Rapid Johne's Test (rjt) (Canada)

Manufacturer: ImmuCell

U.s. Vet. Lic. No.

327

Contents

Reagents and materials:

1) One vial 0.25 mL Mycobacterium paratuberculosis antigen, labelled vial #1.

2) One vial 0.40 mL bovine serum positive control, labelled vial #2.

3) 10 agarose gel immunodiffusion plates, (10 tests, 10 controls).

Rapid Johne's Test (rjt) Indications

The ImmuCell AGID test has been designed specifically to aid in the differential diagnosis of cattle with clinical Johne's disease. The test is subject to the limitations and sensitivity stated in the section “Limitations of the Test”.

Test Principle

The agarose gel immunodiffusion (AGID) test is a reliable, easy-to-use method for the detection of serum antibodies to Mycobacterium paratuberculosis, the causative agent of Johne's disease.

The test relies on the diffusion of two components in an agarose gel matrix. The two components are 1) a protoplasmic extract of M. paratuberculosis and 2) positive control or serum sample containing circulating antibodies to M. Paratuberculosis. When these two components combine under the proper conditions, an antibody-antigen precipitate will form in the gel and eventually appear as a diffuse or precipitin line depending upon the relative concentration of the two reactants. This precipitin line will usually form within 24-48 hours after the introduction of sample and antigen into the gel. The presence of a precipitin band, regardless of intensity, is considered a positive result. The absence, however, of a precipitin line at the end of incubation does not mean the animal is free of the disease. A negative test result may be due to serum antibody levels which are insufficient to cause immune precipitation in the gel, a condition commonly referred to as antigen excess. It is recommended that fecal culture be performed in all cases where a negative AGID test is observed in animals with apparent clinical symptoms.

Test Procedure

1. Remove the agar gel plate from the tray.

2. Remove the lid from the plate and place the plate on a smooth, level surface. Be careful not to scratch the bottom of the plate as scratches within the test area may be misinterpreted as positive test reactions.

3. The triangular pattern of wells on the plate should be positioned so that the smallest well is located at the top of the triangle. This well is the antigen well. The remaining large wells at the bottom of the triangle should be marked “S” and “C” (sample and control).

4. Into the antigen well carefully dispense 20 µL of the antigen from vial “1”. Place the pipette directly into the well, thereby preventing accidental discharge of fluid onto the gel surface. Improper filling of the well may effect the test results.

5. The control serum provided with the kit is assurance that the test components are working properly. A positive control should be run with every test. Carefully dispense (as in step #4) 35 µL of positive control serum from vial #2 into the well marked “C”.

6. Dispense 35 µL of serum from a suspect animal into the well marked “S”.

7. Following the addition of antigen, positive control and sample to the appropriate wells, the plate lid should be replaced firmly to prevent dehydration of the gel during incubation.

8. Allow the plate to incubate on a level surface at room temperature for 48 hours. The plate should be located in an area that is free from significant temperature variations and accidental movement.

9. Upon completion of the 48-hour period, the plate(s) are ready to observe for the presence of precipitin lines.

10. Carefully wipe the bottom of the plate with a soft cloth or paper towel to remove any smudges, fingerprints, etc., giving special attention to the center area (test area) of the plate.

11. Remove the lid from the plate and hold the plate between the fingertips in order to avoid smudging the previously cleaned plate bottom.

The results are sometimes easier to observe when backlighting, such as with a desk lamp. When using a light source such as a desk lamp, hold the test plate approximately six (6) inches from a dark background with illumination directed from the side of the plate. Look for a white precipitin band similar to the one generated by the positive control.

Specimen collection: The best results are obtained using fresh bovine serum taken in the proper manner. Slight hemolysis does not interfere with the performance of the test.

The presence of a positive AGID test in addition to the observation of clinical signs of Johne's disease (diarrhea, rapid weight loss) indicates an animal with high probability of Johne's infection. The absence of a precipitin line or “negative result” cannot be interpreted as a negative test result. This is due to the sensitivity limits of the agarose gel test. It is recommended that a “negative” result be followed-up by a fecal culture. During the course of Johne's disease there are usually fluctuations in the serum antibody titer. This variation in titer may result in intense precipitin band(s). The use of backlighting will enhance the visibility of weaker reactions.

