Molecular Characterization of Babesia bigemina Isolates

ROMINA PETRIGH; PAULA RUYBAL; ROSALIA MORETTA; GONZALO GIL; PABLO NUÑEZ; ROBERTO NEUMANN; SILVINA WILCOWSKY; MARISA FARBER

Merida, Yucatan , Mexico

Conferencia; 9th Biennial Conference of the Society for Tropical Veterinary Medicine; 2007

Society for Tropical Veterinary Medicine

Bovine Babesiosis is caused by tick-transmitted intraerythrocytic protozoa Babesia bovis and Babesia bigemina and represents a major constraint to livestock economy. Genetic characterization of B. bigemina isolates contributes to understand vectorparasites interaction, parasite pathogenicity and for applying to epidemiological studies. Previous studies reported genetic polymorphism between Brazilian isolates by means of three different PCR based techniques (Madruga et al., 2002). In addition, polymorphism has been described in both rhoptry-associated protein-1c gene (Suarez et al., 2003) and merozoite surface antigen gp45 gene (Fisher et al., 2001). In this study two strategies were explored to further characterize B. bigeminaBabesia bovis and Babesia bigemina and represents a major constraint to livestock economy. Genetic characterization of B. bigemina isolates contributes to understand vectorparasites interaction, parasite pathogenicity and for applying to epidemiological studies. Previous studies reported genetic polymorphism between Brazilian isolates by means of three different PCR based techniques (Madruga et al., 2002). In addition, polymorphism has been described in both rhoptry-associated protein-1c gene (Suarez et al., 2003) and merozoite surface antigen gp45 gene (Fisher et al., 2001). In this study two strategies were explored to further characterize B. bigeminaand Babesia bigemina and represents a major constraint to livestock economy. Genetic characterization of B. bigemina isolates contributes to understand vectorparasites interaction, parasite pathogenicity and for applying to epidemiological studies. Previous studies reported genetic polymorphism between Brazilian isolates by means of three different PCR based techniques (Madruga et al., 2002). In addition, polymorphism has been described in both rhoptry-associated protein-1c gene (Suarez et al., 2003) and merozoite surface antigen gp45 gene (Fisher et al., 2001). In this study two strategies were explored to further characterize B. bigeminaB. bigemina isolates contributes to understand vectorparasites interaction, parasite pathogenicity and for applying to epidemiological studies. Previous studies reported genetic polymorphism between Brazilian isolates by means of three different PCR based techniques (Madruga et al., 2002). In addition, polymorphism has been described in both rhoptry-associated protein-1c gene (Suarez et al., 2003) and merozoite surface antigen gp45 gene (Fisher et al., 2001). In this study two strategies were explored to further characterize B. bigeminaB. bigemina isolates. Initially, B. bigemina genome (www.sanger.ac.uk/Projetcs/b_bigemina) was analyzed using Tandem Repeat Finder software (c3.biomath.mssn.edu/trf.html). A tandem repeat composed of 11 aminoacids was found in two contigs within an Open Reading Frame (ORF). BLASTP analysis of the translated sequence of both ORFs led to the identification of membrane attack complex/perforin domain (MACPF, a protein found in natural killer and cytolytic T-cells) in the carboxy terminus of the molecule. Moreover, the protein sequence is highly similar to Toxoplasma gondiiB. bigemina genome (www.sanger.ac.uk/Projetcs/b_bigemina) was analyzed using Tandem Repeat Finder software (c3.biomath.mssn.edu/trf.html). A tandem repeat composed of 11 aminoacids was found in two contigs within an Open Reading Frame (ORF). BLASTP analysis of the translated sequence of both ORFs led to the identification of membrane attack complex/perforin domain (MACPF, a protein found in natural killer and cytolytic T-cells) in the carboxy terminus of the molecule. Moreover, the protein sequence is highly similar to Toxoplasma gondiiToxoplasma gondii Perforin-like protein 1 (E Value 2 e-51) and to sporozoite protein with MACPF related domain from Plasmodium berghei (E Value 4 e-47). Specific primers matching the conserved region flanking the tandem repeats were designed and the amplified product was named Perforin-like repeat (PLR). On the other hand, a polymorphism in the 18S rRNA gene was analyzed. This variability was detected as a consequence of an unsuccessful detection of Argentinean isolates by Reverse Line Hybridization. This technique allows the simultaneous detection of multiple tick-transmitted protozoan and rickettsial cattle pathogens by hybridization of PCR products from 16S (Anaplasma/Erhichia) or 18S (Babesia/Theileria) rRNA genes with numerous pathogen specific probes. PLR and 18S rRNA were analyzed in Argentinean (BgM1A, BgS1A, BgS2P, BgS3P, BgM1P, BgM2P), Brazilian and Mexican isolates. PLR pattern of each isolate was variable, and the sequences of the PCR products revealed seventeen different repeat units. For the 18S rRNA the sequence variations resulted in distinct combination of point mutations in relation to the original RLB probe. Furthermore, each isolate showed more than one 18S combination that was confirmed by reverse hybridization and sequence. Only BgM1A and BgS1A showed identical polymorphism pattern (18S and PLR), while pathogenic isolates displayed higher variability. These new molecular tools for B. bigemina isolates typing could be applied to pathogenicity, tick-host interaction and vaccine development studies.Plasmodium berghei (E Value 4 e-47). Specific primers matching the conserved region flanking the tandem repeats were designed and the amplified product was named Perforin-like repeat (PLR). On the other hand, a polymorphism in the 18S rRNA gene was analyzed. This variability was detected as a consequence of an unsuccessful detection of Argentinean isolates by Reverse Line Hybridization. This technique allows the simultaneous detection of multiple tick-transmitted protozoan and rickettsial cattle pathogens by hybridization of PCR products from 16S (Anaplasma/Erhichia) or 18S (Babesia/Theileria) rRNA genes with numerous pathogen specific probes. PLR and 18S rRNA were analyzed in Argentinean (BgM1A, BgS1A, BgS2P, BgS3P, BgM1P, BgM2P), Brazilian and Mexican isolates. PLR pattern of each isolate was variable, and the sequences of the PCR products revealed seventeen different repeat units. For the 18S rRNA the sequence variations resulted in distinct combination of point mutations in relation to the original RLB probe. Furthermore, each isolate showed more than one 18S combination that was confirmed by reverse hybridization and sequence. Only BgM1A and BgS1A showed identical polymorphism pattern (18S and PLR), while pathogenic isolates displayed higher variability. These new molecular tools for B. bigemina isolates typing could be applied to pathogenicity, tick-host interaction and vaccine development studies.Anaplasma/Erhichia) or 18S (Babesia/Theileria) rRNA genes with numerous pathogen specific probes. PLR and 18S rRNA were analyzed in Argentinean (BgM1A, BgS1A, BgS2P, BgS3P, BgM1P, BgM2P), Brazilian and Mexican isolates. PLR pattern of each isolate was variable, and the sequences of the PCR products revealed seventeen different repeat units. For the 18S rRNA the sequence variations resulted in distinct combination of point mutations in relation to the original RLB probe. Furthermore, each isolate showed more than one 18S combination that was confirmed by reverse hybridization and sequence. Only BgM1A and BgS1A showed identical polymorphism pattern (18S and PLR), while pathogenic isolates displayed higher variability. These new molecular tools for B. bigemina isolates typing could be applied to pathogenicity, tick-host interaction and vaccine development studies.B. bigemina isolates typing could be applied to pathogenicity, tick-host interaction and vaccine development studies.