures.
Much of the LPS extraction had been performed with Brucella smooth strains
(Moreno et al., 1981; Caroff et al., 1984; Moriyon and Montanes, 1985; Aragon et
al., 1996) using Moreno’s method. However, this method has some drawbacks.
Therefore, Wu et al., (1987) attempted to modify Moreno’s procedure by employing
quick freezing and thawing to lyse B. abortus cells. This was followed by
ultrasonication to eliminate non-membrane-bound material, extraction with phenol
and washing ten times with water to remove chromogen, polysaccharides and nucleic
acids (Velasco et al., 2000). Protein contamination varied between 16% and 42%
(wt/wt) as estimated by the dye binding test using Coomassie Brilliant Blue (G-250)
11
as suggested by Bradford et al., 1976, and 17% and 60% by using the Lowry phenol
method (Lowery et al., 1951) with bovine serum albumin as a standard. Contrary to
previous techniques used, a higher yield of smooth LPS (3.6% to 7.7%) was obtained.
Suarez et al., (1988) introduced the use of hot saline for LPS extraction from
rough Brucella LPS. The pelleted LPS was analyzed for lipids, sugars, Kdo content,
and by immunoelectrophoresis and SDS-PAGE methodologies.
By the end of 1980, many techniques for extraction of Brucella rough and
smooth LPS had been reported. In an attempt to select the best technique, Malikov et
al., (1989) compared various methods of LPS extraction. LPS was purified from
Brucella virulent and vaccine strains using phenol water extraction. They used
Bovin's method of LPS extraction (Bovin and Mesrobeanu., 1935) and a mild alkaline
hydrolysis method, separately and in conjunction. Each LPS preparation (rough and
smooth) was analyzed for its chemical composition, immunological characteristics
and serological activity. The results suggested that the mild alkaline hydrolysis
method according to Bovin's protocol was optimal and the resulting LPS preparation
from virulent strains yielded a more consistent and sensitive product for use for the
passive hemagglutination tests than LPS from a vaccinal strain. Their work also
showed that the soluble complex of lipid A obtained from Brucella LPS had
serological activity. Garin-Bastuji et al., (1990) also combined several methods for
the extraction of smooth LPS from different biovars of B. abortus, B. melitensis, and
B. suis. They used a combination of hot phenol-water treatment, hot sodium dodecyl
sulfate followed by treatment with proteinase K and extraction with dimethyl
sulfoxide.
All methodologies that had been used up to this point in time for extraction of
either rough or smooth Brucella LPS were quite laborious and time consuming.
Sunsequently, Yi and Hackett (2000) developed a fast method of LPS extraction
using a commercial RNA-isolating reagent that facilitated the separation of LPS or
lipid A from small amounts of bacterial cells. The method did not require specialized
12
equipment and permit the use of large numbers of samples. The major constituents of
the commercial RNA isolating reagent (Tri-Reagent) were phenol and guanidinium
thiocyanate contained in aqueous solution. Bacterial cell membranes were lysed with
guanidinium thiocyanate, which eliminated the need for specialized equipment. LPS
and its main components such as lipid A were analyzed and the results compared to
the conventional hot phenol-water extraction. SDS-PAGE analysis revealed that the
LPS fraction was cleaner and less degraded, although loss of phosphate or fatty acyl
side chains from lipid A occurred. This method also yielded preparations with low
free fatty side chains and phosphate content. The total phosphate content was up to
11% by this method compared to 58% by hot phenol-water extraction. This method
required only two days for the isolation of LPS as compared to the hot phenol-water
extraction that took two weeks. Alina et al., (2007) obtained highly purified LPS
preparations by repurification of commercial or laboratory prepared LPS. They used
three methods for the repurification including; heat-detergent promoted
repurification, heat promoted repurification and acid-solvent promoted repurification
method. The last method was further selected and used. They also used triethylamine-
deoxycholate sodium for repurification after extraction of LPS and major
contaminants removal. This method is applicable to various LPS preparations and
does not require the use of phenol. The integrity and purity of the Brucella LPS
obtained was established by SDS-PAGE and matrix-assisted laser desorption
ionization mass spectrometry.
LPS Purification
Brucella LPS was purified by Zygmunt et al., (1988) using ultrafiltration,
repetitive gel filtration, high-performance liquid chromatography, SDS-PAGE, gas-
liquid chromatography mass spectroscopy, and 13C and 1H NMR spectroscopy.
Phillip et al., (1989) found that purification of LPS using proteinase K-treatment was
advantageous since proteinase K did not change the immunochemical, and other
defining properties of LPS. Butanol was used to extract LPS from smooth B. abortus
(S2308 and S-19). These procedures together yielded LPS preparations containing
less than one percent proteins. The LPS obtained was analyzed by chemical analysis,
SDS-PAGE, cesium chloride gradients, electron microscopy and gel
immunodiffusion. Furthermore, the butanol procedure proved the method of choice
for the extraction of LPS from B. abortus. Analysis of proteinase K treated LPS
preparations from sixteen smooth Brucella strains by SDS-PAGE after periodic acid
oxidation and silver staining revealed two differing profiles. LPS from B. abortus
was exhibited as regularly spaced narrow bands; while for theB. meliensis LPS, gels
contained regularly spaced doublets (Dubray and Limet, (1987).
SDS-PAGE Analysis
Sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
uses an anionic detergent (SDS) to denature proteins. The protein molecules are
“linearized”. One SDS molecule binds to two amino acids. The charge to mass ratio
of the denatured proteins in a mixture is constant. Therefore, the protein molecules
moves toward the anode in the gel based on their molecular weights only, and thus
are separated.
A modified silver staining method for LPS detection was developed by Tsai
and Frasch, (1982). The silver stain is 500 times more sensitive than periodic acid-
Schiff stain of LPS and is able to detect less than 5 ng of R-LPS. Polypeptide and
polysaccharide components of B. abortus smooth LPS (S99) were analyzed by
Dubray and Charriaut, (1983) using SDS-PAGE gels stained with Coomassie blue or
silver staining. Triton X-100 was used to separate the cell wall from cytoplasmic
material prior to LPS isolation. Triton X-100 is a non-inonic surfactant which has a
hydrophilic polyethylene oxide group. The surface active agents (surfactants) have
been known for many years to possess bactericidal activity and in some cases
bacteriolytic activity as well. The bactericidal effects of ionic surfactants were
attributed to their ability to electrostatically bind to, and denature cellular enzymes
(Volko, 1946) or to disrupt the cellular components (Hotchkiss, 1946). The liquid
effects of surfactants on bacteria; however, require a more complex interpretation due
to the presence of rigid peptidoglycan layer surrounding bacterial cells which is not
present in erythrocytes. Triton X-100 may induce cellular lysis by releasing a lipid
inhibitor of the cellular autolytic enzyme. The SDS-soluble fraction revealed two
major components: a high molecular weight broad band (S-LPS) and a 43kDa
polypeptide band. The B. abortus outer membrane was composed of four major
components: LPS (43kDa) and polypeptides (36-37-38kDa) and glycopeptides (25-
26-27kDa). The S-LPS fraction appeared as broad band of high molecular weight as
compared to the multiple regularly spaced bands of high molecular weight found in
Escherichia coli S-LPS gels. A phenol extracted, alkali-treated LPS preparation from
the vaccine strain (S 19) of B. abortus was analyzed by Sowa et al., (1986) using
SDS-PAGE and silver staining. Their gels revealed ten polydispersed bands.
