Clinical Pathology II: Lesson 3 - Immunology

Tests of Humoral Immunity

* Enzyme Immunoassay * Latex agglutination * Immunodiffusion * Rapid immunomigration

Immunoglobulin Classes

* IgM * IgG * IgE * IgA * IgD

Polymerase Chain Reaction (PCR)

- "Amplification assay" - A small amount of a DNA segment detected in the sample is amplified to run the test better and to determine the results. - A PCR test produces many copies of a small, select region of the DNA molecule. - The nucleotide sequence of this section of DNA must be known before the test so that the proper reagents are used. - The region of the DNA that will ID the virus or bacteria is predetermined. - The amplification process consists of three basic steps; denaturation, annealing, and extension. - After amplification, the DNA segments are separated on an electrophoretic gel for ID. - The sample mixture contains the specimen with the original DNA in question (if present), primers, nucleotides, and Taq DNA polymerase.

Cattle - Blood Types

- 11 blood groups - A, B, C, F, J, L, M R, S, T, Z - Group B = polymorphic, with more than 60 different antigens - Anti-J antibodies are the only common natural antibodies in cattle. - J-negative donors may be used to minimize transfusion reactions.

Effector Cells

- A cell of the immune system that performs specific functions to destroy foreign antigens.

DNA Vaccines

- A sequence of DNA representing an antigen - Expected to be safer and more stable than traditional vaccines and quicker to produce - Developed with the use of molecular genetics.

Immunization

- Accomplished by injecting a suspension of microbes into an animal for the purpose of eliciting an antibody response but not causing the disease. - The microorganisms may be attenuated (weakened but still alive) or inactivated (killed). - Vaccines may be given SQ or IM depending on the vaccine. - Some may be aerosolized and given intranasally. - Some vaccines are put into feed or water. - Vet techs at fish hatcheries vaccinate the fish via vaccines put into the water.

Active Immunity

- Animals become actively resistant to disease by having the disease and developing antibodies, or by being vaccinated in order to develop antibodies.

Type II Hypersensitivity Disease

- Antibody mediated diseases - Antibodies directed against animal's own cells or components of the extracellular matrix - Immune-mediated hemolytic anemia (IMHA) & immune-mediated thrombocytopenia (IMT) are both type II hypersensitivity disorders - Mediated by IgG binding to receptors on the cell surfaces. - IgM may also be involved and the resulting immune complexes will serve to activate the complement system. - The activation of complement leads to the initiation of the inflammatory response. - With IMHA, the antibodies bind to several surface receptors on the RBC membrane where they undergo opsonization and are subsequently phagocytized. A similar mechanism occurs with IMT. - Neonatal isoerythrolysis is an IMHA of neonates that occurs most often in foals and kittens. Results from the ingestion of colostrum that contains maternal antibodies against the fetal erythrocytes. - Transfusion reactions are also mediated by antibodies. *Complement and Fc receptor-mediated recruitment and activation of lymphocytes *Opsonization and phagocytosis of cells *Abnormal cellular function; hormone receptor signaling

Humoral Immune System

- B cells are concerned chiefly with the production and secretion of immunoglobulin (Ig) molecules, which are also known as antibodies. - This is called humoral immunity because the antibodies are secreted into the body's fluids or "humors".

IgD

- B lymphocyte surface antigen receptor in some species - When present, only present in small amounts

Blood Typing - Card Agglutination Test

- Blood samples used cannot already show evidence of autoagglutination. - Washing the RBC's with phosphate buffered saline may help to salvage a sample that is showing evidence of agglutination. - The RapidVet-H Canine DEA 1.1 is a blood-typing test card that is used to classify dogs as DEA 1.1 positive or negative. - The typing card contains a monoclonal antibody specific to DEA 1.1. - Each card has visually defined wells for the patient test and controls. - One drop of EDTA whole blood and one drop of phosphate-buffered saline are mixed on the lyophilized reagents within each well. - In a DEA 1.1 positive test well, the monoclonal antibody forms an antiserum which is then mixed with whole blood from the patient. - If present, DEA 1.1 positive erythrocytes react with the antiserum to cause agglutination. - The antiserum in the patient test well does not react with DEA 1.1 negative blood. - RapidVet-H (feline) is similar to classify feline blood as type A, B, or AB. - The assay uses test wells that contain lyophilized reagent, which represents an antibody to the A antigen, and an anti-B antigen, which consists of a lectin. - RBC's from type A cats agglutinate with anti-A monoclonal antibodies (the A well) - RBC's from type B cats agglutinate with anti-B solution (the B well). - RBC's from type AB cats agglutinate with both wells. - The third well on the card serves as an autoagglutination saline screen and must be NEGATIVE for results to be valid. - Samples are FIRST screened for autoagglutination. - If autoagglutination is present, the RBC's may be washed with phosphate-buffered saline and the autoagglutination screen must be repeated. - If the autoagglutination screen is negative, the typing test may be performed.

