Ehrlichia phagocytophila (previously known as Cytoecetes phagocytophila) which causes tick-borne fever (TBF) in sheep and pasture fever in cattle in the UK and mainland Europe is transmitted by the temperate hard tick Ixodes ricinus. The disease in sheep is characterized by fever, leucopenia and immunosuppression. Studies on the pathogenesis and other aspects of the disease have been hampered because the organism has not been cultivated in continuous or primary cell culture systems. This paper describes the first successful cultivation of a European isolate of E. phagocytophila in two continuous cell lines, IDE8 and ISE6, derived from the temperate hard tick Ixodes scapularis. Once adapted to tick cell cultures the organism was serially sub-cultured in new cells by transferring small portions of infected cell suspension every 2 to 3 weeks. The identity of the organism was confirmed by polymerase chain reaction (PCR), with primers specific to the granulocytic ehrlichiae. Sequence analysis of the PCR products amplified from infected tick cells were shown to be identical with those amplified from the blood of sheep infected with the same strain of E. phagocytophila. A susceptible sheep inoculated with a third passage of the tick cell-adapted E. phagocytophila reacted with fever and rickettsiaemia 5 days later, thus satisfying Koch's postulates.
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This project was supported in part by a grant from the Wellcome Trust. We are grateful to Lesley Bell-Sakyi, Centre for Tropical Veterinary Medicine, University of Edinburgh, for her invaluable advice regarding the maintenance of tick-cells in vitro, and to Nigel Jones, University of Liverpool, Department of Veterinary Clinical Science and Animal Husbandry, for providing animal care.
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