Characterization and growth of polymorphic Rickettsia felis in a tick cell line.

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.

Development of an autodissemination strategy for the deployment of novel control agents targeting the common malaria mosquito, Anopheles quadrimaculatus say (Diptera: Culicidae).

BACKGROUND: The reduced efficacy of current Anopheline mosquito control methods underscores the need to develop new methods of control that exploit unique target sites and/or utilizes novel deployment methods. Autodissemination methodologies using insect growth regulators (IGRs) is growing in interest and has been shown to be effective at controlling Aedes mosquitoes in semi-field and field environments, yet little information exists for Anopheline mosquitoes. Therefore, we tested the hypothesis that female-driven autodissemination of an IGR combined with a new mechanism of action insecticide (Kir channel inhibitor) could be employed to reduce Anopheline populations. METHODOLOGY: We studied the ability of three IGRs to be transferred to the larval habitat during oviposition in laboratory and semi-field environments. Adult mosquitoes were exposed to the chemicals for 4 hours immediately after blood feeding and efficacy was tested using classical methodologies, including adult emergence inhibition and High Performance Liquid Chromatography (HPLC). A complete autodissemination design was tested in a semi-field environment. PRINCIPAL FINDINGS: Larval survivability and adult emergence were significantly reduced in habitats that were visited by novaluron treated adults, but no statistical differences were observed with pyriproxyfen or triflumuron. These data suggested novaluron, but not pyriproxyfen or triflumuron, was horizontally transferred from the adult mosquito to the larval habitat during oviposition. HPLC studies supported the toxicity data and showed that novaluron was present in the majority of larval habitats, suggesting that novaluron can be horizontally transferred by Anopheles quadrimaculatus. Importantly, the combination of novaluron and the Kir channel inhibitor, VU041, was capable of reducing adult and larval populations in semi-field environments. CONCLUSIONS: Novaluron can be transferred to the adult at a greater efficacy and/or is not degraded as quickly during the gonotropic cycle when compared to pyriproxyfen or triflumuron. Pending field confirmation, autodissemination approaches with novaluron may be a suitable tool to manage Anopheles populations.