Rickettsia felis from Cat Fleas: Isolation and Culture in a Tick-Derived Cell Line

Abstract

Rickettsia felis, the etiologic agent of spotted fever, is maintained in cat fleas by vertical transmission and resembles other tick-borne spotted fever group rickettsiae. In the present study, we utilized an Ixodes scapularisderived tick cell line, ISE6, to achieve isolation and propagation of R. felis. A cytopathic effect of increased vacuolization was commonly observed in R. felis-infected cells, while lysis of host cells was not evident despite large numbers of rickettsiae. Electron microscopy identified rickettsia-like organisms in ISE6 cells, and sequence analyses of portions of the citrate synthase (gltA), 16S rRNA, Rickettsia genus-specific 17-kDa antigen, and spotted fever group-specific outer membrane protein A (ompA) genes and, notably, R. felis conjugative plasmids indicate that this cultivatable strain (LSU) was R. felis. Establishment of R. felis (LSU) in a tick-derived cell line provides an alternative and promising system for the expansion of studies investigating the interactions between R. felis and arthropod hosts.Spotted fever group rickettsiae (SFGR) are obligately intracellular gram-negative bacteria that are typically associated with ticks, with one exception being the etiologic agent of flea-associated spotted fever, Rickettsia felis, described predominately in the cat flea, Ctenocephalides felis. There are a growing number of reports implicating R. felis as a human pathogen. Serological analysis and PCR amplification of rickettsial DNA in human samples coupled with clinical manifestations, including fever and maculopapular rash, characterize rickettsiosis (reviewed in reference 24). In colonized cat fleas, vertical transmission of R. felis is thought to be the primary route of maintenance (3, 43); however, the acquisition mechanism of R. felis by vertebrates and uninfected fleas in nature is unknown. Studies on the ecology of R. felis identified a role for opossums in the transmission cycle (6,32,45). Additionally, a role for companion animals, rodents, and, specifically, their fleas as the potential source of human exposure has been suggested (2,8,27).The relatively recent identification and characterization of R. felis yielded novel aspects for the genus Rickettsia. A rickettsia-like organism, first observed by electron microscopy in the midgut epithelial cells of colonized adult cat fleas, was designated the "ELB agent" after the source of the fleas, the Elward Laboratory (El Soquel, CA) flea colony (1). Amplification of rickettsial genes encoding citrate synthase (gltA) and the genus-specific 17-kDa antigen confirmed the presence of rickettsiae in the Elward Laboratory colony (3), and the name R. felis was proposed (10). Subsequent amplification of the genes encoding the 17-kDa antigen and the 190-kDa antigen (ompA) from a colony of fleas maintained at Louisiana State University (LSU) (Baton Rouge, LA) confirmed the molecular characterization of the bacteria and further classified R. felis as an SFGR (7). Although attempts were unable to produce a sustained culture of either the ELB ...

Tick cell lines: tools for tick and tick-borne disease research

“…of the spotted fever group by cocultivation with midgut tissues from an A. americanum tick [35]. Very recently, the ISE6 cell line was used to isolate previously uncultivated strains of Rickettsia felis from cat fleas [36] and A. phagocytophilum from I. scapularis ticks [37].…”

Transposon Insertion Reveals pRM, a Plasmid ofRickettsia monacensis

“…Demonstration of the existence of plas-mids in other Rickettsia species would strengthen the concept that rickettsiae in general, rather than R. felis in particular, might be amenable to genetic manipulation with a vector based on a rickettsial plasmid. Pornwiroon and colleagues (38), using PCR assays, confirmed the presence of pRF in R. felis strain LSU but could not detect pRF␦, while PCR assays by Ogata and colleagues (32) did not detect pRF-like plasmids in other Rickettsia species.…”

Acquisition ofRickettsia felisby Cat Fleas During Feeding

“…Adult fleas were provided a blood meal via an artificial dog (Wade and Georgi 1988), and eggs, not separated from feces, were reared to adults on sand with artificial diet as previously described (Lawrence and Foil 2002). R. felis (LSU), originally isolated from the Louisiana State University cat flea colony, was propagated in an Ixodes scapularis-derived cell line (ISE6) maintained in modified L15B growth medium, and cellular infection was examined by Diff-Quik (Dade Behring) staining as described previously by Pornwiroon et al (2006).…”