Serological diagnosis

Serological testing is generally less expensive and easier to perform than the isolation or detection of an infectious agent, particularly virus. Clinicians often request serological screening as the first step in a disease investigation. Serology testing only requires clotted blood samples which are relatively easy to collect from large numbers of animals. Many of the classic serology techniques have been employed for decades and have a proven track record in the field. They are very reliable and the results are accepted internationally. Traditional serological diagnosis of infection is based upon identification of specific antibody. When an animal encounters an infectious agent or foreign substance for the first time, antibody to that agent or substance is usually (but not always) detectable in the serum within 10–14 days. The time required for antibody production is influenced by the nature, amount and route of exposure to the agent, the age and immune status of the animal and the sensitivity of the assay used to demonstrate circulating antibody. For some infectious organisms the production of detectable antibody may take considerably longer than 14 days, for example, antibodies to equine infectious anaemia virus may not be detected for more than 60 days post exposure. The first antibody response to antigen is referred to as the primary response. The interval between exposure and production of antibody is referred to as the latent period (or lag period). If exposure to the same antigen occurs again some time later, the animal responds with a stronger and more sustained response and the latent period is shorter due to the presence of memory B cells. This is termed the secondary response (Fig. 3.1). The memory response of an animal for an agent it has previously been exposed to is called the anamnestic response.

Figure 3.1 A comparison of specific antibody production during primary and secondary responses to infectious agents or to vaccination. Natural infection usually results in higher levels of antibody for a longer time period.

When blood sampling animals for serological testing for infectious diseases, two samples may be required to demonstrate an increasing level of circulating antibody. A fourfold or greater increase is considered significant in many serological tests and is known as seroconversion. This is indicative of recent exposure to the agent. The testing of paired samples collected 10 to 14 days apart frequently leads to a retrospective diagnosis which is of limited value in the management of acute illness. The presence of serum IgM antibody is generally accepted as evidence of a current or very recent infection but the detection of specific viral IgM antibody is rarely feasible for routine diagnosis in veterinary laboratories. However, an IgM ELISA is used extensively for the diagnosis of West Nile fever (WNF) in horses.

Serological examination of a single sample may be performed to determine if an animal has previously been exposed to an agent, to assist in the identification of a carrier or to evaluate the animal’s immune status. However, the presence of antibody does not always correlate with protective immunity. Antibody-based immunity is the major defence system against extracellular bacteria and viruses as well as against bacterial exotoxins but cell-mediated immunity plays a more prominent role in host resistance to intracellular pathogens, for example, herpesviruses and Salmonella spp.

The majority of serological assays do not differentiate between exposure by natural infection and exposure by vaccination. Some serological assays do not allow the unequivocal differentiation of antigenically related organisms. Such assays may be used for screening purposes and the laboratory can revert to a type-specific assay when a positive result is obtained. In recent times the incorporation of highly purified recombinant antigens or synthetic peptides in some assays has improved their specificity and aided standardization of interpretation. However, in some instances this has led to a decrease in sensitivity. If a specific serological assay is not available then agent detection or isolation is a prerequisite for identification.

Serological assays depend on the amount of antibody present, the binding of this antibody to the antigen in the test and the detection of the antigen–antibody complexes. These complexes can be detected by a biological effect, for example, neutralization or a physical effect such as precipitation. Alternative detection systems use immunoglobulins labelled with a chromagen (immunohistochemistry, ELISA), a radioisotope (radioimmunoassay) or a fluorochrome (immunofluorescence). It is usual to dilute the serum of the test animal to determine the concentration of specific antibody present and this is expressed as the titre of that serum. Titre may be defined as the highest dilution of a serum which gives a demonstrable reaction in a defined test procedure.

International reference sera have been developed for many serological tests. Such sera are made available to national reference laboratories for the purpose of establishing national standards. Diagnostic laboratories should whenever possible use these national standards or prepare working standards against them for incorporation in the routine tests. Similarly, the use of reference strains for diagnostic antigen production promotes international harmonization of diagnostic testing.

Many of the immunological techniques described below can be used to detect antibody or antigen (see also Chapter 5).

Precipitation

When soluble antigen is added to homologous antibody at the correct ratio insoluble antigen–antibody complexes are formed. If the immune complexes are of sufficient size they precipitate out of solution. This form of immunological precipitation is termed the precipitin reaction. When diluted antigen is layered carefully over homologous antibody a band of precipitate forms where the ratio of the reagents is at optimal proportions. This method is termed the ring precipitin test (Fig. 3.2) and has found application in identifying streptococci and assigning them to their appropriate Lancefield groups. A limitation of this method is the requirement for high levels of antibody in the antiserum employed.

Figure 3.2 Ring precipitin test: negative (left) and positive (right).

Precipitation reactions can take place in semisolid media such as agar gels. When soluble antigen and antibodies are placed in wells cut in a gel, they diffuse towards each other and where they meet at or near optimal proportions, a precipitin line forms in the gel. Immunodiffusion in agar (Fig. 3.3) is applied in diagnostic tests for infectious diseases particularly in virology and the Coggins test for equine infectious anaemia is based on this method. The agar gel immunodiffusion test for caprine arthritis/encephalitis and for maedi-visna or ovine progressive pneumonia is a cost-effective method of identifying persistently infected carriers and is the prescribed test for international trade. It is also the traditional test for the detection of antibody to virus-infection-associated antigen (VIAA) of foot-and-mouth disease (FMD) virus and has been used extensively in FMD eradication in South America.