Brucella antigen & co-agglutination presentation

1. BY Ajay Gurung
3RD Year BSc.MLT

2.  Brucella are non-
motile,non-
sporing,gram negative
cocobacilli
 David Bruce first
isolated a small
microorganism in 1887
from the spleen of fatal
cases of British soldiers

3.  MAJOR SPECIES:-Brucella melitensis,Brucella abortus &
Brucella suis
 OTHER SPECIES:-Brucella canis,Brucella ovis & Brucella
neotomae

4.  Brucellosis is a zoonosis primarily of domestic animals,
causing a chronic debilitating septicemic disease leading to
abortion.
 The disease is prevalent worldwide and is particularly
common in the Mediterranean and Middle Eastern countries,
and in parts of Africa and South America.

5.  Brucella does not posses any classical virulence factor
 Lipopolysaccharide is major virulence factor
 They undergo antigenic variation where colonies
switch from smooth to rough morphology
 Brucella posses smooth phase they contain S-LPS which
are virulent and resist killing by macrophages

6.  Abortus and Melitensis antigens are common to 3
mains Brucella spp.
 B.melitensis has the highest concentration of M antigen
and causes the most serious infections.
 The difference between species is related to the amount
of the two main antigen:

7.  B.abortus : A:M=20:1
 B.melitensis: A:M=1:20
 B.suis : A:M=2:1

8.  The S-LPS O (somatic antigen) chain from both smooth
B.melitensis and smooth B.abortus strains are composed
of polymers 4,6 dideoxy-4-formamido-D-mannose
(i.e.,N-formyl-D-perosamine)
 Cross reactivity is observed between brucella species
and E.coli O:116,O 197,Salmonella khauffman white
group N (O:30 antigen),V.cholerae O:1 &Y.enterocolitica
O:9.

9.  The serodominant A antigen tends to be rod-shaped
due to the five consecutive α-1,2-linked residues
 The serodominant M antigen is “kinked” shape
because the fourth residue is linked to the fifth by an α-
1,3 linkage

10.  Serological test are generally done for diagnosis for
brucella in many cases
 General serological test done for Brucella species are as
follows:-
1.Rose bengal agglutination test
2.Standard agglutination test
3. Indirect Coombs test
4.Enzyme immuno assays

11. -Most frequently Utilized test
-It is generally done by tube method
-A standardized bacterial suspension of whole
bacteria is treated standardized diluting serum
ranging from 1:20 to 1:1280
-After 24 hours of incubation at 37°C,visible
agglutination of cells at the bottom is observed
-Test usually turn positive after 7-10 days of
infection

12.  Upon infection at first IgM level increases but after 2nd
week becomes decline then starts production of IgG
antibodies
 The sensitivity and the specificity of the test depends
on the background of the level of disease in population
and cutoff value
 They increase if the cutoff value is near to the Brucella
endemic level

13.  The presumptive diagnosis of the test can be made by
the four-fold rise in the titer
 Active Brucella infection the titer level is more than
equal to the 160
 B.abortus is used as the antigen employed which cross
reactive B.melitensis or B.suis
 Prozone phenomenon is common
 Serial dilution of the serum is tested

14.  It is a Rapid agglutination test
 The test is based on the agglutination of whole
smooth based B.abortus cell stained with rose
bengal dye at low pH which inhibit nonspecific
agglutinating of bacterial cells
 It detects both agglutinating and non-
agglutinating substance
 Due to low specificity of this test and large
number of false positive it is not specific for the
endemic region

15.  Since S-LPS show cross reactivity against other gram
negative bacteria it further decreases its specificity due
to high number of false positive result

16.  Its helps in the detection of blocking antibodies or Non
agglutinating IgG antibodies
 Its as same standard as the SAT
 Standard tube dilutions that are negative for whole of
B.abortus or B.melitensis is centrifuged
 It is washed several times and resuspended
 Anti-IgG is added to the suspension

17.  The tubes are incubated at 37◦C for 24 hours
 Agglutination is visually observed
 This test is also useful for the detection of chronic or
relapsing disease since it detects both agglutinating or
non agglutinating antibodies

18.  This test are valuable and very sensitive
 Its is seen as higher sensitivity than SAT and Indirect
Coomb test
 Its can distinguish IgG,IgM and IgA
 B.abortus S-LPS is used as an antigen
 It distinguish acute infection with chronic infection

20.  It is a simple slide test first devised by Kronwald in
1973
 The test is based on the presence of protein A on the
outer surface of Staphylococcus aureus(Cowan 1 strain)
 Protein A of the S.aureus contain a large amount of
antibody binding protein.
 The antibody binds by Fc terminal to protein A leaving
Fab region the antigen combining remain free

21.  Due to its functional activity with the IgG molecules
Staphylococcal Co-agglutination is used for the
detection of antigen
 This method is used for the serological beta hemolytic
grouping of streptococci
 When killed S.aureus cells with A protein is mixed with
antiserum
 Staphylococcal cells binds IgG molecules of Antibody
by Fc region and visible agglutination is observed

23.  It is best perfomed on a cardboard slide and the
agglutination occurs in 1 minute
 Commercially coagglutination is used for cultural
confirmation of the Neisseria gonorrhea & Staphylococcus
aureus

24.  P Chakraborty textbook of microbiology
 Koneman color atlas and diagnostic of
microbiology
 Bailey & scott diagnosis of microbiology