Molecular Diagnostics

- Can be used to identify a number of diseases such as Leptospira spp. - Molecular diagnostic testing is based on the analysis of DNA or RNA. - Most commonly used type of molecular diagnostic test is the polymerase chain reaction (PCR). - PCR tests detect the segment of interest in the specimen submitted and amplifies its amount. ADVANTAGES - Increased sensitivity and specificity. - Amount of specimen needed for the test can be very small. - The tests are safe. - Certain factors affecting other tests do not affect molecular diagnostic tests (age and condition of sample, fastidious growth requirements, organism viability) - Newer techniques have faster turnaround times as well. - Molecular diagnostic testing can be accomplished in a matter of hours. DISADVANTAGES - Contamination leading to false-positive results - High level of technical expertise needed - More than one room needed for testing - High cost

Immunology Analyzers

- Capable of performing tests on multiple patients simultaneously. - Most are completely automated and only require patient information and for the test sample to be loaded.

Antigen-presenting cells (APCs)

- Cells such as B cells and macrophages - Displays a portion of the antigen on its surface and presents it to the helper T lymphocytes. - This stimulates the helper lymphocytes to release cytokines. - These cytokines are the chemical signals that help the APC to further phagocytize the ingested microbe.

Real-Time Polymerase Chain Reaction

- Compared with PCR: * Decreases risk of contamination * More easily automated * Generally faster and easier to run - A fluorescent probe is added to the sample mix - This probe attaches to the DNA segments; as thee quantity of segments is amplified, fluorescence increases. - The sample is considered positive at a set amount of fluorescence.

Complement System

- Consist of a group of proteins found in the blood. - Collectively referred to as complement; integral in the innate and adaptive systems. - When activated, a series of chemical reactions known as the complement cascade occurs. - Activated by one of three pathways, but the later steps are the same for all three pathways. - Components labeled C1-C9, with some having subunits designated by letters.

Tuberculin Skin Test (TB test)

- Correlates with a specific cell-mediated immune reaction. - Animals infected with Mycobacterium spp. bacteria develop characteristic delayed hypersensitivity reactions when exposed to purified derivatives of the organism called tuberculin. - Commonly performed on cattle and primates. - Tuberculin is injected ID at a site in the cervical region or in a skinfold at the base of the tail (large animals). - A delayed local inflammatory reaction is observed if the animal has been exposed to Mycobacterium. - The reaction is delayed because a day or more passes before the T lymphocytes migrate to the foreign antigen injected into the dermis.

Crossmatching

- Crossmatching a blood donor and a recipient reduces the risk of a transfusion reaction. - The two-part procedure (minor and major crossmatching) requires a serum sample and a whole blood sample. - RBC suspensions are prepared. - Major Crossmatching: * A few drops of serum are added from the recipient to a few drops of washed packed RBC's from the donor. * The mixture is incubated and centrifuged. * Macro/microscopic evidence of hemolysis or agglutination indicates a blood-type mismatch. - Minor Crossmatching: * Donor serum and recipient RBC's are used. - Both procedures should be done in animals with unknown blood types that require transfusion. - Two controls are used, which consists of using donor cells with donor serum and recipient cells with recipient serum. - Agglutination reactions may be graded: * 0 = no evidence hemolysis/agglutination * 1 = Many small agglutinates and some free cells * 2 = Large agglutinates and smaller clumps of cells * 3 = Many large agglutinates * 4 = Solid aggregate of cells

Interferons

- Cytokines that elicit other cellular reactions such as the prevention of viral replication. - Influences the actions of NK cells. - Active in the adaptive immune response.

Coombs Testing

- Done to detect the presence of inappropriate antibodies against the own body's tissues. - The direct Coomb's reaction is used to detect antibody that has attacked the body's own erythrocytes. DIRECT COOMBS TESTING - Positive Direct Coomb's Test = Evidence of immune-mediated hemolytic disease. * The procedure involves incubating the suspect sample with antisera, which reacts with the species' immunoglobulins. * If the RBC's in the sample are coated with immunoglobulin, then the antisera and the immunoglobulin on the RBCs will react and result in visible agglutination of the erythrocytes. INDIRECT COOMBS TESTING - A positive indirect Coomb's Test = Indicates the presence of circulating antibodies against the body's own tissues. * Patient serum is incubated with RBC's from a normal animal of the same species to visualize the reaction. * If the antibody is present in the patient's serum, it will bind to these RBC's just like it would to its own. * The subsequent addition of an anti-gamma globulin for the species being tested results in hemagglutination.