Nitrocellulose immunoblots showed that all ten reacted with bovine anti-B. abortus
polyclonal sera. Only six bands were antigenically reactive with anti-B. abortus O-
antigen murine monoclonal antibody. These findings are attributed to differences in
either the core or O-antigen side chain structure and covalently bound protein.
Schurig et al., (1991) suggested that the SDS-PAGE profile of Brucella rough (RB51)
LPS was not sufficient evidence for the absence of O-chain. Therefore, they
performed Western blot analysis with a monoclonal antibody (BRU 38) specific for
the O-chain of smooth Brucella LPS. Their results established that R-LPS lacked O-
chain as compared to the parenteral smooth strain 2308, although rough RB51
resembled its parental 2308 strain in its ability to metabolize erythritol. Moreover,
intraperitoneal inoculation of RB51 strain into mice or undergoing in vitro passages
did not convert it back to the smooth form. Freer et al., (1995) measured the
molecular size of each LPS component (O-polysaccharide, core oligosaccharide and
lipid A). SDS-PAGE and Western blotting revealed antigenic heterogeneity in
Brucella LPSs. Three dense high-molecular-weight bands related to S-LPS, a low-
molecular-weight band corresponding to the O-antigen were absent in rough LPS. B.
15
abortus R-LPS displayed four bands. On other hand, Cloeckaert et al., (2002) found
that RB51 strain produced low levels of a M-like O-antigen. This group used
monoclonal antibodies directed against O-polysaccharide and performed SDS-PAGE
along with Western blots to show that B. abortus RB51 lacked the O-side chain.
Although the increasing periodate concentration and reaction time of LPS
with sodium periodate can affect efficacy of this assay, still it is an improved method
for LPS quantification (Quesenberry and Lee 1996). The utility of this method is
shown by low Kdo content attributed to incomplete hydrolysis as well as conversion
of a portion of the Kdo to inert molecular species during hydrolysis (Brade et al.,
1983; McNicholas et al., 1987). These problems were circumvented by using the
purpald assay. Lee and Tsai, (1999) for first time used the purpald assay for the
quantification of lipopolysaccharide. This technique was based on the oxidation of
vicinal glycol groups in Kdo. After reaction with purpald reagent, the formaldehyde
released was measured at 550 nm.
Neutrophil Isolation
Neutrophils are the first line of defense against infections and are considered
the major cells involve in resisting invading Brucella. Several methods for the
isolation of neutrophils from different animal species have been suggested. Isolation
of neutrophils from human blood using enzymes such as Escherichia fmundii endo-β-
galactosidase and neuraminidase along with direct probe mass spectrometry is
possible, but the results are not comparable with the routine dextran isolation method
(Macher and Klock, 1981). A fast and simple magnetic cell sorting system based on
the principle of using specific cell surface markers could be helpful for separation of
large numbers of neutrophils (Forsell et al., (1985). The use of gradient magnetic
columns has been suggested by Milteny et al., (1990). The isolated cells, after
staining with biotinylated antibodies (fluorochrome-conjugated avidin and
superparamagnetic biotinylated-microparticles), were passed through magnetic
column. The labeled cells retained while the unlabelled cells passed through the
column. More than a million cells could be isolated in fifteen minutes. The use of
flow cytometry employing fluorochrome tagged cells and light scattering fluorescent
parameters did not alter the viability and proliferation of the isolated neutrophils. The
17
findings of Cotter et al., (2001) also suggested the potential of negative selection for
the immunomagnetic separation for the isolation of viable, highly pure and unaltered
neutrophils. They suggested that this technique could be used for in vitro and in vivo
inflammatory studies and provided neutrophils. Blood was incubated with antibodies
specific against surface markers of non-desired cells and subsequently with secondary
antibody-coated magnetic beads. The purity of the neutrophils in the effluent was
>95%, and had with <97% viability. Differences in surface L-selectin and CD18
expression of isolated neutrophils were compared with neutrophils in whole blood
using flow cytometry. On the basis of a comparison of the magnetic separation to
conventional percoll density gradient techniques in terms of neutrophil function,
purity, yield, morphology, oxidative burst and pre-activation, supports the use of
magnetic separation as a simple and fast method. This method yields highly purified
(99%), functional neutrophils. Neutrophils isolated by the percoll method contained
6% eosinophils. Each method yielded preparations contaminated with platelets. Using
magnetic separation, CD11b expression of neutrophil activation was lower than that
found with ficoll separation, but magnetic separation did not change oxidative burst
(Zahler et al., 1997). Another comparative study by Watson et al., (1994) showed that
cells isolated by combined dextran/ficoll procedure had a greater ability to produce
reactive oxygen species (ROS) than cells isolated by a one-step procedure using
Mono-Poly Resolving Medium (M-PRM). Cells isolated by the M-PRM method
could be primed in vitro more efficiently by GM-CSF than cells isolated by ficoll.
The ability of neutrophils for ROS generation and CD11 expression in response to
fMet-Leu-Phe was increased 10-fold if blood was pre-incubated with GM-CSF, rather
than the neutrophils in whole blood.
In order to circumvent problems associated with density gradient material, Oh
et al., (2005) used commercially available separation medium containing sodium
metrizoate and Dextran 500. After layering whole blood over the density gradient
medium, the samples were centrifuged and the residual erythrocytes lysed. The
neutrophils were washed, counted, and resuspended in buffer to desired
concentration. This modified method provided preparations with >95% purity and
>95% viability.
