Ecology of Rickettsia felis: A Review

FORUM Ecology of Rickettsia felis: A Review KATHRYN E. REIF AND KEVIN R. MACALUSO 1 Department of Pathobiological Sciences, Louisiana State University, School of Veterinary Medicine, Skip Bertman Dr., Baton Rouge, LA 70803 J. Med. Entomol. 46(4): 723Ð736 (2009) ABSTRACT It has been two decades since the Þrst description of Rickettsia felis, and although a nearly cosmopolitan distribution is now apparent, much of the ecology of this unique microorganism remains unresolved. The cat ßea, Ctenocephalides felis, is currently the only known biological vector of R. felis; however, molecular evidence of R. felis in other species of ßeas as well as in ticks and mites suggests a variety of arthropod hosts. Studies examining the transmission of R. felis using colonized cat ßeas have shown stable vertical transmission but not horizontal transmission. Likewise, serological and molecular tools have been used to detect R. felis in a number of vertebrate hosts, including humans, in the absence of a clear mechanism of horizontal transmission. Considered an emerging ßea-borne rickettsiosis, clinical manifestation of R. felis infection in humans, including, fever, rash, and headache is similar to other rickettsial diseases. Recent advances toward further understanding the ecology of R. felis have been facilitated by stable R. felis-infected cat ßea colonies, several primary ßea isolates and sustained maintenance of R. felis in cell culture systems, and highly sensitive quantitative molecular assays. Here, we provide a synopsis of R. felis including the known distribution and arthropods infected; transmission mechanisms; current understanding of vertebrate infection and human disease; and the tools available to further examine R. felis. KEY WORDS Rickettsia felis, Ctenocephalides felis, cat ßea, review 1 Corresponding author, e-mail: kmacaluso@vetmed.lsu.edu. Although a number of bacteria are associated with blood-feeding arthropods, members of the genus Rickettsia are recognized for their unique relationship with the vector. One such agent is Rickettsia felis, a gramnegative bacterium predominately described in the cat ßea, Ctenocephalides felis. First identiþed in cat ßeas in 1990 (Adams et al. 1990), R. felis has since been identiþed in a variety of ßeas. Within the last 20 yr, there also have been a growing number of reports implicating R. felis as a human pathogen. Despite the increasing appearance of R. felis in the literature, the basic transmission mechanisms of R. felis in nature is unknown. Research progression toward an understanding of the epidemiology and pathogenesis of R. felis has been hampered by the limited knowledge of basic R. felis biology. However, the persistence of R. felis in colonized cat ßeas and more recently the cultivation of several R. felis isolates from arthropod hosts have aided the characterization of R. felis transmission. Phylogenetically, rickettsiae belong to the -sudivision of Proteobacteria, with arthropod-borne Ehrlichia and Anaplasma as close relatives (Dumler et al. 2001). Within the genus Rickettsia, the organization of the disease-causing organisms were traditionally grouped into the typhus group (TG) and spotted fever group (SFG). Recent genome analyses have determined that additional groups exist within the genus; R. felis is currently placed in the transitional group (TRG) (Gillespie et al. 2007, Weinert et al. 2009); however, existence and placement of R. felis in this group has been debated (Fournier et al. 2008). Structurally, rickettsiae are coccobacilli (rodshaped) bacteria that average 0.7Ð2.0 m by 0.3Ð0.5 m in size. The physical composition of Rickettsia includes a typical trilaminar, gram-negative cell wall structure consisting of inner and outer membranes separated by a peptidoglycan layer (Pang and Winkler 1994). A sizable body of literature describing polymorphic rickettsiae exists; however, the signiþcance of the morphologically distinct forms to rickettsial biology is unknown (Gulevskaia et al. 1975, Philip et al. 1983, Sunyakumthorn et al. 2008). Among the more recently recognized emerging rickettsial infections, R. felis has proved challenging to characterize. A rickettsia-like organism, Þrst observed by electron microscopy in the midgut epithelial cells of colonized adult cat ßeas, was designated ELB agent after the source of the ßeas, the Elward Laboratory cat ßea colony (El Soquel, CA) (Adams et al. 1990). AmpliÞcation of rickettsial genes encoding citrate synthase (glta) and the genus-speciþc 17-kDa antigen genes conþrmed the presence of rickettsiae in the Elward Laboratory ßea colony (Azad et al. 1992), and the name R. felis was proposed (Higgins et al. 1996). Subsequent ampliþcation of rickettsial genes 0022-2585/09/0723Ð0736$04.00/0 2009 Entomological Society of America

724 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 46, no. 4 Fig. 1. Reported distribution of R. felis. R. felisðpositive arthropods have been identiþed in the following countries (gray): Afghanistan, Algeria, Argentina, Australia, Canada, Chile, Croatia, Cyprus, Democratic Republic of Congo, Ethiopia, Gabon, Indonesia, Israel, Japan, New Zealand, Peru, Portugal, United Kingdom, and Uruguay. R. felisðpositive arthropods and cases of ßea-borne spotted fever have been identiþed in the following countries (black): Brazil, Egypt,* France, Germany, Laos, Mexico, South Korea, Spain, Taiwan, Thailand, Tunisia,* and the United States (*not reported from arthropods in this country). encoding the 17-kDa antigen and the 190-kDa antigen (ompa) genes from a colony of cat ßeas maintained at Louisiana State University (LSU; Baton Rouge, LA) conþrmed the original molecular characterization and further classiþed R. felis as an SFG Rickettsia (Bouyer et al. 2001). Although initial attempts to produce a sustained culture of either the ELB or the LSU strains of R. felis failed (Radulovic et al. 1995, Bouyer et al. 2001), later attempts to isolate and propagate R. felis from cat ßeas maintained by Flea Data (Freeville, NY) proved successful (Raoult et al. 2001). The Flea Data isolate was designated strain Marseille-URRWXCal 2 (also reported as strain California 2; Ogata et al. 2005) and is the current type strain for R. felis (LaScola et al. 2002). Additional strains of R. felis have been isolated from colonized and wild-caught cat ßeas and propagated using various arthropod cells lines (Horta et al. 2006b, Pornwiroon et al. 2006). The genome and proteome of R. felis (Marseille-URRWXCal 2 ) recently have been described (Ogata et al. 2005, Ogawa et al. 2007), and unique characteristics include the presence of conjugative plasmids prf and prf and the expression of several proposed virulence factors, respectively. To further exemplify this truly dynamic organism, it seems that plasmid content can vary among isolates of R. felis and among Rickettsia species (Baldridge et al. 2008, Fournier