Brucellosis: My Post graduate Thesis( www.ubio.in)

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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

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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. 5

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. 6

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. 7

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 8

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 9

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 10

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 microorganisms. 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 11

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 12

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-dihydroxybenzoic 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 13

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. 14

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. 15

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). 16

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 17

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 18

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 19

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 20

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 21

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 noninfected 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

Discussion Discussion Host defence and pathogenic mechanism of bacteria are continuously in- teracting and constantly evolving. As such, the microorganisms have evolved mechanisms 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 intermediates (ROI) and reactive nitrogen intermediates (RNI), the production of which is triggered by phagocytosis or exposure to certain inflammatory mediators including 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 intracellular survival is due to specific factors produced by the bacteria, that interfere with 45

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 components 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

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 generation 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 complement 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 association of lipopolysaccharide with the protein in Brucella. As the protein antigen has been speculated to be the choice antigen for overcoming many of the associated 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

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 following 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 respect 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

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 reported 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 radicals 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 stimulation 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

(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 suppression 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 associated 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

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 after opsonizing the brucellae with normal heat inactivated sheep serum. It was observed 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 virulence 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 references 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 disease 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

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 development in infected host cell as has been proposed for some other intracellular pathogens (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 promote 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- production 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

suppression of mitochondrial cytochrome C release necessary for caspase activation in cytoplasm. This inhibit several apoptotic pathways. Modulation of host cell apoptosis could eliminate key defence cells that are necessary to eliminate pathogen, inhibit or allow bacterial replication, facilitate the release of intracellular bacteria 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 antigen 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

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 produced four peaks. A 39 kDa protein was highly reactive to antisera in western blotting 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

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 production. 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 nonsignificantly, 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

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 reactivity 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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Appendix MEDIA 1. Potato infusion agar Thinly sliced potatoes (250 g) in distilled water (1000ml) was kept overnight in waterbath at 600C. It was filtered through gauze the next morning and the volume was made upto 1000 ml. To this the following ingredients were added: Sodium chloride : 5g Peptone : 10 g Beef extract : 5g Agar : 30 g Dissolved the above ingredients in 1000 ml potato infusion. Final pH was adjusted to 7.0 to 7.2 after adding 20 ml glycerol.Sterilized by autoclaving at 1210C for 15 min. 2. Glycerol dextrose agar (GDA) Agar : 20 g Peptone : 10 g Sodium chloride : 5g Beef extract : 5g Glycerol : 20 ml D-glucose : 5g Dissolved the above ingredients in 1000 ml distilled water and pH was adjusted to 7.0 to 7.2 .Sterilized by autoclaving at 1210C for 15 min. 3. Complete RPMI media RPMI-1640 (sigma) : 10.41g HEPES (24 mM) : 3.90 g Sodium bicarbonate (25 mM) : 3.70 g Streptomycin (100 µg/ml) : 0.10 g Pencillin (100 IU/ml) : 0.06 g 71

Distilled water : 1000 ml pH : 7.2 Newborn calf serum : 10% Sterilized by filtering through membrane filter (0.45 µ) and stored at 40C. BUFFERS AND REAGENTS 1. Phosphate buffered saline (0.01M, pH 7.2-7.4) NaCl : 8.0 g KCl : 0.2 g Na2HPO4 : 1.15 g KH2PO4 : 0.2 g Distilled water : 1000 ml The pH was adjusted to 7.2-7.4 with 1 N HCl/ 1 N NaOH and sterilized by autoclaving for 15 min at 1210C. 2. Hanks balanced salt solution (HBSS) KCl (5.4mM) : 0.4 g Na2HPO4 (0.3 mM) : 0.053 g KH2PO4 (0.4 mM) : 0.05 g NaHCO3 (4.2 mM) : 0. 353 g MgSO4 (0.6 mM) : 0.148 g NaCl (137 mM) : 8.00 g D-glucose (5.6 mM) : 2.00 g Distilled water make upto 1000 ml. pH was adjusted to 7.4. Filter sterilized and stored at 40C. 3. Trypan blue solution (0.1%) Trypan blue : 0.1 g PBS : 100 ml The solution was filtered through Whatman filter paper and stored at 40C. 72

