Sensitive, quantitative detection of and related parasites in intermediate hosts and to assess felids as definitive hosts for known and as-yet undescribed related parasite species.

and are closely related coccidian parasites with cats as definitive hosts. While uses opossums as intermediate hosts, and have been described in Southern Plains woodrats () from the USA and in domestic rabbits from Argentina, respectively. A comparison of the Internal Transcribed Spacer-1 (ITS-1) region of the ribosomal DNA (rDNA) of these spp. showed only a few differences. The present study aimed at developing a real-time PCR to detect and in tissues of intermediate and in faeces of definitive hosts in order to support studies of these organisms' epidemiology and pathogenesis. The established PCR was based on primer regions distinct from the ITS-1 sequences of ungulate spp. and made use of a Besnoitia universal probe. To monitor inhibition, a heterologous internal control was established based on the enhanced green fluorescent protein gene. The real-time PCR reacted with , and while the novel PCR did not recognize ungulate spp. (). DNA of Apicomplexa ascribed to other Besnoitia-related genera, including other gut parasites of cats (, , ), was not recognized. The real-time PCR had an analytic sensitivity of less than 1 tachyzoite per reaction. In feline faeces spiked with oocysts, the limit of detection was a DNA amount equivalent to 1 oocyst per PCR reaction. In infected ɣ-interferon knock-out mice, the lung was identified as the predilection organ. In conclusion, this real-time PCR should advance further studies on these parasites and may inspire research on related species, not only in the Americas, but also in other parts of the world.