et al. 2008). The recognition of R. felis as an emerging pathogen, in conjunction with its intriguing biology, provides exciting avenues for research. As detection of R. felis in arthropods and vertebrate hosts becomes more widespread, the distribution of human ßea-borne rickettsiosis cases also expands (Fig. 1). Here, we summarize the current knowledge regarding the ecology of R. felis, including the expanding range of invertebrate and vertebrate hosts; the potential routes of transmission; the tools and techniques used to diagnose R. felis infection; and the characteristics and dynamics of R. felis rickettsioses. We will also present topics that require further study. Rickettsia felis Infection of Arthropods The cat ßea serves as the primary vector and reservoir of R. felis. Consequently, most of our current understanding of R. felis infection in arthropods comes from the R. felis/c. felis model. The distribution of R. felis within ßeas has been examined using microscopic (transmission electron microscopy [TEM]) and molecular methods (polymerase chain reaction [PCR]). SpeciÞc tissues in which R. felis has been identiþed in adult cat ßeas include the midgut epithelial cells (also identiþed in larval ßeas), muscle cells, fat body, tracheal matrix, ovaries, epithelial sheath of testes, and salivary glands (Adams et al. 1990, Bouyer et al. 2001, Macaluso et al. 2008). Within the epithelial cells of the salivary glands, R. felis were typically free in the cytosol, often surrounded by a halo (cleared cytoplasmic contents) consistent with previous ultrastructural descriptions of R. felis in tick-derived cell culture (Pornwiroon et al. 2006) and in other ßea tissues (Adams et al. 1990). The wide dissemination of R. felis within the ßea host suggests a close association between ßeas and the bacteria; however, a correlation between rickettsial distribution in ßea tissues and the distinct transmission route has not been determined. Quantitative real-time PCR (qpcr) assays have been developed for use as a diagnostic tool and to

July 2009 REIF AND MACALUSO: REVIEW OF Rickettsia felis 725 Table 1. Prevalence of R. felis in institutional and commercial flea colonies Institution Year assessed R. felis prevalence Reference Ag Research Consultants a 1994 70% Higgins et al. 1994 American Biological Supply 1990 0% Adams et al. 1990 1992 0% Azad et al. 1992 Elward Labs (EL) 1992 100% Azad et al. 1992 1994 83% Higgins et al. 1994 2007 0% Pornwiroon et al. 2007 Flea Data 2001 20Ð100% b Raoult et al. 2001 Heska 1998 0% Noden et al. 1998 2008 0% Macaluso et al. 2008 Louisiana State University (LSU) a 1994 86% Higgins et al. 1994 2000 65% Wedincamp and Foil 2000 2002 65% Wedincamp and Foil 2002 2007 94% Pornwiroon et al. 2007 2007 100% Henry et al. 2007 2008 96%, 67%, 35% Reif et al. 2008 Merck a 1994 66% Higgins et al. 1994 Nu-Era Research Farms 1994 83% Higgins et al. 1994 Professional Laboratory Research Service (PLRS) a 1994 50% Higgins et al. 1994 2007 16% Pornwiroon et al. 2007 Texas A&M University b 1994 43% Higgins et al. 1994 University of California 1990 0% Adams et al. 1990 University of Maryland at Baltimore (UMAB) 1992 0% Azad et al. 1992 USDA, MAVERL b 1994 93% Higgins et al. 1994 a Created from or supplemented with originally R. felisðinfected EL Lab ßeas. b Potentially contaminated with originally R. felis-infected EL Lab ßeas. quantitatively assess rickettsial load within ßeas. Both SYBR Green and probe-based qpcr assays have been described as methods for detecting and quantifying R. felis load in ßeas. In an antibiotic susceptibility study, replication of multiple isolates of R. felis were measured in XTC-2 cells treated with various antibiotics; however, assay details and R. felis load calculation methods in this study were not clear (Rolain et al. 2002). In another study, a probe-based method targeting a portion of the ompb gene, which encodes rickettsial outer membrane protein B, was able to differentiate between R. felis and R. typhiðinfected ßeas and had an assay sensitivity of 1 copy/ l (determined using serial dilutions of a plasmid containing a portion the ompb gene) (Henry et al. 2007). Comparison of crude (boiled ßea lysate) versus kit-based DNA extraction methods determined crude extraction of DNA was insensitive, resulting in limited detection of Rickettsia (Henry et al. 2007). A recently developed SYBR Green assay was used to describe the kinetics of R. felis infection in cat ßeas during the metabolically active periods of ßea bloodmeal acquisition and oogenesis (Reif et al. 2008). In this study, a mean rickettsial load of 3.9 10 6 rickettsiae per ßea with a range of 1 10 3 Ð1.6 10 7 rickettsiae per ßea was identiþed. R. felis infection density was also determined by comparing the ratio of rickettsiae to ßea genes. During 9 d of ßeabloodmeal feeding, rickettsial load remained relatively stable; larger-sized, female ßeas had greater rickettsial burdens than male ßeas (Reif et al. 2008). Tissue-speciÞc R. felis infection burden has not been examined in ßeas but may provide clues to the speciþc tissues involved in R. felis transmission. Additional studies examining molecular interactions between R. felis and the arthropod host will provide valuable information to decipher R. felis biology and epidemiology. The presence of R. felis in commercial and institutional ßea colonies has been extensively studied (Table 1). After the initial identiþcation of R. felis in the Elward Laboratory cat ßea colony, a 1999 survey examining the prevalence of R. felis infection in commercial and institutional colonies showed that R. felis infection was widespread in controlled cat ßea colonies (Higgins et al. 1994). In addition to cat ßeas, R. felis has been detected molecularly in a panoply of blood-feeding arthropods worldwide, in 28 countries spanning Þve continents (Table 2). As the only currently deþned biological vector, the infection of cat ßeas by R. felis is concerning from a public health perspective because of the cat ßeasÕ habit of indiscriminate host selection (Azad et al. 1992). In contrast to other described rickettsial species, R. felis is unique because it may potentially infect both insect and acarine hosts and has been detected in both ticks and mites (Ishikura et al. 2003, Choi et al. 2007, Tsui et al. 2007, Oliveira et al. 2008). The prevalence of R. felis infection in surveyed wild-caught arthropods ranges from 0.8 to 100%, depending on species and geographic location, but typically is 25% (Table 2). Although R. felis has been detected molecularly in numerous arthropod species, there exists the potential for arthropods that have just consumed an R. felis-infected bloodmeal to appear positive for infection, despite being a noncompetent vector. Therefore, vector competence must be assessed, and additional studies will be required to discern the biological signiþcance of R. felis infection in these various arthropod hosts.

