Immunology and Serology
It has been known that animals in the recovery phase of an infectious disease are resistant to re infection. Immunity (from the Latin immunis, meaning safe) was the word used to describe this state of heightened resistance.
The goal of the body’s immune response is to combat the effects of foreign substances on vital bodily processes. To accomplish this, the body must be able to recognize minute differences among foreign substances and it. After a substance is recognized as foreign, there must be a means of responding to it physiologically, thus elimination or limiting any harmful effects of the agent. To facilitate a future immune response and encounter with the same agent, memory components for the foreign material are required.
Occasionally, the immune system malfunctions, resulting in such disorders as allergy, immunodeficiency, neoplasia or auto- immune disease.
The Immune Response
Vertebrate species have 2 major internal defense systems: The innate immune system and the adaptive immune system. A major function of the innate system is phagocytosis, a nonspecific response. Phagocytic cell (The chief phagocyte is the macrophage) ingest and destroy inert particles, viruses, bacteria and cellular debris. Macrophages are found throughout the body. In the blood they are called monocytes. From the blood they migrate to various tissues and organs, and are then called macrophages or other specialized names. They locate in connective tissue, liver, brain, ling, spleen, bone marrow and lymph nodes and together comprise the mononuclear phagocytic system.
The adaptive immune system is more sophisticated. It has the ability to respond specifically to foreign substances. These substances, or antigens, can be bacterial, viral, fungal or altered endogenous cell of he host’s body. Their presence initiates humoral and cellular responses that neutralize, detoxify and eliminate these foreign materials from the host.
Lymphocytes and their progeny are the cell types largely responsible for the adaptive immune system. This line of defense is not, however, divorced from the innate immune system. It is now clear that macrophages process antigens and present them to antigen- reactive lymphocytes.
Lymphoid stem cell develop first in the yolk sac and then in the fetal liver. The bone marrow assumes this responsibility near parturition and serves as the source of these cells throughout postnatal life. The lymphoid stem cells are destined to further develop in 1 of 2 places: the bone marrow or the thymus.
The adaptive immune system is divided into 2 components: the humoral immune system and the cell mediated immune system.
The Humoral Immune System
The bone marrow – derived lymphocytes (B-cell) are concerned chiefly with production and secretion of immunoglobulin (lg) molecules, which are also known as antibodies. Many clones of B-cell differentiate, each of which is programmed to respond to a specific antigen. Their maturation processes consists of 3 stages: the lymphoblast, the prolymophocyte and the mature lymphocyte. The mature cells leave the bone marrow to secondary lymphoid organs, chiefly the spleen and lymph nodes.
When a foreign antigen enters the body, an antigen presenting cell, such as a macrophage, is the first immune-system cell to confront it. These antigen-presenting cells process the antigen and then fix it to its surface for presentation to a helper T-lymphocyte. These cells “help” B-cells to proliferate and differentiate into antibody secreting plasma cells. The B-cells may also differentiate into memory B-cells, which respond faster to a second exposure to antigen.
Antibodies (immunoglobulin) are proteins consisting of 2 distinct functional portions. One portion of the antibody molecule, the variable portion, is specific for the antigen. The other portion of the antibody molecule, the constant portion, is the same in all antibodies of the same class. When an antigen is encountered by an appropriate antibody, the antibody binds to the antigen to form a complex.
Immunoglobulins (Ig) are divided into distinct classes, each with unique biologic properties. The most abundant of these classes is IgG, the major immunoglobulin in serum. It plays the major role in humoral immunity.
IgM, the largest antibody, is the first antibody to appear in response o exposure to an antigen. Its concentration, rapidly declines, followed by and increase in IgG concentration, which is also specific for the same antigen. Functions of IgG and IgM include bacterial toxin neutralization, activation of complement and phagocytic enhancement.
IgA prevents attachment of pathogens to mucosal surfaces and is important in protection of the respiratory, intestinal and urogenitlal tract. High levels of IgE are found in allergic and parasitized individuals.
Cell – Mediated Immunity
Lymphoid stem cells that mature in the thymus develop into T-cell lymphocytes. Like that of B-cells, their maturation process consists of 3 morphologically distinct stages: lymphoblast, prolymphocyte and lymphocyte. As these cells mature, they develop receptors to specific antigens and become “immunocompetent lymphocytes.” Then, after contact with a specific antigen, the cell proliferates and differentiates into either memory cells or effectors cells.
Memory cell recognize antigens to which they have previously been exposed. Upon a subsequent encounter, they elicit a more rapid immune response.
There are different types of T-effectors cells, such as helper T-cells and cytotoxic T-cells.T-helpers function in humoral immunity but they also participate in cell-mediated immunity. For example, in delayed hypersensitivity reactions, such as cell –mediated immune response to intracellular bacteria, T- helpers recognize protein antigen and then secrete cytokines, cytokines are chemicals that attracts white blood cells to the area and activate macrophages. The macrophages then eliminate the foreign antigen.
