Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

VI. Laboratory Diagnosis of Diseases with an Immunological Pathogenesis
169
K . Interpretation of Immune Responses to
Pathogens
Diagnosis of infection with viral, bacterial, or parasitic pathogens
can be determined by isolation of the agent, identification
of pathogen nucleic acid using PCR methods, or by
examination of the immune response to the suspected pathogen.
The assays performed to detect an immune response
to pathogens utilize the immunological principles described
previously in this chapter. When immunological methods
are used, it is most often necessary to obtain paired serum
samples. These are acute and convalescent; samples are
taken when the animal is first presented to the veterinarian
and again in 2 to 3 weeks. Performance of the assay on both
samples at the same time eliminates much variation from
day-to-day fluctuations. Generally an increase in titer that
is greater than two dilutions is evidence for induction of an
immune response and hence (in the absence of recent vaccination)
infection. When the initial sample is negative for
antibodies to the pathogen and then becomes strongly positive,
the patient is said to have undergone seroconversion.
Evaluation of the immune response to vaccination is
currently of particular interest because immunologists are
advising against routine vaccination annually. Yet it is
important that immunity to diseases for which vaccination is
performed is present. Thus, monitoring the titer of the antibody
response has become more common (Schultz, 2006).
When evaluating the immune response to virus, the ELISA
is often used and so is the serum neutralization test. These
assays evaluate different aspects of the immune response.
The ELISA is a primary binding assay, which evaluates
antibody levels without regard to function. The serum neutralization
assay measures functional antibody and is better
suited to determine protective levels of antibody to viral
pathogens. Thus, for determination of protective antibody
in response to vaccination, use of the serum neutralization
(SVN) assay is most likely to equate to protection.
Many reference laboratories now have data that allow for
a prediction for immune status based on serological titers.
Determination of antibody levels against Sarcocystic
neurona is done using a combination of Western blot on
serum and cerebrospinal fluid and indirect immunofluorescence
with patient serum for diagnosis of equine protozoal
myelopathy. For this disease, the presence of antibodies
against S. neurona in the serum indicates exposure but not
necessarily infection. Evaluation of the cerebrospinal fluid
for the presence of antibody is preferable to detect infection.
VI . LABORATORY DIAGNOSIS OF
DISEASES WITH AN IMMUNOLOGICAL
PATHOGENESIS
A . Autoimmune Diseases
The use of the diagnostic clinical immunology tools is generally
focused on the diagnosis or prevention of infectious
disease, diagnosis of immune deficiency, detection of autoimmune
responses, and diagnosis of hypersensitivity conditions.
A variety of immune tests are used to help establish a
diagnosis of autoimmune disease. Several examples follow.
Autoimmune hemolytic anemia is characterized by the
presence of a Coombs’ positive anemia. The Coombs’ test
evaluates the presence of autoantibody bound to patient
erythrocytes by provision of a cross-linking antibody to
facilitate agglutination of the antibody-bearing cells. This
assay allows for the detection of so-called incomplete antibody.
In the indirect Coombs’ test, the patient’s serum is
examined for the presence of antibody that will bind to
erythrocytes and agglutinate them when a secondary antiglobulin
is added. Both tests can be employed to evaluate
immune-mediated anemia.
The often-related disease autoimmune thrombocytopenia
(AITP) can be tested for by staining a bone marrow biopsy
with fluorescein-conjugated antisera specific for immunoglobulin
(from the species of interest). The stained section is
then observed for fluorescence of megakaryocytes, indicating
the presence of antimegakaryocyte antibodies.
The systemic autoimmune disease systemic lupus erythematosus
(SLE) requires a positive antinuclear antibody test
diagnosis. Although the ANA test is sensitive, it is not specific.
Nevertheless, antinuclear antibody titers of greater than
50 are present in SLE . Sometimes a lupus erythematosus
(LE) cell may be observed; these are neutrophils that have
engulfed opsonized nuclei. Such an observation is essentially
pathognomic for SLE. In human medicine, a variety of
other measurements for the presence of antinuclear antibodies
are made. Some of these have been occasionally reported
in domestic animal species (Gershwin, 2005; Smee, 2007).
Development of autoantibodies against components of
the thyroid gland is associated with hypothyroidism caused
by autoimmune thyroiditis, although sensitized T lymphocytes
are also important in pathogenesis of the disease.
Recognition of the antithyroid antibodies using indirect
immunofluorescence with serum from the patient and normal
thyroid tissue has been used to demonstrate the presence
and titer of these antibodies. Similar assays can be
performed on pancreas to detect the presence of anti-islet
cell antibodies in patients with type I diabetes mellitus.
One form of the neuromuscular disease myasthenia gravis
develops as a result of immune destruction of the acetylcholine
receptor at the neuromuscular junction. Detection
of autoantibodies binding these receptors can be accomplished
using patient sera in indirect immunofluorescence
or immunohistochemistry.
B . Primary Immune Deficiency Diseases
Primary immune deficiency diseases are caused by a
genetic defect that creates a malfunction or absence of a
component or components of the immune system. Several
such diseases were discussed previously in this chapter.
Clinical signs of the disease generally in the young animal