AN

assignment
PRESENTATION
ON
RECENT ADVANCES IN DIAGNOSIS AND
MANAGEMENT OF BRUCELLOSIS WITH
SPECIAL REFERENCES OF ERADICATION in
bovine

Presented By: Dr. Dhiren Bhoi

 Introduction of Brucellosis

It is a zoonotic disease of economic importance

with world wide distribution and infecting all
domestic animals. (Renukaradhya et al., 2002)

Also called as Bang’s Disease, Contagious

Abortion or Infectious Abortion. In human it
causes Malta fever/Undulant fever
In cattle it is caused by B. abortus, which has 9
biotypes.Biotype-1 is most predominant in
cattle. (Renukaradhya et al., 2002)
Brucella abortus
• Gram-negative coccobacillary (0.5-0.7 × 0.6-1.5 µm)
• Non-motile,
• Non-spore forming
• Partially acid fast, not decolorized by 0.5% acetic acid in the
Modified Ziehl-Neelsen (MZN) stain (Nielsen, 2002)
• Biochemical properties
– Aerobic
– Capnophilic (carboxyphilic, require 5-10% CO2 for growth )
(Bandara and Mahipala, 2002)
– Catalase positive,
– Oxidase positive
– Urease positive
– Not grow on MacConkey agar
• Facultative intracellular organism with
shedding in reproductive and mammary
secretions. (Dey et al., 2000)

• Brucellosis is primarily a reproductive

disease characterized by abortion,
retained placenta and impaired fertility in
cattle. (Muskens et al., 1996)
Recent Advance Diagnostic
Technique For Brucellosis in
Cattle
 Diagnostic
Diagnostic test
test for
for B.
B. abortus
abortus

1. Rose Bengal Test (RBT)

2. Milk Ring Test (MRT)
3. Complement Fixation Test (CFT)
4. Enzyme Linked Immuno Sorbent Assay (ELISA)
5. Polymerase Chain Reaction (PCR)

6. Skin Delayed Type Hypersensitivity test (SDTH)

 Rose
Rose Bengal
Bengal Test
Test (RBT)
(RBT)

• Spot test for brucella diagnosis

• Detects specific antibodies (IgM and IgG types)
but more effectively detect IgG-1 than IgM and
IgG 2 (Levieux, 1974)
• Low pH (3.6) of the antigen enhances the
specificity of the test, the temperature of the
antigen and the ambient temperature influence
the sensitivity and specificity of the RBT.
(MacMillan,
1990)
Principle

The buffered acid Rose Bengal antigen

interacts with serum antibody to produce
agglutinin, which is used for the early
detection of Brucella specific antibodies.
(Nielsen,
2002)
Procedure
1. Place a drop (30 μl) of undiluted serum on a slide.

2. Add a drop of the reagent (Rose Bengal Brucella antigen)

next to the drop of the serum.

3. Mix both drops by the disposable stirring stick, spreading

them over the full surface of the circle.

4. Observe for the result

Reading
1. No agglutination = (- Ve) Negative result.
2. Agglutination = (+Ve) Positive results
3. (Singh,
2004)

Positive test Negative test

 Milk
Milk Ring
Ring Test
Test (MRT)
(MRT)
 Cheap, Easy, Simple and Quick to perform
 Detects lacteal anti - Brucella IgM and IgA bound to milk
fat globules
 False positive - when milk that contains colostrum,
- milk at the end of the lactation period
- cows suffering from hormonal disorder
- milk from cows with mastitis
(Bercovich and Moerman, 1979)

 False negative - milk with low conc. of IgM and IgA

- milk lacking the fat-clustering factors
(Patterson and Deyoe, 1978)
 Used to detect brucella antibody in milk
samples.
Principle
Based on the principle of agglutination
between antibodies contained in milk and
colored bacterial antigen of brucella to form
antigen-antibody complexes that are
progressively carried by the fat towards the
surface of the milk and formed a blue violet
ring. (Singh, 2004)
Procedure
 Take 10 ml of milk sample in a test tube.

 Add 50 µl of the brucella colored antigen and mix carefully.

