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Exp Parasitol. 2014 Jun;141:98-105. doi: 10.1016/j.exppara.2014.03.016. Epub 2014 Mar 25.

Typification of virulent and low virulence Babesia bigemina clones by 18S rRNA and rap-1c.

Author information

1
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.
2
Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria Rafaela, Ruta 34 km 227, CC 22, CP 2300 Rafaela, Santa Fe, Argentina.
3
Instituto Nacional de Tecnología Agropecuaria, Centro Nacional de Investigaciones Agropecuarias Castelar, Los Reseros y Las Cabañas, CP 1712 Castelar, Buenos Aires, Argentina.

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.

KEYWORDS:

18S rRNA; Babesia bigemina; Biological clones; HRM; rap-1c

PMID:
24681200
DOI:
10.1016/j.exppara.2014.03.016
[Indexed for MEDLINE]

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