Although the density gradient separation and magnetic separation are the two
most common techniques in use they are not easy to perform because of the laborious
methodology. In order to replace these techniques Dodek et al., (1991) developed a
one step method for neutrophil separation. Perfusate (0.2% gelatin and 0.1 % glucose
in phosphate buffer saline) was pumped into an eluriator rotor at 2370 rpm followed
by loading with twenty milliliters of anticoagulated porcine blood mixed with 60 mL
perfusate. The concentration of neutrophils was measured in each fraction. Percentage
of total neutrophils was plotted against flow rate. This method yielded neutrophil
preparations of 95.1% that retained their morphology and contained intact granules
and lobulated nuclei. The isolated neutrophils produced superoxide in the presence of
phorbol myristate acetate (PMA) and phagocytosed zymogen particles. The
characteristics of neutrophils isolated by this method were not different from cells
obtained by conventional sedimentation methods. Using this method, human
neutrophil preparations were 94% pure.
Redbruch and Recktenwald (1995) isolated peripheral neutrophils, and
reviewed techniques for their isolation and analysis. Most isolation studies for
neutrophils have been performed on human blood samples (Saeed and Georgina
1998; Stickle, 1996).
Murine neutrophils were partially characterized by Gaines et al., (2005). They
studied multiple functional responses such as chemotaxis, adhesion, transmigration
across endothelial cells, phagocytosis, and pathogen destruction.
Stie and Jesatis, (2007) used two isolation procedures for the isolation of
neutrophils: a gelatin-based method and a pyrogen-free dextran-based method.
Neutrophils isolated by the gelatin method stimulated more superoxide generation as
compare to the neutrophils isolated by dextran method. Similarly the percentage of
19
cell adherence was more in the neutrophils isolated by gelatin based method than the
dextran based method. Similar results were obtained after neutrophil stimulation with
LPS, TNF- and GM-CSF. The authors also concluded that stimulation of suspended
and circulating neutrophils had a relationship with reorganization of the plasma
membrane.
The effect of different anticoagulants such as EDTA, citrate and heparin on
isolation of human neutrophils was studied by Freitas et al., (2008). Each of three
anticoagulant tested yielded different populations of neutrophils. On the basis of their
calcium levels and reaction to phorbol myristic acetate (PMA), EDTA was suggested
to be a superior reagent for the isolation of neutrophils. Rezapour and Majidi., (2009)
found that due to contamination with lymphocytes, commonly used isolation methods
for neutrophils were not acceptable and proposed a new and easy method for
neutrophils isolation using meglumine compound. The neutrophils were not deformed
and of high purity (79.5%) and viability (>97.5%).
Isolation and Culturing of Bovine Macrophages
Price et al., (1990) isolated bovine peripheral blood macrophages and
evaluated their role in natural resistance to bovine brucellosis. They used mammary
and blood derived macrophages from 22 animals including 11 heifers and 11 bulls.
They suggested that macrophages from naturally resistant cattle exhibited a superior
ability to regulate in vitro intracellular replication of B. abortus. Furthermore,
mononuclear phagocytes from more than 80% of resistant cattle regulated
intracellular replication of B. abortus better than mononuclear phagocytes from
susceptible cattle. After isolation and use of various experimental indicies including
phagocytosis, and ROS production, the recovered cells were deemed satisfactory for
experimental use (Bounous et al., 1993). Macrophages from one animal exhibited
decreased levels of phagocytic activity, intracellular killing and ROS production. The
differential ability of macrophages to phagocytize, and to kill B. abortus was not
related to each other, or to oxidant production. Qureshi et al., (1996) obtained
20
peripheral blood monocyte-derived macrophages from cows for the study of in vitro
replication of Brucella abortus in bovine macrophages. The macrophages from
resistant cows were able to prevent the intracellular growth of Brucella abortus than
the macrophages from susceptible cows. These resistant and susceptible cows were
also challenged with in vivo injections of Brucella abortus strain S2308. The in vivo
results showed that the susceptible cows became more resistant for Brucella infection
after in vivo injection of Brucella abortus strain S2308.
Lysozyme Release Assay
Lysozyme is an enzyme and can catalyze the hydrolysis of the bacterial cell
wall. Cell lysis, or bursting, that usually follows is the basis for lysosome bactericidal
activity in vivo. Most assays for lysozyme are turbidometric, measuring the clearing
of a suspension of dead bacterial cells as their walls break down. The Petri dish
method is somewhat similar in principle to the widely used agarose gel diffusion
assay for antibiotics. The lysozyme produced by macrophages and neutrophils are
diffused into Micrococcus lysodeikticus cells that are spread on agarose producing a
visibly cleared circle within which bacteria have been lysed.
Factors influencing lysozyme production such as phagocytosis were
determined by (Gordon et al., (1974). They noted 70% of lysozyme was sedimented
with azurophilic and specific granules of rabbit neutrophils. High concentrations of
lysozyme were also detected in alveolar macrophages. Further, investigation showed
that lysozyme production increased upon BCG stimulation and phagocytosis. On the
other hand, mouse peritoneal macrophages and human monocytes also secreted
substantial levels of lysozyme. Riley and Robertson, (1984) compared lysozyme
production from azurophil and specific granules of bovine and human neutrophils
stimulated with Brucella smooth-intermediate 45/0 and rough 45/20 strains. They
observed that bovine neutrophils had a greater killing ability against a smooth-
intermediate strain of B. abortus (45/0) than did human neutrophils. However, both
types of neutrophils killed rough Brucella at the same rate. They further found that
smooth-intermediate strains were more resistant to intraleukocytic killing in each type
of neutrophil than were rough strains. B. abortus could not stimulate an effective
level of degranulation for neutrophil stimulation after ingestion as compared to
extracellular organisms such as Staphylococcus epidermidis. Moreover,
myeloperoxidase and lactoferrin released from neutrophils infected with S.
epidermidis were four and two times higher respectively, than neutrophils infected
with B. abortus 45/0. Evidence was presented that B. abortus LPS and lipid A
22
induced lower levels of ROS and lysozyme in human neutrophils in dose dependent
manner. The low levels of lysozyme induced by Brucella LPS was also confirmed by
Rasool et al., (1992). They postulated that this low induction of lysozyme by Brucella
LPS could contribute to Brucella survival inside phagocytes. Lysozyme production
was increased in the presence of autologus plasma. Brucella LPS and lipid A
stimulation resulted in a 100 fold lower lysozyme release than did Salmonella LPS.
