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Chapter 2 Principles of immunity and diagnostic techniques.


After completion of this chapter, the learner should be able to

* Differentiate between innate and acquired immunity

* Describe different types of innate immunity

* Describe how fever is induced

* Describe the steps involved in inflammation

* Describe the steps involved in phagocytosis

* Describe the different types of acquired immunity

* Differentiate the types of T lymphocytes and their functions

* Differentiate the types of immunoglobulins and their functions

* List examples of cytokines and their functions

* Describe humoral immunity

* Describe cell-mediated immunity

* Differentiate the primary and secondary immune responses

* Describe immunodiagnostic tests

* Describe diagnostic techniques involving genetic material

Key Terms

acquired immunity

agglutination test



complement fixation

complement system




assay (ELISA)


fluorescent antibody


humoral immunity





innate immunity






natural killer cells



polymerase chain

reaction (PCR)

precipitation reaction

primary immune





reverse transcriptasepolymerase


reaction (RT-PCR)

secondary immune




vasoactive amines

Western blot test


Animals and humans have survived on earth for hundreds of thousands of years because they have developed naturally occurring nonspecific defense mechanisms against pathogens as well as complex interactions among different types of immune cells and cellular secretions that target specific pathogens. The ability of any animal species to resist foreign invaders and recover from disease is a result of both innate and acquired immunity. Innate immunity is often thought of as "inborn immunity" and is a mechanism of defense that does not depend upon prior exposure to an infectious agent to be effective. Innate immunity consists of nonspecific defense mechanisms that come into play immediately or within hours of the appearance of the antigen (molecule that triggers an immune response) in the body. Innate immunity is different for each species. The fact that dogs do not get human immunodeficiency virus (HIV) is a result of innate immunity. Acquired immunity, sometimes referred to as adaptive immunity, is often thought of as the immunity acquired as one goes through life and is specific to a particular foreign infectious agent, requires time to develop, and occurs more quickly and vigorously upon second exposure to that particular agent. It is the combination of these two types of immunity that work in conjunction with each other to protect living organisms from becoming diseased.
Innate immunity is nonspecific
immunity that consists of a set of
disease-resistance mechanisms that
are not specific for a particular antigen.
Acquired immunity is specific immunity
that displays a high degree of specificity
as well as the property of memory.


There are many different innate defense mechanisms that are nonspecific and help protect the body from pathogens. Innate defense mechanisms can be as simple as a physical barrier to prevent initiation of infectious agents into the body and can become more complex as is the case with substances such as complement and cytokines. An animal's defenses are sometimes categorized as occurring in stages; the first two lines of defense are nonspecific and the third line of defense is specific.

Anatomic and Physiologic Properties

Animals and humans are constantly in the process of defending themselves against microbial invaders. The first line of defense against infectious agents includes any barrier that prevents an organism from entering the body. This first line of defense limits access to the internal tissues and organs of the body. These first lines of defense include:

* Anatomic properties. Intact (unbroken) surfaces serve as an anatomic barrier to infectious agents because few pathogens can penetrate unbroken skin or mucous membranes. Hard outer surfaces like skin have an outer layer of dead epithelial cells that have been cornified and keratinized (the epithelial cells are compacted, cemented together, and have an insoluble protein in them called keratin). Desquamation (flaking) of skin also helps skin rid the animal or human of potential pathogens. Soft outer surfaces such as the lining of the digestive, urinary, and respiratory tracts are usually protected by a layer of mucus, which lubricates the surface and helps dislodge particles from it. Mucus producing cells rapidly divide, are constantly produced, and are constantly released from mucous membranes taking bacteria adhering to them out of the body. These soft surfaces may have other adaptations to further protect the animal or human. Other adaptations include hairs and cilia, which can help trap particles and keep them from entering the body or they may propel entrapped particles either cranially out of the body or caudally into another body part. For example, the epithelial lining of the gastrointestinal tract has cilia and mucus. If a foreign particle gets trapped in this cilia and mucus, it can either be expelled cranially out of the esophagus or caudally into the stomach where it can be destroyed by the stomach acid.

* Physiologic properties. The wide variety of surfaces found in living organisms has unique properties that protect an animal or human from disease.

* Skin provides an anatomical barrier against microbe invasion, but it also has physiologic properties that help the skin resist pathogens. These physiologic properties include dryness, acidity, and temperature of the skin; these inhibit the growth of many microbes. Oil produced by sebaceous glands contains fatty acids that are toxic to some pathogens. Sweat produced by sweat glands flushes microbes from pores and skin surfaces and contains the enzyme lysozyme, which degrades part of bacterial cell walls. Sweat also contains salts, urea, and lactic acid that discourage microbe growth.

* Mucus produced at mucous membranes contains many substances that can kill or inhibit the growth of bacteria. Lysozyme, lactoferrin (a protein that binds iron that is needed by all pathogens), and lactoperoxidase (an enzyme that produces highly reactive superoxide radicals that are toxic to bacteria) are all examples of substances found in mucus.

* Many body systems have pH levels that can alter the rate of microbe growth. Hydrochloric acid production in the stomach produces an acid environment that helps retard the growth of some ingested microbes. The small intestine contains digestive enzymes and bile (an alkaline substance) that destroy microbes. In the reproductive systems, vaginal fluid has a low pH and semen contains an antimicrobial chemical that inhibits bacterial growth. Tears and saliva contain lysozyme, a chemical that can degrade bacterial cell walls and has a basic pH to hinder bacterial growth.

* Gravity's role in disease prevention is a result of its ability to hinder organisms from gaining a foothold in many body systems. Urination flushes microbes from the urethra and helps reduce the number of microbes that can colonize the urinary tract. Peristalsis and expulsion of feces help remove microbes from the intestine. Lacrimation flushes the eye's surface with tears and carries irritants from the eye.

