Multi-locus sequence analysis of Anaplasma in the common warthog (Phacochoerus africanus) from South Africa

The prevalence and diversity of Anaplasma in common warthog (Phacochoerus africanus) was investigated using a multi-locus sequence analysis (MLSA) approach targeting the 16S rRNA, citrate synthase (gltA) and heat-shock operon (groESL) genes. PCR screening of 100 warthog samples from the Kruger National Park in South Africa with eight published assays identified 50 positive animals, all of which were initially identified with the 16S rRNA assay. In contrast, the gltA and six groESL assays recovered PCR-positivity rates of 2 % and 0 %-4 %, respectively. As optimisation did not improve Anaplasma detection rates, an alternative groESL assay targeting a 923 bp region was designed. This new assay detected 45 positive animals, all of which were positive with the 16S rRNA assay. Nucleotide sequencing of the three MLSA gene targets confirmed that 50 % (50/100) of warthogs were Anaplasma-positive. Juvenile warthogs displayed a significantly higher infection rate (15/18; 83.3 %) than adults (35/82; 42,68 %). Phylogenetic analyses of individual and concatenated gene datasets confirmed that the Anaplasma species in warthogs is closely related to the species detected in Ornithodoros soft ticks from Zambia. This, together with the high levels of nucleotide sequence identity (≥98.97 %), suggests the likely existence of a host-restricted cycle involving warthogs and the soft ticks that inhabit their burrows. Based on the distinctiveness and monophyly of the Anaplasma species in warthogs and Ornithodoros soft ticks, confirmed through genetic characterisation of three gene regions, we propose that Candidatus status be assigned and suggest "Candidatus Anaplasma ornithodorii".