- Email: [email protected]
4.14 Humoral immune response to parasites in Bufo viridis
388
Session 4
demonstrated high binding capacity to both. Whether this reflects an isotype shift, as can be demonstrated during an ongoing immune response in mammals, is not known, but specificity studies of the MABs and further characterization of Ig determinants will be undertaken.
4.13
IMMUNOGLOBULIN HETEROGENEITY MONOCLONAL A N T I B O D Y I~6
IN
THE
TROUT
DEFINED
WITH
C. Sanchez, J. Dominquez and J. Coll, Departamento de Sanidad Animal, Embajadores 68, 28012 Madrid, Spain. Monoclonal antibodies against purified immunoglobulins from the trout Salmo aairdneri were obtained as described (see Abstract 1.8). Immunoglobulins defined by ELISA using monoclonal antibody IA6, were about 30% of the total immunoglobulins, as defined by ELISA using polyclonal antibodies, in healthy trout, S. qairdneri (n = 19) or S. trutta (n = 7). In trout obtained from farms with a previous history of viral infectious diseases, IA6immunoglobulins were only about 14% (n = ii). Several serum pools from trout could be totally depleted of iA6-immunoglobulins (about 12% of total immunoglobulins) after affinity chromatography over Sepharose-immobilized monoclonal antibody IA6. Preliminary results by gradient polyacrylamide electrophoresis under denaturing conditions, of affinity purified total immunoglobulin, IA6immunoglobulins and no-iA6 immunoglobulins, showed that a ii kDa polypeptide preferentially associate with the IA6 immunoglobulins. Two light chains of approximately molecular weights 22 kDa and 24 kDa could be resolved in all three cases. 4.14
HUMORAL
IMMUNE
RESPONSE
TO
PARASITES
IN BUFO
VIRIDIS
G.A. Ingram, *Fadwa Ai-Yaman and **K.G. Wooldridge, University of Salford, Salford, UK, Yarmouk University, Irbid, Jordan and "'University of Leicester, Leicester, UK. The primary humoral immune response of the green toad (B. viridis) following injection with the choanomastigotes of Crithidia fasciculata, a trypanosomatid flagellate parasite, was studied. Complement-fixing antibodies (CFA), indirect haemagglutinins (IHA), direct agglutinins (DA) and antibodies detectable by ELISA were demonstrated in sera 7 days post-injection, reached maxima after 49 days (P<0.01), and declined to background levels by day 70. Concurrently, serum lysozyme levels paralleled the rise in antibody titres and increased overall two-fold (P<0.001) compared to the controls. Highly significant correlation coefficients (r) were calculated for control sera when I H A a n d ELISA titres were compared (r>0.79; P<0.001) and when comparison were made between IHA and CFA or ELISA for immunised animals (r>0.96; P<0.001). The highest mean ELISA titre was approximately 1.5-fold to 3.5-fold greater than those determined for CFA, DA and IHA. Furthermore, both IHA and ELISA titres were affected by different crithidial antigen
Humoral and cell-mediated immunities
389
extracts and CFA values were influenced by complement source. Moreover, the use of two different e n z y m e - s u b s t r a t e systems in ELISA gave significant correlation of titres for both control (r = 0.73; P<0.001) and immune (r = 0.95; P<0.001) toads. The stimulated antibodies, possibly HMW IgM, were antigen-specific, exhibited 71 -electrophoretic mobility, sensitive to dithiothreitol, relatively heat-labile, effectively fixed complement and displayed precipitin activity. Parasite antigen(s) was localised in mainly the spleen red pulp from 1 to 4 weeks postinfection and was not detected in GALT, liver or kidney. To the authors' knowledge, this is the only report of the use of ELISA to detect antibodies and of the immune response to parasites in amphibians.
4.15
T-CELL FUNCTION INCUBATION
IN
CHICKENS
BURSECTOMIZED
AT
60
HR
OF
T. Veromaa, O. Vainio, E. Eerola, L. Lehtonen, S. Jalkanen and P. Toivanen, Department of Medical Microbiology, Turku University, 20520 Turku, Finland. Chickens surgically bursectomized in ovo (Bx) at 60 hours of embryonic development offer a unique model for selectively studying the influence of the bursa of Fabricius to t h y m u s - d e p e n d e n t immune functions, because the lymphoid cells of these animals develop in the total absence of the bursal microenvironment. The Bx chickens have been shown to be unable to respond to antigenic stimulation by specific antibody production. In the present study, we have c h a r a c t e r i z e d different aspects of T-cell mediated immunity in Bx chickens. Our results indicate existence of a normal, functional T-cell system in these animals. Peripheral blood leucocytes from Bx chickens were able to induce normal g r a f t - v e r s u s - h o s t reactions and mixed lymphocyte reactions. The capacity of peripheral blood T-cells from Bx animals to produce interleukin-2 was indistinguishable from that of their normal counterparts suggesting normal functioning of T helper cells. We demonstrated that peripheral blood leucocytes from Bx birds have normal in vitro proliferative responses to nonspecific T cell mitogens, concanavalin A and phytohemagglutinin, and to a specific antigen, keyhole limpet hemocyanin. The inability of the Bx chickens to respond to specific antigens is therefore restricted to B-cells and to p r o d u c t i o n of specific antibodies. Our findings indicate that the bursa of Fabricius is not necessary for the development of t h y m u s - d e p e n d e n t immune functions and support the suggestion that the specific function of the bursa is the creation of antibody diversity.
