Tick cell culture isolation and growth of Rickettsia raoultii from Dutch Dermacentor reticulatus ticks

M Pilar Alberdi,Ard M Nijhof,Frans Jongejan,Lesley Bell-Sakyi

doi:10.1016/j.ttbdis.2012.10.020

M Pilar Alberdi, Ard M Nijhof + Show 2 more

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https://doi.org/10.1016/j.ttbdis.2012.10.020

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Abstract

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.

Highlights

Tick cell lines play an increasingly important role in research on ticks and tick-borne pathogenic and symbiotic microorganisms (Bell-Sakyi et al, 2007)
Less is known about the pathogenicity of the Eurasian species R. raoultii, which has been found in Dermacentor reticulatus, D. silvarum, D. nuttalli, and D. marginatus ticks (Mediannikov et al, 2008)
In the 6 surviving primary cultures, cytopathic effects began to appear after 7 months in vitro; Giemsa-stained cytocentrifuge smears revealed the presence of an intracellular rod-shaped Rickettsia-like bacterium infecting varying proportions (90%) of the cells in each culture (Fig. 2A)

Summary

Introduction

Tick cell lines play an increasingly important role in research on ticks and tick-borne pathogenic and symbiotic microorganisms (Bell-Sakyi et al, 2007). Continuous cell lines are available from 4 New World Dermacentor species, some of which have been used for the isolation, propagation, and study of tick-borne Rickettsia spp. Considering the importance of D. reticulatus as a vector of human and livestock disease, we set up primary cultures from embryonic tissues derived from field ticks collected in The Netherlands, with a view to establishing continuous cell lines. Unsuccessful in this aim, we were able to isolate a Rickettsia from the D. reticulatus primary cultures. We describe its isolation, propagation in established tick cell lines, and partial characterisation as the SFG Rickettsia species R. raoultii, confirming previous PCR-based detection in Dutch field ticks (Nijhof et al, 2007a)

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