Nitroblue Tetrazolium Assay
Reactive oxygen species (ROS) produced by activated macrophages
and neutrophils reduce Nitroblue tetrazolium (NBT) dye, a colorless compound, to a
dark blue formazan by oxygen metabolites found in immune cells. NBT gives an
indirect measure of oxidative metabolism; as ROS are produced; more formazan is
produced as is indicated by increased absorbance. Activated cells produce increased
levels of reactive oxygen species. Therefore, this assay is used as a parameter to
determine macrophage and neutrophil activation. Canning et al., (1985) reported that
in vitro interaction of Brucella S-LPS with bovine neutrophils lowered NBT
reduction and inhibited the myeloperoxidase-hydrogen peroxide-halide system. This
may be a possible reason for the escape of smooth Brucella from intracellular killing
by neutrophils. On other hand, ROS production was not down regulated by heat-
killed B. abortus. Oxidative metabolism of neutrophils and peritoneal macrophages
increases after experimental infection with sporotrichosis in contrast to non-infected
neutrophils and macrophages (Rodolfo and Mendoza, 1986). In 1988, Canning et al.,
evaluated the ability of nonopsonized B. abortus to stimulate superoxide anion
production in bovine neutrophils. B. abortus stimulation was dependent on presence
of bacterial associated opsonins. The ability of Brucella species to inhibit ingestion
by neutrophils or macrophages explained why the oxidative burst had a significant
role in antibacterial process of phagocytic cells and Brucella survival was related to
evasion from cellular defenses (Liautard et al., 1996). Further, in vivo and in vitro
23
studies by Baldwin and Parent (2002) also revealed anti-Brucella activity of reactive
oxygen intermediates.
Nitric Oxide Production in Macrophages and Neutrophils
Nitric oxide (NO2) is a product of immune cells and directly involved in
killing of intracellular Brucella. The Griess reaction may be used to determine NO2
concentrations (Becker et al., 2000). The reaction is based on enzymatic conversion
of nitrate to nitrite by nitrate reductase. Stimulated macrophages and neutrophils
produce elevated levels of nitric oxide to counteract infection. Nitric oxide is thought
to be involved as a defense mechanism against Brucella (Margaret et al., 2002), but
the efficiency of Brucella killing is not same in all the cells and also varies in
different species. Gross et al., (1998) described that in mice B. suis was sensitive to
NO2 killing whereas in rat macrophages, B. abortus LPS did not induce high amount
of NO2 and could not kill Brucella. These results explained the acute outcome of
Brucella infection in the rat, the low frequency of septic shock and prolonged
intracellular survival of Brucella (Lupis-Urrutia et al., 2000). The production of nitric
oxide also varies with different stimuli within the same species or same types of cells.
Intracellular survival of B. abortus revealed that as compared to E. coli LPS, it
released less nitric oxide. Nitrite production was increased by 140 μM after 72 hrs
exposure to 10 ng/mL E. coli LPS, while in B. abortus infected macrophages, nitrite
concentration was 60 μM after 72 hrs of infection. The number of surviving Brucella
decreased, from 6 to 24 hrs, in the presence of nitrite accumulation and NO2 increased
killing of intracellular B. abortus Gangtsetse et al., (2003).
When scientists recognized that nitric oxide had remarkable antibrucella
activity, research was conducted to confirm this fact in a number of cell types,
especially Brucella carrying phagocytes. Gross et al., (2003) reported that in human
macrophages, nitric oxide was not effective against Brucella since the NO2
production was not sufficient. Thus, lack of NO2 production in isolated human
macrophages infected with Brucella could not eliminate Brucella. To further explore
the relationship of enhanced nitric oxide production and Brucella survival, three
strains of B. abortus and two macrophage cell lines were studied by Serafino et al.,
(2007). It was found that NO2 production was higher in macrophages infected with
rough RB51 strain than macrophages infected with smooth Brucella strains S19 and
2308. Since these observations were limited to a single bacterial species, Petra et al.,
(2008) compared in vitro nitric oxide induction in rat, bovine and porcine
macrophages, and in vitro upon stimulation with LPS and other stimulators including
phorbol myristate acetate, ionomycin and recombinant interferon gamma. The Griess
reaction showed differences in NO2 production in pulmonary alveolar macrophages
in all the species tested. The highest amount of NO2 was produced by rat
macrophages and the lowest in bovine macrophages. Porcine macrophages failed to
produce NO2.
Cytokine ELISA
Cytokines are protein, and are classified into two categories: pro-
inflammatory cytokines and anti-inflammatory cytokines. The pro-inflammatory
cytokines include TNF-α, IL-1α, IL-1β, IL-6, IL-12 and IFN-γ. The most important
anti-inflammatory cytokine is IL-10.
TNF-α was considered to be necessary for full expression of macrophage
antibrucella activities. Macrophages were able to kill intracellular Brucellae in 12 to
24 hrs following infection (Jiang et al., 1993b). Maurin et al., (2001) observed that
B. suis Omp25 could not induce TNF- in human macrophages due to some non-
identified protein. Negative regulation of TNF- was observed by Bruce et al., (2002)
in human macrophages. They found that B. abortus rough LPS activated the same
mitogen-activated protein kinase signaling pathways (ERK and JNK) for TNF-α as
did E. coli LPS. However, Brucella LPS was found to be a weak agonist of TNF-α as
were most of other pro-inflammatory cytokines. The production of cytokines differs
in different species. For example in mice (infected with Brucella) all Th1 type
cytokine responses helped in reducing infection (Dornand et al., 2002). The human
25
macrophages infected with B. suis only produced IL-1 and IL-6 cytokines. There was
no production of TNF-α. Other than cellular differences, the type of stimuli used was
also important for cytokine induction. Kariminia et al., (2002) reported an elevated
induction of IL-10 by smooth Brucella LPS. Flow cytometric studies showed that
LPS alone could not stimulate IL-12 expression. Similarly, murine macrophage line
J774 infected with B. abortus smooth strain (S19) produced less IL-6 than when
infected with B. melitensis vaccinal, and virulent strains (Khatammi and Ardestani,
2005).
Numerous PCR-based assays have been developed for the diagnosis of
brucellosis. Leal-Klevezas et al., (1995) introduced a novel method for the extraction
of Brucella DNA for PCR studies from body fluids (lymph node aspirates, blood and
milk) of infected animals. They synthesized two oligonucleotide regions of omp-2
gene and used PCR methodology to detect DNA of Brucella species in carrier cattle,
goats and humans. This method is sufficiently sensitive for the diagnosis of
brucellosis in animals and humans. Later, in 2000, Sreevatsan et al., (2000)
developed a multiplex PCR method for the detection of B. abortus DNA. This
method was highly sensitive and economical. It was used as an alternative to
serological tests. Differential PCR based assays are more complex and difficult to
carry out. Therefore, AMOS-PCR (named on basis of species it identifies: abortus,
melitensis, ovis) was developed (Bricker and Halling 1994). The genetic element
targeted is IS711. This work was extended by Hinic et al., (2008) who devised a
novel PCR assay for the rapid detection of Brucella. The assay was able differentiate
all six Brucella species. This method is highly specific for Brucella detection.
