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

Abstract

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.

Molecular survey of Besnoitia spp. (Apicomplexa) in faeces from European wild mesocarnivores in Spain. Short running title: Molecular survey of Besnoitia spp in faeces from wild carnivores.

“…besnoiti . In addition, the later study (Schares et al, 2021) suggests that a so far unknown Besnoitia species closely related to B. darlingi , B. neotomofelis , B. oryctofelisi ,B. akodoni or B. jellisoni is cycling in Namibian wildlife.…”

“…On the other hand, two studies have managed to detect by molecular techniques Besnoitia spp. DNA in faeces from pond bat (Myotis dasycneme ) in the Netherlands (Hornok et al, 2015) and in faecal matter from cheetahs (Acinonyx jubatus ) in Namibia (Schares et al, 2021); in the first report, authors hypothesized that B. besnoiti -like sequences might have originated from French cattle via bloodsucking dipterans (Stomoxys calcitrans, Tabanus spp.). In this regard, bats frequently use cattle stables for roosting, where they can prey on the mechanical vectors ofB.…”

An update on epidemiology and clinical aspects of besnoitiosis in livestock and wildlife in sub-Saharan Africa: A systematic review

“…Natural infection of horses with B. bennetti were documented in Kenya ( Bwangamoi, 1972 ) and South Africa ( Van Heerden et al, 1993 ). Besnoitia darlingi -like species was found naturally infecting cheetah ( Schares et al, 2021 ), and only in Namibia. Besnoitia infections by unidentified species ( Besnoitia spp.)…”

“…Although infections in Africa has only been detected based on clinical signs, histological and pathological examination, exposure or infection of B. bennetti in equids in Europe were detected based on clinical signs, serological test (IFAT and western blot) ( Gutiérrez-Expósito et al, 2017 ) and partial rDNA sequencing ( Liénard et al, 2018 ), which have not been able to conclusively distinguish between species ( Villa et al, 2021 ). Schares et al (2021) isolated and identified B. darlingi -type naturally occurring in cheetah in Botswana based on conventional and real-time PCR. Although reviewed studies showed that this is the only report of B. darlingi -type in sub-Saharan Africa, natural infections of B. darlingi -like have been documented in opossum ( Didelphis marsupialis ) in Panama and Brazil, and D. virginiana in North America) and lizard species ( Basiliscus basiliscus , Ameiva ameiva , A. festiva , A. leptophrys ) from Panama, ( Basiliscus vittatus ) from Belize, ( Ameiva ameiva ) from Brazil, and in rodents ( Akodon montensis ) from Brazil ( Dubey et al, 2003 ; Elsheikha et al, 2003 ; Elsheikha et al, 2004 ; Kiehl et al, 2010 ).…”

New Insights into Gastrointestinal and Pulmonary Parasitofauna of Wild Eurasian lynx (Lynx lynx) in the Harz Mountains of Germany

“…To monitor the inhibition of the real-time PCRs, a heterologous plasmid with DNA sequences resembling the enhanced green fluorescent protein (EGFP) gene was added to the reaction mix in all real-time PCRs. The internal control PCR included the primers EGFP1-F, EGFP2-R and the probe EGFP1 as previously reported [83]. Cycling condition for qPCR was as follows.…”