Agglutination reactions are among the most easily performed of immunological tests. Their usefulness in presumptive identification of bacteria has long been recognized and for bacterial fish pathogens has been reconfirmed and docu-mented. With as few as a dozen antisera, it is possible to confirm the identity of the majority of known bacterial fish pathogens. Agglutination has provided valuable information on the serological relation of bacterial fish pathogens, including species within genera and strains of the same species.
In this reaction, the antigen is particulate (e.g., bacteria and red blood cells) or is an inert particle (latex beads) coated with an antigen. Because it is divalent or multivalent, the antibody cross-links the antigenically multivalent particles and forms a latticework, and clumping (agglutination) can be seen (Figure 10-1). The procedure is used both to demonstrate the presence of antibodies in serum and to identify antigens on microbial cell surfaces. The principle is the same in both applications. If the serum contains antibodies against a surface antigen, they will agglutinate the bacterial cells. Using a variety of typing sera,
bacteria can be identified and classified. If a constant amount of a bacterial cell suspension is mixed with graded volumes of dilutions of homologous anti-serum, one obtains a measurement of the concentration of antibodies in the serum. The term used to describe the concentration of antibody in serum is “titer”, which is the reciprocal of the highest dilution producing a definite reac-tion.
For use in agglutination tests, bacteria harvested from broth or agar medium may be resuspended adequately. Sometimes bacteria have a tendency to aggre-gate spontaneously, or “autoagglutinate”, making their use in agglutination tests unfeasible. If spontaneous agglutination is observed with a bacterial sus-pension to be used as a test organism, there are several modifications that can be used to eliminate this reaction. If the preparation was formalinized before removal from the growth medium, sometimes washing the bacteria in neutral buffered saline before formalinization reduces non-specific agglutination. If agglutination still occurs, resuspension of the organisms in a 0.1% protein so-lution such as non-fat dry milk to block sites of adherence may be a remedy. Further autoagglutination may necessitate heating of bacterial suspension by immersion in a boiling water bath for 1 to 10 minutes. If none of these proce-dures eliminate the autoagglutination problem, the particular organism may not be amenable to this procedure.
Agglutination test has been widely used in detecting bacterial fish pathogens belonging to the genera Vibrio, Pasteurella, Aeromonas,Yersinia, Edwardsiella and Pseudomonas (Toranzo et al., 1987).