Enzyme-Linked Immunosorbent Assay (ELISA)

- ELISA has been adapted to many agents commonly tested for in the veterinary laboratory. - With monoclonal antibodies, the specificity of ELISA is high, meaning little cross reactivity occurs with other agents. - ELISA's high specificity makes it an accurate way to detect specific antigens (viruses, hormones, bacteria, parasites) in serum. - ELISA may be used to test for an antibody in the serum. - During ELISA testing, monoclonal antibody is bound to the walls of the wells in a test tray, to a membrane, or to a plastic wand. - If the antigen is present in the sample, it will bind to the antibody and to a second enzyme-labeled antibody that is added to help with the detection of the antigen. - This is followed by rinsing. - When a chromogenic substrate is added to the mixture, it reacts with the enzyme to develop a specific color, which indicates the presence of antigen in the sample. - If there is no antigen, the enzyme labeled antibody will be washed away and no color reaction will develop. - ELISA antibody detection is done similarly. - Kits available include HWT, FELV, FIV, Parvo, and progesterone.

Urticaria and Angioedema Causes (K9, Feline)

- Foods - Drugs * Penicillin, cephalexin, ampicillin, tetracycline, vitamin k, propylthiouracil, amitraz, ivermectin, moxidectin, radiocontrast agents, HyLyt*efa shampoo, vincristine, azathioprine - Antisera, bacterins, vaccines * Panleukopenia, lepto, distemper-hepatitis, rabies, feline leukemia - Stinging and biting insects * Bees, hornets, mosquitoes, black flies, spiders, ants - Hairs from processionary caterpillars - Allergenic extracts - Blood transfusions - Plants * Nettles, buttercups - Intestinal Parasites * Ascarids, hooks, tapes - Infections * Staphylococcal pyoderma, canine distemper - Sunlight (K9) - Excessive heat/cold (K9) - Estrus (K9) - Dermatographism (K9) - Atopy (K9) - Psychogenic factors (K9) - Vasculitis, food allergy induced (K9)

Blood Typing - Tube Method

- Gold standard - Requires use of antisera, which consists of antibodies specific for each possible blood type of a given species. - Commercial antisera for canine and feline testing is available for a few blood groups. - Tube method requires a whole blood sample w/EDTA, heparin, or acid-citrate-dextrose anticoagulant. - Blood centrifuged at 1000 g for 10 min - Plasma and the buffy coat is removed and the erythrocytes are washed three times in a saline solution, centrifuged, and resuspended. - The RBC suspension is distributed in as many tubes as required for the number of blood type antisera being tested. - Usually 0.1 mL of the antisera is added to the tube. - The tubes are incubated for 15 min at room temp and recentrifuged for 15 seconds and 1000 g. - Each tube is examined macro- and microscopically for evidence of agglutination or hemolysis. - Weak positives may require repeat testing. - Blood typing of large animals is impractical; thousands of different antisera would be required because of the large number of different blood groups in the sheep, cow, and horse.

Adaptive Immune System

- If foreign bodies invade the innate immune system, they then encounter the adaptive immune system. - Divided into two components: * Humoral Immune System * Cell-Mediated Immune System - Has the ability to respond specifically to foreign substances; antigens such as bacteria, viruses, fungi, or altered endogenous cells of the host's body. - Foreign materials are neutralized, detoxified, and eliminated from the host. - Lymphocytes primarily responsible for the adaptive immune system. - Macrophages process antigens and present them to antigen-committed lymphocytes. "Antigen-presenting cells" - Lymphoid stem cells develop first in the yolk sac and then in the fetal liver. - Bone marrow serves as a source of these cells throughout postnatal life and assume this responsibility near parturition. - Lymphoid stem cells further mature in either the bone marrow or the thymus. - B lymphocytes mature in the bone marrow - T lymphocytes mature in the thymus.

Type 1 Hypersensitivity Disease

- Immediate - Occurs when chemical mediators from mast cells are released - Allergies and anaphylactic shock are type 1. - Occurs when IgE antibodies are formed in response to a previously encountered antigen. - When the antigen is re-encountered, the IgE binds to receptors on mast cells, which results in the cross-linking of IgE and the release of mast cell mediators. - Mast cell mediators cause smooth muscle contraction and increase the permeability of the vasculature within minutes. - Mast cell mediators are cytokines that attract cells of the inflammatory response to the area. - A variety of clinical signs will be present depending on the location of the hypersensitivity reaction. *Mast Cell Derived *Cytokine mediated inflammation

Type III Hypersensitivity Disease

- Immune complex disease - Occurs when antibodies and antigens form complexes that are deposited in various blood vessels. - Glomerulonephritis is an example of type III hypersensitivity. Occurs when antibody-antigen complexes are deposited in the kidney. - Systemic lupus erythematosus is another example, characterized by the production of a large amount of autoantibodies to a diverse population of cells and tissues. *Complement and Fc receptor-mediated recruitment and activation of leukocytes

Immunogenic

- Invading microbes = immunogenic. - They will interact with their own specific naive lymphocytes which then proliferate and differentiate into effector cells that destroy the foreign microbes.

