Highly prevalent co-infections and genetic diversity of Anaplasma species in asymptomatic small ruminants in a Mediterranean context revealed by a gltA-based nested multiplex PCR assay

Anaplasmosis is a widespread tick-borne disease affecting various host species. Small ruminants (sheep and goats) can be infected by multiple Anaplasma species:. To evaluate the circulation and co-infection rates of five Anaplasma species (A. ovis, A. capra, A. bovis, A. marginale and A. phagocytophilum) in asymptomatic small ruminants in Corsica--a Mediterranean island of France--we designed a singleplex, duplex, and multiplex nested PCR (nPCR) scheme based on the gltA gene. Anaplasmosis was found to be highly endemic on the island, with 85.2% of the tested small ruminants infected by one or more Anaplasma species (82.8% of sheep and 89.5% of goats). We confirmed the presence of A. ovis and A. capra, and, for the first time in Corsica, detected A. bovis and A. phagocytophilum in both sheep (respective infection rates: 64.3%, 34.3%, 4.3%, and 7.1%) and goats (respective rates: 84.2%, 13.2%, 18.4%, and 5.3%). A. marginale was not detected. Approximately one-third of the infected animals were co-infected (35.1% of infected sheep and 32.4% of infected goats). A. ovis was always involved in co-infections, most often associated with A. capra (54.8% of co-infections). Importantly, A. bovis was never detected as a mono-infection in asymptomatic animals, but was always found associated with A. ovis (32.3% of co-infections). Sequence analysis of the gltA and 16S rRNA genes confirmed the species identification of all detected Anaplasma species. A probably new Anaplasma species was detected for the first time in Corsica using the 16S rRNA nPCR. Furthermore, we found several circulating A. phagocytophilum genetic variants. Sequence analysis of A. ovis 16S rRNA revealed a single SNP that delineates two sequence groups (Ao1goat and Ao2sheep), which were significantly linked to the host of origin. Additionally, two groups of A. bovis gltA sequences were identified (Ab1 and Ab2, with 87.4% sequence homology), forming two novel clades. While the Ab1 clade is closely related to the six previously described clades, the Ab2 clade formed a separate phylogenetic group that also contained isolates from Corsican symptomatic goats. In conclusion, Anaplasma species are highly prevalent in the small ruminant population of Corsica, confronted with a great diversity of co-infecting species and genetic variants.