Interpreting Diagnostic Serology

Diagnostics based on serological analysis have long been a complex task for veterinarians and diagnosticians alike. Serologic testing is frequently chosen, owing to the ease of blood sample collection and handling when compared to the alternative of isolation or identification of an etiological disease agent. Pathogen identification generally requires specimens of whole blood or swabs, which have exceedingly more stringent sample handling requirements than serum samples and sometimes require lengthy culturing and/or expensive molecular procedures. Thus, clinicians and laboratory personnel are often asked to make what can be tenuous interpretations of serological assay results.

One notion must be recognized above all others when discussing serologic reports: a result on one serum sample drawn on a particular day is not necessarily indicative of infection or disease. Most serological tests do not discriminate between recent exposure, past exposure, or vaccination.

Serological testing on a single sample can be helpful in regulatory and surveillance efforts for a particular agent in a naive population where risk of exposure to that agent is considered absent or extremely low. In such instances, a serologic assay should, theoretically, test negative for antibody for most, if not all, of the population. Additionally, vaccination against these agents must be essentially nonexistent due to the inability of most serology tests to discriminate between vaccination and natural exposure. Serological testing for exposure to vesicular stomatitis virus (VSV) fits into this category because the majority of horses in the United States are immunologically naive with respect to the virus and because vaccination is not performed. Therefore, a positive serologic test for VSV only indicates exposure, be it past or present.

Equine herpesvirus types 1 and 4 (EHV-1, EHV-4) serology is an example of a much more complex situation. When infectious agents are intimately related antigenically, as are EHV-1 and EHV-4, serologic tests are often unable to discriminate between antibodies developed against either virus. Confounding the situation, EHV-1 and EHV-4 are ubiquitous viruses in the United States, and exposure to or vaccination against either or both viruses is essentially a certainty. These circumstances foster a situation in which virtually all horses have been exposed to the viruses, have mounted immune responses, and are, therefore, antibody positive. Such conditions make it virtually impossible to consider a positive serologic test on a single serum sample as diagnostic for disease, regardless of the antibody titer. Additionally, since conventional serologic tests used in diagnostic laboratories have a limited capacity in differentiating between antibodies developed against EHV-1 and EHV-4, it is extremely difficult to assert whether the test result is specific for the virus in question.

Finally, regardless of the disease agent in question, serological testing of acute and convalescent serum samples is crucial in identifying a recent exposure. When clinical signs are first observed, it is imperative that a serum sample be drawn immediately and followed by a second sample two to three weeks later. A significant difference in the antibody titer of these two samples in a side-by-side analysis must be observed to indicate an acute exposure. Diagnostic laboratory personnel can offer information in determining whether any difference observed on the serologic tests is significant. Each testing method has specific limits of specificity and sensitivity; these affect the level at which a difference may be interpreted as being significant. A two-fold difference in titer as observed on a virus neutralization test, for example, is not considered significant due to the limited sensitivity of the test. Furthermore, with each testing method having unique specificities and sensitivities, it is virtually impossible to compare antibody titers between the methods. For example, an equine arteritis virus (EAV) neutralizing titer of 1:4 is considered significant for exposure to the virus. In comparison, a West Nile virus (WNV) IgM capture enzyme-linked immunosorbent assay (ELISA) positive at a 1:400 dilution is considered significant for exposure to WNV.

An exception to the rule of testing both acute and convalescent sera would be the use of IgM capture ELISA for detecting a subclass of antibodies developed during an initial exposure to an antigen in a single serum specimen. Such tests may be used to identify recent exposure to an infectious agent and are extremely helpful for clinicians in making diagnoses when accompanied with clinical signs of disease. Therefore, providing case and vaccination histories is extremely important when submitting any sample for diagnostic testing, given that proper interpretation of any serologic result requires these key factors be considered.

The development and use of more antigen specific as well as antibody class and subclass specific tests is critical for the advancement of serological diagnostics. These developments will enhance not only equine veterinary medicine, but the equine industry as a whole.

This is an excerpt from Equine Disease Quarterly, funded by underwriters at Lloyd's, London, brokers, and their Kentucky agents.

Contact: Dr. Morgan H. McCoy, 859/253-0571,, Livestock Disease Diagnostic Center, University of Kentucky, Lexington, Kentucky

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Equine Disease Quarterly

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