Historically, veterinarians have had four options for diagnosing most infectious agents. One is to look under a microscope at a patient's blood or tissue sample for the organisms themselves, but this can be a highly insensitive method, says Edward Breitschwerdt, DVM, Dipl. ACVIM, professor of medicine and infectious diseases at North Carolina State University College of Veterinary Medicine and adjunct professor of medicine at Duke University Medical Center. A second option is to culture the organisms, growing them in special media to identify the specific infectious agent. However, most vector-borne organisms have been successfully grown only in specialized research laboratories, Breitschwerdt says.

The third option is an antibody test to indicate whether an animal was exposed to a particular organism (also known as an antibody titer). And the fourth is a polymerase chain reaction (PCR) test that detects an antigen or an infectious agent's DNA, both of which confirm active infection in most instances. The diagnostic advantage of PCR testing, which copies specific sections of an organism's DNA, over serology (antibody titers) is in diagnosing disease in the very early stages of infection, before the development of antibodies. PCR testing is generally considered as sensitive and specific as culture, is significantly faster and, for some organisms, is more reliable.

"Every diagnostic test has its limitations," Breitschwerdt says. "It's as important to know what a test will not tell you as what it will tell you."

The main use of antibody titers in companion-animal medicine is to help diagnose a number of different infectious diseases. These titers are useful for indicating exposure to a bacterial, viral or fungal agent, says Craig Datz, DVM, Dipl. ABVP, assistant teaching professor at the University of Missouri's College of Veterinary Medicine. Some of the common diseases that titers may help diagnose are distemper, ehrlichiosis, anaplasmosis, Rocky Mountain spotted fever, leptospirosis, borreliosis, blastomycosis, histoplasmosis, cryptococcosis, coccidioidomycosis, toxoplasmosis, feline heartworm disease and feline immunodeficiency virus. They also measure protection from infectious diseases, such as after vaccinations, and are sometimes used in healthy animals as screening tests to evaluate possible exposure to an infectious agent.

Veterinarians can run titer results in-house in some cases, but most submit titers to reference laboratories where results can take a few days, Datz says.

Titer interpretation

Interpretation of results is the most difficult and controversial aspect of using titers for diagnosing disease, Datz says. Different infecting organisms produce different antibody responses, and animals do not always develop a disease just because they have been exposed to an infectious agent. Also, a high antibody titer does not prove that disease is present or that treatment is needed.

Technicians obtain an antibody titer by doubling dilutions of the reactive blood (1:4, 1:8, 1:16, 1:32, etc.) until the test becomes nonreactive or negative. The titer that is reported to veterinarians is the last dilution that gave a reactive or positive antibody detection point. Although dilution ranges vary from laboratory to laboratory, the laboratories typically stop the titer at 1:256 and report the result as > 1:256 if the pet's blood is still positive. For example, if the titer was reported as 1:32, it means the serum was diluted serially out, and the 1:32 dilution gave the last positive response for presence of antibody, says Richard A. Hesse, MS, PhD, associate professor and director in the department of Diagnostic Virology at Kansas State University's College of Veterinary Medicine. If the titer is reported as ≤ 1:8, it means that the lab didn't test at a 1:4 or 1:2 dilution.

Sometimes the terms negative and positive are in themselves distracting. "Instead of reporting serology results as a positive or negative test for an antibody, we report serologic results as being reactive or not reactive," Breitschwerdt says. "This means the technician noted reactivity in the serum sent to the lab against the test antigen grown in the lab. This may or may not equate to the organism causing a disease in the patient, the patient being actively infected with that organism at the time of testing or the reactivity being specific for the infectious agent that is being targeted by the test."

The problem with using the term negative in reporting a titer is that the veterinarian's interpretation often is that the dog is not infected with the specific pathogen and, therefore, does not have the disease, Breitschwerdt says. However, with many diseases, the test may not detect a positive serologic response until the animal has been symptomatic for at least a week, so even a negative test, if done early in the course of infection, does not rule out the disease.

Conversely, the problem with using the term positive when reporting a titer is that veterinarians often interpret that positive report as the animal having the disease. "In reality, over half of the flea-infested cats in the Southeast United States have antibodies to Bartonella henselae, but many are not infected with the organism," Breitschwerdt says. As another example, dogs that are seroreactive to Ehrlichia canis antigens have been exposed, but in many cases their immune systems have cleared the infection.

Another challenge is that antibody titers often increase or decrease over time, and taking two or more samples at different time points may be necessary for diagnosis. A good practice is to draw serum samples when a sick animal is first presented, Datz says. Samples can be saved in a refrigerator for a few days or in a freezer for longer. If the veterinarian later suspects a specific infection, he or she can submit the stored sample.

For some diseases, the veterinarian should obtain titers on acute and convalescent samples. If the veterinarian stored the acute serum sample, he or she should submit it at the same time as the convalescent sample, Datz says. This reduces the variability in testing results that can occur when samples are run at different times. With these titers, a disease can be diagnosed based on comparing the results, looking for a large increase in antibodies as the disease progressed.