* Normal flora or microflora is a complex mixture of microbes (bacteria, fungi, protozoa, and viruses) that reside on or within an animal. The animal body provides a favorable habitat for microbes because it is a constant source of nourishment and moisture, relatively stable pH and temperature, and extensive surfaces on which to live. Animals acquire normal flora at birth and it may fluctuate to some extent during the animal's life. The uterus is normally sterile during embryonic development until prior to an animal's birth at which time the fetus is exposed to microbes with the breaking of the fetal membranes. At this time exposure to many microbes occurs and continues with the process of birth through the vagina. The nature of the intestinal normal flora depends initially upon the type of milk (either mother's milk or milk replacer) being consumed, but in time will be influenced by the environment, feed, and contact with other animals. Normal flora protects an animal from infection by transient microbes because these nonresident microbes must compete with the normal flora for space and nutrients of a particular body area. Many times there are just not enough space and nutrients to go around. Normal flora may also produce substances that help kill transient bacteria thus protecting the environment of normal flora. Body systems that do not contain normal flora such as the nervous system and blood can be especially vulnerable to infections once a microbe gets into these areas.

Cellular and Chemical Protection

Microbes that are able to penetrate the first line of defense are usually destroyed by nonspecific responses. These responses are cellular and chemical in nature and act rapidly at both the local and systemic levels once the first line of defense has been broken. The second line of defense includes:

* Fever. Many animals have specific normal body temperatures. An elevation of body temperature above the normal range is referred to as a fever. Normal body temperature is maintained by the hypothalamus, a control center located in the brain. Initiation of fever occurs when a pyrogen (a fever-stimulating chemical) resets the hypothalamic thermostat to a higher level. This resetting of the internal thermostat signals muscles to increase heat production and peripheral arterioles to decrease heat loss through vasoconstriction. Pyrogens may be produced in the body (endogenous pyrogen) or they may be produced outside the body (exogenous pyrogen). Interleukin-1 is an example of an endogenous pyrogen because it is produced by activated macrophages found in the body. Bacteria, viruses, parasites, and fungi are all examples of exogenous pyrogens. The benefits of fever include the inhibition of replication of certain temperature-sensitive microbes, the stimulation of white blood cells to destroy microbes, the increase in metabolism of certain cells, the increase in phagocytosis, the reduction in iron available for replication of bacteria, and the enhancement of the effects of interferon. The disadvantages of fever include increased heart rate, increased demand for calories, seizures, and dehydration. Figure 2-1 provides an example of fever induction.

* Complement. The complement system, named because it complements the immune reaction, is a group of approximately 30 different proteins found in blood plasma in an inactive form. Complement is an important defense against bacteria and some fungi. The proteins in the complement system interact with one another in a step-wise manner known as the complement cascade. The complement system is activated by the classical pathway (named because it was discovered first) when complement proteins come in contact with a foreign substance. A series of reactions follow in a sequential manner and as each complement protein is activated, it activates the next complement protein, until the final protein is activated. The classical pathway depends upon an antibody attaching to an antigen; therefore, it responds to the specific immune system covered later in this chapter. In the alternate pathway, the first protein is activated spontaneously in blood and binds to foreign cell surfaces. This binding initiates a cascade of activations, ultimately destroying the foreign cell. The main function of the alternate pathway is rapid lysing of foreign cells, especially viruses and gram-negative bacteria, in the absence of specific immunity. In addition to destroying foreign cells, the complement system also enhances the inflammatory process and phagocytosis.

* Interferons. Interferons are a group of glycoproteins released by a variety of cells in response to invasion by intracellular parasites (including viruses) and other stimuli. There are three known types of interferon: alpha (?) a product of lymphocytes that is induced by infection with viruses, bacteria, and other agents; beta (?) a product of fibroblasts, epithelial cells, and macrophages in response to viruses; and gamma (?) a product of T lymphocytes and natural killer cells that function in immune regulation. Alpha and beta interferon are important in the nonspecific immune response, whereas gamma interferon is important as part of the specific immune response to antigens. Virus-infected cells that produce interferons are unable to save themselves from destruction; however, the interferon produced by these cells attaches to membranes of surrounding cells and prevents viral replication from occurring in those cells. Thus, interferon inhibits the spread of infection. Interferons are not virus-specific (they are effective against a variety of viruses, not just a particular type) but are species-specific (they are effective only in a particular species and ineffective in another species). In addition to cell protection, alpha interferon produced by T lymphocytes also activates a subset of cells called natural killer cells (NK cells). NK cells are nonspecific cytotoxic cells that work against any foreign antigen or abnormal cell, but particularly well against tumor and virus-infected cells. NK cells kill animal cells by releasing various cytotoxic molecules, some of which create holes in the target cell's membrane causing cell lysis, whereas others enter the target cell and fragments its nuclear DNA.

* Inflammation. Inflammation is a reaction to any traumatic event in the body. Local injury, irritation, microbe invasion, or toxin release are all examples of traumatic events in the body that can trigger the inflammatory response. Once the initial traumatic event has occurred, a chain reaction takes place at the site of damaged tissue, calling beneficial cells and fluids into the injured area. Some of the earliest changes occur in the vasculature near the damaged tissue. These changes are controlled by the nervous system and cytokines (chemical mediators) released by blood and tissue cells. The initial reflex response to damaged tissue is vasoconstriction (narrowing of the blood vessels). Vasoconstriction only lasts for a few seconds or minutes and is rapidly followed by vasodilation (widening of the blood vessels). Vasodilation is caused by vasoactive agents such as histamine and prostaglandin released from damaged cells. Vasodilation causes increased blood flow to the damaged area, which in turn causes the redness and heat associated with inflammation. Vasodilation also causes the endothelial cells lining the capillaries to stretch and form gaps through which blood components can leak into the extracellular spaces. The exudate (fluid) that escapes is typically plasma and it accumulates in the tissues causing edema (local swelling and hardness because of exudate accumulation). The accumulation of fluid (dilutes toxic substances), cells, and cell debris signals the infiltration of neutrophils (a phagocytic white blood cell) to the area. After a period of time, more slowly reacting phagocytic cells such as monocytes and macrophages come to the damaged site. Clearing of fluid, cellular debris, dead neutrophils, and damaged tissue is done by the macrophages. Lymphocytes react by producing antibodies or kill intruders directly (see specific immune response described later in this chapter). Figure 2-2 summarizes the steps involved in inflammation.