726 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 46, no. 4 Table 2. Geographic distribution of R. felis in wild-caught arthropods Country Surveyed vector for R. felis Prevalence of R. felis-infection Surveyed mammalian hosts with R. felis vector Reference Algeria Archaeopsylla erinacei and 100% Rodents and hedgehogs Bitam et al. 2006 C. canis Afghanistan rodent ßeas 9% Gerbils Marie et al. 2006 Argentina C. felis 22.6% Dogs Nava et al. 2008 Australia C. felis and Echidnophaga ND Dogs and cats Schloderer et al. 2006 gallinacea Brazil Ctenocephalides spp. ND Dogs and cats Oliveira et al. 2002 C. felis 28Ð80% Dogs Horta et al. 2005 Ctenocephalides sp. ND Dogs and horses Cardoso et al. 2006 Rhipicephalus sanguineus and Amblyomma cajennense C. felis and C. canis 36% C.f., 19.1% C.c. Dogs Horta et al. 2006a C. felis and Polygenis 41% C.f., 3Ð8% P.a. Opossums, dogs and Horta et al. 2007 atopus cats R. sanguineus and C. felis ND Dogs and horses Oliveira et al. 2008 Canada C. felis 18% Cats Kamrani et al. 2008 Chile C. felis 70% Cats Labruna et al. 2007b Croatia Haemaphysalis sulcata 20Ð26% Sheep and goats Duh et al. 2006 Cyprus C. felis 5.6% Rats Psaroulaki et al. 2006 Democratic Republic Pulex irritans, E. gallinacea, 10.7% Collected off host Sackal et al. 2008 of Congo Xenopsylla brasiliensis, Tunga penetrans Ethiopia Fleas ND ND Raoult et al. 2001 a France C. felis 8.1% Cats Rolain et al. 2003 C. felis, C. canis, A. erinacei 17% C.f., 27% C.c., 100% Dogs and cats Gilles et al. 2008a A.e. Gabon C. felis ND Monkey Rolain et al. 2005 Germany C. felis, A. erinacei 100% A.e., 9% C.f. Dogs and cats Gilles et al. 2008b Indonesia Xenopsylla cheopis ND Rodents and shrews Jiang et al. 2006 Israel C. felis 1Ð13% Cats Bauer et al. 2006 Japan Ixodes ovatus, Haemaphysalis flava, Haemaphysalis kitasatoe ND None collected by ßagging Ishikura et al. 2003 Mexico C. felis ND Dogs Zavala-Velazquez et al. 2002 New Zealand C. felis 15% Dogs and cats Kelly et al. 2004 Peru C. felis ND Dogs Blair et al. 2004 Portugal A. erinacei and 3.5% Rodent and hedgehog DeSousa et al. 2006 Ctenophtalmus sp. South Korea Chigger mites ND Wild rodents Choi et al. 2007 Spain ND 40% Dog Perez-Arellano et al. 2005 C. felis ND Dogs and cats Marquez et al. 2002 C. felis ND Dogs and cats Marquez et al. 2006 C. felis and C. canis 28.4% Dogs and cats Blanco et al. 2006 Taiwan Mesostigmata mite ND Rodents Tsui et al. 2007 C. felis 18.8% Tsai et al. 2009 Thailand C. felis and C. canis 4.4% Dogs and ferret-badger Parola et al. 2003 United Kingdom C. felis 6Ð12% Dogs and cats Kenny et al. 2003 C. felis 9Ð21% Dogs and cats Shaw et al. 2004 United States C. felis 1.8% Opossums Williams et al. 1992 C. felis 3.7% Opossums Schriefer et al. 1994b P. irritans ND Dog Azad et al. 1997 C. felis 3.8% 1993, 2.1% 1998 Opossums Boostrom et al. 2002 Anomiopsyllus nudata 0.8% Rodents Stevenson et al. 2005 Carios capensis 1.5% Brown pelican Reeves et al. 2006 C. felis ND Cats Hawley et al. 2007 X. cheopis 24.8% Mice Eremeeva et al. 2008 Uruguay C. felis and C. canis 41% Dogs and cats Venzal et al. 2006 a Reported as unpublished data in this reference. ND, not determined. Transmission Routes of R. felis Global dissemination of C. felis is contributing to the vast distribution of R. felis. Although R. felis has been identiþed molecularly in a number of arthropod species, the cat ßea is currently the only arthropod associated with biological transmission of R. felis. Maintenance of R. felis in the environment is most likely a function of stable vertical transmission, through transstadial and transovarial transmission within cat ßea populations (Azad et al. 1992, Wedincamp and Foil 2002). Mechanisms of possible R. felis transmission routes were postulated with the discovery of R. felis in

July 2009 REIF AND MACALUSO: REVIEW OF Rickettsia felis 727 ßea reproductive tissue (Azad et al. 1992), because other rickettsial species had been shown to be vertically transmitted (Farhang-Azad et al. 1985, Azad and Beard 1998). Initial reports describing R. felis vertical transmission within ßea populations used PCR to detect R. felis in freshly deposited cat ßea eggs, showing that R. felis could be transovarially transmitted, a Þnding that correlated with the high prevalence ( 90%) in the Elward Laboratory ßea colony (Azad et al. 1992). A subsequent study examined the prevalence of R. felis in newly emerged unfed adult cat ßeas from eight colonies in the United States and identiþed R. felis infection in all colonies, with prevalence ranging from 43 to 93% (Higgins et al. 1994). The authors attributed high R. felis infection prevalence in colonized ßeas to efþcient vertical transmission among ßea cohorts and horizontal transmission between ßeas through co-feeding. Because newly emerged, unfed adult ßeas were examined, acquisition of R. felis through vertical transmission is the most reasonable conclusion, because horizontal routes of transmission by ßeas co-feeding or acquisition at immature stages were not deþnitively shown. The most comprehensive R. felis vertical transmission study was performed by Wedincamp and Foil (2002), in which the efþciency of R. felis vertical transmission in cat ßeas over 12 generations without the aid of an infectious bloodmeal or vertebrate host was described. It was reported that R. felis infection prevalence waned from 65 to 2.5% by the 12th generation in ßeas fed bovine blood using an in vitro feeding system