Veterinary Immunology

Veterinary Immunology Type IV Hypersensitivity Dr. Chi-Young Wang

The Tuberculin Reaction • A skin test for an identification of animals suffering from tuberculosis • Purified protein derivative (PPD) tuberculin: growing organisms in synthetic medium, killing them with steam, and filtering • PPD is precipitated with TCA

The Tuberculin Reaction • The major antigenic component is the heat-shock protein HSP65 • A normal animal-no apparent response for tuberculin • Animals infected with TB, a delayed hypersensitivity occurs (indurated swelling)

The inflammation begins between 12 and 24 hours, reaches its greatest intensity by 24 to 72 hours, and may persist for several weeks before fading gradually • The lesion is infiltrated with lymphocytes and macrophages, although neutrophils are present

in the early hours of the reaction • When tuberculin is injected intradermally, it is taken up by Langerhans cells (macrophages), which then migrate to the draining lymph node • They present antigen to memory T cells that respond by generating Th1 effector cells

The circulating Th1 cells accumulate around the antigen deposit; the injected site is infiltrated with γ/δ+, WC1+ T cells; no B cells • Therecruited Th1 cells secrete IFN-γ, IL-2, and IL-16; The first two up-regulate adherence molecules; IL-2 stimulates CXCL8, CCL5, and XCL1

which activates Tcells; IL-16 attracts CD4+ T cells • The macrophages release serotonin and CXCL1 and CCL2 which attract basophils; T cell-derived chemokines CCL2 and CCL3 can induce mast cell degranulation • By 60-72 hours, the predominate lymphocytes are α/β+, CD4+, and CD8+ ; macrophages accumulate as a result of the production of CXCL8 and IFN-γ

Tuberculin Reactions in Cattle • 0.05 ml of PPD tuberculin derived from M. tuberculosis or Mycobacterium bovis is injected into one anal fold and the injection site is examined 72 to 96 hours later (The single intradermal test; SID test)-A firm lump

Tuberculin Reactions in Cattle • In USA, two shots (one in the vulva and the other into an anal fold); other countries, the side of neck (neck site is more sensitive than the anal fold) • But the cross-reaction such as Mycobacterium avium or Norcardia

Tuberculin Reactions in Cattle • If animals exposed to nonpathogenic mycobacteria, false positive occurs • False-negative SID tests with advanced tuberculosis, in animals with very early infection, claved within the preceding 4 to 6 weeks

Tuberculin Reactions in Cattle , in very old cows, and in animals tested during 1 to 10 weeks • The lack of reaction (anergy) seen in Johne’s disease due to the blocking factor • The comparative test involves intradermal inoculation of avian and bovine tuberculins; each tuberculin

Tuberculin Reactions in Cattle is injected into the side of neck at separate sites..(90% sensitive and 99% specific) • Short thermal test: a large volume of tuberculin solution is given subcutaneously and examined for a rise in temperature between 4 and 8 hours later (due to IL-1 from macrophage)

Tuberculin Reactions in Cattle • Stormont test: giving 2 doses of tuberculin at the same injection site 7 days apart • Both used in postpartum cows and heavily infected animals • Repeated tuberculin testing results in a period of decreased reactivity and the induction of antibodies anti-HSP 70

Pathological Consequences • Mycobacterium tuberculosis is resistant to intracellular destruction until M1 macrophages are activated by Th1 and dead organisms are very slowly removed because they contain large quantities of metabolized waxes • Many macrophages fail to prevent bacterial growth so they die

Pathological Consequences • Other macrophages fuse to form multinucleated giant cells • After 4-5 weeks, microscopic granulomas enlarge and coalesce. The lesion is consisted of necrotic debris, a layer of fibroblasts, lymphocytes, and macrophages, which are called epithelioid cells; The entire called “tubercle”

Pathological Consequences

Pathological Consequences • The mycobacteria are unable to multipy within the caseous tissue because of its low pH and lack of oxygen • If the host mounts an Th1 responses-sufficient to control; if Th2 responses-the organisms spread to lung, and results in liquefaction of the caseous center of tubercle • Granuloma-a common chronic inflammation