* Phagocytosis. Animal cells must be able to recognize when a substance does not belong in the body. The recognition of nonself is important in the process of phagocytosis, the engulfment of an invading particle and abnormal cells (such as dead cells) via invagination of the cell membrane. Phagocytosis begins when phagocytes migrate to the needed site as a result of chemical attraction. This chemical attraction is called chemotaxis. A variety of cells produce chemotactic agents during the complement cascade and during inflammation. Phagocytes move across a concentration gradient (they move from an area of low concentration of chemotactic agents to an area of high concentration of chemotactic agents) and are attracted to the site where they are needed. Different chemotactic agents attract different leukocytes (white blood cells). There are several categories of cells capable of phagocytosis. Types of phagocytes include:

* Granulocytes. Granulocytes are leukocytes that originate from stem cells in the bone marrow and have prominent cytoplasmic granules that can be seen after staining with the appropriate dyes. Granulocytes are divided into neutrophils, eosinophils, and basophils. Neutrophils and eosinophils are phagocytic. Neutrophils are the most abundant and efficient circulating phagocyte and provide the first line of phagocytic defense in an infection. Neutrophils react early in the inflammatory response to bacteria and a high neutrophil count in blood (neutrophilia) is a common finding with bacterial infections. Eosinophils are attracted to sites of parasitic infections and antigenantibody reactions and an increase in eosinophils (eosinophilia) indicates certain types of parasitic infections or allergic reactions. Basophils are not phagocytic and are involved in allergic and inflammatory reactions.

* Monocytes. Monocytes are leukocytes that originate from stem cells in the bone marrow and enter the peripheral blood stream. As monocytes leave the blood and spread through tissues they differentiate into active phagocytes called macrophages. Monocytes become macrophages in lymph nodes, spleen, lungs, and nervous system; they become Kupffer cells in the liver; they become alveolar macrophages in the air sacs of the lungs, and they become microglial cells in the central nervous system (CNS). Macrophages are extremely efficient phagocytes and engulf foreign particles such as cellular secretions, dead leukocytes, erythrocytes (red blood cells), and tissue cells. Macrophages live longer than neutrophils and have the ability to replicate. Macrophages also play an important role in the specific immune response.
Interferons interfere with viral




Phagocytosis continues when the phagocyte attaches to the foreign particle. Phagocytes can only ingest foreign particles to which they can attach. For this attachment to occur sometimes a process called opsonization is needed. Opsonization is a process that facilitates phagocytosis by the deposition of opsonins such as antibodies or complement fragments that coat the surface of foreign particles. This coating of the surface of foreign particles facilitates the recognition and engulfment of the foreign particle because the phagocyte possesses surface receptors for antibodies and complement fragments, thus allowing the phagocyte to attach to the foreign particle. Following attachment of the phagocyte to the foreign particle, the phagocyte then surrounds the particle with pseudopodia causing fusion of the phagocyte and foreign particle. The foreign particle is then engulfed (this process may be referred to as endocytosis). When a phagocyte engulfs a particle, the particle becomes a vacuole enclosed by the cell membrane and is destroyed by the phagocyte's lysosomes. Lysosomes are membrane-bound cellular structures that are filled with digestive enzymes that destroy the foreign particle when they fuse with the vacuole and release these enzymes. Figure 2-3 summarizes the steps involved in phagocytosis.


When innate defense mechanisms fail to protect an animal from a foreign particle, the acquired defense mechanisms need to be activated. An acquired immune response is activated by a specific foreign particle called an antigen. Antigens are usually proteins and are usually (but not always) foreign to the host. There are two types of acquired immunity, the humoral and cellular, which are both mediated by different components of the immune system and function in the elimination of distinct types of microbes. Humoral immunity is based on antibodies found both on cell surfaces and dissolved in blood and lymph, whereas cellular immunity is associated with cell surfaces. The two types of acquired immunity do not occur as isolated events but rather communicate and interact with each other.

Acquired immunity against a particular microbe can be induced by the host's response to the microbe or by the transfer of antibodies specific for that microbe. There are four terms involved with understanding acquired immunity: active, passive, natural, and artificial (Figure 2-4). The terms active and passive describe whether the individual's immune system responds to the antigen (active) or whether the individual receives immunity from another source (passive). Active immunity occurs when an individual's own body makes activated lymphocytes to a particular antigen; therefore, the host's immune system plays an active role in responding to the antigen. Active immunity takes days to weeks to develop and lasts for a long time because memory cells have been produced. Examples of active immunity include the body responding to antigens during an infection or via vaccination. Passive immunity occurs when the immune components develop in another animal and are transferred to an individual who was not previously immune; therefore, the recipient becomes immune without having been exposed to or having responded to that particular antigen. Passive immunity provides immediate protection to a specific antigen but lasts for only a short time because memory cells were not produced. Examples of passive immunity include transfer of antibodies across the placenta or the introduction of antibodies in antisera. The terms natural and artificial refer to how the immunity is obtained. Natural immunity occurs when the immunity is acquired unintentionally through everyday living. Artificial immunity occurs when deliberate action is taken to acquire the immunity such as getting a vaccine. Combining these terms provides four types of acquired immunity: naturally acquired active immunity (the body responding to antigens that enter unintentionally such as during infection), artificially acquired active immunity (the body responding to antigens that are intentionally introduced into the body such as vaccines), naturally acquired passive immunity (the unintentional transfer of antibodies from mother to offspring across the placenta or through colostrum/breast milk), and artificially acquired passive immunity (the intentional injection of antitoxins or antisera (obtained from immune individuals) into an animal). Figure 2-4 provides examples of acquired immunity.