compared with the steady 65% infection prevalence for cat-fed ßeas. Although it is plausible that, in the cat-fed colony, R. felis infection prevalence was boosted by occasional rickettsemias, uninfected ßeas fed on these same cats did not acquire infection. R. felis transmission also was not observed by copulation or direct contact between infected and uninfected ßeas. With the exception of vertical transmission among colonized cat ßeas, biological transmission of R. felis by other arthropods has not been described; R. typhi and R. felis are the only pathogenic rickettsial species transovarially transmitted in arthropods other than ticks or mites (Azad et al. 1992). Horizontal transmission of viable R. felis to juveniles by adult ßeas has not been shown; however, the potential for horizontal transmission between arthropod and vertebrate or arthropod and arthropod is likely. Observed by ultrastructural studies in the salivary glands of cat ßeas (Macaluso et al. 2008), saliva produced during blood feeding may provide one route of R. felis transmission to a susceptible vertebrate host. Evidence for rickettsiae transmission from ßea to host through salivary secretion is supported by PCR ampliþcation of R. felis DNA in the blood of laboratory cats exposed to R. felis-infected cat ßeas and their subsequent seroconversion (Wedincamp and Foil 2000). The containment of the ßeas in a feeding capsule ensured that cat hosts did not become exposed to infection during grooming procedures (e.g., ingesting infected ßeas or ßea feces), strongly suggesting horizontal transmission through ßea feeding. Additional studies examining veterinary clinic cats infested with R. felis-infected ßeas were unable to amplify R. felis DNA but could detect antibodies to R. felis, indicating possible past infection or exposure (Hawley et al. 2007, Bayliss et al. 2009). Although DNA and serological analyses do not directly address the viability of R. felis in the mammalian host, large amounts of dead rickettsiae would be required for a mammalian host to generate a strong antibody response. Transmission of rickettsiae in saliva during blood feeding has been shown with other rickettsial species (reviewed in Azad and Beard 1998) and likely is a mechanism of R. felis transmission. Co-feeding between R. felis-infected and uninfected ßeas or other susceptible arthropods poses another possible horizontal transmission route of R. felis. In this scenario, an infected ßea would be able to transmit R. felis in saliva or by regurgitation of rickettsiae to an uninfected ßea feeding nearby. Pathogen transmission through co-feeding has been described for numerous arthropods including Ixodes scapularis in the transmission of Borrelia burgdorferi (Patrican 1997) and in Culex species with the transmission of the West Nile virus (McGee et al. 2007). Rickettsial species, including Rickettsia massiliae in Rhipicephalus turanicus and Rickettsia rickettsii in Dermacentor andersoni, have also been documented to use co-feeding as a transmission strategy (Philip 1959, Matsumoto et al. 2005). In one study, Rickettsia-free ßeas did not acquire R. felis infection after feeding with R. felisinfected ßeas for 5 d on an artiþcial feeding system (Wedincamp and Foil 2002). Neither the presence of rickettsiae in the shared meal or the susceptibility of the uninfected ßeas to infection were determined. Although R. felis transmission through co-feeding was not observed, additional studies are needed before this potential transmission route is ruled out. Larval feeding may be another potential route for R. felis horizontal transmission. Cat ßea larvae feed on adult ßea feces as their main source of nutrition because 80Ð90% of total blood proteins are retained in adult ßea feces (Silverman and Appel 1994). In addition to their regular diet, cat ßea larvae lead cannibalistic lives preying on eggs and other larvae (Silverman and Appel 1994, Lawrence and Foil 2000, Hsu et al. 2002), all of which can lead to a greater success during larval and adult maturation (Lawrence and Foil 2000). Feeding on R. felis-infected feces and/or cannibalism of R. felis-infected eggs/larvae may facilitate R. felis transmission to uninfected ßeas. In one study, uninfected larvae allowed to feed on PCR-positive R. felis-infected feces, eggs, and earlier-instar larvae were unsuccessful in acquiring R. felis; however, R. felis viability and persistence in ßea feces and infective dose had not been assessed (Wedincamp and Foil 2002). Additional studies examining R. felis viability, persistence, and infectivity in adult ßea feces are needed. Transmission of rickettsiae to vertebrates through infected feces is a common transmission strategy used by the TG Rickettsia. The predominant mode of R. typhi transmission is through ßeas feeding on rickett-

