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8/14/2019 Brucellosis - Dr.dhiren Bhoi
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AN
assignmentPRESENTATIONON
RECENT ADVANCES IN DIAGNOSIS ANDMANAGEMENT OF BRUCELLOSIS WITH
SPECIAL REFERENCES OF ERADICATIONin bovine
Presented By: Dr.Dhiren Bhoi
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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 Bangs Disease, Contagious
Abortion or Infectious Abortion. In human
it causes Malta fever/Undulant fever
In cattle it is caused byB. abortus, which has 9biotypes.Biotype-1 is most predominant in
cattle. (Renukaradhya et al., 2002)
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Brucella abortus
Gram-negative coccobacillary (0.5-0.7 0.6-1.5 m) 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
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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 incattle. (Muskens et al., 1996)
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Recent AdvanceDiagnostic Technique For
Brucellosis in Cattle
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Rose Bengal Test (RBT)
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)
Diagnostic test forB. abortus
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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 influencethe sensitivity and specificity of the RBT.(MacMillan, 1990)
)Rose Bengal Test )RBT
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Principle
The buffered acid Rose Bengal antigeninteracts with serum antibody to produce
agglutinin, which is used for the early
detection of Brucella specific antibodies.
(Nielsen,
2002)
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Procedure Place a drop (30 l) ofundiluted serum on a slide.
Add a drop of the reagent (Rose Bengal Brucella antigen)
next to the drop of the serum.
Mix both drops by the disposable stirring stick, spreading
them over the full surface of the circle.
Observe for the result
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Reading
Noagglutination= (- Ve) Negative result. Agglutination= (+Ve) Positive results (Singh,
2004)
Positive test Negative test
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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)
Milk Ring Test (MRT)
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Used to detect brucella antibody in milksamples.
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)
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Procedure
Take 10 ml of milk sample in a test tube.
Add 50 l 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.
10.Ring of cream less colored than the underlying milk
= Negative result.
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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)
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COMPLEMENT FIXATION
(TEST (CFT
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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 andspecific in animals with naturally infected of
brucellosis. (Nielsen, 2002)
CFT detects specific antibodies of the IgM andIgG-1 type, but more sensitive against the IgG-1
type than that of IgM (Levieux, 1974 )
Complement Fixation Test (CFT)
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Gives false negative result with antibodies ofIgG-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 toreduce the number of samples that need to be tested by
(CFT).
CFT is recommended by the OIE as a test prescribed forinternational trade
(Nielsen, 2002 and OIE Manual, 2000c)
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Principle:Antigen and Antibody fixed complement willnot be available to lyse the target RBC so nohemolysis will occur in positive test while in the
absence of an antibody, complement will beavailable to lyse the target cells and hencehemolysis in the negative tests.
(Nielsen,2002)
No Haemolysis Positive testHaemolysis negative test
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Complement Fixation
Ag mixed with test serum to be assayed for AbStandard amount of complement is added
Erythrocytes coated with Abs is added
Amount of erythrocyte lysis is determined
Ag
Patientsserum
Ag No Ag
Ag
Methodology
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Enzyme Linked Immuno Sorbent
(Assay (ELISA
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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.
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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 IgG1conjugated with
peroxidase(OIE
Manual,2000a)
OIE prescribed Indirect ELISA test as international trade
(OIEManual,2000a)
Indirect ELISA is enable to differentiate the Ab produced
by vaccination and infection (Nielsen and Gall, 1994)
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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
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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
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1. Add
antigens
Ag-coated
well
3. Add anti-Ab2. Add mouse serum
Ag-Abcomplex
OpticalOptical
DensityDensity
)OD)OD
ReadingReading
4. Add enzyme-4. Add enzyme-
substrate mixsubstrate mix
5.Let colorize5.Let colorize
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.
cont
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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)
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Polymerase Chain Reaction
((PCR
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What is PCR??
In vitro DNA synthesis
Use to differentiate among Brucella species
and/or biovars (Bricker, 2002a)
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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 betweenspecies)
PCR amplify these variable regions and thensequence our amplified fragments to determinethe identity of unknown organisms
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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
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The basic protocolwhats in the tube
Target DNA
5 3
3 5
primers
AB Free
nucleotides
Taq DNA
polymerase
Mg2+Mg2+
Mg2+
Mg2+
Mg2+
Mg2+
Buffer
containing
magnesium
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Primers
Two oligonucleotides of different sequences.
Each are typically 18-25 nucleotides long.
Primers complementary base pair (hybridizeor anneal) to template DNA.
