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In vitro chemiluminescence studies of marine bivalve defence mechanisms and responses against specific pathogens
$102
The Scientific & Social Program: Vth ISDCI Congress
J8 I N V I T R O CHEMILUMINESCENCE STUDIES OF MARINE BIVALVE DEFENCE MECHANISMS AND RESPONSES AGAINST SPECIFIC PATHOGENS. E. Bachere, .V Boulo, P. Godin, L. Goggin, D. Hervio, G. Le Gall, A. Morvan and E. Mialhe. IFREMER, Unit~ Pathol. Immunol. G~n~t. Mol., BP 133, 17390 La Tremblade, France. Because of the impact of infectious diseases in marine mollusc aquaculture, researches have been progressively developed in the field of immunology, specially in an attempt to define the defence response related to the variable infection course, naturally or experimentally, observed among animals. In order to analyse this individual response at the cellular and molecular levels, emphasis was on the development of in vitro experiments. Thus, the technique of chemiluminescence (CL) was developed for quantitative analyses of the oxygen-dependent microbicide processes associated with the phagocytic activities of bivalve hemocytes. Individual variability was demonstrated for chemiluminescence assays performed with an equivalent number of hemocytes, suggesting an influence of different hemocyte-type proportions. Subsequent CL experiments concerned, on one hand, separated hyalinocytes and granulocytes, and on the other hand, statistical analyses of CL values in relation to hemograms. Some recognition and stimulation mechanisms involved in the triggering of hemocyte respiratory burst were investigated for several specific pathogens. In the case of Bonarnia ostreae, an obligatory intrahemocytic parasite of Ostrea edulis, it was established that the respiratory burst was not triggered during the host hemocyte phagocytosis. Moreover, it was shown that some biochemical constituents of this parasite may be related to the inhibition of hemocyte microbicide activity. Similar results were observed for other pathogens, such as Perkinsus atlanticus parasite of Tapes phillzppinarum and Rickettsiales-like organism of Pecten maximus. On the basis of these results, it appears that chemiluminescence could constitute a well-adapted method for estimating individual immune capacity, specially in relation to a determined pathogen.
J9 MONOCLONAL ANTIBODIES AGAINST HEMOCYTES OF THE JAPANESE OYSTER C R A S S O S T R E A GIGAS (MOLLUSCA : BIVALVIA) A. Morvan, V. Boulo, B. Despres, D. Hervio, E. Bach~re and E. Mialhe, IFREMER, Unit~ Pathol. Immunol. G~n~t. Mol., BP 133, 17390 La Tremblade, France. For a few gastropod species, monoclonal antibodies (MABs) prepared against hemocytes have permitted to undertake the identification of hemocyte subpopulations. Such an approach was applied to the Japanese oyster Crassostrea gigas in order to determine its hemocyte types and lineage and also to investigate the immunobiological functions of the specifically recognized antigens. To enhance chance for the production of different cell-type specific MABs, granulocyte and hyalinocyte populations were separated by isopycnic centrifugation in Percoll gradients and subsequently used as different sources of immunogens. The lymphocytic hybrization method was performed for each immunization type. The screening of specific hybridomas was accomplished by using an indirect immunofluorescence assay (IIFA)..A panel of 21 MABs was harvested. Their binding characteristics were assessed by IIFA on both hemocyte primary cultures and cytocentrifugated preparations of f'Lxed and washed hemocytes. Four MABs were pan-hemocytic and reacted also with extra cellular components. Seventeen MABs would discriminate hemocyte subpopulations, seven of them being strictly specific for cellular epitopes and the others reacting with extra cellular components. All these MABs were tested against hemocytes of several species belonging to different bivalve families and showed some common epitopes. Finally these MABs were used according to chemiluminescence technique in an attempt to identify possible immunobiological functions of epitopes.
The Scientific & Social Program: Vth ISDCI Congress
J8 I N V I T R O CHEMILUMINESCENCE STUDIES OF MARINE BIVALVE DEFENCE MECHANISMS AND RESPONSES AGAINST SPECIFIC PATHOGENS. E. Bachere, .V Boulo, P. Godin, L. Goggin, D. Hervio, G. Le Gall, A. Morvan and E. Mialhe. IFREMER, Unit~ Pathol. Immunol. G~n~t. Mol., BP 133, 17390 La Tremblade, France. Because of the impact of infectious diseases in marine mollusc aquaculture, researches have been progressively developed in the field of immunology, specially in an attempt to define the defence response related to the variable infection course, naturally or experimentally, observed among animals. In order to analyse this individual response at the cellular and molecular levels, emphasis was on the development of in vitro experiments. Thus, the technique of chemiluminescence (CL) was developed for quantitative analyses of the oxygen-dependent microbicide processes associated with the phagocytic activities of bivalve hemocytes. Individual variability was demonstrated for chemiluminescence assays performed with an equivalent number of hemocytes, suggesting an influence of different hemocyte-type proportions. Subsequent CL experiments concerned, on one hand, separated hyalinocytes and granulocytes, and on the other hand, statistical analyses of CL values in relation to hemograms. Some recognition and stimulation mechanisms involved in the triggering of hemocyte respiratory burst were investigated for several specific pathogens. In the case of Bonarnia ostreae, an obligatory intrahemocytic parasite of Ostrea edulis, it was established that the respiratory burst was not triggered during the host hemocyte phagocytosis. Moreover, it was shown that some biochemical constituents of this parasite may be related to the inhibition of hemocyte microbicide activity. Similar results were observed for other pathogens, such as Perkinsus atlanticus parasite of Tapes phillzppinarum and Rickettsiales-like organism of Pecten maximus. On the basis of these results, it appears that chemiluminescence could constitute a well-adapted method for estimating individual immune capacity, specially in relation to a determined pathogen.
J9 MONOCLONAL ANTIBODIES AGAINST HEMOCYTES OF THE JAPANESE OYSTER C R A S S O S T R E A GIGAS (MOLLUSCA : BIVALVIA) A. Morvan, V. Boulo, B. Despres, D. Hervio, E. Bach~re and E. Mialhe, IFREMER, Unit~ Pathol. Immunol. G~n~t. Mol., BP 133, 17390 La Tremblade, France. For a few gastropod species, monoclonal antibodies (MABs) prepared against hemocytes have permitted to undertake the identification of hemocyte subpopulations. Such an approach was applied to the Japanese oyster Crassostrea gigas in order to determine its hemocyte types and lineage and also to investigate the immunobiological functions of the specifically recognized antigens. To enhance chance for the production of different cell-type specific MABs, granulocyte and hyalinocyte populations were separated by isopycnic centrifugation in Percoll gradients and subsequently used as different sources of immunogens. The lymphocytic hybrization method was performed for each immunization type. The screening of specific hybridomas was accomplished by using an indirect immunofluorescence assay (IIFA)..A panel of 21 MABs was harvested. Their binding characteristics were assessed by IIFA on both hemocyte primary cultures and cytocentrifugated preparations of f'Lxed and washed hemocytes. Four MABs were pan-hemocytic and reacted also with extra cellular components. Seventeen MABs would discriminate hemocyte subpopulations, seven of them being strictly specific for cellular epitopes and the others reacting with extra cellular components. All these MABs were tested against hemocytes of several species belonging to different bivalve families and showed some common epitopes. Finally these MABs were used according to chemiluminescence technique in an attempt to identify possible immunobiological functions of epitopes.