Propagation of the Israeli vaccine strain of Anaplasma centrale in tick cell lines

Anaplasma centrale has been used in cattle as a live blood vaccine against the more pathogenic Anaplasma marginale for over 100 years. While A. marginale can be propagated in vitro in tick cell lines, facilitating studies on antigen production, immunisation and vector-pathogen interaction, to date there has been no in vitro culture system for A. centrale. In the present study, 25 cell lines derived from 13 ixodid tick species were inoculated with the Israeli vaccine strain of A. centrale and monitored for at least 12 weeks by microscopic examination of Giemsa-stained cytocentrifuge smears. Infection of 19 tick cell lines was subsequently attempted by transfer of cell-free supernate from vaccine-inoculated tick cells. In two separate experiments, rickettsial inclusions were detected in cultures of the Rhipicephalus appendiculatus cell line RAE25 28-32 days following inoculation with the vaccine. Presence of A. centrale in the RAE25 cells was confirmed by PCR assays targeting the 16S rRNA, groEL and msp4 genes; sequenced PCR products were 100% identical to published sequences of the respective genes in the Israeli vaccine strain of A. centrale. A. centrale was taken through three subcultures in RAE25 cells over a 30 week period. In a single experiment, the Dermacentor variabilis cell line DVE1 was also detectably infected with A. centrale 11 weeks after inoculation with the vaccine. Availability of an in vitro culture system for A. centrale in tick cells opens up the possibility of generating a safer and more ethical vaccine for bovine anaplasmosis.