IgE

- Involved in immediate hypersensitivity reactions such as allergy and anaphylactic shock. - Coats helminth parasites for destruction by eosinophils - Usually present in very small amounts - Similar in structure to IgG.

Reasons for False-Positive ID Test Results

- Irritant test allergens (ie glycerin, house dust, feather, wool, mold, food preps) - Contaminated test allergens - Skin-sensitizing antibody only (prior clinical or present subclinical sensitivity) - Poor technique - Substances that cause nonimmunologic histamine release (narcotics) - Irritable skin (large reactions seen to all injected substances including the saline control) - Dermatographism - Mitogenic allergen

Inactivated vaccines

- Killed - Generally safer and have less ability to cause disease - Vaccine associated sarcomas have been an issue in cats with inactivated vaccines.

Attenuated vaccines

- Live - Cause a longer lasting and more potent immune response.

Cell-Mediated Immune System

- Lymphoid stem cells that mature in the thymus become T-Cells. - Three morphologically distinct stages of maturation: * Lymphoblast * Prolymphocyte * Lymphocyte - As the cells mature they develop receptors to specific antigen-committed T lymphocytes. (this stage is called naive lymphocyte) - After contact with specific antigens these cells proliferate and differentiate into either clones of memory cells or clones of effector cells against those antigens. - As with B cells, memory cells recognize previously encountered antigens and launch a more rapid immune response.

Latex Agglutination

- Makes use of small, spherical, latex particles that are coated with antigen and suspended in water. - If serum containing the corresponding antibody is added to the mixture, the formation of antibody-antigen complexes causes clumping, or agglutination. - If no antibody is present in the patient sample, then the mixture of latex and serum remains evenly dispersed.

Adjuvant

- May be added to a vaccine to enhance the normal immune response. - Some adjuvants do this by simply slowing the rate of antigen elimination from the body so that the antigen is present longer to stimulate antibody production. - Because killed vaccines require more adjuvants, it is suspected that this could be why killed vaccines have a potential to cause vaccine induced sarcomas.

Antibody Titers

- May be needed to distinguish between active infection and prior exposure to certain antigens. - Titer refers to the greatest dilution at which a patient sample no longer yields a positive result for the presence of a specific antibody. - The test involves making serial dilutions of a sample - Each dilution is examined for the presence of the antibody. - The reciprocal of the greatest dilution that still elicits a positive test result is the titer. - A high titer often indicates active infection - Low titers usually indicate previous exposures to the specific antigen. - Some in house testing kits exist that use ELISA technology.

Monoclonal Antibodies

- Monoclonal antibodies are laboratory-made proteins that mimic the immune system's ability to fight off harmful pathogens such as viruses.

Monocytes

- Monocytes follow neutrophils to the inflammatory sites. - Monocytes ingest and destroy inert particles, viruses, bacteria, and cellular debris via phagocytosis. - When monocytes migrate from the blood to various tissues and organs to react with specific cytokines, they become macrophages. - Macrophages may be derived from other tissues as well; connective tissue, liver, brain, lung, spleen, bone marrow, and lymph nodes. These make up the mononuclear phagocytic system.

Blood Types - Dogs

- More than a dozen different canine blood groups have been identified. - DEA is the nomenclature used to designate the blood groups in canines (dog erythrocyte antigen). A number follows. - For DEA systems other than DEA 1, the erythrocytes are designated as positive or negative for the specific antigen. DEA 1 GROUP Subgroups * DEA 1.1, DEA 1.2, DEA 1.3 - K9 erythrocytes may be positive or negative for each of the DEA 1 subgroups DEA 3, 4, 5, 7 GROUPS - Designates the other major bloodgroups - The blood groups considered to be clinically significant are DEA 1.1 and DEA 7. - DEA 1.1 elicits the greatest antigen response and causes the most serious transfusion reactions. - 50% of dogs are positive for DEA 1.1. - Transfusion reactions to other blood groups are less likely to cause clinical signs. - Anti-DEA 1.1 antibodies are not known to exist. The first transfusion of DEA 1.1 positive blood into a DEA 1.1-negative recipient may not cause clinical signs until up to a week later. - If a DEA 1.1 negative dog previously received DEA 1.1 positive blood and receives it again, a severe reaction can occur in less than an hour. - Dal is another canine antigen that has been described

IgA

- Mucosal immunity - Protection of respiratory, intestinal, and urogenital tracts - Comprises 20% of circulating antibodies.