An antigen is any substance that will
stimulate an immune response.

Components of Acquired Immunity

The basis for acquired immunity is the recognition of self versus nonself. Nonself recognition involves proteins imbedded in the cell surface called major histocompatibility complexes (MHC). There are two classes of MHC proteins: class I is found on the surface of almost all cells, whereas class II is found on certain cells that have a role in the immune system. MHC proteins are recognized by two basic types of molecules: antibodies and cells that have T-cell receptors.

Lymphocytes are types of leukocytes involved in acquired immunity. There are two main categories of lymphocytes known as B and T lymphocytes and they function differently. Both types of lymphocytes originate in the bone marrow from the same basic stem cell, but develop into two distinct types. Maturation of B lymphocytes is believed to occur in the bone marrow (or some believe they mature in lymphoid tissue of the gut), whereas T lymphocytes mature in the thymus gland. The process of maturation commits each B or T lymphocyte to one specific type. Both types of lymphocyte then migrate to precise areas in lymphoid tissue until they are needed. B lymphocytes have antibody molecules in their surface and give rise to plasma cells. Plasma cells actively secrete antibodies into blood. T lymphocytes have surface receptors that bind antigens. Table 2-1 describes a variety of different types of T lymphocytes. When lymphocytes become activated they are stimulated to move from a stage of recognition where they bind with particular antigens to a stage where they proliferate and differentiate into cells that function to eliminate antigens.

Antibodies are proteins called immunoglobulins that are produced when B lymphocytes become sensitized to a specific antigen, multiply, and mature into plasma cells. The rate of antibody production is extraordinary; one functional plasma cell can make approximately 2,000 antibody molecules per second for about the first 5 days of its activation. The basic structure of an antibody molecule resembles the letter Y and consists of two identical light polypeptide chains, two identical heavy polypeptide chains, two antigen-binding sites, and an FC region (Figure 2-5). The stem region of the Y, known as the FC region, is always constant and consists of macrophage-and complement-binding sites. The FC region is the portion of the antibody that allows it to bind to cells such as neutrophils, macrophages, basophils, and mast cells that possess surface receptors able to recognize this region. Attached to the stem region are two projections that produce the arms of the Y. Each arm of the Y consists of a light and heavy polypeptide chain. The light polypeptide chains contain fewer amino acids than the heavy polypeptide chains; therefore, they are shorter and lighter in weight than the heavy polypeptide chains. The end of each arm contains pockets called antigen-binding sites that are varied in shaped to accommodate a wide variety of antigens. This variability to the end of the antibody is because of the presence of a variable region in which the amino acid composition is varied from one clone of B lymphocyte to another. The rest of the light and heavy polypeptide chain is known as the constant region because its amino acid composition does not vary greatly from one antibody to another. Figure 2-5 shows the basic structure of an antibody.


There are five different classes of antibody, referred to as IgM, IgG, IgA, IgD, and IgE. The class of antibody determines the role of the antibody in the immune response, but not the antigen that it recognizes. Table 2-2 summarizes the classes of immunoglobulins and their functions.

Antibodies can destroy foreign particles in a variety of ways (Figure 2-6). Some ways include:

* Opsonization. Foreign particles become coated with IgG so that they can be more easily recognized by phagocytes. When the foreign particles become coated macrophages are stimulated to engulf the particle.

* Neutralization. IgG and IgM can neutralize some viruses and toxins secreted by bacteria, whereas IgA neutralizes toxins in digestive and respiratory secretions. Neutralization masks the dangerous parts of bacterial toxins and viruses. Immunoglobulins can bind to a virus' envelope and prevent the virus from attaching to host cells thus preventing it from functioning normally.

* Complement activation. The classical pathway of the complement cascade is activated by bound antibody. Bound antibody fixes and activates complement, which leads to antigenic cell lysis.

* Precipitation. Antibodies produce clumping of proteins around antigen so that the antigens are contained within an area. This containment enhances the process of phagocytosis.

* Agglutination. Antibodies produce clumping of red blood cells or microbial cells because each basic antibody has two antigen-binding sites. Each antigen-binding site can attach to two antigenic determinants at once resulting in several antibody molecules binding with two cells. Agglutination enhances the chance of phagocytosis and hinders the activity of phagocytic organisms.


Cytokines are protein hormones that serve as chemical communicators helping the body mount cooperative mechanisms against foreign particles. There are several different types of cytokines such as monokines (cytokines produced by monocytes and macrophages), lymphokines (cytokines produced by lymphocytes), inflammatory peptides (cytokines produced by neutrophils), and vasoactive amines (cytokines produced by platelets and mast cells). Some cytokines act during inflammation and allergic reactions, whereas others function as part of the acquired immune system. Table 2-3 lists a variety of cytokines.