 Place in incubator for 1 hour at 37 OC or for 18-20 hours at

4 OC, and then read the result.
Results
1. Ring of cream equal or more colored than the
underlying milk = Positive result.
2. Ring of cream less colored than the underlying milk
= Negative result.
• MRT titer — in infected animals → 1 : 25 or above
— in vaccinated animals → 1 : 10 or above
(Singh and Pathak, 1975)

• MRT is recommended by the OIE as a screening

test for bovine brucellosis (OIE Manual,
2000a)
COMPLEMENT
COMPLEMENT FIXATION
FIXATION
TEST
TEST (CFT)
(CFT)
 Complement
Complement Fixation
Fixation Test
Test (CFT)
(CFT)
• The complement fixation test (CFT) is widely used
for the diagnosis of brucellosis in cattle, sheep and
goat.
• Relatively insensitive to antibody produced in
response to vaccination but highly sensitive and
specific in animals with naturally infected of
brucellosis. (Nielsen, 2002)

• CFT detects specific antibodies of the IgM and

IgG-1 type, but more sensitive against the IgG-1
type than that of IgM (Levieux,
1974 )
• Gives false negative result with antibodies of IgG-2 type
(MacMillan,
1990)

• The test is some what complex and in mass testing a

screen test such as Rose Bengal test is often used to
reduce the number of samples that need to be tested by
(CFT).

• CFT is recommended by the OIE as a test prescribed for

international trade
(Nielsen, 2002 and OIE Manual, 2000c)
Principle:
Antigen and Antibody fixed complement will
not be available to lyse the target RBC so no
hemolysis will occur in positive test while in the
absence of an antibody, complement will be
available to lyse the target cells and hence
hemolysis in the negative tests.
(Nielsen,
2002)

No Haemolysis → Positive test

Haemolysis → negative test
 Complement Fixation
• Methodology
– Ag mixed with test serum to be assayed for Ab
– Standard amount of complement is added
– Erythrocytes coated with Abs is added
– Amount of erythrocyte lysis is determined

Ag No Ag
Ag
Patient’s
serum
Ag
Enzyme
Enzyme Linked
Linked Immuno
Immuno Sorbent
Sorbent
Assay
Assay (ELISA)
(ELISA)
• ELISA – an immunological test, using an enzyme as a
label to determine presence of target antibody/antigen.

• The enzyme linkage or labeling allows to follow target

protein and if present (qualify) and at what amounts
(quantify).

• An enzyme conjugate is an enzyme bound or joined with

an antibody which binds with target antigen. This
enzyme labeling is a safe and effective way to track
antibody.
• Commonly SLPS antigen & peroxidase or alkaline
phosphatase are used
(Nielsen et al., 1994; Vanzini et al, 1997, 2001)

• OIE approved purified SLPS antigen, serum dilution 1:50,

MAB specific for bovine IgG1 conjugated with peroxidase
(OIE Manual,2000a)

• OIE prescribed Indirect ELISA test as international trade

(OIE Manual,2000a)

• Indirect ELISA is enable to differentiate the Ab produced

by vaccination and infection (Nielsen and Gall, 1994)
 Direct ELISA

• Micro wells are coated to antigen

• Enzyme conjugated Antibody (Ab-E) is added and binds
with antigen.
• Wells are washed to remove any excess (Ab-E).
• Substrate is added and color development is observed.

Ag + Ab-E + Substrate
 Indirect ELISA
• Antigen (Ag) are coated to micro wells.
• Antibodies (Ab) is added and binds with antigens.
• Excess Ab is washed away.
• Enzyme conjugate (Ab-E) is added and binds with
antigen to form the double antibody sandwich.
• Wells are washed to remove any excess (Ab-E).
• Substrate is added and color development is compared
in terms of OD at 492 nm (Sharma et al., 2003).

(Nielsen et al., 1994)

Ag + Ab + Ab-E + Substrate
 cont…

ELISA test is a technique for detecting & measuring antigen or antibody.

:-It is one of the most reliable techniques to detect
antibody against brucella infection.
:-Its procedure is the principal for development of recent
rapid diagnostic kits.
:-This technique is widely used in laboratories & hospitals.

Ag-Ab
complex
Optical
Density
Ag-coated
well
(OD)
Reading

1. Add 2. Add mouse serum 3. Add anti-Ab

antigens
4. Add enzyme- 5.Let colorize
substrate mix
 dot – ELISA

 B. abortus S-19 whole cell antigen is used

(Chand et al., 1990)
 dot – ELISA 89% agreed with RBPT and STAT,
and its sensitivity is 95-98 %
(Shrivastava et al., 1991)
 Its titer ranges from 1:160 to 1:320
(Shrivastava et al., 1991)
Polymerase
Polymerase Chain
Chain Reaction
Reaction
(PCR)
(PCR)
 History of PCR
• PCR technique was invented by
Kary Mullis in 1983 (Nobel Prize in
Chemistry)

• Make millions of copies of DNA from

trace amounts of DNA starting
material.

•Only specific pieces of DNA are amplified.

• This process acts as a “copying machine” for DNA

 What is PCR…??