728 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 46, no. 4 semic vertebrate hosts and the subsequent shedding up to 10 d of infectious R. typhi in their feces (Azad and Beard 1998). In addition to R. typhi being horizontally transmitted to a vertebrate host through infected ßea feces, vertical transmission within ßea populations has also been described, although at a lower rate (Farhang-Azad et al. 1985). The ability of ßeas to transmit R. typhi both horizontally and vertically suggests similar mechanisms of transmission for R. felis. Although the ability of ßeas to acquire R. felis during blood feeding and then shed viable R. felis in their feces has not been fully examined, in vitro studies have shown an infectious extracellular state of R. felis (Sunyakumthorn et al. 2008). The increased availability of more sensitive detection methods will allow for detection of low-level transmission by other postulated transmission routes. Along with Rickettsia species, numerous bacterial endosymbionts have been described in domestic and wild-caught ßeas (Gorham et al. 2003, Murrell et al. 2003, Pornwiroon et al. 2007, Jones et al. 2008). Abundant endosymbionts in arthropod populations may regulate the ability of R. felis to colonize through either similar tissue tropism or nutrient competition. In ticks, the interference phenomenon, the establishment of one species of Rickettsia inhibiting the transovarial transmission of a second rickettsial species (Burgdorfer 1988, Macaluso et al. 2002), and niche competition have been described. In ßeas, an examination of R. felis and R. typhi co-infection prevalence, where R. felis-infected cat ßeas were fed blood containing R. typhi for 9 d, showed the ability of cat ßeas to acquire dual infection; however, infection rates were at a lower prevalence than in either single infection, indicating that in cat ßeas previously infection with R. felis may inhibit R. typhi infection (Noden et al. 1998). Complete inhibition of R. typhi infection in R. felis-infected ßeas was not observed in all ßeas, and whether or not R. felis is able to inhibit vertical transmission of R. typhi to ßea progeny or inhibit transmission to a susceptible vertebrate host is not known. During the initial description of R. felis, Wolbachia were described in the ovaries of cat ßeas (Adams et al. 1990); subsequently, Wolbachia species have been described in several species of ßeas (Gorham et al. 2003, Rolain et al. 2003, Dittmar and Whiting 2004, Luchetti et al. 2004). The possibility of R. felis and Wolbachia interactions are especially interesting because both organisms occupy many of the same host cells (niches) and possibly compete for similar host resources. In insects, the inßuence of microbial interactions on Wolbachia abundance has been shown (Bordenstein et al. 2006, Goto et al. 2006). SpeciÞcally in ßeas, assessment of microbiota in the R. felisðfree Elward Laboratory cat ßea colony showed Wolbachia to be the predominant bacteria compared with decreased detection of Wolbachia in the LSU colony with an R. felis prevalence of 94% (Pornwiroon et al. 2007). In addition to Wolbachia, R. felis-infected arthropods have also been infected with either Bartonella clarridgeiae (Rolain et al. 2003), Bartonella henselae (Shaw et al. 2004), Bartonella quintana (Rolain et al. 2003, Marie et al. 2006), and Hemoplasma sp. (Shaw et al. 2004) or Spiroplasma, Stenotrophomonas, and Staphylococcus (Pornwiroon et al. 2007). Occupying the same cells or organs in the arthropod host suggests R. felis must be able to contend with other vertically maintained endosymbionts and microbiota acquired during feeding (Pornwiroon et al. 2007). For example, Spiroplasma negatively affected the population of Wolbachia during co-infection of Drosophila melanogaster (Goto et al. 2006). The biological impact of the interspeciþc relationship of co-infecting rickettsial and other bacterial species in the ßea host requires further examination. Bacterial species, such as Wolbachia and Spiroplasma, are readily able to manipulate their hostõs biology, e.g., cytoplasmic incompatibility, male-killing, feminization, to facilitate their transmission (Werren 1997, Dobson et al. 1999, Fry et al. 2004, Montenegro et al. 2006, Duron et al. 2008). The Þtness cost associated with R. felis infection of ßeas is not clear and needs to be examined because other pathogenic Rickettsia, such as R. rickettsii and R. prowazekii, have been shown to negatively impact the Þtness of their host arthropod (Snyder and White 1945, Burgdorfer and Brinton 1975, Niebylski et al. 1999). Like Wolbachia and Spiroplasma, other insect-speciþc Rickettsia species can negatively impact the Þtness of their arthropod hosts, such as the rickettsial species in the twospotted lady beetle, Adalia bipunctata, which is associated with male embryo mortality (Werren et al. 1994, Perlman et al. 2006). The ability of R. felis to inßuence the biology of the arthropod vector may delineate which arthropod species are competent vectors (Pornwiroon et al. 2007). Regulation of R. felis transmission within a vector population has also been hypothesized. One of the most used R. felisðinfected cat ßea colonies is at LSU, with studies reporting R. felis infection prevalence ranging from 35 to 100% (Higgins et al. 1994, Wedincamp and Foil 2000, Henry et al. 2007, Pornwiroon et al. 2007, Reif et al. 2008). In a recent study, the prevalence of R. felis infection in a cat ßea colony was inversely correlated to R. felis infection loads in individual ßeas (Reif et al. 2008). In three trials, ßea- and Rickettsia-naõ ve cats were exposed to 100 cat ßeas, with varying prevalence of R. felis infection, for 9 d, and daily R. felis infection load and infection density were assessed in individual ßea lysates. When colony R. felis infection prevalence was greatest, R. felis infection load in individual ßeas was low. However, as colony R. felis infection prevalence decreased, individual ßea R. felis infection loads increased by as much as 4.75-fold, suggesting that R. felis infection is regulated in the vector population. Regulating infection prevalence and load in a vector population may represent a maintenance strategy for R. felis infection in LSU colonized cat ßeas, whereby the prevalence of waning infection signals greater infection burdens in individual ßeas in an effort to facilitate transmission in a higher percent of progeny or in a susceptible

