Abstract

Specific immune responses are divided into cell-mediated immunity and humoral immunity. Humoral immunity is mediated through soluble proteins (antibodies) found in body fluids. Serology is the measurement of antigen-antibody interactions for diagnostic purposes.³ The practical application of serologic methods for evaluation of vaccine responses in zoo animals is the focus of this presentation.

Overview of Serologic Tests

Serologic tests can be used to detect antigen or antibody. The specific methodology determines whether the result reflects the presence of an antigen or the immune response to that antigen.³ Results are usually expressed as “titers” using an endpoint dilution of test material or as “positive” or “negative” at a specified dilution. However, a titer is not a titer—in other words, knowledge of the methodology used in a specific test to measure a titer is important in being able to correctly interpret the results.⁵ Another confounding factor is that humoral immunity is only one aspect of a complete immune response. In some infections, cell-mediated immunity plays a more important role, so measuring the humoral response might not reflect the level of protective immunity.^5,6 In other cases, antibodies to particular parts of an organism may be present but might not play a role in elimination of the infection. Even when measuring antibodies to a single organism, different test methods can result in variable titers or only detect specific types of immunoglobulins (e.g., IgM vs. IgG).³ A particular problem working with exotic animals is the lack of validation of most serologic tests for these species. In most cases, “protective” titers are unknown.

Despite all these difficulties, serologic tests can be useful in assessing response to vaccination or exposure to an antigen. Current discussions in veterinary medicine have focused on developing strategic vaccination programs based on risk assessment and duration of immunity.^2,4,6 Use of serologic tests to individually tailor vaccination programs in individual animals have been proposed. Advantages include cost savings in vaccine, avoiding the potential for adverse reactions, and scheduling vaccinations to maintain effective levels of immunity while decreasing the potential stimulation of autoimmune disease. Disadvantages include increased cost associated with serologic testing, multiple veterinary visits, inability to obtain samples at regular intervals (e.g., in many zoo species), lack of validation for species, and difficulty determining “effective” immunity. Since this presentation will focus on application of serologic testing to vaccination, some commonly used assays that detect antibody will be reviewed.

Immunofluorescence Assays

Indirect fluorescent antibody test⁵ (IFA) is used to detect antibodies. Antigen produced in the laboratory specifically for this test can be in the form of a tissue smear, section, or cell culture on a slide. The patient’s serum is incubated with the slide, washed, and immune complexes are visualized using a fluorescent-labelled antiglobulin. The quantity of antibody in the test serum can be estimated by using increasing dilutions of serum to detect the endpoint. The technique used in radioimmunoassay (RIA) is similar but uses radiolabels to detect complexes.

Immunoenzyme Assays

Indirect enzyme-linked immunosorbent assay³ (ELISA) is one of the most common serologic methods used in veterinary medicine. The indirect ELISA technique utilizes an antigen bound to a well or tube, to which the patient’s serum is added at varying dilutions (or a single dilution in the case of positive/negative test methodology commonly employed in in-clinic kits). The presence of bound antibody from the patient’s serum is detected after adding an enzyme-labeled antiglobulin, followed by enzyme substrate. The amount of color change is proportional to the amount of bound serum antibody. Results can be visualized as positive/negative color change or can be quantified using a spectrophotometer.

Western Blot

This technique is often used when dealing with a complex mixture of protein antigens.⁵ Antigens are separated using electrophoresis and then transferred to a nitrocellulose membrane. The membrane is incubated in the patient’s serum with antibodies binding to specific protein bands. Detection of the antigen-antibody complexes are detected using enzyme-labeled antiglobulin (or radioisotope-labeled antiglobulin). A dot blot is a variation of the Western blot technique.

Precipitation

These tests require a solution of soluble antigen mixed with antiserum.³ When the ratio of antibody to antigen is in optimal proportions, a visible precipitate is formed. Results are often read visually as positive or negative at a specific dilution.

Agar Gel Immunodiffusion (AGID)

Instead of using tubes to detect precipitation of antibody-antigen complexes, this technique uses diffusion of soluble antigen and antibody from separate areas of an agar plate.⁵ An opaque line of precipitate appears where the reagents meet in optimal proportions. In the case of complex antigens, multiple lines of precipitation may be visible since the optimal proportions of each component occur at different positions. The Coggin’s test used to detect antibodies to equine infectious anemia virus uses this method.

