Skin Testing

Skin testing aids characterization of im­mune diseases and helps determine what an­tigens are involved, especially in dermatologic and pulmonary disorders.

Types of Skin Tests

Skin testing can be accomplished in several ways. In the scratch test, the skin is abraded and antigen applied to the area. Patch tests in­volve application of antigen to intact skin, fol­lowed by application of a patch over the area. In the skin window test, antigen is applied to intact skin and the area is covered with a glass cover slip. The most commonly used skin test is intradermal injection of an antigen. The character of immune disease can be deter­mined by evaluation of the time until onset of the skin reaction, gross appearance of the le­sion, and histologic appearance of the affected skin.

Skin-testing extracts, such as pollens or tu­berculin, are commercially available or can be made from suspected antigens in the horse's environment. An extract can be made by add­ing 10 g antigen to 100 ml pyrogen-free saline, homogenizing the mixture in a blender, filter

ing, and sterilizing the filtrate. The extract should be diluted several times and tested in normal experimental horses prior to clinical use.

Technic: The skin must be carefully pre­pared for skin testing. Depilatories should not be used to remove hair from test areas. Hair should be removed by carefully clipping the area using a #40 blade. Rough, careless clip­ping may result in dermographia, a nonspecific reaction (wheals) over the clipped area.

Because intradermal injection is the most commonly used type of skin testing, it will be discussed in detail. The intradermal injection of 0.05 ml of extract should be made with a 25-ga needle attached to a tuberculin syringe. The diameter of the subsequent reaction (wheal) is measured in millimeters at 30 minutes, 4 hours and 24 hours. Erythema does not occur unless the horse's skin is normally pink. In ad­dition to gross examination of the reaction, his­tologic examination of a punch-biopsy skin specimen aids evaluation of the reaction.

The most useful and convenient injection site is the lateral aspect of the neck. However, the ventral aspect of the chest may be used if, for cosmetic reasons, the cervical site cannot be used.

Skin Test Reactions

Immediate Reaction: Immediate reactions are characterized by a sharply defined, slightly pruritic, firm wheal 30 minutes after intra­dermal injection (Fig 1). Pseudopodia, which are inflamed lymphatics radiating away from the wheal, may be present. The wheal softens and is less defined within 4 hours, and gener­ally disappears by 24 hours. Agents causing immediate reactions include extracts of Par-ascaris equorum and Gasterophilus larvae.

Histologically, immediate reactions to anti­gen injection are type-I reactions characterized by edema and infiltration of eosinophils.

Arthus Reaction: In Arthus reactions, a small, nonsensitive wheal may or may not form within 30 minutes of intradermal injec­tion. Approaching 4 hours, the wheal enlarges and develops a firm center, but is not as well-defined as an immediate reaction. Depending on the sensitivity of the horse and the amount of antigen injected, by 24 hours the wheal is smaller or may be very swollen, with a necrotic area at the center of the reaction. Bovine serum albumin, chicken serum and fungal ex­tracts produce Arthus reactions.

Fig 1. Firm wheals and pseudopodia are evident 30 minutes after intradermal injection of histamine,

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Fig 2. The Prausnitz-Kustner reaction.

Histologically, Arthus reactions to antigen injection are type-Ill immune reactions char­acterized by necrotizing vasculitis and perivas-cular neutrophilia, with few eosinophils present.

Delayed Reaction: In delayed hypersensitiv-ity reactions there is no reaction 30 minutes and 4 hours after intradermal injection. By 24 hours a soft, slightly sensitive wheal forms. Antigens producing delayed reactions include tuberculin, johnin, and extract of Candida. Tu­berculin is not a useful diagnostic extract be­cause most normal horses react to tuberculin injection.⁶

Histologically, delayed reactions to antigen injection are type-IV immune reactions char­acterized by perivascular monocytosis, with minimal vascular damage and few eosinophils.