July 2009 REIF AND MACALUSO: REVIEW OF Rickettsia felis 729 Table 3. Detection of R. felis infection in mammals Country Mammalian hosts positive for R. felis (% positive) Tissue tested Assay Reference Chile Cat 73% a Serum IFA Labruna et al. 2007a Germany Dog NA Serum MIF, Western blot Richter et al. 2002 Peru Rat NA Blood ND Blair et al. 2004 b Spain Dog NA Serum PCR glta, ompa, ompb Oteo et al. 2006 Taiwan Cat Serum IFA Tsai et al. 2009 United States Opossums 30% a Serum DFA Williams et al. 1992 Opossums 33% a Blood PCR 17 kda, glta, IFA Schriefer et al. 1994b Cat 8% Serum ND Higgins et al. 1996 b Cat 81 a,c Serum Serology, PCR Wedincamp and Foil 2000 Cat 100 c Serum IFA Wedincamp and Foil 2002 Opossums 33% 1993 and 22% Serum IFA Boostrom et al. 2002 in 1998 a Cat 15% Serum IFA Boostrom et al. 2002 b Cat 11% Serum IFA Case et al. 2006 Cat NA a Blood PCR glta, ompb Hawley et al. 2007 Cat 3.9% Serum IFA Bayliss et al. 2009 a Infested with R. felis-infected C. felis. b Reported as unpublished data in this reference. c Infected under experimental conditions. NA, not available. mammalian host. Further studies examining the population dynamics of R. felis are required to identify the mechanisms of R. felis infection at the vector population level. Rickettsia felis Infection of Mammals and Humans Serological-based studies have tried to deþne the prevalence and incidence of R. felis infection in speciþc populations of domestic and wild animals. A de- Þnitive mammalian host(s) has not been identiþed in the epidemiology of R. felis and may vary by geographic location and distribution of arthropod vectors. Several peri-domestic species associated with the cat ßea vector have been implicated, including cats (Higgins et al. 1996, Boostrom et al. 2002, Case et al. 2006, Labruna et al. 2007b, Bayliss et al. 2009); dogs (Richter et al. 2002, Oteo et al. 2006); and opossums (Williams et al. 1992, Schriefer et al. 1994b, Boostrom et al. 2002), all of which have been seropositive for R. felis infection outside of laboratory experiments (Table 3). Serological testing of domestic animals in areas with R. felis-infected ßeas showed that infestation of local animals with R. felisðinfected ßeas did not always correlate to seropositive animals (Williams et al. 1992, Horta et al. 2005, Pinter et al. 2008). Examination of cat serum from veterinary clinics by indirect immunoßuorescent assay (IFA) found Þve febrile cats and two control cats reactive against R. felis antigen (Bayliss et al. 2009); however, in a similar study, R. felis DNA could not be ampliþed from cats naturally infested with R. felis-infected cat ßeas (Hawley et al. 2007). A study in Chile examined ßeas from local cats by PCR and cats by serology for R. felis infection and reported that 70% of ßeas were positive for R. felis infection and that 70% of cats had a titer 1:64 for R. felis. Under laboratory conditions, previously ßea- and Rickettsianaõ ve cats were infested with R. felis-infected cat ßeas and most cats either continuously or intermittently exposed to R. felis-infected cat ßeas seroconverted by 4 mo after infestation (Wedincamp and Foil 2000). Although these studies showed horizontal transmission from ßeas to cats, transmission from mammal to arthropod has not been shown, and doubts have been raised about whether the latter occurs (Weinert et al. 2009). However, only occasional horizontal transmission from mammal to arthropod may be needed to enhance or maintain R. felis in nature (Weinert et al. 2009). The lack of a description of a deþnitive mammalian host impedes epidemiological studies of R. felis. Human cases of R. felis infection have been reported in 12 countries worldwide (Fig. 1). The Þrst human case of R. felis infection was reported from Texas in 1994 (Schriefer et al. 1994a); since then, additional human cases have been reported from Spain (Perez-Arellano et al. 2005, Bernabeu-Wittel et al. 2006, Nogueras et al. 2006, Oteo et al. 2006), Germany (Richter et al. 2002), France (Raoult et al. 2001), Egypt (Parker et al. 2007), Brazil (Raoult et al. 2001, Galvao et al. 2006), Mexico (Zavala-Velazquez et al. 2000, Galvao et al. 2006, Zavala-Velazquez et al. 2006), Thailand (Parola et al. 2003), Taiwan (Tsai et al. 2008), South Korea (Choi et al. 2005), Laos (Phongmany et al. 2006), and Tunisia (Znazen et al. 2006) (Table 4). Interestingly, only in Tunisia and Egypt were there reported human R. felis infection cases and no additional reports of R. felisðpositive arthropods. Serosurveys to detect R. felis infection in humans have also been conducted. In Spain, serum samples tested by IFA to survey two populations of people for past exposure to R. felis reported up to 7.1% of people had antibodies to R. felis (Bernabeu-Wittel et al. 2006, Nogueras et al. 2006). Misdiagnosis of R. felis infection as another rickettsial infection, such as murine typhus or Mediterranean spotted fever, is also common (Schriefer et al. 1994a, Raoult et al. 2001), implying a higher prevalence of R. felis infection in areas where multiple rickettsial species overlap.

