Canine Brucella Antibody RSAT Test

BACKGROUND:

Dog brucellosis, primarily caused by the bacterium Brucella canis, is a contagious disease that affects both domestic dogs and wild canids. The bacterium spreads through contact with infected animals or their bodily fluids, including during mating, from mother to puppies, and through exposure to aborted fetal materials or vaginal discharges. Infected dogs often exhibit reproductive issues such as infertility, failure to conceive, and abortion, with females typically aborting between 45 to 55 days of gestation. Males may suffer from epididymitis, testicular atrophy, and poor semen quality. General symptoms like lethargy, swollen lymph nodes, and fever can also occur, though they are less common.

Diagnosis of dog brucellosis involves serological tests to detect antibodies, culture tests to isolate the bacterium, and PCR to identify bacterial DNA. Treatment is challenging and often involves extended courses of antibiotics, though it may not fully eradicate the infection. Preventive measures include routine testing of breeding dogs, maintaining strict hygiene in kennels, and careful regulation of dog imports from regions with lower breeding standards. While the disease is zoonotic, transmission to humans is rare, typically resulting in symptoms such as fever and joint pain. Preventive practices and good hygiene can significantly reduce the risk of human infection.

TEST PRINCIPLE:

The BioFAST Canine Brucella Antibody RSAT Test designed to detect antibodies against Brucella canis using a Rapid Slide Agglutination Test (RSAT). It is used to confirm suspected Brucella canis infections in dogs. 

The test involves two steps:

• Step 1- Initial Antibody Detection: The serum sample is mixed with Brucella canis antigens inactivated and coated on polymer nanospheres. If Brucella canis antibodies are present in the sample, they will react with the antigen, leading to visible agglutination. This step detects antibodies of 1 to 5 weeks infection.

• Step 2 - Exclusion of False Positives: 2-mercaptoethanol helps removing non-specific agglutinins in the serum of non-infected dogs. These agglutinins bind to 2-mercaptoethanol and prevent them from reacting with the Brucella canis antigens.

MATERIALS PROVIDED:

• 2 Reusable Glass Slides with 6 wells each
• 2.5 mL Antigen Dropper Bottle
• 1.5 mL 2-Mercaptoethanol Dropper Bottle
  
• 0.75 mL Positive Control Dropper Bottle
• 0.75 mL Negative Control Dropper Bottle
• 25  Mixing sticks
• 75  Dropper Pipettes
• 25  Reaction Tubes
• 1 Manual Instruction

SAMPLE REQUIREMENTS:

• The test requires 1 drop of serum sample.
• Plasma, whole blood or whole blood with anticoagulant cannot be used.
• The serum samples should be at room temperature before use. Don't use cooled or freezed samples. 
• Mix well the serum samples before use. 
• Serum samples can be stored for few days at +4°C. 
• Serum samples can be stored for long term at -20°C.

TEST PROCEDURE - STEP 1

• Remove the glass slide from the box kit just before use.
• Place the glass slide on a flat and clean surface.
  
• Use the first well of the glass slide as a Negative Control, the second well as a Positive Control, and the remaining four wells for sample tests (Fig.01).
  
• Add 1 drop of antigen on each well (Fig.02).
  
• For subsequent steps, add additional drops next to the antigen drop without touching or mixing the different reagents to avoid bottles contamination (Fig.03).
  
• Add 1 drop of negative control on the first well and 1 drop of positive control on the second well (Fig.03).  
  
• Using the dropper pipette, draw the sample up to the dark line (20 μL) and add the entire sample to the designated sample test wells. Repeat this procedure for other samples in the remaining wells (Fig.03).
  
• Always handle the dropper bottles and dropper pipettes vertically.
  
• Use a different dropper pipette for each sample.
  
• Dropper pipettes are single-use and must be disposed of after use.
  
• Using the mixing sticks, thoroughly mix the drops added to each well, covering the entire well area without crossing the well lines.