Intracellular Survival of Brucella
Most in vivo studies concerned with the intracellular survival of Brucella have
been performed in mice (Limet et al., 1989). Macrophages kill a majority of Brucella
cells at an early stage of infection, but the surviving bacteria are capable of avoiding
26
brucellacidal action at later stages of infection (He et al., 2006). Most of the active
brucellacidal activity occurred between 0 and 4 hrs post infection. Virulent smooth B.
abortus strain S2308 inhibited apoptosis in murine RAW 264.7 macrophages. B.
abortus strain RB51 induced both apoptotic and necrotic cell death. Similar results
were obtained in in vivo studies in mice immunized intraperitonially with vaccine
strain RB51. Inhibition of macrophage apoptosis may be a factor in the survival of
smooth Brucella cells inside macrophages (Chen and He, 2009).
LPS Mediated Immunity
Wu et al., (1988) studied B. abortus LPS and reported it as the most
immunodominant component among antigens of B. abortus examined. They
demonstrated that its immunochemical reactivity was quite stable and was not altered
prior to, or after non-enzymatic treatment. Goldstein et al., (1992) extracted LPS from
B. abortus by butanol extraction. The product was contaminated with less than 2%
protein, 1% nucleic acid and 1% ketodeoxyoctanic acid. These authors concluded that
B. abortus LPS could be a vaccine component because of its ability to serve as a
superior carrier. It could activate human B cells without involving T cells and was 10,
000 times less toxic than E. coli LPS. It is interesting to note that properties of B.
abortus LPS are similar to those of the whole bacterium.
Materials and Methods
Experimental Design:
A series of six sets of experiments were conducted to study and explore the
project as described in Fig 1 below.
FLOW CHART OF THE STUDY
Lipopolysaccharides
Brucella abortus rough attenuated strain RB51 and smooth virulent strain
S2308 were procured from Dr Gerhardt G. Schurig’s laboratory in Virginia Tech,
USA.
3.1.2 Cultivation
Freeze dried cultures of B. abortus were activated in a safety cabinet (class II
BSL lab, University of Michigan, Ann Arbor), in screw capped test tubes containing
tryptic soy broth (TSB), (Annex 01). TSB was prepared following the manufacturer’s
instructions. Tubes were placed in a shaking incubator (New Brunswick Scientific,
Edison NJ, USA) overnight at 37°C. The active cultures (100 µL each) were
transferred to Falcon tubes containing 10 mL of TSB and shaken at 37ºC for 24 hrs.
Brucella cultures (10 mL) were transferred to sterile plastic flasks containing one liter
of TSB and shaken at 37ºC at 180 rpm for 48 hrs, and were centrifuged at 4ºC at
4000 x g for one hour. The supernatant was discarded and the pellet was used for
lipopolysaccharide (LPS) isolation (Fiaori et al., 2000).
3.1.3. Differentiation of Rough and Smooth Brucella abortus Strains:
Differentiation of rough and smooth B. abortus strains was performed as
described by Bandara et al., (2009) using crystal violet staining. Tryptic soy agar
(TSA), (Annex 02) was prepared according to instructions of the manufacturer.
Autoclaved agar was poured in Petri plates and the plate sterility checked in an
incubation at 37ºC for 48 hrs. Plates free from contamination were streaked with
strains of B. abortus and cultured for 48 hrs. Crystal violet stock solution (Annex 03)
was diluted to 1:40 with sterile deionized distilled water and the surface of each plate
flooded with one milliliter of the diluted crystal violet solution and allowed to stand
for 30 seconds to one minute. The crystal violet solution was carefully removed
29
Rough colonies stained dark blue and the smooth colonies were unstained (white).
3.1.4 Gram Staining
Smears of Brucella abortus were prepared and heat fixed on glass slides.
Crystal violet solution was poured onto each smear for one minute and then the slide
was washed with water. Gram’s iodine solution was applied to the smears for one
minute followed by decolorization with alcohol for 5-6 seconds. The slides were
washed with water and counter stained with safrinin for 30 seconds. Microscopic
examination of slides revealed red colored coccobacilli rods indicating the Gram
negative character of Brucella.
3.1.5 Biochemical Profile
3.1.5a Catalase Test:
One drop of a fresh solution of 30% hydrogen peroxide was placed onto a
glass slide and a small number of Brucella cells were transferred into the solution.
The release of bubbles indicated a positive reaction.
3.1.5b Urease Test:
This test was used to test for the presence of urease in Brucella abortus
cultures. Brucella cultures were inoculated onto urea agar plates (Annex 04) using a
sterilized inoculating loop and the plates incubated at 37oC for 24 hrs. A change in the
color of the medium to deep pink color was considered positive assay for urease.
3.1.6a Lipopolysaccharides (LPS) Extraction from Brucella abortus Smooth
Strain
LPS from B. abortus smooth strain (S2308) was extracted using the protocol
of Apurba et al., (2002). The Brucella culture from the smooth S2308 strain was
pelleted at 4ºC at 2000 x g for 20 min in a GSA angle head rotor in a Sorvall RC-5B
refrigerated superspeed centrifuge. The Brucella pellet was resuspended in extraction
buffer (Annex 05) at 1:10 ratio. The extraction buffer containing the pellet was stirred
at room temperature for 24 hrs and kept at 5°C for five days until material in the
30
pellet changed into a colloidal suspension. This suspension was centrifuged at 4°C at
10,000 x g for 20 min, and supernatant obtained was dialyzed extensively against
cold distilled water. The dialyzed crude extract was filtered through a YM-10
membrane (Cat # 13651, 90 mm ultrafilteration membrane, Amicon corporation,
Ireland). The resulting filterate (25 mL) was centrifuged at 4°C at 105,000 x g for 16
hrs to obtain LPS pellet. The LPS pellet obtained was resuspended in a minimal
amount of distilled water and lyophilized (Section 3.1.8). The lyophilized LPS was
suspended in chloroform/methanol (2:1) and centrifuged at 4°C at 1800 x g for 20
min. The supernatant was poured off and the process was repeated. The resulting
pellet was lyophilized and the dried residue was suspended in water saturated
chloroform/water (1:1) and centrifuged at 4°C at 1800 x g for 15 min. The water
phase was decanted and an equal volume of distilled water added, this suspension was
mixed, re-centrifuged (at 4ºC at 1800 x g for 15 min) and the water phase was
removed. The water phase from the chloroform/water extraction was lyophilized to
obtain partially purified LPS.