4. Normal saline solution (NSS) NaCl : 8.5 g Distilled water : 1000 ml Sterilized by autoclaving at 1210C for 15 min. 5. 2.7% EDTA solution EDTA : 2.7 g PBS (0.01M, pH 7.2) : 100 ml Sterilized by autoclaving at 1210C for 15 min and stored at 40C. 6. Isotonic ammonium chloride solution Ammonium chloride : 8.32 g Sodium bicarbonate : 0.84 g Distilled water : 1000 ml Sterilized by autoclaving at 1210C for 15 min. 7. MTT solution MTT : 50 mg PBS : 10 ml Filter sterilized and stored at 40C. 8. ELISA reagents a. Coating buffer (Carbonate-bicarbonate buffer, 0.05 M, pH 9.5) Sodium carbonate (15 mM) : 1.6 g Sodium bicarbonate (35 mM) : 2.9 g Sodium azide (3.1 mM) : 0.2 g Distilled water : 1000 ml b. Washing buffer (PBS-T) PBS : 1000 ml Tween -20 : 500 µl 73

c. Blocking buffer PBST : 100 ml Skim milk powder : 5g d. Citrate buffer (0.1 M pH 4.6) SOLUTION A Citric acid : 2.1 g Distilled water : 100 ml SOLUTION B Sodium citrate : 2.94 g Distilled water : 100ml 25.5 ml of solution A and 24.5 ml of solution B were mixed to make 50 ml and stored at 40C. e. Substrate solution Citrate buffer : 10 ml H2O2 (30%) : 1.2 µl O-phenylene diamine : 10 mg It was prepared freshly. f. Stopping solution (5 N H2SO4) Conc. H2SO4 : 1.37 ml Distilled water : 10.0 ml 9. Reagents for Nitrite estimation a. Stock solutions Solution A Naphthylethylene diamine dihydrochloride (0.1%) : 100 mg Distilled water : 100 ml Solution B Sulphanilamide : 1g 5% Concentrated phosphoric acid : 100ml 74

b. Working solution Solution A : 10 ml Solution B : 10 ml These solutions were mixed and used fresh for each experiment. c. 10 mM sodium nitrite (stock solution) Sodium nitrite : 64 mg Distilled water : 100 ml 10. Reagents for Hydrogen peroxide estimation a. Potassium phosphate buffer (0. 01M, pH 7.0) KH2PO4 : 1.3609 g Distilled water : 1000 ml pH adjusted to 7.0 with 1 N KOH. b. Stock solution NaCl (140 mM) : 8.186 g Dextrose (5.5mM) : 0.4908 g Potassium phosphate buffer : 1000 ml Filter sterilized and stored in flask covered with aluminium foil at 40C untill used. c. Working solution Stock solution : 50 ml Phenol red : 5 mg HRPO : 2.5 mg Working solution was prepared freshly at time of each experiment. d. Stock hydrogen peroxide solution H2O2 (30%) : 1.02 ml Distilled water : 1000 ml Stored at 40C in brown coloured bottles. 75

11. Reagents for SDS-PAGE a. Upper buffer (Tris -HCl 2 M, pH 8.8) Tris : 24.2 g DW : 50. 0 ml pH adjusted to 8.8 with NaOH and made volume to 100 ml with distilled water. b. Lower buffer (Tric-HCl, 1 M, pH 6.8) Tris : 12.10 g Distilled water : 50.0 ml Adjusted pH to 6.8 with NaOH and made volume to 100ml c. Electrophoresis buffer Tris : 3.025 g Glycine : 14.413 g SDS : 1.00 g Distilled water to make 1000 ml. d. Sample buffer (5 X) 1 M Tris-HCl, pH 6.8 : 0.6 ml 50% glycerol : 5.00 ml 10% SDS (w/v) : 2.00 ml 2- mercapto ethanol : 0.5 ml 1% Bromophenol blue : 1.00 ml Distilled water : 0.9 ml e. Acrylamide (30%) stock solution Acrylamide : 29.2 g Methylene bis-acrylamide : 0.80 g Distilled water to make 100.00 ml 76

f. Stacking gel 4.5% Upper buffer : 1.5 ml Acrylamide : 0.9 ml SDS 10% : 60 µl Distilled water : 3.6 ml TEMED : 20µl 10 % Ammonium persulphate (APS) : 10µl g. Separating gel –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– –– Separating gel 5% 12.5% 15% –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– –– Lower buffer 4.5 ml 4.5 ml 4.5 ml Acrylamide 3.0 ml 7.5 ml 12.0 ml Distilled water 10.5 ml 6.0 ml 1.5 ml 10% SDS 180 µl 180 µl 180 µl TEMED 20 µl 20 µl 20µl 10% APS 70 µl 70 µl 70µl –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– –– 12. Reagent for silver staining of polyacrylamide gels a. Fixer Methanol : 50 ml Acetic acid : 12 ml Distilled water to make 100 ml b. Staining solution Silver nitrate : 0.2 g Formalin : 50 µl Distilled water : 100 ml 77

