title: Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine.
abstract: The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein-blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves. Collectively, these data show the efficacy of a transfected HlGST-Cln B. bovis parasite to induce detectable anti-glutathione-S-transferase antibodies and a reduction in tick size and fecundity of R. microplus feeding in experimentally inoculated animals.
authors.affiliation: Centro de Biotecnologia Universidade Federal do Rio Grande do Sul, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Daiane P
authors.initials: DP
authors.lastname: Oldiges
authors.affiliation: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, United States of America.
authors.forename: Jacob M
authors.initials: JM
authors.lastname: Laughery
authors.affiliation: Faculdade de Veterinária Universidade Federal do Rio Grande do Sul; Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Nelson Junior
authors.initials: NJ
authors.lastname: Tagliari
authors.affiliation: Faculdade de Veterinária Universidade Federal do Rio Grande do Sul; Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Ronaldo Viana
authors.initials: RV
authors.lastname: Leite Filho
authors.affiliation: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, United States of America.
authors.forename: William C
authors.initials: WC
authors.lastname: Davis
authors.affiliation: Centro de Biotecnologia Universidade Federal do Rio Grande do Sul, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.|Faculdade de Veterinária Universidade Federal do Rio Grande do Sul; Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Itabajara
authors.initials: I
authors.lastname: da Silva Vaz
authors.affiliation: Centro de Biotecnologia Universidade Federal do Rio Grande do Sul, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.|Departamento de Bioquímica Universidade Federal do Rio Grande do Sul; Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Carlos
authors.initials: C
authors.lastname: Termignoni
authors.affiliation: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, United States of America.|Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, Washington, United States of America.
authors.forename: Donald P
authors.initials: DP
authors.lastname: Knowles
authors.affiliation: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, United States of America.|Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, Washington, United States of America.
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Suarez
mesh_terms: D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics* / Q000276:immunology; D016793:Babesia bovis / Q000235:genetics* / Q000276:immunology; D001404:Babesiosis / Q000276:immunology* / Q000469:parasitology / Q000517:prevention & control / Q000635:transmission; D002417:Cattle; D002418:Cattle Diseases / Q000276:immunology / Q000469:parasitology / Q000517:prevention & control*; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics* / Q000276:immunology; D049452:Green Fluorescent Proteins / Q000235:genetics; D026863:Ixodidae / Q000201:enzymology*; D008297:Male; D048494:Rhipicephalus / Q000469:parasitology; D014162:Transfection; D014612:Vaccines / Q000235:genetics / Q000276:immunology
chemical_list: D060829:Arthropod Proteins; D014612:Vaccines; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase