Type IV Hypersensitivity Reaction in Veterinary Microbiology

TUBERCULIN TEST Submitted to- Dr Sundareshan Dr P Sheela Dept of Veterinary microbiology H A BHARAT I MVSC DEPT OF PATHOLOGY

Tuberculin is the dermatological test based on delayed type of hypersensitivity used for screening animals for tuberculosis

Hypersensitivity refers to increased reactivity or exaggerated immune response of a host to an antigen to which it has been previously exposed.

According to the most widely accepted classification of Gell and Coombs, there are four main types of hypersensitivity reactions as type I, II, III and IV

DELAYED TYPE OF HYPERSENSITIVITY Type IV hypersensitivity reaction also known as cell mediated hypersensitivity or delayed type of hypersensitivity is the T lymphocytes mediated destruction of cells along with dendritic cells, macrophages and cytokines playing major roles Delayed hypersensitivity reactions are mainly mediated by T cells and natural killer (NK) cells

The reaction is mediated by specific subsets of CD4+ helper T cells (Th-1 and Th-17 cells) or by CD8+ cytotoxic T cells. Type IV hypersensitivity occurs 24-72 hours after contact with an antigen, usually starting at 2 or 3 days and often last for many days. For this reason, type IV hypersensitivity reaction is termed as delayed hypersensitivity

In type IV hypersensitivity reaction, when a sub- population of CD4 Th1 cells encounter certain type of antigens, they produce cytokines which induce a localized inflammatory reaction mediated by non- specific inflammatory cells most prominently marcrophages. The antigens involved may be either intracellular pathogens such as Mycobacterium tuberculosis, Listeria monocytogens, Histoplasma capsulatum, Herpes Simplex virus etc. or contact antigens such as Nickel salts, Poison ivy etc.

The reaction is accomplished in two phases: the initial sensitization phase and the later effector phase. In the sensitization phase, the primary contact with the antigen is established. During this period, specific Th cells are sensitized and are clonally expanded. In the effector phase, a subsequent exposure to the same antigen induces the delayed type hypersensitivity response.

Cytokines such as IL-2 and interferon gamma is released inducing the further release of other Th1 cytokines, which mediates the immune response activating macrophages and other non-specific inflammatory cells. Activated CD8+ T cells on the other hand, destroy target cells on contact, whereas activated macrophages produce hydrolytic enzymes and on presentation with certain intracellular pathogens, transform into multinucleated giant cells.

TUBERCULIN Tuberculin is the name given to extracts of mycobacteria used to skin-test animals in order to identify those suffering from tuberculosis.

PURIFIED PROTIEN DERIVATIVE Purified protein derivative (PPD) tuberculin is prepared by growing organisms in synthetic medium, killing them with steam, and filtering. The PPD tuberculin is precipitated from this filtrate with trichloroacetic acid, washed, and resuspended in buffer ready for use Its major antigenic component is heat-shock protein (HSP)65 Many of its proteins are shared among different mycobacterial species, thus PPD tuberculin tests are relatively nonspecific Specificity of the tuberculin test maybe increased with a defined mycobacterial protein like early secretory antigenic target-6(ESAT-6) ESAT-6 is a mycobacterial protein of unknown function that is recognized strongly by T cells

SINGLE INTRADERMAL TUBERCULIN TEST the injection site should be clipped and cleansed injection site should be situated at the border of the anterior and middle third of the neck 0.1ml /5 TU of ppd injected intradermally to the site More than 4mm increase in skin thickness is considered as positive reactor

SINGLE INTRADERMAL TUBERCULIN TEST

COMPARATIVE INTRADERMAL TUBERCULIN TEST 0.1 ml/5 TU Avian and mammalian tuberculin ppd are injected on the same side of the neck 12 cm apart one above another The animal is considered to be positive reactor if the increase in skin thickness at the bovine site of injection is greater than the avian reaction with a difference of more than 4 mm

COMPARATIVE INTRADERMAL TUBERCULIN TEST

SHORT THERMAL TEST Inject 4 ml of tuberculin ppd subcutaneously to the neck region Measure rectal temperature before injecting and 4- 8hrs post injection Temp above 104F animal is classified as positive reactor

STORMONT TEST Single intradermal test repeated at same site after 7 days The animal is positive reactor if greater than 4mm thickness is observed 24 hrs post second intradermal injection

INTRAVENOUS JOHNIN TEST Johnin ppd is injected intravenously and temperature is measured 6 hrs post injection 1.8 F increase in temperature nuetrophilia indicates animal is a positive indicator

OPTHALMIC TEST in tuberculosis opthalmic test is performed by dropping the antigen solution(PPD) into an eye. Transient conjunctivitis develops in 24-48 hrs if the test is positive.

In pigs and dogs, the best test is an SID test given in the skin behind the ear, In cats, the short thermal test is being followed In sheep and goats, the antigen is usually given in the anal fold But the results are usually unreliable in these species as well. Horses appear to be unusually sensitive to tuberculin and the dose used must be reduced accordingly. The results obtained do not always correlate well with the disease status of the animal.

TUBERCULIN TEST IN POULTRY In birds good results can be obtained by inoculating 0.05ml tuberculin/ johnin into the wattle A swollen/ soft , edematous wattle 48hrs post injection indicates a positive reactor

OTHER TESTS BASED ON DTH The extracts of Brucella abortus have been used to diagnose brucellosis. Brucellin, a filtrate of a 20-day broth culture and brucellergen, a nucleoprotein extract is currently used for SID

A culture filtrate of Burkholderia mallei, termed mallein, is used for skin testing. Mallein can be used in either a short thermal test or an ophthalmic test

Intradermal skin testing with microbial extracts is also employed in the diagnosis of many fungal diseases Histoplasmin is used for histoplasmosis, coccidioidin for coccidioidomycosis, toxoplasmin is used in attempts to diagnose toxoplasmosis In these cases, the tests are not very specific, and the test procedure may effectively sensitize the tested animal, causing it to become serologically positive.