title: Typification of virulent and low virulence Babesia bigemina clones by 18S rRNA and rap-1c.
abstract: The population structure of original Babesia bigemina isolates and reference strains with a defined phenotypic profile was assessed using 18S rRNA and rap-1c genes. Two reference strains, BbiS2P-c (virulent) and BbiS1A-c (low virulence), were biologically cloned in vitro. The virulence profile of the strains and clones was assessed in vivo. One fully virulent and one low-virulence clone were mixed in identical proportions to evaluate their growth efficiency in vitro. Each clone was differentiated by two microsatellites and the gene gp45. The 18S rRNA and rap-1c genes sequences from B. bigemina biological clones and their parental strains, multiplied exclusively in vivo or in vitro, were compared with strain JG-29. The virulence of clones derived from the BbiS2P-c strain was variable. Virulent clone Bbi9P1 grew more efficiently in vitro than did the low-virulence clone Bbi2A1. The haplotypes generated by the nucleotide polymorphism, localized in the V4 region of the 18S rRNA, allowed the identification of three genotypes. The rap-1c haplotypes allowed defining four genotypes. Parental and original strains were defined by multiple haplotypes identified in both genes. The rap-1c gene, analyzed by high-resolution melting (HRM), allowed discrimination between two genotypes according to their phenotype, and both were different from JG-29. B. bigemina biological clones made it possible to define the population structure of isolates and strains. The polymorphic regions of the 18S rRNA and rap-1c genes allowed the identification of different subpopulations within original B. bigemina isolates by the definition of several haplotypes and the differentiation of fully virulent from low virulence clones.
authors.affiliation: Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria Rafaela, Ruta 34 km 227, CC 22, CP 2300 Rafaela, Santa Fe, Argentina. Electronic address: thompson.carolina@inta.gob.ar.
authors.forename: C
authors.initials: C
authors.lastname: Thompson
authors.affiliation: Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria Rafaela, Ruta 34 km 227, CC 22, CP 2300 Rafaela, Santa Fe, Argentina.
authors.forename: M E
authors.initials: ME
authors.lastname: Baravalle
authors.affiliation: Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria Rafaela, Ruta 34 km 227, CC 22, CP 2300 Rafaela, Santa Fe, Argentina.
authors.forename: B
authors.initials: B
authors.lastname: Valentini
authors.affiliation: Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria Rafaela, Ruta 34 km 227, CC 22, CP 2300 Rafaela, Santa Fe, Argentina.
authors.forename: A
authors.initials: A
authors.lastname: Mangold
authors.affiliation: Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria Rafaela, Ruta 34 km 227, CC 22, CP 2300 Rafaela, Santa Fe, Argentina.
authors.forename: S
authors.initials: S
authors.lastname: Torioni de Echaide
authors.affiliation: Instituto Nacional de Tecnología Agropecuaria, Centro Nacional de Investigaciones Agropecuarias Castelar, Los Reseros y Las Cabañas, CP 1712 Castelar, Buenos Aires, Argentina.
authors.forename: P
authors.initials: P
authors.lastname: Ruybal
authors.affiliation: Instituto Nacional de Tecnología Agropecuaria, Centro Nacional de Investigaciones Agropecuarias Castelar, Los Reseros y Las Cabañas, CP 1712 Castelar, Buenos Aires, Argentina.
authors.forename: M
authors.initials: M
authors.lastname: Farber
authors.affiliation: Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria Rafaela, Ruta 34 km 227, CC 22, CP 2300 Rafaela, Santa Fe, Argentina.
authors.forename: I
authors.initials: I
authors.lastname: Echaide
mesh_terms: D000818:Animals; D001118:Argentina / Q000453:epidemiology; D001403:Babesia / Q000145:classification / Q000235:genetics / Q000472:pathogenicity*; D001404:Babesiosis / Q000453:epidemiology / Q000469:parasitology / Q000662:veterinary*; D001483:Base Sequence; D002417:Cattle; D002418:Cattle Diseases / Q000453:epidemiology / Q000469:parasitology*; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D004912:Erythrocytes / Q000469:parasitology; D005838:Genotype; D060005:Genotyping Techniques; D006239:Haplotypes; D008297:Male; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics*; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D016415:Sequence Alignment; D014774:Virulence
chemical_list: D016054:DNA, Protozoan; D015800:Protozoan Proteins; D012337:RNA, Ribosomal, 18S; C097551:rhoptry-associated protein-1, Babesia
keywords: 18S rRNA; Babesia bigemina; Biological clones; HRM; rap-1c