Brucellosis and immunity by Prof. Dr. Mohamed Refai Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. firstname.lastname@example.org
Discovery of Brucella organisms • In the late 1887, Bruce isolated an organism he named Micrococcus melitensis from individuals withMediterranean 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 namedBrucella.
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):443-448 • 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
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 are made by all nucleated cells in the body and possess a deep groove that can bind peptideepitopes, 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
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)
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.
Differentiation of B-lymphocytes into Plasma Cells and B-Memory Cells
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. Detectionof 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 detectionof affinity purified anti-B. abortus antibody isotypes Assay IgM IgG1 IgG2 AgA • SAT 20* - 125 650 • BAPA - 550 5500 - • RBT - 600 7500 - • RIV - 1550 2750 - • 2ME - 1500 2600 - • CFT - 210 - ____________________________________ • * 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.
Bang, 1906 Natural infection gives life-long immunity This means the best immunity is achieved by using live vaccines
Peculiarities of brucellosis 1. Acute infection is revealed by abortion 2. Subsequent infection may not lead to abortionbut 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 mayremain infected and excretors
A good, strong and long-lasting immunity against Brucella requires that: • 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 normalmacrophages 3. Activated macrophages can kill Brucella 4. In order to be able to persist for a period sufficientto induce immune response, virulence determinants must be expressed
Question 1:What immunological mechanisms protectagainst brucellosis ? 5. Immunity in brucellosis is mediated by bothhumoral 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 firststage of infection restricting dissemination to lymph nodes and professional organs
Question 1:What immunological mechanisms protectagainst brucellosis ? • T-cells are responsible for killing of intracellular bacteria • * Microorganisms engulfed by a macrophage • may be induced to synthesize unique antigensand/or virulence factors which permit them tosurvive and also may induce CMI
Question 1:What immunological mechanisms protectagainst brucellosis ? • Thus killed vaccines may not have the • crucialantigens necessary of induction of • CMI • Ideally, is to develop an attenuated organismwhich is able to persist, multiply and synthesizethe unique antigens necessary for induction ofprotective 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 : • * 94-98 KDa OMP's or group 1 • * 36-40 KDa OMP's or group 2 • * 26-34 KDa OMP's or group 3