Brucellosis - Dr.dhiren Bhoi

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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Brucellosis - Dr.dhiren Bhoi

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