Differential antibody response towards rhoptry-associated protein-1 in bovines experimentally infected with an attenuated and a pathogenic strain of Babesia bovis

ASENZO G; DOMINGUEZ M; RODRIGUEZ, A; ECHAIDE I; TORIONI DE ECHAIDE S; WILKOWSKY, S; FLORIN CHRISTENSEN, M

Palermo, Italia

Congreso; II Babesia World Summit; 2007

Instituto Zooprofilactico de Sicilia

Differential antibody response towards rhoptry-associated protein-1 in bovines experimentally infected with an attenuated and a pathogenic strain of Babesia bovis Purpose of the job: Babesia bovis is a bovine tick-transmitted apicomplexan protozoon that causes important economic losses in vast tropical and subtropical areas of the world. Vaccination of bovines with live attenuated strains is used in Argentina and other countries to prevent outbreaks. The process of attenuation is achieved by successive passages of a pathogenic isolate through splenectomized calves. It is still unknown which features are lacking in an attenuated strain that prevents it from causing disease and even death, while still rendering long-term immunity. As a first approach to investigate this process, we have studied the early immunological responses developed in Babesia bovis-experimentally infected bovines with a pathogenic and an attenuated strain from Argentina, against two antigens known to be expressed in in vitro cultured merozoites and sporozoites:  Rhoptry Associated Protein-1 (RAP-1) (1) and Merozoite Surface Antigen 2-c (MSA-2c) (2,3). Materials and Methods: Two bovines were inoculated with an Argentine B. bovis vaccinial strain (R1A) and two bovines with an Argentine pathogenic strain (S2P); and serum samples were collected at different time points up to 66 days post infection. Recombinant forms of MSA-2c and RAP-1 were produced in the pBAD/thioTOPO prokaryotic expression system and purified by affinity chromatography on Ni-agarose. Indirect ELISA tests were developed for each of these antigens, optimizing antigen concentration, incubation conditions, blocking reagents and colorimetric substrate. The recombinant antigens were bound to ELISA plates and after blocking, serum samples, previously adsorbed with E. coli lysates to reduce unspecific reactions, were added to the wells in serial dilutions. Detection of antibody binding was carried out by incubation with anti-bovine IgG-peroxidase, followed by OPD-H2O2 and absorbance was read at 492 nm. Titers were calculated as the maximal dilution that doubled the values obtained with negative serum samples. Results: Specific antibodies against MSA-2c and RAP-1 appeared around day 15 post infection and peaked around day 20. In the case of MSA-2c, titers remained high until at least 66 days post infection, both in the case of R1A and S2P. However, there were important differences in the patterns of anti-RAP-1 antibody levels between infections with R1A and S2P. In the infections with the attenuated R1A strain, anti-RAP-1 titers decreased to background levels around day 55 and remained undetectable until at least day 66. On the other hand, in S2P infections, anti-RAP-1 titers did not decrease. Conclusions: This different antibody behavior could reflect antigenic variations in the parasites during infection or a different modulation of the bovine immune response to infections with an attenuated or a pathogenic strain. More experiments to discern between these possibilities are now under way and can throw light on an almost unexplored subject, i.e. the changes that occur in parasites during attenuation. References: 1. Mosqueda, J., McElwain, T.F., Stiller, D., Palmer, G.H. (2002) Babesia bovis merozoite surface antigen 1 and rhoptry-associated protein 1 are expressed in sporozoites, and specific antibodies inhibit sporozoite attachment to erythrocytes. Infect Immun. 70:1599-603. 2. Wilkowsky, S., Farber, M., Echaide, I., Torioni de Echaide, S., Zamorano P.I., Dominguez, M., Suarez, C.E., Florin-Christensen, M. (2003) Babesia bovis Merozoite Surface Antigen-2c contains highly immunogenic, conserved B-cell epitopes that elicit neutralization-sensitive antibodies in cattle.  Mol. Biochem. Parasitol. 127: 133-141. 3. Mosqueda et al. Mosqueda, J., McElwain, T.F., Palmer, G.H. (2002) Babesia bovis merozoite surface antigen 2 proteins are expressed on the merozoite and sporozoite surface, and specific antibodies inhibit attachment and invasion of erythrocytes. Infect. Immun. 70: 6448-55.   (This work was supported by ANPCyT PICT 98-083838 and Fundacion Antorchas, Argentina; and the European Commission, INCO 0003691, MEDLABAB)