Isolation, propagation and preliminary characterisation of Anaplasma phagocytophilum from roe deer ( Capreolus capreolus) in the tick cell line IDE8

Abstract

Anaplasma phagocytophilum is an obligate intracellular bacterium causing granulocytic anaplasmosis in dogs, horses, and humans and tick-borne fever of ruminants. The bacterium has been detected in a variety of other mammals including wild ruminants without overt clinical signs of disease. Isolates in cell culture have been obtained from humans, dogs, horses, sheep, and ticks, but no strain from wild ruminants exists in cell culture in Europe. From September to November 2010, EDTA blood samples were collected from the jugular vein of 19 shot roe deer from a forest in southern Germany. The presence of specific A. phagocytophilum DNA was demonstrated with a real-time PCR targeting the msp2 gene in all 19 animals. Subsequently, blood cells were used to inoculate the tick cell line IDE8. The first infected IDE8 cells were detected in Giemsa-stained smears 57 days post inoculation. Only one roe deer yielded a positive culture which has been propagated for 9 consecutive passages thus far representing 228 days in culture. Further analysis of the A. phagocytophilum strain was performed by PCR followed by sequencing for the partial 16S rRNA, groEL, msp2, and msp4 genes. Phylogenetic topology of groEL and msp4 sequences placed the roe deer isolate in close proximity to sequences available from roe deer and goats from the neighbouring Alpine regions of Austria and Switzerland, and of msp2 with other ruminant species. This represents the first isolation of A. phagocytophilum in a tick cell line directly from an infected wild ruminant reservoir host, Capreolus capreolus, in Europe. The availability of a cultured A. phagocytophilum strain isolated from roe deer will allow us to study the biological characteristics and the pathogenic potential of this strain as well as to compare its host tropism and its genetic and antigenetic properties with those of other A. phagocytophilum strains from other animal species.