Kevin R. Macaluso

Ecology of Rickettsia felis: A Review

ABSTRACT It has been two decades since the first 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 flea, Ctenocephalides felis, is currently the only known biological vector of R. felis; however, molecular evidence of R. felis in other species of fleas as well as in ticks and mites suggests a variety of arthropod hosts. Studies examining the transmission of R. felis using colonized cat fleas 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 flea-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 flea colonies, several primary flea 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.

Geographic Association of Rickettsia felis-Infected Opossums with Human Murine Typhus, Texas

Application of molecular diagnostic technology in the past 10 years has resulted in the discovery of several new species of pathogenic rickettsiae, including Rickettsia felis. As more sequence information for rickettsial genes has become available, the data have been used to reclassify rickettsial species and to develop new diagnostic tools for analysis of mixed rickettsial pathogens. R. felis has been associated with opossums and their fleas in Texas and California. Because R. felis can cause human illness, we investigated the distribution dynamics in the murine typhus–endemic areas of these two states. The geographic distribution of R. felis-infected opossum populations in two well-established endemic foci overlaps with that of the reported human cases of murine typhus. Descriptive epidemiologic analysis of 1998 human cases in Corpus Christi, Texas, identified disease patterns consistent with studies done in the 1980s. A close geographic association of seropositive opossums (22% R. felis; 8% R. typhi) with human murine typhus cases was also observed.

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 scapularis-derived 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.

Infection and Transovarial Transmission of Rickettsiae in Dermacentor variabilis Ticks Acquired by Artificial Feeding

In this study we examined the efficiency of an in vitro feeding technique using glass microcapillaries as a method of establishing rickettsiae-infected lines of ticks. To quantify the volume ingested by ticks during microcapillary feeding, the incorporation of radiolabeled amino acids in tick gut and hemolymph was calculated. Fifteen of 18 ticks consumed between 0.06 and 6.77 microl. However, ingestion of fluid was not correlated to weight gain during capillary feeding. Uninfected and partially fed laboratory-reared female Dermacentor variabilis ticks were exposed to either Rickettsia montana- or Rickettsia rhipicephali-infected Vero cells via microcapillary tubes, returned to rabbit hosts, and allowed to feed to repletion. All tissues collected from ticks allowed to feed overnight on rickettsiae-infected fluids were found to be infected when examined by IFA. When rickettsiae-infected and uninfected capillary-fed ticks were allowed to feed to repletion and lay eggs, no significant differences in mean engorgement weight or fecundity was observed. When we assessed the efficiency of transovarial transmission of rickettsiae by ticks that imbibed rickettsiae-infected cells by polymerase chain reaction (PCR) and IFA, infection was detected by PCR in the eggs from 85% of the ticks exposed to R. montana and 69% of the ticks exposed to R. rhipicephali. Rickettsial genes were not amplified in samples of the uninfected controls. Examination by IFA of egg samples from females exposed to rickettsiae-infected cells identified rickettsiae in 100% of the samples tested, while the uninfected controls were negative.

Differential Expression of Genes in Uninfected and Rickettsia-Infected Dermacentor variabilis Ticks as Assessed by Differential-Display PCR

ABSTRACT Ticks serve as both the vector and the reservoir for members of the spotted fever group rickettsiae. The molecular interaction(s) that results from this close relationship is largely unknown. To identify genetic factors associated with the tick response to rickettsial infection, we utilized differential-display PCR. The majority of upregulation appeared in the infected tissue. We cloned and sequenced 54 differentially expressed transcripts and compared the sequences to those in the GenBank database. Nine of the 54 clones were assigned putative identities and included a clathrin-coated vesicle ATPase, peroxisomal farnesylated protein, Ena/vasodilator-stimulated phosphoprotein-like protein, α-catenin, tubulin α-chain, copper-transporting ATPase, salivary gland protein SGS-3 precursor, glycine-rich protein, and Dreg-2 protein. Confirmation of the rickettsial influence on the differential expression in the ovaries for a number of these clones was demonstrated by semiquantitative reverse transcription-PCR and Northern blot analyses, resulting in confirmation of six out of nine and three out of four assessed clones, respectively. Further characterization of the clones identified tissue-dependent expression in the midguts and salivary glands. The potential roles of these molecules in the maintenance and transmission of rickettsiae are discussed.

