Molecular investigation and phylogeny of Anaplasma spp. in Mediterranean ruminants reveal the presence of neutrophil-tropic strains closely related to A. platys

Abstract

Few data are available on the prevalence and molecular typing of species belonging to the genus Anaplasma in Mediterranean ruminants. In this study, PCR analysis and sequencing of both 16S rRNA and groEL genes were combined to investigate the presence, prevalence, and molecular traits of Anaplasma spp. in ruminants sampled on the Island of Sardinia, chosen as a subtropical representative area. The results demonstrate a high prevalence of Anaplasma spp. in ruminants, with animals infected by at least four of six Anaplasma species (Anaplasma marginale, A. bovis, A. ovis, and A. phagocytophilum). Moreover, ruminants host a number of neutrophil-tropic strains genetically closely related to the canine pathogen A. platys. The high Anaplasma spp. prevalence and the identification of as-yet-unclassified neutrophil-tropic strains raise concerns about the specificity of serological tests routinely used in ruminants and provide additional background for reconstructing the evolutionary history of species genetically related to A. phagocytophilum.

FIG 1

16S rRNA-based phylogenetic analyses of the strains identified in the present study and of eight sequences representative of the different species of the genus Anaplasma. Even though both character-based and distance-based evolutionary analyses generated coinciding trees, only the evolutionary history inferred using the neighbor-joining method is shown, with sum of branch length of 0.32723954. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. All positions containing gaps and missing data were eliminated. There were a total of 760 positions in the final data set. E. canis and R. rickettsii were used as outgroups.

FIG 2

groEL-based phylogenetic analyses of the strains identified in the present study and of 16 sequences representative of the different species of the genus Anaplasma. As in the case of 16S rRNA-based analyses, both character-based and distance-based evolutionary analyses generated coinciding trees, but only the evolutionary history inferred using the neighbor-joining method is shown, with the sum of the branch length being 0.82216554. The evolutionary distances were computed using the Kimura two-parameter method and are in the units of the number of transitional substitution per site. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. All positions containing gaps and missing data were eliminated. There were a total of 470 positions in the final data set. E. canis and R. rickettsii were used as outgroups.

FIG 3

Detection of A. platys-like strains in goat neutrophil granulocytes. Buffy coats obtained from PCR-positive goats were layered in slides by centrifugation and probed with TRITC (tetramethyl rhodamine isothiocyanate)-labeled A. platys DNA probes alternatively combined with fluorescein isothiocyanate (FITC)-labeled anti neutrophil antibodies (upper row) or FITC-labeled antiplatelet antibodies (lower row). DAPI was used to counterstain peripheral blood mononuclear cell nuclei.

FIG 4

Identification of A. platys-like strains by network analyses (right) and cellular distribution in neutrophil granulocytes (lower left). Neutrophil granulocytes were contemporarily probed with a FITC-labeled A. platys DNA-probe and with TRITC-labeled antineutrophil antibodies.