General Example ofPrimers
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Primers
Target DNA
5 3
3 5
forward
reverse
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Primers
Designed according to the region to beamplified
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
Target DNA
5 3
3 5
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Heat-stable DNA polymerase
Taq(isolated from the bacterium Thermus acquaticus),
Pfu(from Pyrococcus furiosus) and Vent(from
Thermococcus litoralis) polymerase
Pfuand Ventare more efficient than the Taq
polymerase but Pfuis slowerthan Taqand more
expensive. (Singh,2004)
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Taq polymerase
Most enzymes would be denatured at 95oC
Taq was isolated from Thermus aquaticus, a bacteriathat grows in hot springs (~75oC)
This organisms enzymes have adapted to the hightemperature, so they can survive cycling through thehigh temperatures
Taq polymerase is stable at the high temperatures(~95oC) used for denaturing DNA.
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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)
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1. Denaturation of DNA to single
strands
2. Annealing of primers to DNA3. Extension by polymerase
4. Repeat 30-35 times
The basic protocol
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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 forTaq DNA polymeraseactivity.
(Singh,2004)
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How does PCR work?
One PCR Cycle:
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Denaturation
Target DNA
95oC
5 3
3 5
5 3
3 5
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Annealing
~55oC
5 3
3 5
5 3
3 5
5
5
Target DNA
A
B
primers
A
B
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Extension
72oC
5 3
3 5
5 3
3 5
5
5
Target DNA
Taqpolymerase
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Extension cont..
72oC
5 3
3 5
5 3
3 5
5
5
Target DNA
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How does PCR work?
One PCR cycle: What the products
really looks like
4 DNA strandsTemplate Strand
Template Strand
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Two cycles: What the products really looks
like
8 DNA strands
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Three cycles
16 DNA strands
The number of DNA strands doubles after each cycle.
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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
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PCR Thermocycler
Very rapidly changes
the temperature
between the variousstages of the PCR
process
Programmable for use
with many differentcycling parameters
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DemonstrateDemonstrate delayed hypersensitivitydelayed hypersensitivity
combined use of SAT and SDTH tests increase the reliabilitycombined use of SAT and SDTH tests increase the reliability
of brucellosis diagnosisof brucellosis diagnosis
The antigenThe antigen (B. abortus S-19 strain)(B. abortus S-19 strain) is a filtrate of a culture ofis a filtrate of a culture of
brucella organisms (brucella organisms (1 mg protein ml1 mg protein ml-1-1)) (Bercovich(Bercovich et al.,et al., 1990)1990) InjectsInjects intradermally (0.1 ml)intradermally (0.1 ml)
The test is positive if localThe test is positive if local rednessredness with thickness of skin bywith thickness of skin by
more than 1 mmmore than 1 mm afterafter24-4824-48 hourshours (Muskens, 1996)(Muskens, 1996)
Antigen can provoke an antibody response or aAntigen can provoke an antibody response or a significantsignificantriserise in a pre-existing responsein a pre-existing response
Skin Delayed Type Hypersensitivity test (SDTH)
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CONTROL/ERADICATION
OF BRUCELLA
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First think about..!
Govt. and public support
Correct guideline and policies
The cost and economic benefits must be
assessed
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Eradication of Bovine Brucellosis in Australia
1st phase starts in 1930s only Tasmania get freefrom brucella
2nd phase starts with the development ofBrucella
abortusStrain 19 vaccine In 230 farms ofVictoria, the abortion rate in heifers dropped from
37% to under 5% within one year. This encourage other states
also to adopt vaccination.
3rd phasestarted in 1970 vaccination alongwith test
and slaughteruntil two tests at least 6 months interval
gives ve result
Australia declare itself free of bovine brucellosis in 1992
(Bunn, 2002)
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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
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Cont.
PD-ADMAS since 1994, conducting large scale of RBPT
and SAT tests, and since 1997 use A-B ELISA
(Islooret 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, conductedserological tests on 47,775 bovine in 24 states and in1
UT (Renukaradhya et al., 2002)
Project Directorate on Animal Disease
Monitoring and Surveillance (PD-ADMAS)
B i B ll i P i C t l
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Bovine Brucellosis Progressive Control
Programme (BBPCP)
Three major components of BBPCP are
Biannual village level screening of pooled milk
samples
5 year vaccination programme with B. abortus S-19
vaccine
Scanning and castration of infected bulls
(Renukaradhya et al., 2002)
32 ELISA laboratories have been set in each district headquarters
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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
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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
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Vaccination Programme
Increases resistance and decreases the sourceof 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
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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 ageof 4-10 months and a second conjunctival vaccination with the samedose after six months
(Plommet,1991)
Disadvantages: Induces abortion in pregnant animals
Excreted in milk
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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 annualbooster (Plommet, 1991)
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B. abortus strain RB51 vaccine
Compared with S-19 vaccine, RB51 vaccinecauses less abortion (Cheville et al., 1996)
Protective effect ofRB51 vaccine in cattle
is similar to that of S-19
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