Strain
Brucella abortus rough strain (RB51) (50g, wet weight) were mixed with 200
mL of extraction mixture (Annex 06), and the suspension was homogenized with an
ultra-Turrax homogenizer (Janke and Kunkel) for two minutes with cooling in ice so
that the temperature remained between 5-20°C. The mixture was centrifuged at 4°C at
2000 x g for 15 min and filtered through filter paper. The yellow brown bacterial
residue was removed by centrifugation and placed in a rotary evaporator at 30-40°C
to remove the remaining petroleum ether and chloroform. To the phenol crystallized
residual material, sufficient water was added to dissolve it. The solution was
transferred to a glass centrifuge tube and water added drop wise until the LPS was
precipitated. The precipitated LPS was centrifuged at 4°C at 1000 x g for 10 min. and
the supernatant was decanted. The precipitate was washed 2-3 times with small
31
portions of 80% phenol (about 5 mL). Finally, the precipitate was washed three times
with ether to remove residual phenol, and dried in a vacuum. The LPS was diluted to
50 mL with distilled water, warmed to 45°C and placed under vacuum to remove air.
The resulting viscous solution was placed in an ultra vibrator for five minutes and
centrifuged at 100,000 x g for four hrs. The LPS fraction in the clear, transparent
pellet was dissolved in distilled water and was freeze-dried (Section 3.1.8), (Galanos
et al., 1969).
3.1.6c Lipopolysaccharides (LPS) Extraction by Using a Commercial Kit
LPS was also extracted from smooth and rough Brucella abortus with the
used of a commercial kit. The packed cells were extracted of LPS as described by the
manufacturer (Intron Biotechnology, Cat # 17141).
The kit contained a Lysis buffer 100 mL and a Purification buffer 80 mL. The kit
components were stored at 4°C until used.
The yield of extracted LPS was proportional to the volume of culture. A
maximum yield was obtained when 5 mL of culture medium was used (OD of 0.8-
1.2). The Brucella broth culture was centrifuged at room temperature at 15,7000 x g
for 10 min. On average, 5 mL of packed bacterial cells were obtained. The
supernatants were decanted and one milliliter of lysis buffer was added and
vigorously mixed (vortex F/S-16, Telron Biotech). Chloroform (200 µL) was added,
and the mixture vortexed for 10-20 seconds and incubated at room temperature for
five minutes. Upon the addition of chloroform, a white line formed just beneath the
upper blue layer. This region was comprised of mixture of cell debris, protein, and
DNA and RNA. The addition of chloroform allowed separation of the phenol layer
from aqueous layer. Purification buffer (800 µL) was added with mixing following by
incubation at -20 °C for 10min. The mixture was centrifuged at 4°C at 15,7000 x g
for 10min and 400 µL of the supernatant were transferred to 1.5 mL Eppendroff tubes
and centrifuged. Two layers were formed. A white precipitate containing protein and
DNA was observed at the interface between the two layers. The supernatant was
carefully removed, avoiding the white precipitate. The supernatants were centrifuged
at 4°C at 15,7000 x g for 15 min and the pellets containing the crude LPS were
washed with one milliliter of 70% ethanol and dried.
3.1.7 LPS Purification
The crude LPS pellets were dissolved in a solution containing 5 mM MgCl2 in
0.1 M Tris-HCl, pH= 7.0 at a level of 10 mg/mL. Each LPS preparation was digested
at 37°C with 50 µg/mL each of DNase and RNase for 30 min. This was followed by
digestion at 55°C with 50 µg/mL of proteinase K for three hours. Digestion with
proteinase K was repeated three times and each time the sample was reisolated after
exposure to proteinase K. The final incubation volume was brought to 26 mL with
distilled water and the suspension centrifuged at 4°C at 100,000 x g for six hours. The
resulting clear gelatinous pellet served as purified LPS (Velacso et al., 2000).
3.1.8 Lyophilization
Purified LPS was suspended in distilled water, transferred to a glass tube,
frozen in a dry ice acetone bath, and lyophilized in a lyophilizer (Freezemobile 12)
(Jones et al., 1976b).
Partial characterization of Brucella LPS was performed bySodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using the method of
Laemmli, (1970). The methods for preparation of the resolving gel buffer, stacking
gel buffer, and sample buffer are given in Annex 07, 08 and 09 respectively. The
resolving gel contained 15% and the stacking gel contained 4.9% polyacrylamide
(Annex 10 and 11). The composition of tank buffer and gel fixation solution is given
in Annex 12 and 13, respectively. The wells were loaded with a known protein
standard, Precision Plus Protein (Cat # 161-0375, Kaleidoscope Bio RAD) and
various volumes of samples. Electrophoresis was carried out at 80V.
3.2.1a Silver staining
The gels were stained using a silver staining kit as recommended by the
manufacturer (Bio-RAD). The resulting bands on the gels were scanned with a
imager (Bio-RAD).
Colloidal Coomassie stain (G-250, National Diagnostics) and 95% ethanol were
mixed in 9:1 ratio to prepare the final stain. The gel was placed overnight in this
solution. The staining solution was poured out and water added to overlay the gel for
washing, and the gel was visualized by scanning with HP Scanjet G4050.
3.3 Quantification of LPS
LPS was quantified by the Parpald assay procedure described by Lee and
Tsai. (1999). Fifty microliters of LPS samples were added in duplicate wells in a 96-
well culture plate. Fifty microliters of 32 mM sodium periodate (NaIO4) (Annex 14)
were added, and plates were incubated at 37ºC for 25 min. Parpald reagent (50 µL)
(Annex 15) in 2N sodium hydroxide (NaOH) was added to each well and the plates
were incubated for 20 min. Finally, fifty microliters of 64 mM sodium periodate
(NaIO4) were added and the plates were incubated for another 20 min. Formation of
bubbles in the wells was eliminated by addition of 20 µL of 2-propanol. Absorbance
was measured with an ELISA plate reader (Synergy Tech) at 550 nm. Standard
curves were generated using glycine (70 to 420 µg/mL).
3.4 Stimulation of Bovine Neutrophils and Macrophages
Samples of blood were collected from one hundred healthy bovines (Holstein)
at Green Meadow Farms Inc. Michigan, USA. The samples (20 mL) were taken
aseptically and the syringes contained EDTA for anticoagulation.