Isolation of a Rickettsial Pathogen from a Non-Hematophagous Arthropod

Rickettsial diversity is intriguing in that some species are transmissible to vertebrates, while others appear exclusive to invertebrate hosts. Of particular interest is Rickettsia felis, identifiable in both stored product insect pests and hematophagous disease vectors. To understand rickettsial survival tactics in, and probable movement between, both insect systems will explicate the determinants of rickettsial pathogenicity. Towards this objective, a population of Liposcelis bostrychophila, common booklice, was successfully used for rickettsial isolation in ISE6 (tick-derived cells). Rickettsiae were also observed in L. bostrychophila by electron microscopy and in paraffin sections of booklice by immunofluorescence assay using anti-R. felis polyclonal antibody. The isolate, designated R. felis strain LSU-Lb, resembles typical rickettsiae when examined by microscopy. Sequence analysis of portions of the Rickettsia specific 17-kDa antigen gene, citrate synthase (gltA) gene, rickettsial outer membrane protein A (ompA) gene, and the presence of the R. felis plasmid in the cell culture isolate confirmed the isolate as R. felis. Variable nucleotide sequences from the isolate were obtained for R. felis-specific pRF-associated putative tldD/pmbA. Expression of rickettsial outer membrane protein B (OmpB) was verified in R. felis (LSU-Lb) using a monoclonal antibody. Additionally, a quantitative real-time PCR assay was used to identify a significantly greater median rickettsial load in the booklice, compared to cat flea hosts. With the potential to manipulate arthropod host biology and infect vertebrate hosts, the dual nature of R. felis provides an excellent model for the study of rickettsial pathogenesis and transmission. In addition, this study is the first isolation of a rickettsial pathogen from a non-hematophagous arthropod.

Prevalence and Infection Load Dynamics of Rickettsia felis in Actively Feeding Cat Fleas

Background Rickettsia felis is a flea-associated rickettsial pathogen recurrently identified in both colonized and wild-caught cat fleas, Ctenocephalides felis. We hypothesized that within colonized fleas, the intimate relationship between R. felis and C. felis allows for the coordination of rickettsial replication and metabolically active periods during flea bloodmeal acquisition and oogenesis. Methodology/Principal Findings A quantitative real-time PCR assay was developed to quantify R. felis in actively feeding R. felis-infected fleas. In three separate trials, fleas were allowed to feed on cats, and a mean of 3.9×106 R. felis 17-kDa gene copies was detected for each flea. A distinct R. felis infection pattern was not observed in fleas during nine consecutive days of bloodfeeding. However, an inverse correlation between the prevalence of R. felis-infection, which ranged from 96% in Trial 1 to 35% in Trial 3, and the R. felis-infection load in individual fleas was identified. Expression of R. felis-infection load as a ratio of R. felis/C. felis genes confirmed that fleas in Trial 3 had significantly greater rickettsial loads than those in Trial 1. Conclusion/Significance Examining rickettsial infection dynamics in the flea vector will further elucidate the intimate relationship between R. felis and C. felis, and facilitate a more accurate understanding of the ecology and epidemiology of R. felis transmission in nature.

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

ABSTRACT 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.

Rickettsia felis, an Emerging Flea-Borne Rickettsiosis

Rickettsia felis is an emerging insect-borne rickettsial pathogen and the causative agent of flea-borne spotted fever. First described as a human pathogen from the USA in 1991, R. felis is now identified throughout the world and considered a common cause of fever in Africa. The cosmopolitan distribution of this pathogen is credited to the equally widespread occurrence of cat fleas (Ctenocephalides felis), the primary vector and reservoir of R. felis. Although R. felis is a relatively new member of the pathogenic Rickettsia, limited knowledge of basic R. felis biology continues to hinder research progression of this unique bacterium. This is a comprehensive review examining what is known and unknown relative to R. felis transmission biology, epidemiology of the disease, and genetics, with an insight into areas of needed investigation.

Identification of Rickettsia felis in the Salivary Glands of Cat Fleas

Rickettsia felis, a flea-associated rickettsial pathogen, has been identified in many tissues, including the digestive and reproductive tissues, within the cat flea, Ctenocephalides felis. We utilized transmission electron microscopy and polymerase chain reaction to identify R. felis in the salivary glands of fed fleas and further define the distribution of R. felis within the arthropod host. We identified Rickettsia-like organisms in salivary glands using electron microscopy. Sequence analysis of portions of the Rickettsia genus-specific 17-kDa antigen gene and R. felis plasmid confirmed the morphological identification of R. felis in cat flea salivary glands. This is the first report of R. felis in tissues critical for horizontal transmission of rickettsiae.