Brucellosis and immunity by Prof. Dr. Mohamed Refai Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.

Discovery of Brucella organisms In the late 1887, Bruce isolated an organism he named Micrococcus melitensis from individuals with Mediterranean fever. In 1895, Bang in Copenhagen isolated what he called Bacillus abortus from cattle that were undergoing spontaneous abortions In 1914, Traum isolated Micrococcus suis from an aborted piglet

Brucella species In 1918, B. abortus, B. melitensis and B. suis were recognized to be closely related and they were placed in a genus named Brucella.

The Genus Brucella Brucella melitensis (Bruce, 1887) Brucella abortus (Bang, 1897) Brucella suis (Traum, 1914) Brucella ovis (Buddle& Boyes 1953) Brucella neotomae (Stoenner&Lackman, 1957) Brucella canis (Carmichael and Bruner,1968) Brucella marins Brucella dolphine

The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc Natl Acad Sci U S A 2002 Jan 8;99(1): DelVecchio VG, Kapatral V, Redkar RJ, Patra G, Mujer C, Los T, Ivanova N, Anderson I, Bhattacharyya A, Lykidis A, Reznik G, Jablonski L, Larsen N, D'Souza M, Bernal A, Mazur M, Goltsman E, Selkov E, Elzer PH, Hagius S, O'Callaghan D, Letesson JJ, Haselkorn R, Kyrpides N, Overbeek R.

Complete genome sequencing of Brucella melitensis 16M The brucellae possess two independent chromosomes Based on DNA-DNA hybridization studies, it has been proposed that there is only a single species, B. melitensis

Epitopes of an Antigen (Polysaccharide)

Epitopes of an Antigen (Protein)

: Folding Domains of an Antibody

Epitope-Specific Receptors on the Surface of B- and T-Lymphocytes

Binding of Epitopes to Corresponding Molecules of sIg on the Surface of B--Lymphocytes

MHC-I Molecules

MHC-I Molecules MHC-I molecules are made by all nucleated cells in the body and possess a deep groove that can bind peptide epitopes, typically 8-9 amino acids long, from endogenous antigens Endogenous antigens are proteins being produced within the host cell such as proteins produced by intracellular bacteria

Binding of Epitopes to Corresponding Molecules of sIg on the Surface of B--Lymphocytes

Animation of Opsonization of a Bacterium

Binding of Peptide Epitopes from Exogenous Antigens to MHC-II Molecules

Binding of Peptide Epitopes from Exogenous Antigens to MHC-II Molecules by a B-Lymphocyte

A T4-Lymphocyte Recognizing Epitope/MHC-II on an Antigen-Presenting Cell (APC)

T4-Lymphocyte Recognizing Epitope/MHC-II on a B- Lymphocyte

Activation of a T4-Lymphocyte by an APC

An Activated T4-Helper Lymphocyte Reacting with an Activated B-lymphocyte

Activation of B cells by Th2 Th2-lymphocytes recognize antigens presented by B-lymphocytes. produce cytokines such as interleukins 4, 5, 9, 10, and 13 that enable activated B-lymphocytes to proliferate, differentiate into plasma cells, secrete antibodies, and switch classes of antibodies.

Proliferation of a T4-Lymphocyte after Activation

Proliferation of B-Lymphocytes

Differentiation of B-lymphocytes into Plasma Cells and B-Memory Cells

Anamnestic Response

B. Ways in Which Antibodies Help to Defend the Body 1. Opsonization 2. Cytolysis 3. Antibody-dependent cellular cytotoxicity (ADCC) by NK cells 4. Neutralization of Exotoxins 5. Preventing Bacterial Adherence 6. Agglutination of Microorganisms

Cell-mediated immunity (CMI) Activation of macrophages and NK-cells, enabling them to destroy intracellular pathogens; Production of antigen-specific cytotoxic T- lymphocytes, that are able to lyse body cells displaying epitopes of foreign antigen on their surface Release of various cytokines) in response to an antigen, that influence the function of other cells involved in adaptive immune responses

