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Appl. Environ. Microbiol. doi:10.1128/AEM.00025-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Characterization and Growth of Polymorphic Rickettsia felis in a Tick Cell Line

Department of Pathobiological Sciences, Louisiana State University, School of Veterinary Medicine, Skip Bertman Drive, SVM-3213, Baton Rouge, Louisiana 70803; Department of Comparative Biomedical Sciences, Louisiana State University, School of Veterinary Medicine, Skip Bertman Drive, Baton Rouge, Louisiana 70803; Department of Technology and Industries, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand

^* To whom correspondence should be addressed. Email: kmacaluso{at}vetmed.lsu.edu.

	Abstract

Morphological differentiation in some arthropod-borne bacteria is correlated to increased bacterial virulence, transmission potential, and/or as a response to environmental stress. In the current study, we utilized an in vitro model to examine Rickettsia felis morphology and growth under varying culture conditions and bacterial densities to identify potential factors that contribute to polymorphism in rickettsiae. We utilized microscopy (electron microscopy and immunofluorescence), genomic (PCR amplification and DNA sequencing of rickettsial genes), and proteomic (western blotting and LC-MS/MS) techniques to identify and characterize morphologically distinct, long-form R. felis. Without exchange of host cell growth medium, polymorphic R. felis was detected at 12 days post-inoculation when rickettsiae were seeded at a multiplicity of infection (MOI) of 5 and 50. Compared to short-form R. felis, no change in membrane ultrastructure in long-form polymorphic rickettsiae was observed and rickettsiae were up to six times the length of typical short-form rickettsiae. In vitro assays demonstrated that short-form R. felis entered into and replicated in host cells faster than long-form R. felis. However, when both short- and long-form R. felis were maintained in cell-free media for 12 days, the infectivity of short-form R. felis was decreased compared to long-form R. felis which were capable of entering host cells, suggesting that long-form R. felis is more stable outside the host cell. The relationship between rickettsial polymorphism and rickettsial survivorship should be examined further as the yet undetermined route of horizontal transmission of R. felis may utilize metabolically and morphologically distinct forms for successful transmission.