Leszek Guz
Immunogenicity of Aeromonas strains. Experimental vaccines against MAI/MAS
Introduction. Bacteria of the Aeromonas genus are common in freshwater. They can be primary, secondary or opportunistic pathonegens causing diseases in many types of animals, both in invertebrates and vertebrates. Developing an effective and inexpensive vaccine for carp MAI/MAS as an alternative to the generally applied antibiotic therapy is a challenge for research laboratories throughout the world.
The aim of the study. Taking into consideration the frequent occurrence in Poland of this disease and the economic losses it causes, an attempt at describing their immunogenic qualities has been undertaken.
Material and methods. The study was performed on virulent for carp Aeromonasspp. strains. The methods used for preparation of protein and LPS samples, immunobloting, ELISA, gel preparation and staining have been described previously [Caldwell i Pigman 1965, Westphal i Jann 1965, Laemmli 1970, Tsai i Frash 1982, Hitchock i Brown 1983, Waterstrat i in. 1989, Rahman i Kawai 2000]. The gels were documented with Bio-1D++ system (Viber Lourmat). Cluster analysis were performed using the UPGMA or UPGMC method. The vaccine trial was conducted at the Department of Fish Diseases and Biology, Life Sciences University in Lublin. Fish were intraperitoneally immunized 
(100 ml vaccine or 100 ml PBS as control) with experimentally prepared vaccines. To determine serum antibody response after vaccination, fish sera were collected from vaccinated fish at 30 days post-vaccination. Thirty days post-immunization, the fish were challenged via subcutaneous injection with homological or heterological strain. For all vaccinations, the fish were monitored for mortality for 30 days post-challenge, and probability of survival and relative percent of survival were calculated.
Results and discussion. The application of cluster analysis can significantly facilitate the selection of a vaccine strain that would induce a high level of antibodies after the application of a vaccine from oil adjuvants. It was proved that the greater the similarity of the protein patterns used to immunize strains from the Aeromonas genus, the greater the survival rate of the fish in the challenge test. Moreover, a high level of post-vaccine antibodies correlated in a positive manner with the survival rate of the fish in the challenge test with the homological strains and the heterological strains of a similar antigenic profile.
The analysis of the strains demonstrated that the examined strains of the Aeromonas genus possess varying electrophoretic patterns of OMP protein. The applied cluster analysis method for the examination of the electrophoretic patterns of proteins enables the selection of vaccine strains and also facilitates the selection of strains for the preparation of polyvalent vaccines.
The analysis of the results demonstrated that within bacteria of the Aeromonasgenus there is a great structural and heterogenic LPS variety, which influences its immunogenicity and immunomodulatory qualities.
The analysis of the results demonstrated that it is possible to gain a post-vaccine tolerance against carp MAI/MAS disease, evoked by bacteria of the Aeromonasgenus, after applying a vaccine based on whole-cell bacteria. Vaccines containing ECP, OMP and LPS induce a weaker post-vaccine response than vaccines containing whole-cell bacteria. Results suggest that the cross-reactivity of OMPs commonly exists in the studied bacteria, which may be a promising choice for the development of polyvalent OMP vaccines. Cluster analysis will help to understand the relation of cross-antigenic OMPs among the studied bacteria.
The analysis of the results demonstrated that the initial level of antibodies in vaccinated carp influences the post-vaccine level of antibodies. The obtained results can significantly restrict the risk level connected with the foreseeable protective effect on infecting fish that undergo protective vaccination.
Conclusions. The investigated Aeromonas strains represent high antigenic diversity. Vaccines prepared from whole cells of bacteria of the genus Aeromonasinduce a higher level of immunity against the MAI/MAS disease than vaccines prepared from OMP proteins and LPS. The high level of post-vaccination antibodies obtained after vaccination with whole bacterial cells correlates positively with fish survival in a challengetest using a homologous strain and heterologous strains with antigenic profiles similar to those of the strain with which the fish have been immunized. Use of cluster analysis for grouping bacteria on the basis of electrophoretic profiles of immunogenic proteins may considerably facilitate the choice of a vaccine strain and selection of strains for a polyvalent vaccine that would induce a high level of antibodies and give high levels of post-vaccination immunity not only against a homologous strain but also against heterologous strains. The level of "natural" antibodies which occurs in non-vaccinated fish has effect on the level of post-vaccination serum antibodies.