730 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 46, no. 4 Table 4. Human cases and associated clinical manifestations of flea-borne rickettsiosis caused by R. felis Country No. cases Rash Fatigue Acute Fever Headache Arthralgia Myalgia Other Reference Brazil 2 2/2 ND 2/2 ND ND ND Stupor 2/2, coma 1/2, Raoult et al. 2001 thrombocytopenia 1/2, vomiting 2/2, elevated liver enzymes 1/2 Egypt 1 ND ND 1/1 ND ND ND ND Parker et al. 2007 France 2 2/2 ND 2/2 ND ND ND ND Raoult et al. 2001 Germany 1 1/1 1/1 1/1 1/1 ND ND Splenomegaly, elevated liver enzymes Richter et al. 2002 Laos 1 ND ND 1/1 ND ND ND ND Phongmany et al. 2006 Mexico 3 3/3 3/3 2/3 2/3 1/3 2/3 Nuchal pain 1/3, leucopenia 1/3, leucocytosis 1/3, anemia 1/3, thrombocytosis 1/3, abdominal pain 2/3, nausea 1/3, vomiting 1/3, diarrhea 1/3, photophobia 2/3, hearing loss 1/3, conjunctivitis 1/3 Zavala-Velazquez et al. 2000 1 0/1 ND 1/1 ND ND ND Skin lesions, cough, pulmonary inþltrates Zavala-Velazquez et al. 2006 South Korea 3 ND ND 3/3 ND ND ND ND Choi et al. 2005 Spain 5 0/5 ND 5/5 4/5 4/5 4/5 Dry cough 3/5, abdominal pain 1/5, elevated liver enzymes 5/5, conjunctivitis 1/5 Perez-Arellano et al. 2005 2 1/2 ND 2/2 ND 2/2 ND Malaise 2/2, elevated liver Oteo et al. 2006 enzymes 2/2 Taiwan 1 0/1 1/1 1/1 1/1 0/1 ND Chills, pyuria, acute Tsai et al. 2008 polyneuropathy Thailand 1 0/1 ND 1/1 1/1 ND ND Chills, hepato-splenomegaly, Parola et al. 2003 leukopenia, vomiting Tunisia 8 8/8 ND 8/8 ND ND ND Peripheral adenopathy 2/8, Znazen et al. 2006 interstitial pneumopathy 1/8 USA 1 0/1 ND 1/1 ND ND ND ND Schriefer et al. 1994a ND, not determined. The clinical disease caused by R. felis infection has been designated several names including ßea-borne spotted fever, cat ßea typhus, and R. felis rickettsiosis. Clinical manifestations of R. felis infection are similar to symptoms caused by other rickettsial organisms and can range in severity (Table 4). Typical symptoms can include fever, rash, headache, myalgia, and eschar at the bite site (Brouqui et al. 2007). More severe symptoms can result from visceral (abdominal pain, nausea, vomiting, and diarrhea) and neurologic (photophobia and hearing loss) involvement (Zavala-Velazquez et al. 2000, Galvao et al. 2006). The variable presentation of clinical disease can make diagnosis difþcult (Azad and Radulovic 2003), and reþnement of the full spectrum of clinical disease associated with R. felis infection will expedite accurate diagnoses. Detection and Diagnosis of R. felis Infection in Vertebrate and Invertebrate Hosts Detection of R. felis infection can be accomplished by serological or molecular diagnosis (Tables 3 and 5). Serological assays, such as direct immunoßuorescent assay (DFA), can be used to detect the presence of rickettsiae in humans and animals; however, periods of rickettsemia may quickly subside, making detection difþcult. Rickettsial infection is routinely diagnosed using serological methods that use the human or animalsõ antibody response to rickettsial antigens. The most common methods of serological diagnosis include microimmunoßuorescence (MIF), IFA, enzyme-linked immunosorbent assay (ELISA), Western blotting, and cross-adsorption assays. Despite many serological assays being rickettsial group speciþc, multiple serologic tests are often required to conþrm diagnosis of the speciþc infecting rickettsiae because cross-reactivity between rickettsial species is common. Also, because these assays rely on development of speciþc antibodies that appear at the earliest 7 d after the onset of symptoms, these methods are more commonly used to conþrm suspected rickettsial diagnosis. R. felis has been detected serologically in humans and domestic animals; however, diagnosis through these methods can be a challenge because of lag-time in diagnosis until speciþc antibody development; cross-reactivity with other rickettsial species; variable cut-off titer values; and reagent availability. Further compounding the issues of accurate diagnosis is the clinical similarity of many rickettsial diseases. In areas where R. felis-infected arthropods (speciþcally infected ßeas) were identiþed, several retrospective studies, using serological diagnosis, have been conducted to determine R. felis infection prevalence among local human and animal populations (Bernabeu-Wittel et al. 2006, Nogueras et al. 2006, Pinter et al. 2008). Ideally, serological results should be veriþed by molecular tests, although this is not always possible,

July 2009 REIF AND MACALUSO: REVIEW OF Rickettsia felis 731 Table 5. Assays used to diagnose human cases of flea-borne rickettsiosis Country No. cases Diagnostic method Tissue tested Reference Brazil 2 MIF, nested PCR glta Serum Raoult et al. 2001 1 Diagnostic methods not provided NA Galvao et al. 2006 Egypt 1 PCR 17 kda Blood Parker et al. 2007 France 2 MIF Serum Raoult et al. 2001 Germany 1 MIF, Western blot, nested PCR PS120 protein gene Serum Richter et al. 2002 Laos 1 MIF, Western blot, cross-adsorption Serum Phongmany et al. 2006 Mexico 3 Skin biopsy, PCR 17 kda, serological test Serum, skin Zavala-Velazquez et al. 2000 1 IFA, PCR 17 kda Whole blood Zavala-Velazquez et al. 2006 2 Diagnostic methods not provided NA Galvao et al. 2006 Spain 5 MIF, Western blot, cross adsorption Serum Perez-Arellano et al. 2005 2 PCR glta, ompa, ompb Serum Oteo et al. 2006 33 IFA Serum Bernabeu-Wittel et al. 2006 7 IFA Serum Nogueras et al. 2006 South Korea 3 Multiplex-nested PCR rompb, glta, sequencing and RFLP Serum Choi et al. 2005 Taiwan qpcr groel, 17 kda, ompb, IFA Whole blood Tsai et al. 2008 Thailand 1 IFA, Western blot, cross adsorption Serum Parola et al. 2003 Tunisia 8 IFA, Western blot, cross adsorption Serum Znazen et al. 2006 United States 1 PCR 17 kda, RFLP, Southern blot Whole blood Schriefer et al. 1994a 3 ELISA Serum Wiggers et al. 2005 NA, not available. especially when looking for evidence of past infections. In general, molecular diagnosis of rickettsial infection offers greater sensitivity and speciþcity and can also be made at the time of illness (e.g., biopsy an eschar). Molecular detection usually involves PCR assays to Þrst determine rickettsial infection, followed by restriction fragment-length polymorphism (RFLP) analysis, dot-blot hybridization, or sequencing of rickettsial genes to identify the speciþc infecting rickettsial species. Genes commonly used for rickettsial detection include the Rickettsia genus speciþc 17-kDa antigen gene, the 16S rrna gene, the citrate synthase gene (glta), ompb, and ompa (this gene is truncated in R. felis). Molecular detection is common in arthropod surveys of R. felis infection, and assessment of individual arthropods can help elucidate the prevalence of R. felis in speciþc areas. For example, in a survey of ßeas collected from rodents and a hedgehog in Portugal, R. felis