3.4.1a Isolation of Bovine Neutrophils
A novel means was developed for isolation of bovine neutrophils using a
modification of the method of Robert and Nauseef (2001). Lymphocyte separating
34
media (LSM, Annex 16), (2 mL) was pipetted into a 15 mL Falcon tube and four
milliliters of blood were carefully layered on the LSM. The tubes were centrifuged
(Eppendroff 5810 R) in a swinging bucket rotor and at 4°C at 652 x g for 10 min.
Four distinct layers were formed. The layer at the bottom contained red blood cells,
second layer from bottom had neutrophils followed by lymphocytes and monocytes
together in next layer. The topped layer was only plasma. The sediment was highly
enriched with neutrophils and erythrocytes. The sediment was mixed with equal
volume of 6% dextran contained in 0.9% NaCl. The cell suspension was incubated at
37°C for 45 min to allow sedimentation of the erythrocytes. The erythrocyte fraction
was discarded. The neutrophil-rich supernatant collected was mixed with an equal
volume of Hanks balanced salt solution (HBSS, GIBCO Invitrogen). The reaction
mixture was centrifuged at 4°C at 290 x g for 10 min. The pellet containing the
neutrophils was washed with HBSS and ACK lysing buffer (1 mL, Cat# 10-548E,
I.Onza, Annex 17) was added to lyse any remaining erythrocytes. The tubes were
incubated at 37°C for five minutes. Two volumes of HBSS were added, the tubes
centrifuged at 4°C at 652 x g for 10 min to remove the lysed erythrocyte particles.
3.4.1b Counting of Neutrophils
The neutrophils were resuspended in phosphate buffer saline (PBS) to achieve
an optimal concentration (2 mL). Ten microliters of this suspension were pipetted
onto a hemacytometer and the neutrophils were counted (25 squares inside the central
double lines). The neutrophils were identified by the specific multilobulated structure
of their nuclei. The neutrophil count was multiplied by 0.1mm3 and 1000 to get
number of neutrophils per milliliter (Cotter et al., 2001).
3.4.1c Determination of Neutrophil Viability
Viability of neutrophils was determined as described by Nagahata et al.,
(2004). The neutrophil suspension was mixed with an equal volume of trypan blue
and examined with a microscope. Transparent cells were considered viable. Blue
stained cells were counted as dead. .
Neutrophil purity was evaluated by two methods:
1- The use of hemocytometer employing differential counting of neutrophils
vs non-neutrophils. The neutrophils were identified by their multilobular nuclei by
observation with a microscope at 20X to 40X magnification (Kabbur et al., 1995).
2- Flowcytometry using CH138A antibodies.
3.4.1e Determination of Neutrophil Purity by Flow Cytometry
The technique used was that described by Piepers et al., (2009). The
neutrophils were suspended in 2% formaldehyde (2 mL of formaldehyde and 98 mL
of PBS). The cells were spun at 1000 x g for five minutes. The neutrophil fraction
(sediment) and was divided into two aliquots. Each aliquot was mixed with 100µL of
Flow buffer (Annex 18). One aliquot was mixed with 5µL of Anti-bovine granulocyte
IgM monoclonal antibody CH138A (Cat # CH138A VMRD) at a concentration of 2.5
µg/mL per million cells. The other aliquot was mixed with Mouse IgG APC (BD
Biosciences) and served as a negative control, at a concentration of 2.5 µg/ million
cells. The CH138A coated neutrophils and their negative controls lacking CH138A
antibody were placed in the dark at 4ºC for 30 min. The samples were centrifuged at
4ºC at 1000 x g for five minutes. The sediment obtained after centrifugation was
washed with 1X PBS (5 mL), at least two times. The neutrophils were resuspended in
200 µL of 2% (v/v) formaldehyde in PBS. Analyses were performed in a BD LSR II
Flow cytometer (Department of Pathology, University of Michigan) along with
Winlist 6.0 software to analyze the data.
3.4.2a Isolation and Cultivation of Bovine Macrophages:
Blood (30 mL) containing the anticoagulant citrate dextrose (ACD, Annex 19)
was gently poured into sterile centrifuge tubes (Falcon) and centrifuged at 4°C at
1000 x g for 30 min. The percoll working solution was prepared by diluting percoll
stock solution (Annex 20) with isotonic suspensions of desired specific gravities
36
containing bovine serum albumin (BSA) (5 mg/mL) (Annex 21) and 13 mM citrate
(Annex 22) using the following formula:
X(a) + 0.1 (b) + 0.1 (c) + (0.8 –X) (d)= desired specific gravity -1
X= mL of PBS/mL of final suspension
0.8-X= mL of stock percoll/mL of final suspension
0.1= mL of albumin and citrate/ml of final suspension
a: specific gravity of PBS-1 (a= 1.0056)
b: specific gravity of 0.5% albumin-1 (b= 1.0227)
c: specific gravity of 13mM citrate-1 (c= 1.0219)
d: specific gravity of stock percoll solution-1 (d= 1.1245)
The desired specific gravity for this study was 1.0770
The recipe for one liter working percoll may be found in Annex 23.
Blood was centrifuged at 4ºC at 1000 x g for 30 minutes. To a white cell
suspension (15 mL), was added 15 mL of PBS-citrate solution (PBS-C) (Annex 24).
The suspension was mixed gently, avoiding mixing the cells with percoll in a final
volume of 45 mL. The tubes were centrifuged at 4ºC at 1000 x g for 30 min. The
white cell fractions were transferred to another tube and PBS-C was added to a final
volume of 50 mL (This step eliminates any remaining percoll). The mixture was
mixed gently and centrifuged at 4°C at 500 x g for 10min. The PBS-C was discarded
and the pellet further processed by addition of ten milliliters of plasma and
homogenized. PBS-C was added to bring the volume up to 50 mL and centrifuged at
4°C at 500 x g for 10 min. This step is repeated twice. The pellet was resuspended in
Complete RPMI (CRPMI, Annex 25) containing 4% bovine calf serum. This
suspension was poured into a Teflon flask and incubated in a humified incubator
flushed with 5-10% CO2 at 37 ºC for 24 hrs. The medium was changed every 3-4
days. Six milliliters of fresh medium was added by keeping one milliliter of old
medium. (Qureshi et al., 1996).
3.4.2b Harvesting of Macrophages
After seven days of culture, macrophages were harvested with a cell scrapper.
The culture medium with cells was centrifuged at 4°C at 500 x g for 10 min and the
supernatant discarded. The cell number was counted and adjusted as needed in
CRPMI-10% FCS (Omar et al., 2003).