Agglutination Assays

Because antibodies have multiple binding sites, they are capable of cross-linking particulate antigens, resulting in clumping or agglutination.³ IgM antibodies are more efficient than IgG. Similar to precipitation, there is an optimal zone of antibody and antigen concentrations that result in a visible reaction. Agglutination reactions are more sensitive than precipitation techniques. Passive agglutination tests are performed by chemically linking soluble antigens to red blood cells or latex beads.

Viral Hemagglutination (HA) and Hemaglutination Inhibition (HI)

Certain viruses can bind and agglutinate red blood cells and thus serve as a basis for this assay.^3,5 This reaction can be blocked or inhibited by the presence of antibody that binds the virus. The higher the dilution of serum that can inhibit hemagglutination, the higher the titer of virus-specific antibody.

Complement Fixation Test

Test serum is incubated with a source of antigen and complement. If antibodies are present to the antigen, binding will occur and activate the complement cascade, depleting the amount of free complement.³ In order to detect free complement, an indicator system consisting of antibody-coated sheep erythrocytes is added in the second step. If complement is present, the cells are lysed and the titer is considered negative. If antibodies are present, the titer is considered the highest dilution of serum in which no more than 50% of the erythrocytes are lysed.

Interpreting Titers

Some considerations to keep in mind when interpreting titers:^4-6

• A positive titer does not necessarily mean the animal is actively infected or that the disease is due to this etiologic agent.
• A negative titer does not necessarily indicate that the disease is not present.
• Titers do not always correlate with the presence or absence of a protective immune response in the host.
• Titers to a particular etiologic agent may vary in a single serum sample due to differences in specificity and sensitivity of the test method used.

Often serologic tests are performed early in the course of disease or too soon after exposure and the negative titer is incorrectly interpreted to rule-out the suspected etiologic agent. Ideally, paired serum samples (acute and convalescent) should be compared to determine if there is a rising titer. A positive result is usually defined as a fourfold or greater increase in titer.⁵ Unfortunately, sometimes only a single sample is available. Extrapolation from other animals or previous serologic results may be used as a basis for comparison with careful consideration of the cautions stated above and in light of the clinical situation. The following examples from zoological practice will be used to illustrate the concepts of serologic test interpretation.

Example 1: First Trimester Abortion in a Southern White Rhinoceros (Ceratotherium simum)

A 30-year-old female southern white rhinoceros was examined after detection of an aborted fetus (estimated first trimester). Diagnostic tests for equine herpesvirus (EHV-1) and infectious bovine rhinotracheitis (IBR) virus were performed using the serum neutralization (SN) technique. This assay measures the ability of specific antibodies to neutralize the cytopathic effects of virus in tissue culture. SN may not distinguish between infection and vaccination; therefore, diagnosis should be based on rising titers, as well as other diagnostic results. Hemagglutination inhibition (HI) was performed to detect antibodies to equine influenza virus. Brucella titers were measured using an agglutination test method. Results are shown in Table 1.

Table 1. Serologic results in a southern white rhinoceros

^aEquine influenza virus.

A review of the medical record revealed that she had been vaccinated annually with a commercial equine vaccine (Fluvac EWT; Ft. Dodge Laboratories Inc. Fort Dodge, IA 50501 USA) containing Eastern and Western equine encephalitis virus, tetanus toxoid and equine influenza virus. The positive titer to equine influenza could be attributable to prior vaccination. However, the positive titer to EHV-1 was not due to previous vaccination and should be considered in the differential list for abortion in perissodactylid species. Additional steps would include a convalescent titer (approximately 4 weeks later) and examination of the fetus for evidence of viral infection. If results indicate a possible link, an equine vaccine that includes EHV-1 should be considered for the breeding herd.