Diagnosis of brucellosis *Accordingly, diagnosis depends on: 1. Isolation of Brucella which is conclusive if +, but not when - 2. Detection of antibodies which is conclusive if -, but it is not 100% conclusive when +( false +, false -) *Accordingly, diagnosis depends on: 1. Isolation of Brucella which is conclusive if +, but not when - 2. Detection of antibodies which is conclusive if -, but it is not 100% conclusive when +( false +, false -)

Serological Diagnosis of brucellosis Although the serological diagnosis is not 100% reliable when positive It is the main tool for the rapid recognition of infected herd and individual animals

A positive serology means: field strain infection vaccination infection residual vaccination titre cross-reactivity with other organisms, like Yersinia, Salmonella, Pasteurella etc human errors.

Serological tests for B. melitensis infection no specific serological test for B.melitensis infection in small ruminants has been developed

Serological diagnosis of B. melitensis infection until now : Antigens are prepared from B. abortus Tests are designed for diagnosis of B. abortus infection in cattle The same antigens and tests are used for diagnosis of B. melitensis in small ruminants, buffaloes, camels, swine and other animals.

Analytical sensitivity of classical assays in detection of affinity purified anti-B. abortus antibody isotypes AssayIgM IgG1 IgG2 AgA SAT20* BAPA RBT RIV ME CFT ____________________________________ * ng of isotype required for a positive test

A prominent example is the Rose Bengal (RB) test the standardisation of the antigen is made to be suitable for diagnosis of brucellosis in cattle (21 I.U./ml gives positive and 18.2 I.U./ml gives negative reaction) These limits of sensitivity result in reduced performance for the diagnosis of Brucella melitensis infection in sheep (Blasco et al., 1994)

Polymerase chain reaction (PCR) PCR is particularly useful in case of tissues and fluids contaminated with non- viable or low numbers of Brucella organisms in diagnosis, It can detect Brucella DNA. A good sensitivity of PCR was reported by Fekete et al. (1990 a and 1990b), Baily et al. (1992) and Da Costa et al. (1996).

Polymerase chain reaction (PCR) Several techniques of PCR are used such as AP-PCR, rep-PCR, ERIC-PCR, either alone or together with labelled probes to differentiate some Brucella species and biovars (Feketa et al., 1992, Romer et al., 1995, Matar et al., 1996).

Polymerase chain reaction (PCR) Bricker and Halling (1994) described a multiplex primer assay, designated as AMOS -PCR, It uses a five-primer cocktail It can distinguish Brucella abortus biovars 1,2 and 4, Brucella melitensis biovars 1,2 and 3, Brucella ovis and Brucella suis biovar 1.

Polymerase chain reaction (PCR) Ewalt and Bricker (2000): reported on the successful application of AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51.

Natural infection gives life-long immunity This means the best immunity is achieved by using live vaccines Bang, 1906

Peculiarities of brucellosis 1. Acute infection is revealed by abortion 2. Subsequent infection may not lead to abortion but the animal may remain infected and excretor 3. Calves may be born alive but contaminated with billions of the organism

Peculiarities of brucellosis 4. Animal infection produces * Humoral immune response * Cell-mediated immune response 5. Young animals exposed to infection before sexual maturity are often naturally immunized, but may remain infected and excretors

A good, strong and long-lasting immunity against Brucella requires that: 1.the vaccinal strain persists a time longe enough in lymphoid organs to produce the desired immunity 2. the vaccinal strain has a low but real residual virulence linked to ability to multiply and resist

Question 1: What immunological mechanisms protect against brucellosis ? 1. Brucella is a facultative intracellular parasite 2. Brucella can survive and replicate within normal macrophages 3. Activated macrophages can kill Brucella 4. In order to be able to persist for a period sufficient to induce immune response, virulence determinants must be expressed