Fluorescent Antibody Testing

- Not commonly performed in clinical practice; usually only available at reference labs - Frequently used to verify a tentative diagnosis that has been made. - Direct & Indirect antibody testing available; both detect the presence of a specific antibody in a sample. DIRECT FLUORESCENT ANTIBODY TESTING * Patient sample added to a test slide that has been precoated with a fluorescent-dye-conjugated antigen. * The dye combines with a specific antibody if it is present in the patient sample. * The slide is then examined with a special microscope that has been designed for fluorescent microscopy. * For cellular antigens, the cell will appear to be outlined with fluorescent material. INDIRECT FLUORESCENT ANTIBODY TESTING (IFA) * Patient sample is incubated on a slide that contains the specific test antigen. * The slide is then washed to remove any unbound antibody * Fluorescent labeled antiantibody is added to the system and the slide is examined under microscope. * Any fluorescence indicts a positive test result.

Passive Immunity

- Occurs via maternal antibodies received in the colostrum or by receiving preformed antibodies by injection. - These antibodies will have been produced in a donor animal. - The donor animal is vaccinated with a pathogen. - When the donor animal's serum antibodies reach a high concentration, the animal's blood is drawn and the globulin portion that contains the antibodies is separated and purified. - The protection received from an injection of this immunoglobulin is short lived but immediate.

Neutralization antibody reaction

- Occurs when antibody binds directly with the antigen - When bound, the antigen cannot infect or damage body cells.

Precipitation Reactions

- Occurs when antigens bind with antibodies and form an insoluble complex. - The precipitate forms on surfaces and the precipitant itself may cause pathology. - ie: The precipitation of bacterial fragments in the glomerular membrane can result in glomerular nephritis. (type III hypersensitivity reaction)

Blood Types - Cats

- One blood group system has been identified in cats, and it has been designated as the AB system. - The blood groups of cats are A, B, and AB. - Most cats in the United States have Type A blood, which likely is why blood transfusion reactions in cats are low. - Type B blood is found in certain purebreeds (Devon Rex, British Shorthair) and in certain geographic areas such as Australia. - Cats have naturally occurring antibodies to the erythrocyte antigen that they lack, unlike dogs. - Type B cats have strong anti-A antibodies. - Type A cats have weak anti-B antibodies. - Transfusing type B cats with type A blood may result in a serious reaction and even death. - Blood for transfusions in purebred cats should be typed or crossmatched. - Mik is another blood cell antigen that has been described in cats. - Neonatal isoerythrolysis has been documented in type A and type AB kittens of type B queens with naturally occurring anti-A antibodies.

Horses - Blood Types

- Over 30 blood types in 8 major blood group systems - Major Groups: A, C, D, K, P, Q, T, U - Naturally occurring antibodies exist but antibodies may be present as a result of vaccinations that contain equine tissue or transplacental immunization. - Crossmatching should be done first; transfusion reactions are commonly fatal. - The mare-foal incompatibility test is a crossmatching procedure that detects the presence of antibodies in mare serum or colostrum to foal erythrocytes to confirm or prevent isoerythrolysis.

Helper T Cells

- Produce proteins called cytokines that activate the B cells.

Antibodies/Immunoglobulins

- Protein molecules that consist of two pairs of polypeptide chains configured in a Y shape. - Each Ig molecule contains two variable regions and one constant region. - The variable regions (Fab regions) bind to the antigen - The constant region (Fc region) is responsible for the unique functions of the different antibody classes.

Red Blood Cell Antigens

- RBC Antigens are structures on RBC surfaces in one animal that may react with antibodies in the plasma of another animal. - The specific surface markers in an animal are genetically determined and are referred to as blood group antigens. - The number of blood groups varies among species. - Antigen-antibody reactions can occur with blood transfusions as a result of variations in blood group antigens between the recipient and the donor. - These reactions usually result in the clumping or agglutination of RBC's or they may manifest clinically as RBC lysis. - Some animals have naturally occurring antibodies (alloantibodies) against the RBC antigens that they do not possess.

Rapid Immunomigration and Immunochromatography (RIM)

- RIM is a technology that has been previously called immunochromatography or lateral flow immunoassay. - Colloidal gold, enzymes, and color reagent or agglutinated latex protocols are the signal-generating components of these tests. - The signal generating component is conjugated to antibodies specific for the antigen being tested. - The conjugated antibodies are present in the membrane of the test cassette where the patient sample is applied. - If antigens are present in the patient sample, they bind to the conjugated antibodies and the antibody-antigen complexes migrate along the membrane to another area of the cassette, where the results are read. - Buffer may be added to help with migrational flow of the antibody-antigen complexes. - In the reading area, a second antibody is present in the membrane. - If the antigen is in the sample, it is captured by the second antibody. The accumulation of conjugate in that area causes a color change. - Control antigen is present in another area of the membrane strip. The conjugated first antibody binds to the antigen in the control area. The accumulation will cause a color change whether or not the antibody is present. - A POSITIVE result shows two areas of color change. - A NEGATIVE result only shows one area of color change, the control strip. - If the control strip does not change color the test should be considered invalid, regardless of the color change in the patient area.

