Molecular analysis suggests that Namibian cheetahs ( Acinonyx jubatus ) are definitive hosts of a so far undescribed Besnoitia species

Background Besnoitia darlingi , B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with felids as definitive hosts. These parasites use a variety of animal species as intermediate hosts. North American opossums ( Didelphis virginiana ), North American southern plains woodrats ( Neotoma micropus ) and South American domestic rabbits ( Oryctolagus cuniculus ) are intermediate hosts of B. darlingi , B. neotomofelis and B. oryctofelisi , respectively. Based on conserved regions in the internal transcribed spacer-1 (ITS1) sequence of the ribosomal DNA (rDNA), a real-time PCR for a sensitive detection of these Besnoitia spp. in tissues of intermediate hosts and faeces of definitive hosts has recently been established. Available sequence data suggest that species such as B. akodoni and B. jellisoni are also covered by this real-time PCR. It has been hypothesised that additional Besnoitia spp. exist worldwide that are closely related to B. darlingi or B. darlingi -like parasites ( B. neotomofelis , B. oryctofelisi , B. akodoni or B. jellisoni ). Also related, but not as closely, is B. besnoiti , the cause of bovine besnoitiosis. Methods Faecal samples from two free-ranging cheetahs ( Acinonyx jubatus ) from Namibia that had previously tested positive for coccidian parasites by coproscopy were used for this study. A conventional PCR verified the presence of coccidian parasite DNA. To clarify the identity of these coccidia, the faecal DNA samples were further characterised by species-specific PCRs and Sanger sequencing. Results One of the samples tested positive for B. darlingi or B. darlingi -like parasites by real-time PCR, while no other coccidian parasites, including Toxoplasma gondii , Hammondia hammondi , H. heydorni , B. besnoiti and Neospora caninum , were detected in the two samples. The rDNA of the B. darlingi -like parasite was amplified and partially sequenced. Comparison with existing sequences in GenBank revealed a close relationship to other Besnoitia spp., but also showed clear divergences. Conclusions Our results suggest that a so far unknown Besnoitia species exists in Namibian wildlife, which is closely related to B. darlingi , B. neotomofelis , B. oryctofelisi , B. akodoni or B. jellisoni. The cheetah appears to be the definitive host of this newly discovered parasite, while prey species of the cheetah may act as intermediate hosts. Graphical Abstract