title: Assessment of primers designed for the subspecies-specific discrimination among Babesia canis canis, Babesia canis vogeli and Babesia canis rossi by PCR assay.
abstract: Canine babesiosis is an infectious disease caused by either Babesia gibsoni or Babesia canis protozoans. The latter is also classified under three different phylogenetic groups, referred to as subspecies B. canis canis, B. canis vogeli and B. canis rossi. The objective of the present study was to validate and standardize a PCR assay to discriminate the organisms at the subspecies level. First, the reference sequences of the 18S rRNA, 5.8S rRNA and 28S rRNA genes, including the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of the most common species and subspecies of the genus Babesia were retrieved from the GenBank database. Subspecies-specific primers (BAB3, BAB4 and BAB5) and one genus-specific primer were designed from the alignment of the sequences. The PCR assays were evaluated in three different combinations of primer pairs in order to assure complete specificity for each reaction. The results of the tests had demonstrated effectiveness of the novel primer pairs BAB1/BAB3, BAB1/BAB4 and BAB1/BAB5 for the amplification of the subspecies-specific target fragments of 746 bp (B. c. canis), 546 bp (B. c. vogeli) and 342 bp (B. c. rossi) by PCR. The original enzymatic amplification assays with novel primers reported in this paper were confirmed to be a reliable tool for the specific discrimination among B. canis subspecies by single-step PCR assays.
authors.affiliation: Veterinary School, Federal University of Goiás, Brazil. sabrinacd@gmail.com
authors.forename: Sabrina Castilho
authors.initials: SC
authors.lastname: Duarte
authors.forename: Guido Fontgalland Coelho
authors.initials: GF
authors.lastname: Linhares
authors.forename: Tatiana Nunes
authors.initials: TN
authors.lastname: Romanowsky
authors.forename: Osvaldo José
authors.initials: OJ
authors.lastname: da Silveira Neto
authors.forename: Ligia Miranda Ferreira
authors.initials: LM
authors.lastname: Borges
mesh_terms: D000818:Animals; D001403:Babesia / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D001404:Babesiosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D001483:Base Sequence; D017931:DNA Primers; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology*; D004285:Dogs; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards / Q000662:veterinary; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D021903:DNA, Ribosomal Spacer; D012335:RNA, Ribosomal