Reverse Transcriptase Polymerase Chain Reaction

- RNA is sometimes used as the nucleic acid for the molecular test, such as when testing for RNA viruses. - Similar to PCR, however, the single stranded RNA must first be converted to double stranded DNA before the PCR process can continue.

Reasons for False-Negative ID Test Results

- SQ Injections - Too little allergen * Testing with mixes * Outdated allergens * Allergens too dilute (1000 PNU/mL recommended) * Too small volume injected - Drug interference * Glucocorticoids * Antihistamines * Tranquilizers * Progestational compounds * Any drugs that significantly lower blood pressure - Anergy (testing during peak of hypersensitivity reaction) - Inherent host factors * Estrus, pseudopregnancy * Severe stress (systemic diseases, fright, struggling) - Endoparasitism or ectoparasitism (blocking of mast cells with antiparasitic immunoglobulin E) - Off-season testing (testing more than 1 to 2 months after clinical signs have disappeared) - Histamine "hyporeactivity"

Tolerogenic

- Self antigens = tolerogenic term-33 - Lymphocytes unable to respond when they encounter self antigens (anergy) or they die when they encounter self antigens (apoptosis).

Nonimmunogenic

- Self-antigens may also be ignored by the lymphocyte, at which point they are nonimmunogenic.

Natural Killer Cells (NK Cells)

- Small subset of lymphocytes found in the blood and the peripheral lymphoid organs. - NK cells recognize and destroy host cells that are infected with microbes (ie viruses). - NK cells activate phagocytes by releasing interferon-y.

Regulatory Lymphocytes

- Some T Lymphocytes (formerly called suppressor T cells) become regulatory lymphocytes. - Regulatory T Cells prevent self-reactive lymphocytes from differentiating into effector cells. They are unable to destroy self-antigens.

Memory B Cells

- Some of the antigen stimulated B cells differentiate into memory B cells which respond faster to a second exposure to an antigen.

Type IV Hypersensitivity Disease

- T-Cell mediated disease caused by the reaction of T lymphocytes against self antigens in tissues. - Examples include contact hypersensitivity; contact with food dishes containing plastic, collars, poison ivy contact in humans. - Tissue injury can occur with a delayed response. - The chemicals from these substances react with skin proteins and the immune system recognizes this chemical-protein complex as foreign, resulting in dermatitis. - Type 1 diabetes, rheumatoid arthritis, and chronic infections such as tuberculosis are t-cell mediated autoimmune diseases. * Macrophage activation, cytokine mediated inflammation * Direct target cell lysis, cytokine mediated inflammation

Immunologic Testing

- Test kits are evaluated for their sensitivity and specificity. SENSITIVITY * Refers to the ability of the test to correctly ID all animals that are truly positive for a given reaction procedure. * Large # of false positives = low sensitivity test SPECIFICITY * A measure of the numbers of false positives produced with the given reaction procedure ** no test can provide 100% sensitivity and specificity - Most serologic tests require serum or plasma as the sample. - The vacutainer system is the most practical method of collection for these tests.

Radioimmunoassay

- Test principle is similar to CELISA technique except a radioisotope is used instead of an enzyme. - The assay typically consists of an antigen that is labeled with a radioisotope and an antibody. - When combined with patient serum containing the same antigen, both antigens compete for the antibody. - With increased amounts of patient antigen, more labeled antigen is displaced from the antibody. - The amount of radioactivity left is measured and compared with a standard curve to determine the concentration of antigen in the patient's serum.

Competitive Enzyme-Linked Immunosorbent Assay (CELISA)

- Tests for patient antigen via the use of an enzyme labeled antigen as well as monoclonal antibodies. - If patient antigen is present, it will compete with enzyme-labeled antigens for the antibodies that coat the test wells. - Color developer reacts with the enzyme to produce a color. - The intensity of the color produced varies with the concentration of the patient antigen. - Equine infectious anemia antibodies may be detected in horse serum using CELISA.

Fab/Fc Region Attachment

- The Fab region of the antibody attaches to receptors on the microbial surface. - The Fc region binds to macrophages or neutrophils and the microbe is phagocytized.

Inflammatory Response

- The body's response to infection or tissue injury. - Chemicals released from the infected site alert blood vessels to dilate and allows neutrophils to pass into the tissue. - The neutrophils phagocytize the bacteria and kill the pathogens with chemicals stored in their cytoplasm. CLASSIC SIGNS OF INFLAMMATION * Redness * Swelling * Pain * Heat * Loss of function - Each symptom of inflammation is related to physiologic mechanisms taking place during the inflammatory process. - Inflammation is a protective mechanism of the innate immune system; however, it can overreact and cause tissue damage.