3.4.3 Murine Macrophage Cultivation.
Murine RAW 264.7 macrophages were obtained from the American Type
Culture Collection (ATCC, cell line TIB-71) and as described by Cynthia et al.,
(2002). Macrophages were cultured in 24 well plates in Dulbecco's Modified Eagle
Medium (DMEM) 10% Fetal Bovine serum (FBS) and 1% penicillin, streptomycin
mixture at a concentration of 2.5x105 cells /mL. Composition of DMEM is described
as Annex 26.
Neutrophils and Murine Macrophages
The assay was performed with little modification from that described by
method of Stabili et al., (2009). Agarose gel 1% (Annex 27) containing 0.5 mg/mL of
dried Micrococcus lysodeikticus cells was suspended in a 0.1M phosphate citrate
buffer pH 5.8 (Annex 28). Agarose (25 mL) was poured into each plate. After
solidification, holes (35 mm diameter) were punched in the gel. Bovine macrophages
and neutrophils, each at a concentration of 2.5x105 /mL were cultured for in a 24 well
plate 24 hrs, in CRPMI containing 10% FBS. Murine macrophages (2.5x105 /mL)
were cultured for 24 hrs in 24 well plates in Modified Eagle Medium (MEM)
containing 10% FBS. At varying concentrations (0.02. 0.2, 2, 20, 200 µg/mL,
respectively). each LPS (rough, smooth alone or in combination) were added to each
well. The samples were incubated at 37°C for two hours with shaking. The incubation
mixtures were centrifuged at 4°C at 3000 x g for 10 min and supernatants stored at -
70°C until assayed. Sample suspensions of 25 µL were loaded into the wells in the
agarose plates. The plates were incubated at 37°C for 24 hrs. Each plate was scanned
38
(HP Scanjet G4050) and the radius of clarified zone around each well was measured
and amount of lysozyme released calculated from a generated standard curve. Each
sample was assayed in triplicate. Egg white lysozyme (0.00 to 16 µg/mL) was used to
generate a standard curve. All background values were obtained using media
(CRPMI) containing FBS and these background values were subtracted from
calculated value of each sample.
3.4.5 Induction of Reactive Oxygen Species (ROS) from LPS-Induced Bovine
Macrophages, Neutrophils and Murine Macrophages
A solution of 0.1% NBT of 200 µL (Annex 29) was added to each well and
the plates incubated at 37°C for 60 min. Following incubation, varying concentrations
of LPS (0.02, 0.2, 2.0, 20, 200 µg/mL, respectively) were added to each well.
Samples were incubated against at 37°C for 30 min. The reactions were stopped by
adding an equal volume (500 µL) of 0.1N HCl (Annex 30). The mixture was
centrifuged at 4°C at 800-1000 x g for 15 min. The resulting pellets obtained were
dried at 37°C in dark. Dioxane (1 mL) was added to each pellet and incubated at
85°C for 20 min. After incubation, the mixture was centrifuged at 4ºC at 800-1000 x
g for 15 min. The optical density of the clarified supernatant was determined at 580
nm with a dual beam spectrophotometer (Beckman Coulter, DU 530) at room
temperature. The polystyrene semimicro cuvettes (Cat# 14-385-942, Fisher
Scientific) were used that can read absorbance between 240-750nm. E. coli LPS and
superoxide dismutase (SOD) served as positive and negative controls, respectively
(Zembala et al., 1980).
3.4.6 Induction of Nitric Oxide from LPS-Induced Bovine Macrophages, and
Neutrophils, and Murine Macrophages
Bovine and murine macrophages and bovine neutrophils were cultured in 96
well plates at a concentration of 2.5x105/mL and incubated at 37ºC in 10% CO2 for 24
hrs. Cultured macrophages were treated with varying concentrations (0.02, 0.2, 2, 20,
200 µg/mL, respectively) of all three types of LPSs {rough, smooth and combined
39
(1:1)} and incubated at 37ºC in 10% CO2 for two hours. After incubation, LPS-treated
macrophages were centrifuged at 4ºC at 600 x g for 10 min and supernatant collected.
Supernatants were mixed with an equal volume of Griess reagent (Annex 31) in 96
well plates. The plates were held at 25°C for 10 min and color change (indicative of
nitrite presence) was quantified at OD540 by reading the plates on ELISA plate reader
(Synergy BioTech). Each experiment was performed in triplicate. A standard curve
was generated using increasing concentrations of sodium nitrite (0.3125 to 20 µg/mL)
dissolved in MEM. Macrophages pretreated with E. coli LPS served as a positive
control. Accumulation of nitric oxide was inhibited by NG monomethyl-L-arginine
(L-NMMA) which served as a negative control (Goldstein et al., 1992). Background
values of nitrite production by untreated macrophages were subtracted from sample
values. The absorbance of the standards, controls and test samples was converted to
ng/mL of nitrite by comparison with absorbance of sodium nitrite standards within a
linear curve fit (Waters et al., 2002).
3.4.7 Determination of Cytokines in LPS-Induced Bovine and Murine
Macrophages by Enzyme Linked Immunosorbant Assay (ELISA).
A sandwich ELISA assay was performed to determine the cytokine production
in LPS-activated macrophages (Rittig et al., 2003).
3.4.7a Sample Preparation
Macrophages were cultured at 37ºC in 24 well plates in DMEM containing
10% FBS and 1% penicillin-streptomycin (PS). After 24 to 48 hrs, the medium was
aspirated from the wells and either 200 µg/mL of rough RB51, smooth S2308 or a
combination of rough and smooth Brucella abortus LPSs (100 µg/mL each) were
added and the plates incubated for another 24 hrs. The samples were then centrifuged
at 4°C at 3000 x g for 10 min and the recovered supernatant was frozen prior to the
quantification of cytokines. The following cytokines served as positive controls:
recombinant bovine and murine interleukin-1-beta (IL1-β), interleukin-6 (IL-6),
α) (Bio-Legend).
3.4.7b Coating of ELISA Plate
Unlabelled, captured antibody (10 µL) was diluted in 10 mL of coating buffer
(Annex 32, 1: 1000 dilution) and 100µL pipetted into a 96 well microtiter plate. The
plate was sealed with parafilm and incubated overnight at 4ºC.
3.4.7c Blocking the Plates
The plates were brought to room temperature for 10 min and the antibody
solution removed. Plate were washed three times with PBS/Tween-20 (Cat# BP337-
100, Fisher Scientific), 0.5% v/v, mentioned in Annex 33. Blocking solution (200 µL,
Annex 34) was added to each well. Plate were sealed with parafilm M and incubated
at room temperature for two hours. The blocking solution was discarded; plates dried
on tissue paper, after washi

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