was detected and differentiated from R. typhi in Archaeopsylla erinacei and Ctenophtalmus sp. by ampliþcation and sequencing of glta and ompb (DeSousa et al. 2006). Likewise, in Israel, multiple genotypes of R. felis were identiþed in cat ßeas by detection and sequencing of the 17-kDa antigen gene, glta, ompa, and ompb (Bitam et al. 2006), which shows the speciþcity of molecular assays in differentiating not only rickettsial species but also in differentiating between genotypes of a single rickettsial species. Molecular detection assays have also been used to describe R. felis infection in mammals including humans. In a survey of opossums infested with R. felisinfected cat ßeas, blood samples were collected, and sequencing of ampliþed glta and 17-kDa gene fragments showed R. felis infection in the opossum (Schriefer et al. 1994b). In Germany, R. felis infection was diagnosed in a person and their dog by ampliþcation from serum samples and sequencing of the R. felis PS120 protein gene (Richter et al. 2002). More sensitive molecular tools have been used to identify speciþc rickettsial infections. Development of qpcr assays able to detect as little as 1Ð10 speciþc rickettsial gene copies allow for detection of low-level R. felis infections (Henry et al. 2007, Reif et al. 2008). The development of species-speciþc probes allows for the immediate differentiation and diagnosis of speciþc rickettsial infection (Rozmajzl et al. 2006, Henry et al. 2007). Although molecular assays may be more sensitive and speciþc, limits in machine and reagent availability are common. Molecular assays also only detect current rickettsial infections, whereas serologicalbased assays can diagnose evidence of past infections. Currently, there is no standard protocol for physicians to diagnose R. felis infection in patients, and despite circumstantial evidence of infection, R. felis has not been isolated from a human case of infection. IdentiÞcation of arthropods and mammals infected or able to be infected with R. felis can serve as a platform for developing methods to study R. felis-host interactions in more detail. R. felisðhost interactions can be examined using both in vitro and in vivo systems. Several cell culture systems for in vitro examination of R. felis have been established and provide a valuable tool for examining R. felis biology (Table 6). R. felis can be successfully cultivated in a variety of vertebrate- and invertebrate-derived cell lines, maintained at or below 32 C. Vertebrate-derived cell lines that support R. felis growth include Vero (Radulovic et al. 1995, LaScola et al. 2002), L929 (Radulovic et al. 1995), and XTC-2 (Raoult et al. 2001, LaScola et al. 2002) cells. Arthropod cell lines capable of supporting R. felis growth include ISE6 (Pornwiroon et al. 2006), C6/36 (Horta et al. 2006b), Aa23 (Sakamoto and Azad 2007), and Sua5B (Sakamoto and Azad 2007) cells. The development of bioassays using arthropods and mammals susceptible to R. felis are crucial to study R. felis biology in vivo. Established cat ßea colonies in-

732 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 46, no. 4 Table 6. Cell culture systems for R. felis studies Cell line Type of host cell Temperature R. felis strain Reference Vertebrate Vero African green monkey kidney 34 C NA Radulovic et al. 1995 28Ð32 C Marseille-URRWFXCal 2 Raoult et al. 2001 L929 Murine Þbroblast 34 C NA Radulovic et al. 1995 34 C LSU Sakamoto and Azad 2007 XTC-2 Toad tadpole Xenopus laevis 28 C Marseille-URRWFXCal 2 Raoult et al. 2001 Invertebrate ISE6 Tick Ixodes scapularis 32 C LSU Pornwiroon et al. 2006 C6/36 Mosquito Aedes albopictus 25 C Pedreira Horta et al. 2006b Aa23 Mosquito Aedes albopictus RT LSU Sakamoto and Azad 2007 Sua5B Mosquito Anopheles gambiae RT LSU Sakamoto and Azad 2007 NA, not available; RT, room temperature. fected with R. felis, such as the LSU C. felis colony, provide a valuable tool to study, in vivo, the biology (infection kinetics, tissue tropism, etc.) of R. felis within its primary host and vector. Within the ßea host, the interspeciþc relationship of R. felis with other ßea bacterial endosymbionts that may affect the establishment or transmissibility of R. felis, and vector competence of individual ßeas, can also be addressed. As of yet, no in vivo mammalian model of R. felis infection has been established. Establishment of an animal model will facilitate a more accurate understanding of R. felis, deþning pathogenesis in both animals and humans and delineating the transmission of R. felis from arthropod to animal, and vice versa. IdentiÞcation of key molecules in both the arthropod and mammalian hosts that aid in establishment and maintenance of R. felis infection and subsequent transmission could aid in the development of transmission control strategies within vector populations and vaccine targets for human or animal infection. In summary, R. felis has been detected molecularly in numerous arthropod species around the world; the cat ßea is currently the only deþned biological vector and reservoir. Within the ßea, R. felis infection is disseminated, having been identiþed in the midgut, ovaries, and salivary glands. Under laboratory conditions, maintenance of R. felis in ßea cohorts is primarily by vertical (transovarial and transstadial) transmission. Seroconversion and detection of R. felis DNA in cats infested with R. felis-infected ßeas provides evidence for horizontal transmission; however, deþnitive transmission of viable organisms between mammals and arthropods has not been shown. As the etiologic agent of ßea-borne rickettsiosis, human cases of R. felis infection have been documented in 12 countries around the world, with patients presenting with typical symptoms of rickettsial infection including: fever, rash, and myaliga. Several serological, molecular, and biological assays are available to study the biology and ecology of R. felis. Despite being transmitted by a cosmopolitan vector and listed as an emerging infectious disease, our current understanding of R. felis biology is incomplete, with several issues that need to be addressed. Primarily, the transmission mechanisms of R. felis, speciþcally identiþcation of competent arthropod vectors and vertebrate reservoirs, deþning the alternate routes of R. felis transmission, and developing accurate detection methodology and a deþnition of disease, should receive further attention. Every year there are reports of arthropod, animal, and human cases of R. felis infection from additional countries. Continued development and implementation of molecular tools to delineate the speciþc roles of the arthropod and vertebrate host in the R. felis transmission cycle will guide accurate risk assessment and controls measures. Acknowledgments We thank J.A. 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