c. Developer Sodium carbonate : 6g Sodium thiosulphate : 0.4mg Formalin : 200µl Distilled water : 100ml 13. Reagents for immunoblotting a. Transfer buffer Tris (0.1 M) : 2.42 g Glycine (0.192 M) : 2.88 g 5% Methanol : 10 ml Distlled water to make 200 ml b. Washing buffer (PBS-T) PBS : 1000 ml Tween-20 : 500 ml c. Blocking buffer PBST : 100 ml Skim milk powder : 5g d. Substrate solution PBS : 10 ml Diaminobenzidine : 6 mg H2O2(30% v/v) : 10 µl 14. Buffers and solutions for DNA isolation and agarose gel electrophoresis a. TBE buffer Tris : 2.7 g Boric acid : 1.375 g EDTA : 0.2325 g Distilled water : 500 ml 78

b. 0.8% agarose Agarose : 0.4 g TBE buffer : 50 ml c. Ethidium bromide stock Ethidium bromide : 10 mg Distilled water : 1 ml d. Gel loading dye Bromophenol blue : 0.25% w/v Sucrose : 40% w/v Xylene cyanol : 0.25% w/v In distilled water, stored at 40C. e. 5 M NaCl NaCl : 29.02 g Distilled water to make up 100.00 ml f. 1 M Tris (pH 7.6) Tris base : 12.10 g Distilled water : 80.00 ml Adjust pH to 7.6 with ConC. HCl and volume make upto 100 ml. g. 0.5 M EDTA EDTA : 18.61 g Distilled water : 80.00 ml Adjusted pH to 8.0 with NaOH and volume make upto 100 ml. h. TE buffer 1 M Tris (pH 7.6) : 100 µl 0. 5 M EDTA : 20 µl Distilled water to make 10 ml. 15. Staining dye for apoptosis Acridine orange : 1 mg Ethidium bromide : 1 mg PBS : 10 ml. 79

Fig. 1 : Ion exchange chromatographic profile of Brucella melitensis cytosolic antigen 0 0 0 OD at 280 nm 0 0 0 0 0 00 0 0 0 0 Fraction number 80

Fig. 8 : Immunoreactivity of the various chromatographic fractions on ELISA 0 0 0 ELISA OD 0 0 00 0 0 0 Fraction number 81

Fig. 10 : Nitrite production by PMNs stimulated with heat killed B. melitensis 16M cells 0 0 (µM/2 x 107 cells/hour) 0 Nitrite production 0 0 0 0 00 0 0 1.5 3 6 Concentration of cells (x 107/ml) Fig. 12 : Hydrogen peroxide production by PMNs stimulated with heat killed B. melitensis 16M cells 0 0 (nM/2 x 107 cells/min.) 0 H2O2 production 0 0 0 0 0 1.5 3 6 Concentration of cells (x 107/ml) Fig. 14 : Effect of heat killed B. melitensis 16M cells on MTT metabolism of PMNs 0 MTT metabolism (% of control) 00 0 00 0 0 0 1.5 3 6 84 Concentration of cells (x 107/ml)

Fig. 13 : Effect of LPS and cytosolic antigen on the MTT metabolism by PMNs (2 x 107 cell/ml) 0 MTT metabolism (% of control value) 00 0 0 0 0 0 0 12.5 25 50 75 150 300 Concentration µg/ml 85

Fig. 15 : Phagocytic index of B. melitensis 16 M by ovine PMNs on stimulation with LPS and cytosolic antigen 0 0 0 Phagocytic index 0 0 0 0 0 0 0 86

Fig. 16 : Intracellular killing indices of B. melitensis 16M by ovine PMNs on stimulation with LPS and cytosolic antigen 00 00 Intracellular killing index 00 00 00 00 00 0 0 0 87