Humoral Immunity

Humoral immunity or antibody-mediated immunity involves the production of antibodies (Figure 2-7). Humoral immunity is most effective against bacteria, viruses located outside of body cells, and toxins. When antigen is introduced into the body it is taken up by antigen-presenting cells (APC) such as macrophages. Macrophages digest the antigen and the antigen binds to the MHC receptor. The combined antigen-MHC molecule is then displayed on the macrophage's surface. The macrophage secretes interleukin-1, which stimulates helper T cells (APC present antigen to all helper T cells; however, only those helper T cells that have binding sites complementary to the presented antigen will be activated). The appropriate helper T cells attach to the antigen-MHC molecules and then they divide and secrete interleukin-2. The helper T cells also secrete chemicals such as B-cell growth factor that reach a B lymphocyte and activate it. The activated B cell will divide to produce a clone of identical B cells. The majority of these B-cell clones matures into antibody-producing plasma cells and expels antibodies for several days until the plasma cell dies. Each plasma cell makes only one antibody type that binds to that specific antigen that initiated its production. Each plasma cell can make thousands of antibody molecules per second. Plasma cells have high activity levels and are thus short-lived (most die within a few days of activation); however, antibodies can remain in body fluids for months.


Small amounts of activated B cells initially formed by B-cell proliferation do not become antibody-producing plasma cells but rather become memory B cells. Memory B cells are long-lived cells with receptors to the specific antigen that triggered their production. Memory B cells persist in lymphoid tissue for months or years. These memory B cells are a reserve of antigen-sensitive cells that become active and are able to respond rapidly should the antigen enter the body at a later time. Memory B cells can proliferate and differentiate rapidly into plasma cells without requiring interaction with an APC. These newly differentiated plasma cells produce large amounts of antibody within a few days. Once the antigen is under control, suppressor T cells inhibit antibody production.

Cell-Mediated Immunity

Cell-mediated immunity is used when intracellular pathogens or abnormal body cells are present because antibodies are unable to enter cells. Cell-mediated immunity involves the interactions of many cell types and cytokines (Figure 2-8). Although cell-mediated immunity does not involve antibody production, antibodies produced during humoral immunity may play a role in some cell-mediated responses. Cell-mediated immunity can be described as four distinct types: delayed type hypersensitivity, cytotoxic T-cell response, natural killer cell responses (which are nonspecific), and immediate hypersensitivity. The cytotoxic T-cell response will be described here. Cell-mediated immunity is initiated when a macrophage engulfs and digests an antigen. Antigenic fragments are displayed on the surface of the macrophage. A helper T cell binds to one of the antigenic parts being displayed on the macrophage surface and the helper T cell produces interleukin-2 and gamma interferon. Interleukin-2 and gamma interferon activate those cytotoxic T cells that have T-cell receptors for the antigen. Most activated cytotoxic T cells differentiate into more cytotoxic T cells, whereas a few differentiate into memory T cells that persist for months or years in lymphoid tissue. The "daughter" cytotoxic T cells produce interleukin-2 and become self-stimulating (they no longer need an APC or helper T cell for activation). Cytotoxic T cells have vesicles containing cytotoxins that form channels in the target cell through which enzymes signaling cell death can be transferred. Once the target cell is destroyed the cytotoxic T cell moves to another infected cell.


Activated T cells that have become memory T cells remain inactive until subsequent contact with the same antigen. When memory T cells encounter the same antigen they respond immediately (without interaction with APC) by differentiating to cytotoxic T cells. Because the number of memory T cells is greater than the original number of T cells that recognized the antigen during the initial exposure, the secondary cell-mediated response is much more rapid and effective.

Figure 2-9 summarizes the interaction of the humoral and cell-mediated immune response.

The primary immune response is slower
than the secondary immune response
because there are not any existing
memory cells for that specific antigen.

Primary and Secondary Immune Responses

The initial action of the immune system to a particular antigen is the primary immune response. The primary immune response occurs the first time a specific antigen is identified by either lymphocyte. Following initial exposure to an antigen there is a delayed primary response in antibody production. This delay, known as the lag phase, is a result of the time needed to process antigen. In time, B cells differentiate into plasma cells capable of producing antibody. The primary response typically takes 10 to 14 days for relatively small amounts of antibodies to be produced. As antigen is destroyed, the number of antibodies in blood declines because the plasma cells die marking the end of the primary response. The antigen-stimulated B cells that did not differentiate into plasma cells now become memory B cells. Memory B cells remain dormant in lymphatic tissue until subsequent exposure by the same antigen. Activated memory B cells will differentiate rapidly (approximately 1 to 3 days) and will produce large amount of antibodies. This more rapid response and increased production of antibody following subsequent exposure to the same antigen occurs because so many more cells are able to recognize and respond to the antigen. This rapid recognition and response on subsequent exposure to antigen is called the secondary immune or anamnestic response (ana- means against and mimneskein means to call to mind).

Vaccine Theory and Immune Response

Primary and secondary immune responses play a role in vaccination theory. When an animal receives the first injection of vaccine, the primary immune response takes place. In time, antibody levels are present in the animal as well as memory cells (the amount of time varies for different types of vaccine). When the animal receives the second injection of vaccine, the secondary immune response takes place. Because memory cells to that specific antigen are present in the animal's body, a more rapid and intense reaction against the antigen occurs resulting in higher antibody levels. Therefore, booster vaccines are given weeks to months apart to raise antibody levels, thus providing adequate levels of antibody protection. Figure 2-10 depicts the primary and secondary humoral immune response.