Allergic Contact Dermatitis • Reactive chemicals painted onto the skin, they may bind to skin proteins and resulting complexes are processed by Langerhans cells • The Langerhans cells migatre to draining lymph nodes and secret IL-12 and IL-18 , to which Th1 cells respond

Allergic Contact Dermatitis • These cells produce large amounts of IFN-γand promote the activities of cytotoxic T cells • Following exposure, macrophages and lymphocytes infiltrate the dermis by 24 hours • Cytotoxic T cells destroy and remove the altered cells leading to intraepithelial vesicles

Allergic Contact Dermatitis • The inflammatory reaction as an intensely pruritic skin disease called allergic contact dermatitis and α/β T cells, γ/δT cells, B-1 cells, and natural killer (NK) Tcells are involved • Formaldehyde, picric acid, aniline dyes, plant resins, oils, organophosphates, neomycin, and nickle and berylium

Allergic Contact Dermatitis

Allergic Contact Dermatitis • Ears of dogs treated with neomycin for otitis externa • Carpet dyes exposed to the scrotum and ventral abdomen of dogs • Neck exposed to dichlorvos in flea collars • Milking machine to dairy cow • Muzzle of dogs to component of plastic food bowls

Allergic Contact Dermatitis • Closed patch tests –suspected allergens are used to impregnate gauze swabs that are then attached to the shaved skin with tape. After 48 to 72 hours the dressing is removed and the areas in contact with the swabs examined • A positive-local erythema and vesiculation

Measurement of Cell-Mediated Immunity • In vivo-Intradermal skin test is difficult to quantitate • To measure the ability of an animal to mount cell-mediated immune response in general rather than response to one specific antigen • To give a small skin graft or paint skin

Measurement of Cell-Mediated Immunity with a contact sensitizer such as dinitrochlorobenzene to mount a cell- mediated immune response

Measurement of Cell-Mediated Immunity • In vivo technique-antigen is mixed with peripheral blood lymphocytes and cultured for 48 to 96 hours. Twelve hours before harvesting, thymidine labeled with tritium is added to the culture • Stimulation index is the ratio of radioactivity in the stimulated cultures

Measurement of Cell-Mediated Immunity to the radioactivity in the control • MTT assay is a pale yellow compound that serves as a substrate for active mitochondrial enzymes; it is sensitive to quantify the increase in T cells numbers triggered by antigen or mitogens

Measurement of Cell-Mediated Immunity • To measure T-cell mediated cytotoxicity-the living cells take up and retain chromium ions (51 Cr), but if the cell dies, the chromium is released into the extracellular fluid • Lymphocytes from an immune animals mixed with 51Cr-labeled target cells; the

Measurement of Cell-Mediated Immunity mixture is incubated for 4 to 24 hours at at 37 ℃;the amount of chromium released is directly to the number of target cells killed • Adding tuberculin PPD to heparinized blood and incubating the mixture for 24 to 48 hours at 37 ℃;the plasma is

Measurement of Cell-Mediated Immunity assayed by ELISA; Mycobactriumbovis and Mycobacterium avium PPD (false- positive results) are used; ESAT-6 reduce the incidence of false-positive results • The assay is at least as sensitive as the single intradermal test

Measurement of Cell-Mediated Immunity • ELISpot assay: a capture-antibody directed against the cytokine of interest is coated on the bottom of tissue culture well • The cells to be tested are cultured on this surface and exposed to the antigen of interest

Measurement of Cell-Mediated Immunity • The cells to be tested are cultured on this surface and exposed to the antigen of interest; Once the culture period is completed, the presence of this bound cytokine can be detected by a traditional ELISA using specific detection antibody and enzyme-labeled antiglobulin

Measurement of Cell-Mediated Immunity This results in the development of a pattern of colored spots that each correspond to the location of a cytokine- secreting cell

Veterinary Immunology