In vitro DNA synthesis

Use to differentiate among Brucella species

and/or biovars (Bricker, 2002a)
 Principle of PCR

• All organisms have DNA sequences (rDNA)

which code for ribosomal RNA
– Remember that rRNA is a critical component of
ribosomes so it is necessary for translation
• There are regions in the rDNA which vary
between genera (and in many cases between
species)
• PCR amplify these variable regions and then
sequence our amplified fragments to determine
the identity of unknown organisms
 Components for PCR
– DNA template
– Two Primers (forward and reverse)
– Heat-stable DNA polymerase (Taq or Pfu
polymerase)
– Deoxynucleotides – dATP, dTTP, dCTP, dGTP
– Mg++, buffer components, and water
 The basic protocol—what’s in the tube
5’ 3’
Target DNA
3’ 5’

A
B Free
primers nucleotides

Mg2+ Mg2+
2+
Buffer
Mg
Taq DNA Mg2+ containing
Mg2+
Mg2+ magnesium
polymerase
 Primers

• Two oligonucleotides of different sequences.

• Each are typically 18-25 nucleotides long.
• Primers complementary base pair (“hybridize”
or “anneal”) to template DNA.

General Example of Primers

 Primers

forward

5’ 3’
Target DNA
3’ 5’

reverse
 Primers
• Designed according to the region to be
amplified
• Melting point determined by G-C and A-T
content
– Tm = 4oC (G+C) + 2oC (A+T)
– Ex: a primer with 10 G/C and 10 A/T would have a
Tm of 60oC 4(10) + 2(10)=60oC

5’ 3’
Target DNA
3’ 5’
 Heat-stable DNA polymerase

• Taq (isolated from the bacterium Thermus acquaticus),

Pfu (from Pyrococcus furiosus) and Vent (from
Thermococcus litoralis) polymerase

• Pfu and Vent are more efficient than the Taq

polymerase but Pfu is slower than Taq and more
expensive. (Singh,
2004)
 Taq polymerase
• Most enzymes would be denatured at 95oC

• Taq was isolated from Thermus aquaticus, a bacteria

that grows in hot springs (~75oC)

• This organism’s enzymes have adapted to the high

temperature, so they can survive cycling through the
high temperatures

• Taq polymerase is stable at the high temperatures

(~95oC) used for denaturing DNA.
 DNA template

• Main genetic targets utilized for these

applications are the brucella BCSP-31 gene and
the 16S-23S rRNA operon.

• 16S-23S rRNA operon has been shown in

studies to be more reliable
(Bricker, 2002b)
 The basic protocol
1. Denaturation of DNA to single
strands
2. Annealing of primers to DNA
3. Extension by polymerase
4. Repeat 30-35 times
 Three steps of PCR cycle

• Each PCR cycle includes:

– A denaturation step (92-96oC for 1-2 minutes)
separates the two DNA strands.

– A primer annealing step (40-75oC for 1 minutes)

which is a few degrees below the Tm of the primers.

– A primer extension step (72oC for 1-2 minutes) which

is the optimal temperature for Taq DNA polymerase
activity.
(Singh,
2004)
 How does PCR work?

One PCR Cycle:

 Denaturation
5’ 3’
Target DNA
3’ 5’

95oC

5’ 3’

3’ 5’
 Annealing
5’ 3’
Target DNA
3’ 5’

A
B
primers
~55oC

5’ 3’

B
5’
5’ A

3’ 5’
 Extension
5’ 3’
Target DNA
3’ 5’

72oC

5’ 3’

Taq polymerase 5’
5’

3’ 5’
 Extension cont…..

5’ 3’
Target DNA
3’ 5’

72oC

5’ 3’

5’
5’

3’ 5’
 How does PCR work?

• One PCR cycle: What the products

really looks like…
Template Strand

4 DNA strands
Template Strand
• Two cycles: What the products really looks
like…

8 DNA strands
 • Three cycles…

16 DNA strands

The number of DNA strands doubles after each cycle.

One One billion in about 2 hours!
• At the end of each cycle, the amount of
DNA has doubled
• By the end of 30 cycles, you will have
about 1 billion molecules from the original
one you started with!!