Another value of booster vaccines is to ensure that maternal antibodies that may be present in young animals are not blocking antibody production in the vaccinated animal. Young animals receive passive immunity through placental transfer of antibodies, consumption of colostrum (antibody-rich milk produced by the mother in the first days following parturition), or if immunoglobulincontaining products are given to them in the first hours of life. Maternal antibodies protect them from infectious agents in the short term; however, they interfere with or delay the young animal's ability to protect itself from infectious agents in the long term. Maternal antibodies treat vaccine antigens like real antigens and inactivate them before they get a chance to stimulate the young animal's immune system. Maternal antibodies have varying durations as a result of both the individual and the infectious agent they protect against. This variable rate of maternal antibody decline makes the timing of vaccine administration difficult. For most infectious agents, maternal antibody concentrations fall to nonprotective levels by 2 to 3 months of age; however, any residual maternal antibody could make antibody production in young animals unresponsive for additional weeks or months. Some maternal antibodies can persist in young animals until 6 months of age, thus preventing immune responses to some antigens for a prolonged period of time.



Diagnosing infectious disease can be done in a variety of ways. Culturing bacteria on a variety of agars (solid growth media) and performing staining procedures can aid in the identification of bacterial pathogens. Observing parasite eggs or larvae in stool examinations can be a fast and inexpensive way to identify parasitic pathogens. However, sometimes these tests may take longer than desired or the isolation of some pathogens may be impossible with current technology. Thus, numerous tests have been developed that take advantage of the patient's immune response in the direct or indirect identification of infectious agents. Although some of the tests described below have provided the opportunity to identify infectious agents or an animal's exposure to them, there may be problems that exist with some of these tests. Specificity is the property of a test to detect only a certain antibody or antigen and not react with an unrelated antibody or antigen (Figure 2-11A). Sensitivity is the capability of a test to detect even very small levels of antibody or antigen for which the test was developed (Figure 2-11B). If tests are not very specific nor very sensitive, false-positive or false-negative results can occur. For example, false-positive results may occur when two related agents have antigenic properties similar enough to cross react with antibodies produced against the other. False-positive results typically occur with tests that do not have a high level of specificity. False-negative results may occur when the level of antibody is not high enough to provoke a positive result yet the animal was exposed to the agent in the past. False-negative results typically occur with tests that do not have a high level of sensitivity.


Immunodiagnostic procedures are laboratory procedures that help diagnose infectious disease by the detection of either antigens or antibodies in clinical specimens. Detection of antigen in a clinical specimen serves as an indication that a particular agent is present in the patient, providing direct evidence that the patient is infected with that organism. Detection of antibodies directed against a particular agent serves as indirect evidence of infection with that organism. The presence of antibodies to a particular organism may indicate exposure to an antigen by past infection or vaccination or may indicate current infection. Because the presence of antibodies may indicate several possible explanations and takes approximately 10 to 14 days to develop, the presence of antigens provides the best evidence of infection.

Some ways to increase the value of antibody tests are to specifically test for IgM antibodies and to utilize paired sera tests. Measuring IgM antibodies is valuable because IgM is the first antibody to appear and is short-lived; therefore, its presence indicates current infection or recent exposure. Paired sera tests involve the collection of one serum sample (the acute sample) during the acute stage of the disease and another serum sample (the convalescent sample) approximately 2 weeks after the acute stage. A significant rise in antibody levels between the acute and convalescent samples demonstrates that the patient was actively producing antibodies against the organism during the 2-week period.

Agglutination Tests

Agglutination occurs when antibodies (also called agglutinins) cross-link with insoluble antigens (also called agglutinogens) to form visible clumps (Figure 2-12A). In agglutination tests, the antigens are whole cells such as bacteria or red blood cells (Figure 2-12B). In some agglutination tests, special agglutinogens have been made by attaching inert particles onto the antigen. Sometimes the inert particles are latex beads (known as latex agglutination tests) or red blood cells that react with viral antigens (known as viral hemagglutination tests).

Precipitation in Agar Gel

Precipitation reactions occur when soluble antigen (also called precipitinogen) is made insoluble by an antibody (also called a precipitin). When optimal proportions of antibody and antigen are reached, a precipitate forms. Precipitates are easily disrupted in liquid media therefore most precipitation reactions are carried out in agar gels (Figure 2-13). Agar gels are soft enough to allow antibody and antigen to freely diffuse, yet are firm enough to hold the antibody-antigen precipitate in place.



Complement Fixation

Complement fixation tests are methods to determine whether complement has been bound to an antigen-antibody complex (Figure 2-14). When complement is bound it is termed fixed and is inactive. Some antibodies bound to the surface antigens of cells have the ability to combine with complement and to make complement inactive. Sample serum is heated to destroy naturally present complement and the sample is incubated with the antigen of interest in the presence of added complement, red blood cells, and anti-red blood cell antibodies. If antibody is present in the sample serum, components of complement bind to the antigen-antibody complex. Complement is then unavailable to bind with the anti-red blood cell antibodies that have bound to the red blood cells and therefore cannot lyse the red blood cells (no lysis = positive result). If antibody is not present in the sample serum, the components of complement remain free and inactivated. Complement is then available to bind with the anti-red blood cell antibodies that have bound to the red blood cells and can lyse the red blood cells (lysis = negative result). Lysis of the sensitized red blood cells indicates that complement is activated because it was not fixed (bound) in an antigen-antibody complex because antibody was not present in the sample serum.

Fluorescent Antibody Techniques

Fluorescent antibody techniques use the properties of dyes such as fluorescein and rhodamine to emit visible light in response to ultraviolet radiation (Figure 2-15). Fluorescent antibodies can be used for direct testing or indirect testing of antigen. In direct testing, an unknown antigen is fixed to a slide and exposed to a fluorescent antibody solution for a known antigen. If the antibodies are specific for the unknown antigen, they will bind to it. The slide is then rinsed to remove unbound antibodies and observed with a fluorescent microscope. Fluorescing cells indicate the presence of antigen-antibody complexes and a positive test result. In indirect testing, the fluorescent antibodies are antibodies to a portion of another antibody. A known antigen is then combined with the sample serum. If the sample serum has antibodies to the known antigen, the antibody will bind to the antigen. The fluorescent antibody solution (the antibody to the antibody in the sample serum) is then added and rinsed. Fluorescing cells indicate that the fluorescent antibodies have combined with the antibodies in the sample serum providing a positive test result. In a negative test, no fluorescing complex will be seen.


