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Applied and Environmental Microbiology, May 2008, p. 3151-3158, Vol. 74, No. 10
0099-2240/08/$08.00+0     doi:10.1128/AEM.00025-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Piyanate Sunyakumthorn,1 Apichai Bourchookarn,1,3 Walairat Pornwiroon,1 Connie David,2 Steven A. Barker,2 and Kevin R. Macaluso1*

Department of Pathobiological Sciences, Louisiana State University, School of Veterinary Medicine, Skip Bertman Drive, SVM-3213, Baton Rouge, Louisiana 70803,1 Department of Comparative Biomedical Sciences, Louisiana State University, School of Veterinary Medicine, Skip Bertman Drive, Baton Rouge, Louisiana 70803,2 Department of Technology and Industries, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand3

Received 3 January 2008/ Accepted 16 March 2008

Morphological differentiation in some arthropod-borne bacteria is correlated with 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 various 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 liquid chromatography-tandem mass spectrometry) 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 postinoculation when rickettsiae were seeded at a multiplicity of infection (MOI) of 5 and 50. Compared to short-form R. felis organisms, 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 organisms were maintained in cell-free medium for 12 days, the infectivity of short-form R. felis was decreased compared to long-form R. felis organisms, 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.

* Corresponding author. Mailing address: Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Skip Bertman Drive, SVM-3213, Baton Rouge, LA 70803. Phone: (225) 578-9677. Fax: (225) 578-9701. E-mail: kmacaluso{at}vetmed.lsu.edu

{triangledown} Published ahead of print on 21 March 2008.

Applied and Environmental Microbiology, May 2008, p. 3151-3158, Vol. 74, No. 10
0099-2240/08/$08.00+0     doi:10.1128/AEM.00025-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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