Fig. 1.
Location of the primers and the probe of the Besnoitia darlingi/B.neotomofelis /B. oryctofelisi-specific real-time PCR assay BdanjoRT1 within the ITS-1 region of the rRNA gene. The sequences of the ITS-1 region of B. akodoni (AY545987, bold), B. jellisoni (AF076860, bold), B. neotomofelis (HQ909085, bold), B. darlingi (AF489696, bold) and B. oryctofelisi (AY182000, bold), were aligned relative to sequences of other Besnoitia spp. including B. besnoiti from Portugal, Spain and Germany, B. bennetti reported from the USA and Belgium, B. tarandi from Canada and Finland and those of Neospora caninum, Hammondia heydorni, Toxoplasma gondii and H. hammondi by using Clustal V (DNAStar, Madisin, Wisconsin, USA). Deletions and substitutions in the sequences relative to and within the clade of B. acodoni, B. jellisoni, B. neotomofelis, B. darlingi and B. oryctofelisi are indicated by black background. Sequences of the primer BdanjoRev and the Probe Bb11-12 are displayed in their complementary form. The probe Bb11-12 was established for a real-time PCR to detect B. besnoiti, but it is universal and can be used for the detection of all Besnoitia spp. mentioned here.