The most sensitive immunologic test
methods that detect antibodies or
antigens are those that have a molecular
tag that is easy to detect at extremely
low concentrations. Examples include
immunoassays and Western blotting.

Immunoassays are extremely sensitive methods that accurately and rapidly identify either antigen or antibody. Ways of detecting antigen or antibody include radio-active -isotope labels, enzyme labels, or electronic sensors. Radioimmunoassay RIA) is a technique in which antibodies or antigens are labeled with a radioactive isotope that can be used to detect small amounts of a corresponding antigen or antibody. In RIA the labeled substance competes with its natural, unlabeled one for a reaction site. Large amounts of bound radioactive component indicate that the sample did not have the substance for which the sample was being tested (in other words, it did not have to compete for the reaction site with its natural form). The level of radioactivity is measured with an isotope counter or autoradiograph. Enzyme-linked immunosorbent assay (ELISA) is an easy to perform technique in which antibody is linked to an antigen and antibody is linked to an enzymeantibody complex that produces a color change (Figure 2-16). In direct ELISA, a known antibody is exposed to a sample. If the sample contains the specific antigen, the antibody will bind to it. An enzyme-antibody complex that can react with the antigen is then added. Bound antigen will attract enzyme-antibody and keep it in place. A substrate to the enzyme is then placed in the test. If an enzyme has been affixed to antigen, it combines with the substrate and releases a colored dye. Any color development is a positive test; lack of color is a negative test. In indirect ELISA, an antigen specific for the antibody being measured is adsorbed to the surface of the test plate. The sample is then added. If the sample contains antibodies to the antigen the two will bind. An enzyme-antibody reagent to the antibody being measured is added to the test plate. The substrate to the enzyme is then added. Any color development indicates that all the components reacted because antibody was present in the sample.
Direct ELISA tests for antigen; indirect
ELISA tests for antibody.



Blotting Tests

Blot tests are used to detect deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein using electrophoresis (separation of ionic molecules based on their rate of migration on a medium such as paper or gel that is then stained and quantified). Blot tests used to detect DNA or RNA are covered in the next section. Western blot tests separate proteins by electrophoresis, transfer them to membranes, and identify them through the use of labeled antibodies specific for the protein of interest (Figure 2-17). Test strips containing antigen are incubated with patient serum and if antibodies are present they will bind to their corresponding antigen. Unbound antibody is removed by washing. Colored bands appear in the positions where antigen-specific antibodies are present. Western blot tests are commonly used to detect antibodies to specific parts of electrophoretically separated antigenic components. Western blot tests are often used to confirm the specificity of antibodies detected by ELISA screening tests.


Recombinant DNA technology is a collection of procedures for manipulating genetic material in vitro. The tools of recombinant DNA technology are used in a variety of basic techniques to multiply, identify, isolate, and sequence the nucleotides of genes.

Southern Blot Tests

Blot tests are used to detect DNA, RNA, or protein (covered previously) using electrophoresis. Southern blot tests (developed by Ed Southern in 1975) denature specimen DNA, treat the DNA with restriction enzymes resulting in DNA fragments, and then separate single-stranded DNA fragments via electrophoresis. These fragments are then blotted to a membrane that has radiolabeled single-stranded DNA fragments with sequences complementary to those being sought. The presence of double-stranded DNA bearing radiolabel detected by radiography indicates a positive test.

Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a technique available since 1985 that is used to amplify and analyze DNA (Figure 2-18). PCR is becoming a valuable tool in disease detection because it rapidly increases the amount of DNA in a sample allowing for possible detection of infection from a single gene copy. PCR allows a single DNA molecule to be detected in a group of other molecules and to make unlimited copies of the DNA. In PCR, the DNA to be amplified is denatured, primed, and replicated by a polymerase enzyme that can function at high temperatures. When the DNA sample is denatured it is heated in a machine called a thermocycler. Heating separates the strands of DNA exposing each base. The sample is then cooled. During priming, synthetic short DNA strands attach at the ends of the test DNA strands to promote replication. Priming prepares the two DNA strands for synthesis. In the last phase heat-stable DNA polymerase enzyme and nucleotides are added to make complementary strands of DNA. These steps are repeated until multiple copies of the original DNA are produced. PCR can go through many cycles within 2 to 3 hours, making it a valuable tool for diagnosing disease.

Reverse transcriptase-polymerase chain reaction (RT-PCR) is used when the nucleic acid of interest is RNA rather than DNA (when the virus of interest is an RNA virus). RT-PCR modifies the PCR procedures to include conversion of RNA to DNA using reverse transcriptase in the initial steps.



An animal's ability to resist foreign invaders and recover from disease is a result of both innate and acquired immunity. Innate immunity is a defense mechanism that does not depend upon prior exposure to an infectious agent to be effective, whereas acquired immunity is the protection acquired through life and is specific to a particular foreign invader. Types of innate immunity include physical and chemical barriers, fever, complement, interferons, inflammation, and nonspecific cellular responses such as phagocytosis. Acquired immunity involves the activation of T and B lymphocytes against a particular antigen. Humoral immunity involves the production of antibodies, whereas cell-mediated immunity involves the interaction of many cell types and cytokines. Table 2-4 shows a summary of the phases of acquired immunity.

Immunodiagnostic testing involves detecting disease via antigen-antibody recognition. Some examples of immunodiagnostics include agglutination tests, precipitation reactions, complement fixation, fluorescent antibody techniques, Western blot tests, and immunoassays such as radioimmunoassay and enzymelinked immunosorbent assays. Recombinant DNA diagnostic techniques include Southern blot tests, PCR, and RT-PCR.