Limitations of the test: The test is not intended to be used as a screening tool. The detection of subclinical animals, although desirable, is not a salient feature of the test.

The test is intended to be used as a differential diagnostic tool in the presence of clinical symptoms (e.g., diarrhea and rapid weight loss). In the presence of clinical signs, rjt has a 85% sensitivity rate.

A negative result cannot be interpreted as a true negative and fecal culture is recommended. Although 97 culture negative cows have been tested and found to give a negative AGID reaction, the possibility that infections with organisms other than M. paratuberculosis could yield a positive result cannot be ruled out. Asymptomatic animals which test positive by rjt should therefore be evaluated by fecal culture to confirm M. paratuberculosis infection.

Precaution(s)

1. Do not use reagents beyond the expiration date.

2. Gently swirl both the positive control and antigen vials to ensure proper mixing of the contents.

3. Do not freeze the gel plates, they will be useless.

4. Be certain that the gel plate covers are securely fastened to the plate bottoms to prevent dehydration of the gel.

5. Avoid placing the plates on surfaces that can mar or scratch the plate bottoms, scratched plate bottoms could interfere with the detection of precipitin lines.

6. Dispose of the kit components in the proper manner through autoclaving or the use of bio-bags. Do not use the kit components if a positive control reaction is not visible after 48 hours of incubation.

7. Wash and/or disinfect the hands thoroughly after use, handle the M. paratuberculosis antigen (vial #1) with precautions appropriate to infective material.

8. Store in a refrigerator at 35-45°F (4-7°C).

9. Store the positive control and antigen frozen after the first use.

Warning(s)

The manufacturer makes only an express warranty which is limited to the statement that the product will function as described in the package insert. The express warranty shall not apply if the product has not been used or operated in accordance with the manufacturer's printed instruction, or if it has been used for any purposes other than those described in the printed instructions. The diagnostic test kit is sold as is, with the stated functional limitations in the package insert regarding sensitivity, specific and intended uses, please read the instructions before using.

User Quality Control: Positive control serum should be used with each test. Failure of the control serum to show a distinctly visible precipitin line at 48 hours indicates either component failure or inappropriate use of the components such as accidental over or underfilling of the wells.

Discussion

Johne's disease is a slow-developing often chronic “wasting” disease of ruminant animals which is caused by the bacterium Mycobacterium paratuberculosis.¹ Passed by fecal-oral transmission from animals that are either acutely ill or asymptomatic shedders, M. paratuberculosis organisms are highly resistant to environmental degradation and can remain viable in manure and stagnant water for more than a year.² Identification of animals with clinical or subclinical disease is therefore important in controlling the spread of infection.

In the United States, Johne's disease has its greatest economic impact in cattle where estimated losses in weight gain and milk production among infected herds is significant.³

Symptoms of the disease in cattle include severe weight loss which may or may not be accompanied by persistent diarrhea. When present, diarrhea associated with Johne's disease is characteristically profuse, uniform in consistency and generally free of blood and mucous. Periodic weight loss in the presence of a normal appetite and body temperature are early clinical features of the disease which may precede diarrhea.

Efforts to control Johne's, a largely untreatable disease, have focused on detection of symptomatic and asymptomatic animals shedding infectious bacteria. Segregation or removal of shedding animals from the herd can prevent the spread of the bacteria to those animals at greatest risk of infection such as calves and breeding age heifers.⁴ The most reliable methods for detecting Johne's infections include histopathological examination of intestinal tissue through biopsy or bacteriological culturing of suspect stool. Fecal culture on selective bacteriological media is the most common diagnostic procedure used in screening exposed or suspect animals, however, the 8-12 weeks required for bacterial growth makes culturing impractical for confirming clinical cases.

An alternative to fecal culture is the agar gel immunodiffusion (AGID) test which has been shown to be a reliable means of detecting paratuberculosis in cattle with clinical disease. A recent study of 50 cows with symptoms of weight loss and/or persistent diarrhea indicated that 98% animals fecal culture and/or necropsy positive for M. paratuberculosis were successfully identified by serological AGID testing.⁵

References

Available upon request.

Presentation

10 test kit.

Nac No.

12700040