Session 4
demonstrated high binding capacity to both. Whether this reflects an isotype shift, as can be demonstrated during an ongoing immune response in mammals, is not known, but specificity studies of the MABs and further characterization of Ig determinants will be undertaken.
4.13
IMMUNOGLOBULIN HETEROGENEITY MONOCLONAL A N T I B O D Y I~6
IN
THE
TROUT
DEFINED
WITH
C. Sanchez, J. Dominquez and J. Coll, Departamento de Sanidad Animal, Embajadores 68, 28012 Madrid, Spain. Monoclonal antibodies against purified immunoglobulins from the trout Salmo aairdneri were obtained as described (see Abstract 1.8). Immunoglobulins defined by ELISA using monoclonal antibody IA6, were about 30% of the total immunoglobulins, as defined by ELISA using polyclonal antibodies, in healthy trout, S. qairdneri (n = 19) or S. trutta (n = 7). In trout obtained from farms with a previous history of viral infectious diseases, IA6immunoglobulins were only about 14% (n = ii). Several serum pools from trout could be totally depleted of iA6-immunoglobulins (about 12% of total immunoglobulins) after affinity chromatography over Sepharose-immobilized monoclonal antibody IA6. Preliminary results by gradient polyacrylamide electrophoresis under denaturing conditions, of affinity purified total immunoglobulin, IA6immunoglobulins and no-iA6 immunoglobulins, showed that a ii kDa polypeptide preferentially associate with the IA6 immunoglobulins. Two light chains of approximately molecular weights 22 kDa and 24 kDa could be resolved in all three cases. 4.14
HUMORAL
IMMUNE
RESPONSE
TO
PARASITES
IN BUFO
VIRIDIS
G.A. Ingram, *Fadwa Ai-Yaman and **K.G. Wooldridge, University of Salford, Salford, UK, Yarmouk University, Irbid, Jordan and "'University of Leicester, Leicester, UK. The primary humoral immune response of the green toad (B. viridis) following injection with the choanomastigotes of Crithidia fasciculata, a trypanosomatid flagellate parasite, was studied. Complement-fixing antibodies (CFA), indirect haemagglutinins (IHA), direct agglutinins (DA) and antibodies detectable by ELISA were demonstrated in sera 7 days post-injection, reached maxima after 49 days (P<0.01), and declined to background levels by day 70. Concurrently, serum lysozyme levels paralleled the rise in antibody titres and increased overall two-fold (P<0.001) compared to the controls. Highly significant correlation coefficients (r) were calculated for control sera when I H A a n d ELISA titres were compared (r>0.79; P<0.001) and when comparison were made between IHA and CFA or ELISA for immunised animals (r>0.96; P<0.001). The highest mean ELISA titre was approximately 1.5-fold to 3.5-fold greater than those determined for CFA, DA and IHA. Furthermore, both IHA and ELISA titres were affected by different crithidial antigen
Humoral and cell-mediated immunities
389
extracts and CFA values were influenced by complement source. Moreover, the use of two different e n z y m e - s u b s t r a t e systems in ELISA gave significant correlation of titres for both control (r = 0.73; P<0.001) and immune (r = 0.95; P<0.001) toads. The stimulated antibodies, possibly HMW IgM, were antigen-specific, exhibited 71 -electrophoretic mobility, sensitive to dithiothreitol, relatively heat-labile, effectively fixed complement and displayed precipitin activity. Parasite antigen(s) was localised in mainly the spleen red pulp from 1 to 4 weeks postinfection and was not detected in GALT, liver or kidney. To the authors' knowledge, this is the only report of the use of ELISA to detect antibodies and of the immune response to parasites in amphibians.
4.15
T-CELL FUNCTION INCUBATION
IN
CHICKENS
BURSECTOMIZED
AT
60
HR
OF
T. Veromaa, O. Vainio, E. Eerola, L. Lehtonen, S. Jalkanen and P. Toivanen, Department of Medical Microbiology, Turku University, 20520 Turku, Finland. Chickens surgically bursectomized in ovo (Bx) at 60 hours of embryonic development offer a unique model for selectively studying the influence of the bursa of Fabricius to t h y m u s - d e p e n d e n t immune functions, because the lymphoid cells of these animals develop in the total absence of the bursal microenvironment. The Bx chickens have been shown to be unable to respond to antigenic stimulation by specific antibody production. In the present study, we have c h a r a c t e r i z e d different aspects of T-cell mediated immunity in Bx chickens. Our results indicate existence of a normal, functional T-cell system in these animals. Peripheral blood leucocytes from Bx chickens were able to induce normal g r a f t - v e r s u s - h o s t reactions and mixed lymphocyte reactions. The capacity of peripheral blood T-cells from Bx animals to produce interleukin-2 was indistinguishable from that of their normal counterparts suggesting normal functioning of T helper cells. We demonstrated that peripheral blood leucocytes from Bx birds have normal in vitro proliferative responses to nonspecific T cell mitogens, concanavalin A and phytohemagglutinin, and to a specific antigen, keyhole limpet hemocyanin. The inability of the Bx chickens to respond to specific antigens is therefore restricted to B-cells and to p r o d u c t i o n of specific antibodies. Our findings indicate that the bursa of Fabricius is not necessary for the development of t h y m u s - d e p e n d e n t immune functions and support the suggestion that the specific function of the bursa is the creation of antibody diversity.