Review Questions

Multiple Choice

1. Regulatory T cells ensure that the immune response is effective but not destructive. What two T cells are considered regulatory T cells?

a. memory and suppressor

b. suppressor and cytotoxic

c. cytotoxic and helper

d. suppressor and helper

2. Cyclosporine is a drug that inhibits the production of interleukin-2 and is used to prevent rejection of transplanted organs. Why would cyclosporine be effective in preventing transplant rejection?

a. it inhibits interleukin-2, a substance that stimulates

B-cell production

b. it inhibits interleukin-2, a substance that stimulates T-cell production

c. both of the above

d. none of the above

3. Humans are not able to contract fowl cholera. This is an example of

a. artificially acquired active immunity.

b. naturally acquired active immunity.

c. innate immunity.

d. acquired immunity.

4. What type of immunity can be referred to as antigen-specific immunity?

a. active

b. acquired

c. passive

d. innate

5. Activated macrophages produce what endogenous pyrogen?

a. interferon

b. interleukin-1

c. interleukin-2

d. immunogen

6. Alpha interferon activates what subset of cells?

a. cytotoxic T lymphocytes

b. cytokines

c. pyrogens

d. natural killer cells

7. One advantage to acquired immunity is that

a. it takes time for it to occur.

b. it produces memory cells.

c. it produces antibodies instead of activated cells.

d. it activates cells instead of producing antibodies.

8. Indirect immunodiagnostic testing involves the identification of

a. antigen.

b. antibody.

c. T lymphocyte.

d. B lymphocyte.

9. Because it is the first antibody to be produced in response to an antigen, its presence can be used to indicate current infection. What antibody is this?

a. IgG

b. IgM

c. IgA

d. IgE

10. What diagnostic test method amplifies and analyzes DNA in its identification of antigen?

a. agglutination test

b. precipitation test

c. ELISA test

d. polymerase chain reaction test

Short Answer

11. Describe five physiologic properties of surfaces that protect animals from infection.

12. What is the complement system and what are some advantages to having it?

13. Differentiate between cell-mediated and humoral immunity with regard to the types of antigens they eradicate.

14. What type of test would give you the greatest chance of false-positive results, a test with low specificity or one with low sensitivity? Why?

15. Give two examples of situations where the active response occurs, yet the animal does not develop signs of disease.


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Table 2-1  Types of T Lymphocytes and Their Functions

Type of Lymphocyte           Function

Helper T cell (also known    Assists in immune response and
as CD4+ or [T.sub.H])        secrete substances that
                             stimulate B lymphocytes.

Cytotoxic T cell             Binds tightly to target cell and
                             secretes a protein that causes
                             pores to form in the foreign
                             cell membrane.

Suppressor T cell            Inhibits B lymphocytes and the
(also known as CD8+)         immune response.

Memory T cell                Remembers the specific antigen
                             and stimulates a faster and
                             more intense response if the
                             same antigen is presented.

Table 2-2 Immunoglobulins and Their Functions

Type                     Function

Immunoglobulin M (IgM)   M is for macro; therefore, IgM is a huge
                         molecule with a great capacity to bind
                         antigen. It is the first antibody produced
                         in response to antigen. It circulates in
                         blood and is too large to cross the

Immunoglobulin G (IgG)   IgG is produced by memory cells responding
                         for the second time to a particular antigen.
                         It is the most prevalent immunoglobulin. It
                         circulates in blood and is the only
                         immunoglobulin to cross the placenta.

Immunoglobulin A (IgA)   IgA is a secretory immunoglobulin found in
                         the mucous and serous secretions of the
                         salivary glands, nasal membrane, mammary
                         tissue, lung, urinary tract, and
                         reproductive tract. IgA provides local
                         immunity to the gastrointestinal,
                         respiratory, and urogenital systems and is
                         passed to offspring via nursing.

Immunoglobulin D (IgD)   IgD is found in small amounts in plasma and
                          may play a role in immune suppression.

Immunoglobulin E (IgE)   IgE stimulates an inflammatory response and
                         is involved in allergic and parasitic

Table 2-3 Some Types of Cytokines and Their Functions

Type             Source                   Function

Interleukin-1    Monokine produced by     Activates B and
                 activated macrophages    T lymphocytes; mediates

Interleukin-2    Lymphokine produced by   Growth factor for B and
                 helper T cells           T lymphocytes; enhances
                                          cytotoxic effects of
                                          nonkiller (NK) cells

Interferon       Lymphokine produced by   Activates macrophages;
                 helper and suppressor    promotes B- and T-cell
                 T cells                  differentiation; activates
                                          neutrophils and NK cells

Tumor necrosis   Monokine produced by     Mediator of inflammation
factor           activated macrophages

Table 2-4 Phases of Acquired Immunity

Phase         Action

Recognition   Exposure to a specific antigen resulting in selective
              activation and expansion of those lymphocytes with
              antigenic receptors specific for that antigen.

Activation    Activation of lymphocytes by binding of antigen

Effector      Lymphocytes that have been specifically activated by
              antigen perform their functions that lead to elimination
              of antigen (such as secretion of antibodies in humoral
              immunity or destruction of cells in cell-mediated

Decline       Elimination of cells from the body.

Memory        Generation of memory cells that provide lasting
              protection from a specific antigen.
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Author:Romich, Janet Amundson
Publication:Understanding Zoonotic Diseases
Geographic Code:1USA
Date:Jan 1, 2008
Previous Article:Chapter 1 Zoonotic disease history.
Next Article:Chapter 3 Bacterial zoonoses.

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