Figure 01

Figure 02

Figure 03

READING TEST RESULTS FROM STEP 1:

Gently shake the glass slide for up to two minutes while observing the antigen-control and antigen-serum mixtures for any signs of clumping or agglutination.

Negative Result:

• No Visible Agglutination (Fig.04).
• No Detectable Levels of Brucella Antibodies in the Sample.

Positive Result: 

• Visible Agglutination (Fig.05).
• Process Step 2.

 TEST PROCEDURE - STEP 2

Verification of Positive Results from Step 1

• Using a fresh dropper pipette, dispense 2 drops of serum into a reaction tube (Fig.06).
• Add 2 drops of 2-Mercaptoethanol to the serum (Fig.06) and vortex the content thoroughly.
• With a new dropper pipette, transfer 1 drop of the serum-mercaptoethanol complex (SMC) onto the glass slide well (Fig.07).
• Add 1 drop to the SMC on the slide (Fig.07), ensuring the dropper does not come into contact with the complex.
• Using a freshly cleaned mixing stick, mix the antigen-SMC complex (ASMC) within the glass slide well, ensuring to use the entire area of the well and preventing contact with adjacent wells.
• Gently rotate the glass slide and immediately observe the results.

READING TEST RESULTS FROM STEP 2:

Carefully tilt the glass slide back and forth for up to two minutes while monitoring the antigen-serum-mercaptoethanol complex (ASMC) for any signs of clumping.

Negative Result:

• No Visible Agglutination (Fig.04).
• Could be Early Stage Infection.
• Test Again After 2 weeks.

Positive Result: 

• Visible Agglutination (Fig.04).
• High Probability of Dog Infection.

RESULT INTERPRETATION:

Always interpret test results in the context of the patient's medical history, clinical information, and available treatment and prevention strategies.

Negative Test Result:

• Indicates no exposure to Brucella canis.
• Note that in chronic infections, antibodies might not be detectable in a single test. Therefore, it is recommended that breeding dogs undergo regular serological testing to reduce the risk of venereal transmission.

Positive Test Result after Step 1, but not after Step 2:

• The dog could be in the early stages of brucellosis infection (less than 2 to 4 weeks post-infection) and may not have produced detectable IgG antibodies yet.
• There may be non-specific agglutinins present in the serum that react with Brucella canis (indicating a non-infected status).
• To clarify, obtain a second serum sample after 28 days and repeat the test with 2-mercaptoethanol. A positive result in the repeat test strongly suggests an infection with Brucella canis.

Positive Test Result after Step 2:

• The dog is highly likely infected with Brucella canis.
• To distinguish between an acute or chronic infection, collect two serum samples 2–4 weeks apart and test them using IFAT. A significant increase in antibody titers in these tests indicates an active brucellosis infection.

TEST PRECAUTION:

• It is recommended to wear disposable gloves and other personal protective equipment.
• Wash and disinfect hands after completing the test.
• Label sample material and associated glass slide to ensure precise assignment.
• Use a new pipette, a new stirring rod, and a new glass slide for each sample.
• The glass slides can be disinfected and washed after use for the next test.
• The reagents contain low concentrations of toxic sodium azide as a preservative, so avoid skin/eye contact and ingestion.
• Treat all sample material as potentially infectious and dispose of it accordingly, along with the used test-kit components.
• Brucella canis is infectious to humans, so exercise extreme caution when handling such sera.

NOTE:

• Follow the instructions precisely from the insert included in the kit.
• Results should be interpreted by a qualified veterinarian familiar with the test and Brucella infections in dogs.

Rapid Agglutination Tests FAQs

What is a rapid agglutination test?

• A rapid agglutination test is a diagnostic tool used to detect the presence of specific antigens or antibodies in a sample by observing the clumping (agglutination) of particles.

How does a rapid agglutination test work?

• The test works by mixing a sample (such as blood, serum, or saliva) with reagents that contain specific antigens or antibodies. If the target antigens or antibodies are present, they will bind to the reagent and form visible clumps.