Fig. 10 : Nitrite production by PMNs stimulated with heat killed B. melitensis 16M cells 0 0 (µM/2 x 107 cells/hour) 0 Nitrite production 0 0 0 0 00 0 0 1.5 3 6 Concentration of cells (x 107/ml) Fig. 12 : Hydrogen peroxide production by PMNs stimulated with heat killed B. melitensis 16M cells 0 0 (nM/2 x 107 cells/min.) 0 H2O2 production 0 0 0 0 0 1.5 3 6 Concentration of cells (x 107/ml) Fig. 14 : Effect of heat killed B. melitensis 16M cells on MTT metabolism of PMNs 0 MTT metabolism (% of control) 00 0 00 0 0 0 1.5 3 88 6 Concentration of cells (x 107/ml)

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Institute of Animal Health and Phone 0472 2840262 (O) Veterinary Biologicals, 04829 238302 (R) Palode, Pacha. P.O, Pin 695562 094471 48302 (M) Thiruvananthapuram, Kerala, IndiaFax 0472 2840262 Dr. V. I. BISHOR. M.V.Sc URL www.vbikerala.nic.in Email: bishorvi@yahoo.co.in JOB DESCRIPTION Institute: Institute of Animal health and Veterinary Biologicals, Palode, Trivandrum (Department of Animal Husbandry, Government of Kerala) Designation: Research Assistant (Research and Development Wing) The Research and Development wing of this institute is engaged in the development of cell culture vaccines and diagnostic reagents. Present assignments include 1. Manufacture of Cell culture Rabies vaccine for veterinary use 2. Manufacture of Brucella abortus S19 vaccine (live, freeze dried) 3. Development of rapid diagnostic kits for Leptospira, Anthrax and Bovine Pas- teurellosis 4. Development of Recombinant DNA Anthrax vaccine in collaboration with Rajiv Gandhi Centre for Biotechnology, Trivandrum. ACADEMIC QUALIFICATIONS M. V. Sc (Veterinary Public Health) from FAO/WHO Collaborating Center for Re- search & Training in Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar (U. P) (2001) B. V. Sc & A.H from College of Veterinary & Animal Sciences, Kerala Agricultural University, Thrissur (1999) M.V.Sc THESIS Studies on in vitro effects of Brucella melitensis cytosolic antigen on ovine neutrophils (2001). Indian Veterinary Research Institute, Izatnagar. Employment history September 1999 – August 2001: M.V.Sc Research scholar, IVRI, Izatnagar November 2001 – December 2001: Lecturer in livestock management, Dept. of Vocational Higher secondary Education, Government of Kerala 97 January 2002 – September 2002: Veterinary Surgeon, Government of Kerala September 2002 – till now: Research Assistant, IAH&VB, Government of Kerala

Research Experiance 1. Production and quality control of veterinary vaccines and diagnostics. 2. Experience in molecular biology techniques, cell culture, immunological techniques, microbiology and handling of risk group 3 pathogens 3. Experience in food quality control and Clean room operations 4. Knowledge of Drugs and Cosmetics Rules, schedule M, c GMP regula tions of FDA, TPD, WHO, Orange guide and ICH-GCP. Member of the core committee for implementation of c GMP at the institute of Animal Health &Veterinary Biologicals, Palode. 5. Care and management of laboratory animals and clinical procedures. 6. Working knowledge in Computer , In charge of the Institute Web site, www.vbikerala.nic.in Awards/fellowships 1. Kerala Agricultural University merit scholarship for B.V.Sc studies (1993-1994) 2. I.V.R.I.- Junior Research Fellowship for postgraduate studies (1999). 3. Indian Council of Medical Research (ICMR)- Junior Research Fellowship for Doctoral studies (2001). Memberships in professional Associations Registered Veterinary practioner of Kerala State Veterinary Council. Member of Indian Veterinary Association(IVA), Member of the editorial board of the IVA web site www.jivaonline.com Life member of Indian Society for Veterinary Immunology & Biotechnology. Life member of the Indian Association of Veterinary Public Health Specialists. Life member of the Indian Association of Public Health Veterinarians. 98