Example 2. Rabies Vaccination in Hybrid Tigers (Panthera tigris)

Potential exposure to rabies virus warrants vaccination of susceptible species. However, vaccine-associated sarcomas have occurred in domestic cats.² The benefits of monitoring the response of vaccination by serologic testing, instead of repeat vaccination, will decrease the risk of adverse effects and may result in the decreased need for further immunization. Unfortunately, there is not an established protective titer for most species, and challenge studies in exotic species are unlikely to be done. A threshold titer needs to be designated below which booster vaccination is recommended.

Rabies titers in six female tigers are shown in Table 2. Titers are determined by the rapid fluorescent focus inhibition test (RFFIT). In humans, a titer greater than 1:5 is considered a protective titer.¹ During quarantine, these tigers were bled and then vaccinated with a commercially available veterinary product (Imrab 3, Merial Inc., Athens, GA 30601 USA, 2 ml IM). Vaccination was repeated in 1999 and 2000. The results demonstrate the significant individual variation in immune response as well as the ability of this product to stimulate an antibody response in this species. Rabies titers are currently monitored so that booster vaccines can be individually scheduled. An arbitrary threshold titer of 1:50 is the cutoff value when vaccination would be scheduled. Information on larger numbers of animals is needed to develop scientifically based immunization programs for exotic species.

Table 2. Rabies titers in six hybrid tigers

Example 3. Eastern Equine Encephalitis (EEE) Virus Vaccination in Secretary Birds (Sagittarius serpentarius)

Eastern equine encephalitis virus vaccinations in secretary birds are shown in Table 3. An adult secretary bird was found dead without any premonitory signs. A second bird died acutely approximately 1 week later. Necropsy findings were consistent with viral encephalitis and EEE infection was confirmed on virus isolation. In an attempt to prevent disease in the remaining two birds, a commercially available killed EEE vaccine (Encephaloid, Ft. Dodge Laboratories Inc., 0.5 ml IM) was administered. Titers were measured using hemagglutination inhibition (HI). A greater than fourfold increase in titer to EEE suggests that the vaccine induced an antibody response in these birds (titers to flavivirus, which includes West Nile virus and St. Louis encephalitis virus, were consistently <1:10). Due to waning antibody at 6 months, the dose of vaccine was increased to 1.0 ml IM. Quarterly titers are being measured to determine duration of titers using this dose. Although antibodies were detected, without challenge studies (either experimentally or through natural exposure), it should not be assumed that this equates with protective immunity.

Table 3. Eastern equine encephalitis (EBE) titers in secretary birds

^aVaccinated intramuscularly with 0.5 ml EEE vaccine.
^bVaccinated intramuscularly with 1.0 ml EEE vaccine.

Summary

Technologic advances and increased knowledge of the immune system have enhanced our ability to detect and measure responses to natural exposure, infection, and vaccination. Although the development and validation of serologic tests for exotic species is limited, rational application of currently available techniques can be used to aid in diagnosis and disease prevention. Serologic monitoring should be used to develop scientifically based vaccination programs for zoo animals.

Literature Cited

1.  Human rabies prevention—United States, 1999 recommendations of the advisory committee on immunization practices (ACIP). 1999. MMWR 48 (RR-1): 1–21.

2.  Meyer, E.K. 2001. Vaccine-associated adverse events. In: Ford, R.B. (ed.). Veterinary Clinics of North America Small Animal Practice, Vol. 31 (3). W.B. Saunders Co., Philadelphia, Pennsylvania. Pp. 493–514.

3.  Tizard, I.R. 1996. Veterinary Immunology, 5th ed. W.B. Saunders Co., Philadelphia, Pennsylvania.

4.  Tizard, I., and Y. Ni. 1998. Use of serologic testing to assess immune status of companion animals. J. Am. Vet. Med. Assoc. 213 (1): 54–60.

5.  Young, K.M., and D.P. Lunn. 2000. Immunodiagnostic testing in horses. In: Lunn, D.P., and D.W. Horohov (eds.). Veterinary Clinics of North America—Equine Practice, Vol. 16 (1). W.B. Saunders Co., Philadelphia, Pennsylvania. Pp.79–103.

6.  2000 Report of the American Association of Feline Practitioners and Academy of Feline Medicine Advisory Panel on Feline Vaccines. 2001. Feline vaccine liability and management. Compend. Cont. Educ. 23 (1): 116–126.