Question 1: What immunological mechanisms protect against brucellosis ? 5. Immunity in brucellosis is mediated by both humoral and cell-mediated immune response * A good vaccine must stimulate both arms (HI,CMI) to prepare the host to promptly react to aggression * Antibodies are very active during the first stage of infection restricting dissemination to lymph nodes and professional organs

Question 1: What immunological mechanisms protect against brucellosis ? T-cells are responsible for killing of intracellular bacteria * Microorganisms engulfed by a macrophage may be induced to synthesize unique antigens and/or virulence factors which permit them to survive and also may induce CMI

Question 1: What immunological mechanisms protect against brucellosis ? Thus killed vaccines may not have the crucial antigens necessary of induction of CMI Ideally, is to develop an attenuated organism which is able to persist, multiply and synthesize the unique antigens necessary for induction of protective CMI, but it is safe

ATTENUATED BRUCELLA VACCINES Brucella abortus Strain 19 Spontaneous loss of virulence Brucella suis 2 by in-vitro transfer Brucella melitensis Rev 1 Selective mutagenesis Brucella abortus RB51 through antibiotics

Brucella cell components acting as antigens 1. Purified extracts 2. Cell wall fractions 3. Lipopolysaccharide (LPS) 4. O-polysaccharides (OPS) 5. Outer membrane proteins (OMPs) * conserved in all Brucella species 6. Ribosomal fractions 7. DNA

Major outer membrane proteins Major OMP were initially identified in the early 1980's by selective extraction techniques and classified on the basis of their molecular mass as : * KDa OMP's or group 1 * KDa OMP's or group 2 * KDa OMP's or group 3

Conclusion about Vaccines through genetic engineering Subunit vaccines proved to be not effective in protecting animals from subsequent infection (Confer et al., 1987 and Winter et al., 1988).

Conclusion about Vaccines through genetic engineering recombinant Brucella abortus proteins induced an increased humoral immune response but did not protect Brucella-challenged mice (Pugh. and Tabatabai, 1994).

The failure to obtain an effective subunit or recombinant monovalent Brucella vaccine is attributed, most probably, to the difficulty in answering the question: which antigen or antigens are responsible in eliciting protective immunity in brucellosis

The failure to obtain an effective subunit or recombinant monovalent Brucella vaccine This problem is in great extent related to the antigen processing and presentation events which are rather complex (Schurig, 1994).

The failure to obtain an effective subunit or recombinant monovalent Brucella vaccine Moreover, microorganisms do not express the same antigens at all times (Yura et al., 1993). This is why, the best immunity is commonly achieved by live microorganism.

Vaccines attenuated by transposition mutagenesis 1. Smooth mutants lacking virulence factors * Smooth mutant lacking galactosidase * Smooth mutant lacking erythritol sensitivity * Smooth mutant lacking catalase, urease enzymes * Smooth mutant lacking superoxide dismutase * Smooth mutant lacking cationic peptides sensitivity

Vaccines attenuated by transposition mutagenesis 2. Rough strains 1. VRTM1 derived from virulent B. melitensis 16M 2. VRTS1 derived from virulent B. suis 2570

Rough Brucella vaccines Contain no LPS O-antigens Induce no diagnostic LPS O- antibodies Do not interfere with serodiagnosis Positive serology means infection

Brucella abortus RB51 * It is a laboratory-derived rough mutant of the virulent strain 2308 of Brucella abortus * Rifampin and penicillin resistant * It contains the same OMP as S19 and S2308

The genome sequence of Brucella melitensis strain 16M contains 3,294,935 bp on 2 chromosomes: 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. 2,487 (78%) ORFs were assigned functions

It is recommended 1. To characterize bacteriological and genetic properties of Brucella isolates from the region. Improvement of our knowledge on the bacteriological characterization and molecular epidemiology of the prevailing strains in the region is expected to provide a tool for rationalization of regional control strategies to combat the disease.

It is recommended 3. to molecularly and bacteriologically characterize the Rev. 1 field isolates recovered from different animals in various countries of the region and compare them to the standard Elberg strain.

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