Publications/Presentations 1. Bishor. V. I, Singh. D. K, Sinha. D . K and Goswami. T. K (2004) Inhibition of Apoptosis in sheep monocytes by Brucella melitensis 16M, J. Appl. Ani. Res., 25(1) 2. Bishor, V. I. and Elizabeth, G. (2001) Seafood Toxins, Ind. Food. Industry, 20(6) 3. Singh, D.K., Sinha, D.K. and Bishor, V. I. (2002) An overview on microbial haematuria / Haemoglobinuria in Bovines. In: An Introduction to Bovine haematuric and Haemoglobinuric Diseases, Ed: Somavanshi. R., Jaypee Brothers, New Delhi 4. Singh, D.K., Bachhil, V.N., Sinha, D.K., and Bishor, V. I. ( 2000) Brucellosis: Diagnosis and Control, In: Compendium of ICAR Summer school on Recent trends in diagnosis and control of important zoonoses and food borne infections, I.V.R.I., Izatnagar, India. 5. Singh, D.K., Sinha, D.K. and Bishor, V.I. (2000) Bacillus cereus food poisoning, In : compendium of ICAR Summer school on recent trends in diagnosis and control of important Zoonoses and food borne infections, I.V.R.I, Izatnagar, India. 6. Singh, D.K., Sinha, D.K. and Bishor, V.I. (2001) Brucella vaccine: present and future needs. Presented in Indian Veterinary Congress, Punjab Agricultural University, India. 7. Sinha, D.K, Bishor, V.I., Goswami, T.K. and Singh, D.K (2001) SDS-PAGE and Western blot analysis of Brucella melitensis soluble antigen. Presented in the symposium on “ Status & Strategies of Animal Health and Production” Patna, Bihar, India. 8. Sinha, D.K, Bishor, V.I., Goswami, T.K. and Singh, D.K (2001) Fractionation of Brucella abortus sonicate, Presented in annual meeting of the Indian Society for Veterinary Immunology and Biotechnology, Pantnagar (Uttaranchal) References 1. DR. R. VIJAYAKUMAR Director Institute of Animal Health & veterinary Biologicals, Palode, Pacha.P.O, Trivandrum 2. D. K. SINGH, PH.D. Scientist (Sr. Scale) FAO/WHO Brucellosis centre FAO/WHO Collaborating center for Research & Training in Veterinary Public Health Indian Veterinary Research Institute, Izatnagar, Bareilly, U. P- 243122 3. DR. E. SREEKUMAR. PHD Scientist Animal Biotechnology Wing Rajiv Gandhi Center for Biotechnology 99 Thycaud. P. O, Trivandrum

TRAININGS ‘Current Good Manufacturing Practices’ at Center for GMP, Hyderabad ‘Cell culture methods in Virology Laboratories’ at Biotechnology Laboratory, National Dairy Development Board, Anand, Gujarat ‘Office Management’ at the Institute of Management in Government, Trivandrum ‘Production of diagnostic antigens’ at the Institute of Animal Health and Vet- erinary Biologicals, Hebbal. Bangalore ‘Production and Quality control of Brucella vaccine’ at the Indian veterinary research institute , Izatnagar ‘Rabies vaccine production’ at the Pasteur institute Of India, Coonoor. ‘Meat inspection and Certification’ at the Center of Excellence in Meat Sci- ence, Kerala Agricultural University, Mannuthy, Kerala. ‘Advanced training in diagnosis of infectious diseases’, organized by Govt. of Kerala and Southern Regional Disease Diagnostic Laboratories (SRDDL), Bangalore ‘Molecular methods in diagnosis of Leptospirosis’ by Defense Research and Development Organization, Gwaliar. ‘Advances in meat processing’ at Meat Products of India Ltd. ‘Good laboratory Practices(GLP)’ organized by Drug Control Administration, Kerala and Hindustan latex Ltd. ‘Zoo animal management’ at Zoological gardens, Trivandrum Fifth international meeting on Molecular Epidemiology and Evolutionary Ge- netics of Infectious Diseases, MEEGID-5, Hyderabad, INDIA. 100

BIODATA Name: Dr V I BISHOR Date of birth: 24-09-1975 Sex: Male Marital status: Married Official address: Research Assistant Institute of Animal Health &Veterinary Biologicals, Palode, Pacha. P. O, Thiruvananthapuram, Kerala. Pin - 695562 Residential Address: Varakukalayil Midayikunnam.P.O Thalayolaparambu Kottayam, Kerala Pin 686605 Phone: 0472-2840262(O) 04829- 238302(R) Fax: 0472-2840262 E-mail: bishorvi@yahoo.co.in Dr. V. I. Bishor Date: 25/06/2004 Palode 101

Brucellosis: My Post graduate Thesis( www.ubio.in)

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