Cytoxic T-cells play a role in combating intracellular viruses, in graft rejections, and in reactions to tumor cells. They specifically bind to virus-infected or foreign cells and cause their lysis.
Animals become actively resistant to disease by having the disease and developing antibodies, or by being vaccinated or immunized, in which case they also develop their own antibodies. They become passively resistant by receiving maternal antibodies in the colostrums, or by receiving preformed antibodies by injection.
Vaccine is produced by injecting a suspension of microorganisms into an animal with the purpose of eliciting an antibody response but yet not cause the disease. The microorganisms may be either attenuated (weakened but still alive) or inactivated (killed). Attenuated vaccines normally cause a longer – lasting, more potent immune response. Inactivated vaccines are generally safer and have no ability to cause disease. After some time and when the antibody titer (serum level) is high enough, the injected animal’s serum is collected and processed. An adjuvant may be added to vaccine to enhance the normal immune response. Some adjuvant does this by simply slowing the rate of antigen elimination from the body so antigen is present longer to stimulate antibody production.
Vaccines may be given subcutaneous or intramuscularly, depending on the vaccine. Other vaccine can be aerosolized and given intranasal. Some vaccines can be put in the feed or drinking water. Fish can be vaccinated by putting the vaccine into their water.
Establishing passive immunity requires use of antibodies that have been produced in a donor animal. A donor animal is vaccinated with pathogen. When its serum antibodies reach a high concentration, the animal is bled and the globulin portion containing the antibodies is separated and purified. The protection that an animal receives from and injection of this immune globulin is short lived but immediate.
Disorders of the Immune System
Some immune responses have an adverse effect on the host animal. Among hypersensitivity reactions are: allergies, anaphylactic shock, a severe reaction that may occur within seconds after an antigen enters the circulation; autoimmune hemolytic anemia, a condition causing destruction of red blood cells by the host itself; glomerulonephritis, caused by deposition of antibody-antigen complexes in the kidney; and contact hypersensitivity reactions, such as reactions to contact with poison ivy.
In addition to the hypersensitivity reactions just described, the immune system may also show deficiencies. There may be a deficiency in phagocytes or in immunoglobulin. A condition called combined immunodeficiency affect animals in early life, after serum level of maternally derived antibodies have declined. Arabian foals with this disease often die from opportunistic infection due to an absence or deficiency of immunoglobulin.
Lymphoma, a type of tumor character sized by uncontrolled proliferation of lymphocytes, is another abnormality of the immune system. The immune system normally recognizes and destroys cancer cells before they become established in the body, but sometimes the cancer seems to become resistant and escapes the immune defense mechanisms.
Principles of Common Immunologic Laboratory Tests
Test of Humoral Immunity
The science of detection and measurement of antibodies or antigens is called serology. Detection depends upon the binding of antibodies and antigens. Unfortunately, this binding phenomenon is ordinarily invisible. Visualization, and thus detection, of the antigen- antibody reaction depends on secondary events upon which the union is easily detected and therefore of diagnostic use in veterinary practice.
Commercial production of monoclonal antibodies to many different antigens has resulted in a variety of test kits for use in the veterinary laboratory. Specific antibodies too many different antigens can be produced and used in the laboratory for rapid identification of disease- producing organisms.
Immunization with viruses, bacteria or other entities stimulates antibody production in an animal. The antibody –secreting, transformed lymphocytes (plasma cells) can be isolated from the animal and chemically fused with a type of “immortal” cell that propagates indefinitely, such as mouse myeloma cells. The antibodies these hybrid cells produce, called monoclonal antibodies are collected. Because each monoclonal antibody attaches to only one specific part of one type of molecule (antigen), use of these antibodies in diagnostic kits makes the tests very specific and greatly reduces interpretation problems of the result. For example, the feline leukemia virus antigen only reacts with the feline leukemia virus antibody. A specific reaction is diagnostically significant for this complicated disease. In addition to their specificity, these procedures allow rapid identification of the pathogen.
Many serologic test use monoclonal antibodies. Enzyme linked immunosorbent assay, latex agglutination and immuno diffusion are 3 methodologies used in veterinary laboratories. These are discussed below. Other methods, such as complement fixation, immunofluorescence, immuno electron microscopy and virus neutralization, are used in veterinary reference laboratories and research facilities, and will give you a good basis for understanding.
Reference laboratories offer myriad serologic tests specifically developed for veterinary samples. Tests for equine infectious anemia, bovine leukemia, toxoplasmosis, feline infectious peritonitis and rabies are but a few of the diagnostic procedures available.
ELISA is an acronym for enzyme linked immunosorbent assay.It has been adapted to assays for many agents commonly tested for in the veterinary laboratory. Using monoclonal antibodies, the specificity of ELISA is very high; that is, there is little cross reactivity with other agents. This makes ELISA an accurate way to detect specific antigens, such as viruses, bacteria, parasites or hormones in serum, ELISA may also be used to test for an antibody in the serum, in which case the test contains the specific antigen. Some of the available ELISA kits are for detection of heartworms, feline leukemia virus, feline immunodeficiency virus, canine parvo virus, Escherichia coli and progesterone.
