Laboratory tests extend the clinician’s diagnostic skill and guide his therapeutic efforts. Serological tests represent a large percentage of the laboratory tools available to aid the clinician. These tests include agglutination, precipitation and complement-fixation, which, along with other laboratory procedures, are explained in the following pages. Animal tests and experimentation provide a useful diagnostic tool in some diseases. Although not a laboratory test, skin tests made on the patient aid in determining susceptibility or immunity to certain infections. Certain serological tests are of great value and are widely used in the diagnosis of infectious disease and the identification of microorganisms on the basis of their antigenic components.
The most commonly used serological tests are the agglutination, precipitation, and the complement-fixation tests. Some other tests are the opsonocytophagic index, the quelling reaction, tests fro neutralizing antibodies, and animal-protection tests. According to the Unitarian theory a single antigen may, under different conditions, react in several different ways. For example, if a rabbit is immunized with killed or attenuated cultures of the typhoid bacillus, its blood serum will contain antibodies specific for typhoid bacilli. A portion of this serum mixed with a suspension of typhoid cells will demonstrate agglutinins. If we were to take an extract of the bacilli and mix it with the antiserum, we could demonstrate precipitins. Similarly it is possible to demonstrate lysins and possibly other antibodies produced in response to the same antigen. One specific antibody, immunoglobulin, accounts for all these different reactions under varying conditions. Toxins and antitoxins, being soluble substances, cannot be demonstrated in the same way as agglutinins, lysins or opsonins. In addition, it is important to remember that bacterial cells contain many different antigens, each stimulating production of its own specific antibody. In closely related groups of organisms – organisms of the same genera or species – there may be a common antigen but different serological types because of type-specific antigens not shared by the whole group.
AGGLUTINATION TESTS The agglutination reactions relatively easy to perform, is simple to interpret and is the serological method of choice when a suitable cellular antigen can be prepared. When there is a common antigenic component in the cell, thin-absorption between closely related species can be made by the agglutinin-absorption technique. Among the diseases of man for which agglutination test is of diagnostic value are typhoid fever, salmonellosis, brucellosis, tularemia, typhus fever, and Rocky Mountain spotted fever. Agglutination tests are useful in the diagnosis of many animal diseases also, including brucellosis, glanders, swine erysipelas, and pullorum disease of chickens. Not only can the diagnosis of certain infectious diseases be confirmed in the laboratory by agglutination of known antigens by the patient’s serum, but unknown cultures of bacteria, rickettsiae, or other microorganisms can be identified by the capacity of a known antiserum to agglutinate the suspension of unknown cells. The Widal test was devised specifically to aid in diagnosis of typhoid fever by agglutinating typhoid bacilli with patients’ serum, but the term is sometimes loosely applied to other agglutination tests using heat-killed cultures of organisms other than Salmonella typhosa. Another agglutination test used in laboratory diagnosis is the Weil-Felix reaction. This test is based on the fact that several of the rickettsiae have a common antigen with strains of Proteus spp., therefore serum from patients with rickettsial infections agglutinate suspensions of the Proteus organisms. The strains of Proteus used most commonly are Proteus OX19, OX2, and OXK. The Weil-Felix reaction is different for certain rickettsial diseases because of the selective agglutination of these strains. Agglutination tests are classified as microscopic when the test is carried out in a small test tube referred to as agglutination tubes and microscopic when antigen and antiserum are mixed on a slide and examined under a microscope. An agglutination test for the laboratory diagnosis of syphilis is known as the Treponema pallidum agglutination (TPA) test. Living or dead treponemes are mixed with the subject’s serum. If T.pallidum antibodies are present in the serum (indicating infection), the spirochetes will be clumped. This may develop into a very useful rapid and simple test for the serodiagnosis of syphilis. It is presently widely used for the diagnosis of leptospiral infections and for serological typing of Leptospira. MACROSCOPIC SLIDE AGGLUTINATION TEST This is a quick and convenient method for determining the presence of agglutinating antibodies. A drop of a dense suspension of the organisms (antigen) in saline is placed on a clean glass slide. One to three drops of known antiserum are mixed with the antigen by gentle rotation. Positive agglutination, observable with the unaided eye, occurs in 3 to 5 minutes. The test can be made semiquantitative by using exact amounts of antigen and antibody. Graduated amounts of antibody are placed in separate spots on a slide. Is a constant amount of antigen is added to each, a range of dilutions results. Positive and negative controls must be set up on separate slides. Since this is a screening test, doubtful or positive reactions should be confirmed by the macroscopic tube agglutination technique. MICROSCOPIC AGGLUTINATION TEST For the microscopic agglutination test, serial dilutions of serum and physiological salt solution are prepared, one loopful of each dilution is placed on a cover glass, and to each a loopful of antigen, which may be young, living broth culture of bacteria, is added. For the control, one loopful of salt solution (containing no serum) and a loopful of antigen are used. Each cover slip is placed over a concave slide ringed with petrolatum to prevent drying, incubated for 1 hour, and then examined with the high-dry objective of the microscope for clumping. With the homologous antiserum at 1:20 dilution, agglutination may occur in 15 minutes or less at room temperature. This method can be used for rapid identification of unknown cultures of bacteria using known antiserums. SPECIAL AGGLUTINATION TESTS The serum-plate agglutination test is used fro rapid serological diagnosis of brucella infections of cattle and pullorum disease of chickens. It differs from the tube and microscopic techniques in the type of antigen used and in the method of conducting the test. Agglutination test with whole blood are used in pullorum-disease eradication programs and for the diagnosis of swine erysipelas. The test is similar to the rapid serum test except that whole blood is used rather than serum. It can be carried out quickly and simply in the field. The test for pullorum disease is made by mixing a loopful of freshly drawn blood with a drop of the antigen on a glass plate with a white background. Positive serum from infected birds causes clumping within 2 minutes. A positive test with Erysipelothrix insidiosa antigen may indicate either infection or contact with the causative organisms of swine erysipelas. Hence this test should be used to detect the presence of the disease in a herd rather than an individual.