T-effector Cells

- The cells that carry out most of the cell-mediated immune functions by seeking and destroying foreign materials, such as viruses, fungi, bacteria, and particles. * Helper T-Cells, CD4+ cells * Cytolytic T Cells CD8+ cells - CD4+ and CD8+ refers to surface molecules or markers that are found on helper T and cytolytic T lymphocytes. - HIV, the virus that causes AIDS, has a special affinity for the helper T lymphocytes. - Helper T lymphocytes recognize antigen that has been phagocytized by an antigen-presenting cell (APC), such as a macrophage. - Helper T cells also release cytokines that help B cells differentiate into antibody-producing cells. - Cytolytic T lymphocytes recognize antigen particles that are on the surface of infected body cells. They are able to lyse and kill the infected cells. - Microbe-infected cells, tumor cells, and cells of foreign tissue graft all may be eliminated in this manner.

Blood Typing - Immunochromatographic Assay

- The control band of an immunochromatographic assay detects a separate antigen on the RBC's. - The canine test uses a paper strip impregnated with monoclonal anti-DEA 1.1 antibody and a second antibody to a universal RBC antigen as a control. - An RBC solution diffuses up the strip and if cells express DEA 1.1, they concentrate in the area of antibody impregnation. - The cells concentrate in the area of the control antigen, demonstrating that the cells have successfully traveled up the length of the strip. - The feline test works the same way but it has an area that contains an anti-A monoclonal antibody, anti-B, and a control antibody for a common feline RBC antigen.

Serology/Immunology

- The detection and measurement of antibodies or antigens. - Detection depends on the binding of antibodies and antigens. - Detection of the antigen-antibody reaction depends on secondary events by which the union is easily detected and therefore of diagnostic use.

IgM

- The first antibody type produced in response to an antigen. - Pentameric molecule (contains five monomers) - Comprises 5% of circulating immunoglobulins. - IgM is relatively large and unable to enter tissue spaces. - Activation of complement

Innate Immune System

- The first barrier/line of defense that bacteria, viruss, and fungi first encounter.e BARRIERS * Skin * Physical and biochemical components in the nasopharynx * Gut * Lungs * Genitourinary Tract * Populations of bacteria in the body that compete with invading pathogens * The body's inflammatory response

Immunologic Tolerance

- The immune system is not supposed to destroy its own cells and it should be able to differentiate between own cells versus foreign cells. - Maturing lymphocytes develop antigen receptors for foreign antigens but also for the animal's antigens on its own cells. - These self reactive lymphocytes could attack the self antigens. - Mechanisms are normally in place in healthy individuals to prevent this from happening. - The immune system can distinguish between self and non-self, which results in immunologic tolerance. - When all of the mechanisms of immunologic tolerance fails, autoimmune disease results.

IgG

- The most abundant circulating immunoglobulin (75%) - Remains in circulation for the longest time - Relatively small monomer that is capable of entering tissue spaces - Usually produced during a secondary immune response. - Neutralizes microbes and toxins - Opsonization of microbes for phagocytosis by macrophages and neutrophils. - Activation of complement - Fetal and neonatal immunity by passive transfer across the placenta and in the colostrum.

Denaturation - PCR Testing

- The sample is heated to break apart the double stranded DNA molecule into two separate strands. - Each strand serves as a template to which new nucleotides will attach.

Annealing - PCR Testing

- The temperature is lowered to cause the primers to bind (anneal) to the separated strands. - Primers mark the beginning and the end of the section of DNA to be copied. - This will only happen if DNA is present in the ample that is complementary to the primers.

Extension - PCR Testing

- The temperature is raised again and the Taq DNA polymerase causes new DNA segments to be produced (extended). - Taq DNA Polymerase is the enzyme that can read the DNA code and assemble the nucleotide bases to form new complementary strands. - Portions of two DNA molecules have been obtained at this point, each with two strands. They are not the complete DNA molecule, but they contain the desired segment. - This process is repeated 25-30 times in an automated thermal cycler. The timing, temperature, and number of cycles are regulated by the instrument. - The amount of DNA segments produced is far greater than the original amount of DNA in the specimen. - This is what makes PCR testing useful in detecting minute quantities of the unknown in a mixed specimen. - Agarose gel electrophoresis is used to see if the microbe was present in the specimen. - The DNA segments are negatively charged particles and will move along the gel toward a positive electrode when a current is applied. - The segments separate according to size and they appear as separate bands on the gel. - Controls are run at the same time as the test samples. By knowing the control bands, the test bands can be compared and identified.

Immunodiffusion

- Used to detect diseases such as equine infectious anemia and Johne disease. - Patient serum samples that possibly contain antibodies and the antigen to these antibodies are placed in separate wells in an agar gel plate. - Both components diffuse into the agar and form a visible band of precipitation when they combine. - If no band forms, no antibody exists in the patients serum sample or the patient's antibody levels are insufficient to cause precipitation in the gel.

