blotting is a method of antigen or antibody detection primarily used for complex proteins including microorganisms and parasites. With this technique, protein antigens are separated via electrophoresis and immobilized on a nitrocellulose membrane. The antigens are then visualized through the use of an enzyme or radioimmunoassay (Tizard 2013). Western blotting is commonly used for the detection of antibodies against FIV, Borrelia spp., Ehrlichia spp., Leishmania, and Neorickettsia spp.
Immunohistochemistry can be utilized to identify specific antigens within histopathological tissue specimens. Prepared tissue sections are treated with an enzyme‐labeled antibody that binds to the antigen of interest. When exposed to an enzyme substrate, the labeled complexes produce a distinct brown color which can be identified by the pathologist. Immunohistochemistry has a wide variety of applications but is most commonly used for the diagnosis of viral diseases such as feline infectious peritonitis, canine distemper virus, and canine parvovirus and is frequently performed on postmortem tissue samples.
4.3.4.1.2 Secondary Binding Tests
Serum antibody levels are typically quantified and reported as “titers.” In practice, test serum is diluted in a series of tubes and a known amount of antigen is added to each dilution. The most diluted sample in which a reaction occurs provides an estimate of the amount of antibody within the serum sample. The reciprocal of this dilution is reported as the titer (e.g. a sample in which the 1:32 dilution of serum was the highest sample dilution to display a reaction, has a titer of 32) (Figure 4.1). There are several caveats to the use and interpretation of antibody titers that should be understood:
A titer from one laboratory may not be the equivalent of a titer at another laboratory.
A titer determined by one method may not be the equivalent of a titer determined by another method.Figure 4.1 Antibody titration.
What constitutes a “protective” titer differs for various pathogens and may vary between laboratories.
The method in which a titer was determined to be “protective” may vary between laboratories.
Lack of a “protective” titer may not indicate susceptibility.
In most cases, a single titer measurement cannot distinguish between previous exposure and current infection.
Titer detection alone cannot establish the cause of existing disease.
In puppies and kittens, titers cannot distinguish between maternal antibodies and those derived from vaccination or previous exposure.
In‐house ELISA test kits can be used to measure antibody titers against canine distemper, canine adenovirus, canine parvovirus, and feline panleukopenia (see the discussion in the section on primary diagnostic testing) (Gray et al. 2012; Mazar et al. 2009; Mende et al. 2014); however, laboratory testing methods are considered the gold standard. Practitioners should be aware of the particular method utilized when interpreting clinical results, making case management decisions, and comparing literature references.
Agglutination, or clumping, of red blood cells in a serum sample occurs when antibodies cross‐link large particles of antigen (i.e. one antibody binds to two antigens). In the presence of excess antibody, the antigenic particles become saturated and further agglutination is inhibited. This reaction is the basis for antibody titration through HI. In these tests, a serial dilution of the test serum is exposed to soluble antigen (e.g. viral particles) cross‐linked to red blood cells. Antibodies within the test serum bind to the soluble antigen causing agglutination of the cross‐linked red blood cells—hemagglutination—until the antibodies are exhausted and further hemagglutination is inhibited. The reciprocal of the highest serum dilution that completely inhibited hemagglutination represents the HI titer for that sample. In addition to its use for measuring antibodies, HI can also be used to identify specific viruses (Tizard 2013). HI is commonly employed to measure antibodies against adenoviruses, coronaviruses, herpesviruses, influenza, and parainfluenza. HI is considered the gold standard titer testing method for canine and feline parvoviruses (Ford 2013).
CF testing relies on the classical complement pathway resulting in the binding of antibodies to red blood cells and their subsequent rupture and hemolysis. After the disruption of existing antigen–antibody complexes, test serum is mixed with a known amount of a new complement source, allowed to form new antigen–antibody complexes, and an indicator in the form of antibody‐linked red blood cells is added. If antibody is present in the sample, the complement is consumed and the red blood cells remain intact. In the absence of antibody, the complement binds to the antibody‐linked red blood cells and is lysed. Conducting the reaction within serial dilutions of test serum allows quantification of antibodies. The reciprocal of the highest dilution of serum in which no more than 50% of the red blood cells are lysed represents the CF titer for that sample (Tizard 2013). CF is commonly employed to measure antibodies against adenoviruses, parvoviruses, mycobacterial infections, and coccidioidomycosis.
4.3.4.1.3 Tertiary Tests
Serum neutralization testing or, when in reference to viruses, virus neutralization (VN) is used to estimate the ability of antibodies within a test sample to disrupt (i.e. neutralize) the biological activity of an antigen. Such activity can include hemolysis of red blood cells, lysis of nucleated cells, and disease or death in animals. In these tests, serial dilution of the test serum is exposed to a constant amount of antigen (i.e. live virus). Antibodies within the test serum bind to the antigen, blocking critical attachment sites thus preventing them from infecting live cells. The reciprocal of the highest serum dilution that prevents lysis of 50% of cells, infection in 50% of tissue cultures or infection in 50% of live animals represents the VN titer for that sample. In addition to its use for measuring antibodies, VN can also be used to identify specific viruses (Tizard 2013). VN typically has higher sensitivity compared to secondary binding tests such as HI and CF and is commonly employed to measure antibodies against adenoviruses, caliciviruses, herpesviruses, and parvoviruses. VN is considered the gold standard titer testing method for canine distemper virus (Ford 2013).
4.3.4.1.4 Molecular Assays
The detection of the nucleic acid of an infectious agent can be accomplished through the use of molecular assays such as PCR, reverse‐transcriptase PCR (RT‐PCR), and real‐time PCR. The ability of such assays to detect minute quantities of nucleic acid makes them among the most sensitive diagnostic testing methods available (Tizard 2013). Another important benefit of molecular assays is their ability to detect organisms in animals that are subclinical carriers or latently infected (Evermann et al. 2012), allowing for quicker identification of animals that may present an infectious disease risk to the population. This benefit may be particularly useful during the response to a disease outbreak when quantitative results are available to distinguish between vaccinates and infectious animals. However, as with antibody titer analysis and other methods of antigen detection discussed above, molecular detection of an organism merely indicates its presence. It does not indicate whether the detected organism is alive or dead or whether it is the cause of active disease. For these reasons, interpretation of results should take into account clinical signs and the possibility of latent stages of infection (e.g. herpesviruses, feline retroviruses). The high sensitivity of these assays makes careful sample collection and handling, as well as submission to a reliable laboratory with high‐quality control standards, of the utmost priority as sample contaminants will readily be detected. Molecular methods can be used to detect pathogens in samples of blood and tissue or, increasingly, in culturettes or swabs containing trace amounts of respiratory secretions or feces.
Molecular assays rely on the PCR to detect trace amounts of pathogen DNA. For RNA viruses (e.g. feline calicivirus, canine distemper virus), RT‐PCR may be performed. In this