230=1,073,741,824
 PCR Thermocycler

• Very rapidly changes

the temperature
between the various
stages of the PCR
process
• Programmable for use
with many different
cycling parameters
Skin
Skin Delayed
Delayed Type
Type Hypersensitivity
Hypersensitivity test
test (SDTH)
(SDTH)

• Demonstrate delayed hypersensitivity

• combined use of SAT and SDTH tests increase the reliability
of brucellosis diagnosis
• The antigen (B. abortus S-19 strain) is a filtrate of a culture of
brucella organisms (1 mg protein ml-1 ) (Bercovich et al., 1990)
• Injects intradermally (0.1 ml)
• The test is positive if local redness with thickness of skin by
more than 1 mm after 24-48 hours (Muskens, 1996)
• Antigen can provoke an antibody response or a significant
rise in a pre-existing response
CONTROL/ERADICATION OF
BRUCELLA
 First think about…..!
 Govt. and public support

 Correct guideline and policies

 The cost and economic benefits must be

assessed
 Eradication of Bovine Brucellosis in Australia

 1st phase starts in 1930s – only Tasmania get free

from brucella
 2nd phase starts with the development of Brucella
abortus Strain 19 vaccine
In 230 farms of Victoria, the abortion rate in heifers dropped from
37% to under 5% within one year. This encourage other states
also to adopt vaccination.

 3rd phase started in 1970 – vaccination alongwith test

and slaughter until two tests at least 6 months interval
gives –ve result
Australia declare itself free of bovine brucellosis in 1992
(Bunn, 2002)
 Control of brucella in Sri Lanka
• Vaccination, serological testing, separation and culling of positive
animal (Peiris, 1981)

S-19 vaccine in 1/20th of

recommended dose

After 2 week SAT titer 1:320 in

90-92% of animals

۞ No abortion is observed in pregnant animal immunized with

same dose of vaccine (Bandara and mahipala, 2002)
 Control/eradication Programme for Brucella in India
• Vaccination, serological test and separation of positive cases,
but can not slaughter the cows due to religious sentiment

• No slaughtering, rapidly development of dairy sector and

uncontrolled movement of animals → spread of infection

• Calf hood S-19 vaccine is practice

• Bulls used for production of semen for AI should be regularly

screen for brucella, infected bulls are castrated

• In each district headquarter diagnostic laboratory is stablished

 Cont….
Project Directorate on Animal Disease
Monitoring and Surveillance (PD-ADMAS)

• PD-ADMAS since 1994, conducting large scale of RBPT

and SAT tests, and since 1997 use A-B ELISA
(Isloor et al.,
2001)
• PD-ADMAS develop IgG based ELISA kit to screen milk
samples in milk co-operative

• PD-ADMAS, during the period of 1994-2001, conducted

serological tests on 47,775 bovine in 24 states and in1
UT (Renukaradhya et al., 2002)
 Bovine Brucellosis Progressive Control
Programme (BBPCP)

Three major components of BBPCP are –

i. Biannual village level screening of pooled milk
samples
ii. 5 year vaccination programme with B. abortus S-19
vaccine
iii. Scanning and castration of infected bulls
(Renukaradhya et al., 2002)

۞ 32 ELISA laboratories have been set in each district headquarters

 Strategies to Fight Brucella

1) Collaboration among laboratory, field and

public health services
2) Control the infection
3) Test and slaughter method
4) Quarantine
5) Depopulation
6) Vaccination Programme
 Control the infection
• Source of Infected
• Transmission of infection
• Movement of animals
• Natural service by bulls
• Transmission by carnivores animals and through
milk
 Test and slaughter method
• No effective treatment, so diagnose, if +ve kill the
animals until no reactor animal for three consecutive
tests, carried out at three-month interval is found
(Mathur et. al.,
1974)
• Various diagnostic test, for untagged animal best test is
SDTH test (Bercovich et al., 1992)

• Financial compensation to farmers

 Vaccination Programme
 Increases resistance and decreases the source of
infection

 Different vaccine against the B. abortus are

 Live B. abortus Strain-19 vaccine
 Killed adjuvant B. abortus 45/20 vaccine
 B. abortus vaccine RB51

Make calfhood vaccination compulsory and avoid

vaccination of adult animals
 Live B. abortus Strain-19 vaccine

Vaccination procedure:
I. calves are vaccinated once with 3-10 x 109 CFU at the age of
4-8 month and for the second time with 3-10 x 109 CFU as adults
II. a conjunctival vaccination of calves with 4-10 x 109 CFU at an age
of 4-10 months and a second conjunctival vaccination with the same
dose after six months
(Plommet, 1991)

Disadvantages:
 Induces abortion in pregnant animals
 Excreted in milk
 Killed adjuvant B. abortus 45/20 vaccine

• Not giving lasting immunity

• Not induce detectable agglutinating antibodies
• Gives marked SDTH test
• Not harmful

Two initial vaccinations 1st at 3-4 month of age

and then repeat after 6 month and an annual
booster (Plommet, 1991)
 B. abortus strain RB51 vaccine

• Compared with S-19 vaccine, RB51 vaccine

causes less abortion (Cheville et al., 1996)

• Protective effect of RB51 vaccine in cattle

is similar to that of S-19