Intradermal Tests - Tests of Cell-Mediated Immunity

- Used to diagnose various allergies to allergens in the environment. - Allergies are mediated by IgE antibody molecules. - Extracts of grasses, trees, weed pollens, molds, dust, insects, and other possibly offending antigens are injected intradermally. - Injection sites are monitored for allergic reaction. - Positive = raised welt - Patients with hypersensitivity reactions may manifest with: * Urticaria (hives) * Wheals * Angioedema (edema of the dermis and SQ tissue) - These reactions are triggered when basophils or mast cells release histamine-containing granules and trigger the inflammatory response. - Allergens to test for are chosen on the patient's history and the geographic area. - Common allergens = dust mites, house dust, human dander, feathers, molds, weeds, grasses, and trees. - ID testing for food allergens is not well validated. - Dogs are frequently allergic to multiple substances. - False positives and false negatives can occur. - The patient is placed laterally for the test and the hair is shaved from the lateral thorax. The skin is NOT scrubbed. - A felt tip marker is used to mark the injection sites which should be 2-cm apart. - A 26-g needle is used to inject a small volume (0.05 mL) of suspected allergens. - Most patients do not require sedation. - An ID injection of saline is used as a negative control - An injection of a histamine product is used as a positive control - Injection sites are evaluated at 15 and 30 minutes after injection, and the reactions are graded. - The saline injection is graded as 0, and the histamine as +4. - The test sites are graded in relation to the two controls. - Each test site is evaluated for the presence of erythema. - The diameter of each wheal should be measured. - An ELISA test is also available for the determination of allergen-specific IgE antibodies in dogs, cats, and horses.

Negative Selection

- When naive lymphocytes are destroyed by apoptosis, the immune system is selecting the beneficial lymphocytes that have receptors for foreign antigens and eliminating the self-lymphocytes that would cause self-destruction. - Negative selection takes place in the bone marrow, thymus, and peripheral lymphoid tissues.

Handling Serologic Samples

- When serum is to be submitted, the sample is allowed to clot at room temperature for 20-30 minutes. It is then centrifuged for 10 minutes at a speed of no faster than 1500 rpm. - If little serum has separated after spinning, rimming the tube with a wooden applicator stick to loosen the clot may help. This can cause hemolysis however. - If plasma is desired, the sample may be centrifuged immediately after collection. - After centrifugation, a small pipette is used to aspirate the serum or plasma (the upper layer) off of the packed erythrocytes. - The aspirate is placed into a transfer tube or another sealable test tube and labeled. - The serum or plasma may be tested immediately or frozen/refrigerated for later use. - Most serologic test samples do not need to be frozen but should be shipped cold, especially during hot weather.

Antigens

Any substance that is capable of generating a response from the immune system.

Pathways of the Complement System

CLASSICAL PATHWAY - Mechanism of the adaptive immune system - Activated when C1 is bound to an antigen-antibody complex. OTHER PATHWAYS - Part of the innate immune system - Triggered by microbial surfaces and plasma lectins that bind to microbes. * * * * * - All three initial pathways catalyze a series of reactions of other complement molecules that have numerous physiologic effects (ie opsonization) - Complement activation can result in the stimulation of inflammation and cell lysis by the formation of a membrane attack complex on the surface of the antigen.

B Cells

Maturation Process: * Lymphoblast * Prolymphocyte * Mature lymphocyte - The mature cells leave the bone marrow to seed secondary lymphoid organs (spleen & lymph nodes) , where they encounter antigens. - The humoral immune system can recognize billions of different antigens. - As B cells mature, each B cell develops a specific receptor molecule to a specific antigen. - When an antigen enters the body, a mature B cell that is committed to that particular antigen will react with it. - The stimulation of that B cell to produce antibodies requires specialized T lymphocytes (helper T cells). - The antigen stimulated B cell quickly divides and differentiates to produce a clone of identical B cells that all produce the same type of antigen-specific antibody. - Antibody-secreting B cells are now called plasma cells. - Plasma cells are a type of effector cell. - May also differentiate into memory B cells.

Sheep & Goats - Blood Types

SHEEP - 7 blood groups - A, B, C, D, M, R, X - B = polymorphic - Naturally occurring R antibodies may be present - Neonatal isoerythrolysis may occur in lambs that are administered bovine colostrum, due to the antibodies to sheep erythrocytes in bovine colustrum. GOATS - 5 blood groups - A, B, C, M, J - Naturally occurring J antibodies present.

Cytokines

Soluble proteins secreted by cells to mediate immune responses.

Opsonization

The binding of complement to the antigen. Promotes phagocytosis.