What are the common applications of rapid agglutination tests?

• Rapid agglutination tests are commonly used for diagnosing infectious diseases, blood typing, detecting certain autoimmune disorders, and identifying bacterial or viral pathogens.

How long does it take to get results from a rapid agglutination test?

• Most rapid agglutination tests provide results within a few minutes, typically between 1 to 10 minutes, making them suitable for point-of-care testing.

What are the advantages of using rapid agglutination tests?

• The main advantages include quick results, simplicity, cost-effectiveness, and minimal equipment requirements. These tests are easy to perform and interpret.

Are there any limitations to rapid agglutination tests?

• Yes, limitations include potential false-positive or false-negative results, the requirement for proper sample handling, and sometimes the need for confirmation with more specific tests.

How should a sample be collected and handled for a rapid agglutination test?

• Samples should be collected using sterile techniques and handled according to the test manufacturer's instructions. It's important to avoid contamination and to use fresh samples for accurate results.

What should I do if I get a positive result on a rapid agglutination test?

• A positive result indicates the presence of the target antigen or antibody. It is recommended to confirm the result with additional diagnostic tests and consult with a healthcare professional for further evaluation and management.

Can rapid agglutination tests be used for screening large populations?

• Yes, due to their speed and ease of use, rapid agglutination tests are often used in mass screening programs, especially in outbreak situations or in areas with limited access to advanced laboratory facilities.

What are some common types of rapid agglutination tests?

• Some common types include the Widal test for typhoid fever, the latex agglutination test for detecting bacterial infections, and the direct agglutination test for diagnosing parasitic infections like leishmaniasis.

Canine Brucella Antibody RSAT FAQs

What is the Canine Brucella Antibody RSAT?

• The Canine Brucella Antibody Rapid Slide Agglutination Test (RSAT) is a diagnostic test used to detect antibodies against Brucella canis in a dog's serum, indicating exposure to the bacteria.

How does the RSAT work?

• The RSAT involves mixing a dog's serum with a reagent containing Brucella antigens on a glass slide. If antibodies against Brucella canis are present, they will bind to the antigens and cause visible clumping (agglutination).

What are the signs that my dog might need an RSAT?

• Signs include reproductive issues, such as infertility or spontaneous abortions, swollen lymph nodes, lethargy, and other symptoms consistent with brucellosis. Dogs with a history of exposure to infected animals or environments should also be tested.

How long does it take to get results from the RSAT?

• The RSAT typically provides results within a few minutes, making it a quick diagnostic tool for veterinarians.

What does a positive RSAT result indicate?

• A positive RSAT result indicates that the dog has been exposed to Brucella canis and has developed antibodies against the bacteria. Further confirmatory tests are recommended to verify the infection.

What should I do if my dog tests positive for Brucella canis?

• If your dog tests positive, consult with your veterinarian for further diagnostic testing, treatment options, and management strategies to prevent the spread of the infection to other animals or humans.

Are there any limitations to the RSAT?

• Yes, the RSAT can produce false-positive or false-negative results. False positives may occur due to cross-reactivity with other bacteria, while false negatives might occur in early stages of infection or in chronic cases where antibody levels are low.

How should a sample be collected and handled for RSAT?

• A blood sample should be collected using sterile techniques, and the serum should be separated and handled according to the test manufacturer's instructions to ensure accurate results.

Can RSAT be used as a standalone diagnostic tool for canine brucellosis?

• No, RSAT should not be used as a standalone diagnostic tool. Positive results should be confirmed with additional tests, such as the 2-Mercaptoethanol RSAT, PCR, or culture methods, to accurately diagnose Brucella canis infection.

Is the RSAT suitable for routine screening of breeding dogs?

• Yes, RSAT is suitable for routine screening of breeding dogs to help prevent the spread of Brucella canis in breeding programs. Regular testing can help identify infected animals early and reduce the risk of transmission.