المناعة والامصال
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.
Immunization
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.
Passive Immunity
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.