Tuberculosis (T.B) is a chronic bacterial disease of animals caused by members of the Mycobacterium tuberculosis complex.
It is a major zoonotic disease that can be transmitted from animal to human
So, the perfect diagnosis is very important.
Let's know more about how to perfectly diagnose T.B through this article.
This article will include:
Why is it called tuberculosis?
What's tuberculin?
Types of tuberculin.
Immunological basis of tuberculin.
Types of tuberculin tests.
SID and its disadvantages.
Comparative tuberculin test.
Short thermal tuberculin.
I/V tuberculin and stormant test.
Why is it called tuberculosis?
This disease is called ‘tuberculosis’ as it causes the formation of nodules called ‘tubercles’ in the lymph nodes and other tissues of the affected patient, either human or animal.
It can be diagnosed through a tuberculin test.
What's tuberculin?
Tuberculin is the name given to extracts of Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacteria ovium which are used as antigen in the testing of animals for detection of infected animals with T.B.
Types of tuberculin
Different types of tuberculins can be used, including:
Old tuberculin (O.T)
It is the supernatant fluid of broth culture of mycobacteria concentrated by boiling
Heat concentrated tuberculin (HCTM)
It is the same as O.T but the microorganism grows on a synthetic medium.
Purified protein derivatives. (P.P.D)
It's tuberculin that obtained by:
Grow bacteria on synthetic media.
The killing of bacteria by steam.
Filtration.
Precipitation of filtrate by Trichloroacetic acid.
Washing and finally resuspension in the buffer.
Immunological basis of tuberculin
It can be simply explained through:
Antigen engulfed by macrophages in the skin (Langerhans cells).
Circulating sensitive T. cells recognize this antigen which presents in Langerhans cells due to the presence of antigen marker on T. cells from the previous infection.
T. cells respond by releasing lymphokines which attract mast cells and basophils to secrete vasoactive factors which increase the permeability of blood vessels and open gaps in the endothelium of capillaries so mono-nuclear cells, B. lymphocytes, and more T. cells will migrate to the site of injection.
These migrating T. cells respond to antigen in Langerhans cells by dividing, differentiation, and releasing lymphokines.
These lymphokines are chemotactic factor and migration inhibition factor to:
Macrophages give lysosomes to destroy antigens.
Mast cells and basophils, which give vasoactive factors leading to swelling and inflammation of the tissue.
B. lymphocytes which form antibodies. d) T. lymphocytes, which secrete lymphokines.
Types of tuberculin test:
There are many types of tuberculin tests, including:
Single intradermal (SID).
Comparative.
Sort thermal.
I/V tuberculin.
Stormant.
I — Single I/D tuberculin (SID):
It's injected at specific sites, including:
Cervical area: for greater sensitivity.
Anal fold: for greater specificity.
But the cervical area is preferred due to the reaction being more pronounced due to greater sensitivity.
The middle part of the neck.
Dose of single I/d tuberculin (SID)
0.1ml tuberculin containing 0.1-0.2 mg of P.P.D. equivalent to 5000–10000 I.U. of tuberculin.
Procedures of diagnosis t.b using SID
The procedures include:
Clip the hair at the site of injection not shaving to avoid inflammation caused by shaving, which will increase the thickness of the skin giving false results.
Wash with water and soap.
Measure skin thickness before injection by caliper.
Take 0.1ml tuberculin by I/d tuberculin syringe.
Catch skin between 2 fingers and inject by the syringe 1/d with an angle of 30° between skin and needle.
True injection should be slow and difficult but if the injection is rapid it will be s/c (false injection).
After injection, pea-like swelling is palpated in the skin which results from I/d injection.
Interpretation of the results:
After 72 hours we measure skin thickness with a caliper:
Any swelling showing edema should be regarded as positive (large amounts of proteases cause liquefaction of tissue).
In absence of edema, we use a caliper and measuring skin thickness:
Increased skin thickness less than 3 mm is negative.
If 3 mm increase in thickness: suspicious and the animal should be retested after 2 months.
Increasing skin thickness by 4 mm or more is positive.
Disadvantages of SID:
SID has 3 disadvantages, including:
Lake of specificity and presence of non-visible lesion reactors (false +ve) (NVLs) as mammalian tuberculin is not sufficient to differentiate between:
Mycobacterium bovis, skin T.B, Mycobacteria ovium, Mycobacterium paratuberculosis, vaccination with BCG and Nocardiosis (sharing antigenic structure).
The maximum permissible rate is 10%, this test is accurate when the percentage of non-specific reactions is 10% but if it exceeds this ratio, the results will be inaccurate and then we should use another test (comparative).
SID failed to detect animals of low sensitivity (false negative) in case of:
Early-stage of the disease (before 6 weeks after infection).
Late-stage of the disease.
Recently calved cows (6 weeks after delivery).
Old cow.
Animals desensitized by tuberculin (8-60 days after test), then to overcome this problem we can use stormant or short thermal tests).
The use of diluted tuberculin doesn't increase the specificity of the test.
II-Comparative tuberculin test:
The comparative tuberculin test depends on the greater sensitivity to homologous tuberculin.
Avian and mammalian tuberculin is injected simultaneously into 2 separate sites on the same side of the neck, 12 cm apart.
The procedure of injection is the same as that of single intradermal tuberculin.
Advantages of Comparative tuberculin test
The presence of nonspecific reactions will give positive avian and negative bovine.
Johnin or skin T.B antigens instead of avian ones can be used if they are suspected.
III-Short thermal tuberculin:
This type of tuberculin is used as the following:
4 ml tuberculin is injected subcutaneously into the neck of cattle.
Rectal temperature should not be more than 39 Cº before injection.
Measure rectal temperature 4, 6, 8 hours after injection. If the temperature is above 40 C, the animal is classified positive.
Advantages: the test is very sensitive for the detection of spreaders (false negative).
Disadvantages:
Animals may die at the peak of reaction due to anaphylactic shock, this can be overcome by giving an antipyretic.
It may cause recumbency of the animal, and this can be overcome by providing calcium.
I/v tuberculin:
It's used experimentally only and requires a special type of tuberculin.
Its procedures are as those of short thermal but injected IV.
If the temperature increases by 1.7°C after 6-8 hours, this indicates a positive result.
Stormant test:
This test is recommended for poor sensitized animals only.
Procedures of stormant test
The same as SID, the same dose, site of injection, but after 7 days repeat injection at the same site with the same dose.
If skin thickness increases by 5mm or more after the second injection by 24 hours, the animal is classified positive.
Cattle with M. avium does not give a positive result, but skin T.B (non-pathogenic) may give a positive result.
Anergy (anergic animal) animals with visible lesions and show no reaction with tuberculin, so we should inject 0.2ml PPD. (as animals with extensive pulmonary involvement).
Desensitization occurs due to exhaustion of T. lymphocytes and phagocytic cells in the test, then gives negative results for 60 days after SID and 6 months after stormant test till regeneration of these cells.
In the late stage of pregnancy, animals become poorly sensitized (4-6 weeks) because antibodies go to the colostrum and deposit in it so decrease the immunity of the dam, and subsequently the calf is positive for 3 weeks after parturition.
Finally, the tuberculin test is a very important test for T.B diagnosis, so it requires an accurate, experienced lab technician to ensure the results.
Written by: Dr. Mai Foda
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