Tick cell lines play an important role in research on ticks and tick-borne pathogenic and symbiotic
microorganisms. In an attempt to derive continuous Dermacentor reticulatus cell lines, embryo-derived
primary cell cultures were set up from eggs laid by field ticks originally collected as unfed adults in The
Netherlands and maintained for up to 16 months. After several months, it became evident that cells in
the primary cultures were infected with a Rickettsia-like intracellular organism. Supernatant medium
containing some D. reticulatus cells was inoculated into cultures of 2 Rhipicephalus (Boophilus) microplus
cell lines, BME/CTVM2 and BME/CTVM23, where abundant growth of the bacteria occurred intracellularly
on transfer to both cell lines. Bacterial growth was monitored by light (live, inverted microscope,
Giemsa-stained cytocentrifuge smears) and transmission electron microscopy revealing heavy infection
with typical intracytoplasmic Rickettsia-like bacteria, not present in uninfected cultures. DNA was
extracted from bacteria-infected and uninfected control cultures, and primers specific for Rickettsia 16S
rRNA, ompB, and sca4 genes were used to generate PCR products that were subsequently sequenced.
D. reticulatus primary cultures and both infected tick cell lines were positive for all 3 Rickettsia genes.
Sequencing of PCR products revealed 99–100% identity with published Rickettsia raoultii sequences. The
R. raoultii also grew abundantly in the D. nitens cell line ANE58, poorly in the D. albipictus cell line DALBE3,
and not at all in the D. andersoni cell line DAE15. In conclusion, primary tick cell cultures and cell lines are
useful systems for isolation and propagation of fastidious tick-borne microorganisms. In vitro isolation of
R. raoultii from Dutch D. reticulatus confirms previous PCR-based detection in field ticks, and presence of
the bacteria in the tick eggs used to initiate the primary cultures confirms that transovarial transmission
of this Rickettsia occurs.