For the ELISA antigen detection system, monoclonal antibody is bound to the walls of wells in a test tray or to a membrane. Antigen, if present in the sample, will bind to this antibody, as well as to a second enzyme- labeled antibody that is added to aid in detection of the antigen. When a chromogenic ((color-producing) substrate is added to the mixture, it reacts with the enzymes to develop a specific color, indicating the presence of antigen in the sample. If the sample contains no antigen, the second antibody would be washed away in a rinsing process and no color reaction would develop.
A similar procedure is used for ELISA antibody detection. In this procedure, antigen is bound to the wells or membrane and the patient sample is assayed for the presence of a specific antibody.
Latex Agglutination: This test uses small, spherical latex particulars coated with antibody (or antigen) and suspended in water. If serum containing the corresponding antigen is added to the mixture, formation of antibody antigen complexes causes agglutination (clumping). This changes the appearance of the latex particles have clustered together. If no antigen is present in the sample, the mixture of latex and serum remains evenly dispersed. Serum for canine brucellosis and rheumatoid factor can be tested using this method.
Immunodiffusion: In this procedure, a serum sample (possibly containing antibody) and the antigen to this antibody, (supplied in the test kit) are placed into separate wells in an agar gel plate. Both components diffuse into the agar and form visible band of precipitation when they combine. If no band forms, there is no antibody in the patient’s serum sample. Or, the patient’s antibody levels are insufficient to cause precipitation in the gel. Diseases that can be detected by immunodiffusion include para tuberclosis, equine infectious anemia and bovine leukemia virus infection.
Zinc Sulfate Turbidity: The zinc sulfate turbidity test may be done in a veterinary practice laboratory. It provides a qualitative estimation of maternally derived immunoglobulin (from colostrums) in the serum of neonates. The test is based on the amount of turbidity that results when the neonate’s serum is combined with the zinc sulfate.
Coombs’ Test: The direct Coomb’s test is used for diagnosis of autoimmune hemolytic anemia, in which erythrocytes become coated with antibodies and are subsequently removed from circulation by the reticuloendothelil system. The causative antibodies can be detected on the surface of RBCs by using antiserum that is specific for the antibodies on the RBCs.
Intradermal Tests: Skin tests are used to diagnose various allergies to allergies in the environment and in food or water. Allergies are mediated by IgE antibody molecules and can be detected by using allergic extracts of grasses, pollens, ragweed and other possibly offending antigens. The extracts are injected intradermally and the injection sites are monitored for allergic intradermally and the injection sites are monitored for allergic reactions. A positive reaction indicates the presence of antibodies, meaning that the animal is allergic to that antigen.
Tests of Cell-Mediated Immunity
Whereas test of humeral immunity involve detection of circulation antibodies, evaluation of cell-mediated immunity is much more difficult.
Tuberculin Skin Test: The tuberculin skin test is one test that correlates with a specific cell-mediated immunity reaction. Animals infected with Mycobacterium bacteria develop characteristic delayed hypersensitivity reactions when exposed to purified derivatives of the organism called tuberculin.
In the tuberculin skin test, tuberculin is injected intradermally at a site in the cervical region or in a fold at the base of the tail in large animals. A delayed, local inflammatory reaction is observed if the animal has been exposed to mycobacterium. The reaction to injection to is delayed because it takes a day or more for the T-lymphocytes to migrate to the foreign antigen injected into the dermis.
Collection Sample for Serologic Testing
Nearly all serologic tests require serum or plasma as the sample. Whole blood should not be sent to the diagnostic laboratory when serum or plasma is specified. Reference laboratories have strict requirements concerning specimen type, quality and handling. For each test, read the requirements carefully and submit exactly what is requested. If a blood sample is to be collected in a syringe, a 5-ml syringe and 20-ga needle combination causes the least hemolysis.
Handling Serologic Samples
When serum is to be submitted, allow the blood sample to clot for 20-30 minutes at room temperature and then centrifuge for 10 minutes at a speed not faster than 1500 rpm. If little serum has separated after centrifuging, “rimming” the tube with a wooden applicator stick to loosen the clot may help; however, this may also cause hemolysis. If plasma is desired, the sample can be centrifuged immediately after collection.
After centrifugation, use a small pipette t aspirate the serum off plasma (upper layer) off the packed erythrocytes. Place the aspirate into transfer tube or other seal able test tube and label clearly. The serum or plasma can be tested immediately, or may be frozen or refrigerated for later use.
Samples for most serologic tests need not be frozen but should be shipped cold, especially during hot weather. The major problem with shipping tubes is breakage. The tubes must be packed firmly in place so they do not move around when the package jarred. Use paper towels, packing material or even newspaper, but pack the tubes tightly. Be sure to label each sample clearly and correctly, and enclose the pertinent paperwork to facilitate proper reporting of the results from the laboratory.