Prausnitz-Kustner Reaction: The Prausnitz-Kustner reaction involves the intradermal in­jection of serum from a suspect allergic horse into a normal horse, and is used to determine the type of immune reaction mediating an al­lergic response. By 24 hours postinjection, the IgE in the suspect horse's serum is fixed to the mast cells at the injection site and the ini­tial reaction subsides.⁷ A positive Prausnitz-Kustner reaction, characterized by wheal formation within 30 minutes, occurs when the suspected antigen is injected intradermally at the site of serum injection in the test animal (Fig 2). Injection of the same amount of antigen at a distant site produces the negative control reaction for comparison.

Because IgE is heat-labile, heating the serum prior to injection aids further definition of the antibody involved in the reaction.

Controls: It is extremely important to use control injections to produce reactions with which the above test reactions can be com pared. Saline diluent is a commonly used neg­ative control that causes no reaction in most animals. Intradermal injection of 50 g hista-mine, as a positive control, produces signifi­cant reactions at 30 minutes, 4 hours and 24 hours.

Bronchoprovocation Testing

Another diagnostic test to differentiate the type of immune reaction mediating an allergic response is bronchoprovocation testing, per­formed by having the animal inhale the sus­pect antigen in a nebulized form

(Fig 3). Bronchoprovocation testing is more dangerous

Fig 3. Ultrasonic nebulizer attached to a horse by a face mask.

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Fig. Plasma protein level. PCV and RBC corn in anaphyaxis

MINUTES

SEVERE

MILD

ANAPHYLAXIS

than skin testing because the antigen is ap­plied over a greater surface area (lungs) as compared with small skin sites.

Type-I immune reactions are manifested by clinical signs of bronchoconstriction, including bronchovesicular sounds and hyperventila-tion, within 30 minutes after nebulization, and a return to normal within a few hours. Type-Ill reactions are manifested by similar clinical signs 3-6 hours after nebulization.⁸

Acute Immune Reactions

Anaphylaxls

Anaphylaxis is an immediate, severe al­lergic reaction that causes sudden collapse and possibly death. The lung is the organ primarily affected in anaphylaxis, but the cecum and large bowel are also involved and are more prominently related to signs of shock.

Clinical Signs: Anaphylaxis was induced in a group of sensitized horses.⁹ Within 90 seconds after inoculation with the antigen to which the animals were sensitive, heart rate tripled, res­piratory rate increased markedly, the nostrils flared and the mucosae became cyanotic. Dys­pnea and tachycardia subsided in animals that survived 5 minutes, and salivation, tenesmus

and defecation were apparent. Sweating oc­curred 10-20 minutes postchallenge. Survivors recovered by 1 hour postchallenge but were somewhat weak.

Death may not be immediate in all cases of fatal anaphylaxis. A sensitized pony had early dypsnea followed by profuse diarrhea, but did not die until 24 hours postchallenge.¹⁰

The exhaustion-shock syndrome ("colitis X") has been associated with endotoxemia and may be related to anaphylaxis.¹¹¹³ The signs and pathologic findings of protracted anaphylaxis mimic those of the exhaustion-shock syndrome.

Pathologic Changes: Clinicopathologic changes were proportional to the severity of anaphy­laxis and included increased RBC count, in­creased PCV and increased plasma protein concentration (Fig 4). Severely affected horses had dramatically reduced thrombocyte num­bers that began to return to normal within 30 minutes and elevated prothrombin times at 60 minutes (Fig 5). Leukopenia, mainly due to neutropenia, developed rapidly and circulating eosinophils disappeared (Fig 6).

Pathologic changes of acute anaphylaxis in­clude air-trapping and ecchymoses throughout the lungs, blood-tinged foam throughout the

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respiratory system, mucosal petechiae in the cecum and large colon, enlarged mediastinal and bronchial lymph nodes, adrenocortical hemorrhage and splenic contraction.

Histopathologic changes, most prominent in the lungs and intestines, included bronchiolar constriction, peribronchiolar and alveolar edema, and hemorrhage and edema in the cecum and large colon.

Treatment: Stress should be minimized, and hydration and electrolyte balance maintained. The use of epinephrine and corticosteroids may be beneficial.