Tick-borne haemoparasite occurrence and Anaplasma bovis strain diversity in eastern rock sengis (Elephantulus myurus)

Abstract

Studying the relationships of parasites with their vertebrate hosts is important as they improve our knowledge on the ecology of diseases of veterinary and zoonotic importance. Eastern rock sengis (Elephantulus myurus) are small insectivores endemic to Africa that are known to have higher tick burdens than other small mammal species. Studies have shown that E. myurus from South Africa harbour an Anaplasma bovis-like strain, a rickettsial pathogen of cattle. Anaplasma bovis infects host monocytes causing monocytic- and bovine anaplasmosis. The role of rock sengi as reservoirs of tick-borne pathogens has not been fully investigated. In addition, the phylogenetic position of the described A. bovis-like strain is unclear as genetic data are currently limited to the highly conserved 16S rRNA gene and studies are constrained by difficulties with cultivation of tick-borne Anaplasmataceae. The aim of the current study was, therefore, to determine the tick-borne haemoparasite diversity in rock sengi and to expand molecular characterization of the A. bovis-like strain in rock sengi (henceforth referred to as A. bovis-like (sengi)), using a more phylogenetically informative gene region. The specific objectives were to (i) screen blood samples for the presence of Theileria, Babesia, Ehrlichia and Anaplasma spp. using the Reverse Line Blot (RLB) hybridization assay, and (ii) to determine the taxonomic position of the A. bovis strain present in rock sengis by PCRscreening of additional rock sengi samples using 16S rRNA and GroEL assays, optimised for specific detection of the A. bovis-like (sengi) variant, in combination with Sanger sequencing and phylogenetic analysis. Genomic DNA extracted from 160 eastern rock sengi blood samples collected from the Goro Game Reserve, Limpopo Province (n=112) and Ezemvelo Nature Reserve, Gauteng (n=48), South Africa, as part of previous studies, was subjected to the RLB hybridization assay and further molecular characterization. The RLB hybridization assay results revealed that PCR products hybridized with the Theileria/Babesia genus-specific probe in 5% (n=8) of the samples and with the Anaplasma/Ehrlichia genus-specific probe in 31.9% (n=51) of he samples. A total of 86 (53.8%) of the samples tested negative or below the level of detection of the assay; none of the PCR products hybridized with any species-specific probes. Alignment of the near full-length 16S rRNA gene sequences of the A. bovis-like strain previously identified in rock sengi (Harrison et al., 2013) revealed that the A. bovis RLB probe differed at three nucleotide sites under the probe area explaining why the species-specific A. bovis RLB probe failed to hybridise. A new RLB probe was designed on the basis of available 16S rDNA sequences, to allow for the specific detection of the sengi-associated A. bovis-like strains. This custom-designed A. bovis-like (sengi) probe was subsequently used to screen a subset (n=108) of the original eastern rock sengi samples that previously tested RLB positive for the Anaplasma/Ehrlichia genus-specific probe. Despite the custom-design of the probe, only 17 (15.7%) tested positive. The parasite 16S rRNA and GroEL genes were subsequently amplified, purified and sequenced from the 17 A. bovis-like (sengi) RLB positive samples and two previously negative samples. A total of twelve 16S rDNA and eight GroEL sequences were generated. Gene trees were inferred using the Neighbour-joining algorithm, prior to more rigorous Maximum likelihood and Bayesian inferences, using appropriate models of sequence evolution and priors, respectively. BLASTn homology searches showed that the obtained 16S rDNA sequences had 99% sequence identity with A. bovis (Accession no: U03775) previousl described in South Africa, while the GroEL sequences were 88% identical to an uncultured Anaplasma sp. identified in a raccoon (Accession no: JN588562). The phylogenetic analyses revealed that the sequences were most closely related to A. bovis type sequences, but were sufficiently genetically distinct to represent a novel species. In contrast, the 16S rDNA results revealed that the novel sengi-associated Anaplasma lineage falls within an unresolved, polyphyletic lineage that includes A. bovis. Little is known about this sengi-associated variant that is closely related to A. bovis. However, given the relatedness to a pathogen of animal health concern it is important to establish the pathogenicity of this species in order to determine its potential impact on animal and/or human health. It is also imperative that more phylogenetic studies should be performed to clearly place this A. bovis-like (sengi) species within the broader genus Anaplasma phylogeny.