Download Brucellosis in Animals - Cairo University Scholars

Brucellosis and immunity
by
Prof. Dr. Mohamed Refai
Department of Microbiology, Faculty of
Veterinary Medicine, Cairo University,
Giza, Egypt. [email protected]
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):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

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 BLymphocyte
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 Tlymphocytes, 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 -)
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
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 nonviable 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
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

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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


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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
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 Oantibodies
 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.
‫شكرا علي حسن استماعكم‬
Thank you for your
attention