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Introduction of Brucellosis : 

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 : 

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

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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 in cattle. (Muskens et al., 1996)

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Recent Advance Diagnostic 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 for B. 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 influence the sensitivity and specificity of the RBT. (MacMillan, 1990) Rose Bengal Test (RBT)

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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)

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Procedure Place a drop (30 µl) of undiluted 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 No agglutination = (- Ve) Negative result. Agglutination = (+Ve) Positive results (Singh, 2004) Positive test Negative test

Milk Ring Test (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) Milk Ring Test (MRT)

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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)

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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. 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) : 

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 ) Complement Fixation Test (CFT)

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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)

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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 : 

Complement Fixation 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 Ag Methodology

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Complement fixation test

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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 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 : 

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 : 

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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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)

History of 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. This process acts as a “copying machine” for DNA Only specific pieces of DNA are amplified.

What is PCR…?? : 

What is PCR…?? In vitro DNA synthesis Use to differentiate among Brucella species and/or biovars (Bricker, 2002a)

Principle of PCR : 

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 : 

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 : 

The basic protocol—what’s in the tube Target DNA 5’ 3’ 3’ 5’ primers A B Free nucleotides Taq DNA polymerase Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Buffer containing magnesium

Primers : 

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 : 

Primers Target DNA 5’ 3’ 3’ 5’ forward reverse

Primers : 

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 Target DNA 5’ 3’ 3’ 5’

Heat-stable DNA polymerase : 

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)

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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 : 

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 : 

Denaturation of DNA to single strands Annealing of primers to DNA Extension by polymerase Repeat 30-35 times The basic protocol

Three steps of PCR cycle : 

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? : 

How does PCR work? One PCR Cycle:

Denaturation : 

Denaturation Target DNA 95oC 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’

Annealing : 

Annealing ~55oC 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ Target DNA A B primers A B

Extension : 

Extension 72oC 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ Target DNA Taq polymerase

Extension cont….. : 

Extension cont….. 72oC 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ Target DNA

How does PCR work? : 

How does PCR work? One PCR cycle: What the products really looks like… 4 DNA strands Template 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.

One One billion in about 2 hours! : 

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 : 

PCR Thermocycler Very rapidly changes the temperature between the various stages of the PCR process Programmable for use with many different cycling parameters

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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 Skin Delayed Type Hypersensitivity test (SDTH)



First think about…..! : 

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 : 

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 : 

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 : 

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…. : 

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

Bovine Brucellosis Progressive Control Programme (BBPCP) : 

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

Strategies to Fight Brucella : 

Strategies to Fight Brucella Collaboration among laboratory, field and public health services Control the infection Test and slaughter method Quarantine Depopulation Vaccination Programme

Control the infection : 

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 : 

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 : 

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 : 

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 : 

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 : 

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

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