Dissemination of bloodmeal acquired Rickettsia felis in cat fleas, Ctenocephalides felis
© Thepparit et al.; licensee BioMed Central Ltd. 2013
Received: 17 April 2013
Accepted: 21 May 2013
Published: 24 May 2013
Cat fleas, Ctenocephalides felis, are known biological vectors for Rickettsia felis. Rickettsial transmission can be vertical via transovarial transmission within a flea population, as well as horizontal between fleas through a bloodmeal. The previously undescribed infection kinetics of bloodmeal-acquired R. felis in cat fleas provides insight into the R. felis-flea interaction.
In the present study, dissemination of R. felis in previously uninfected cat fleas fed an R. felis-infected bloodmeal was investigated. At weekly intervals for 28 days, rickettsial propagation, accumulation, and dissemination in gut epithelial cells, specifically in the hindgut and the specialized cells in the neck region of midgut, were observed on paraffin sections of infected cat fleas by immunofluorescence assay (IFA) and confirmed by PCR detection of R. felis 17-kDa antigen gene. IFA results demonstrate ingested rickettsiae in vacuoles during early infection of the gut; lysosomal activity, indicated by lysosome marker staining of freshly-dissected gut, suggests the presence of phagolysosome-associated vacuoles. Subsequent to infection in the gut, rickettsiae spread to the hemocoel and other tissues including reproductive organs. Densely-packed rickettsiae forming mycetome-like structures were observed in the abdomen of infected male cat fleas during late infection. Ultrastructural analysis by transmission electron microscopy (TEM) confirmed the presence and infection characteristics of Rickettsia including rickettsial destruction in the phagolysosome, rickettsial division, and accumulation in the flea gut.
This study intimately profiles R. felis dissemination in cat fleas and further illuminates the mechanisms of rickettsial transmission in nature.
Rickettsia felis has emerged as a cosmopolitan arthropod-borne pathogen infecting various arthropod hosts. First associated with human disease in 1991, this intracellular Gram negative bacteria shares clinical signs with other rickettsial diseases, ranging from fever and generalized maculopapular rash to more severe acute polyneuropathy-like symptoms [1, 2]. Coinciding with the impetus to understand transmission biology, increased recognition of R. felis as a pathogen derives from studies revealing R. felis in approximately 6% of non-malarial, febrile Senegalese and Kenyan patients hospitalized [3, 4].
Rickettsial transmission by arthropod vectors can be vertical, horizontal, or for dynamic organisms such as R. felis, both. In an endosymbiont relationship, some arthropod hosts and their respective Rickettsia spp. are strictly maintained vertically between life cycle stages typically providing a benefit to the host . Alternatively, Rickettsia spp. conferring a negative fitness effect require horizontal transmission for maintenance in arthropod populations. Identification of both transmission paradigms is unique to R. felis. For example, in non-hematophagous insect hosts, R. felis infection is associated with parthenogenesis and maintained 100% transovarially. Clearance of the organism from adult Liposcelis, common booklice, results in decreased longevity, fecundity, and non-viable egg production [6, 7]. Conversely, in the cat flea, Ctenocephalides felis, vertical transmission of R. felis varies, not influencing flea fitness, and horizontal transmission via cofeeding is essential for rickettsial maintenance in flea populations (reviewed in ).
While molecular methods confirm detection of R. felis in a variety of hematophagous arthropods, only cat fleas are known biological vectors. A disseminated infection exists in vertically infected fleas; R. felis has been identified by PCR and microscopy in the midgut epithelial cells (adult and larval fleas), as well as adult flea muscle cells, fat body, tracheal matrix, ovaries, epithelial sheath of testes, and salivary glands [9–11]. Despite the known transmission potential of cat fleas for R. felis, the infection kinetics of horizontally-acquired R. felis in cat fleas remains unexamined. The ability to infect cat fleas with R. felis via an infectious bloodmeal  and show transmission of R. felis between fleas  was recently demonstrated using an artificial host system. To better understand the infection process, the objective of the current study involved monitoring rickettsial infection in naïve fleas as it was acquired via an infectious bloodmeal. A combination of PCR, immunofluorescence assay (IFA), and transmission electron microscopy (TEM) techniques were employed to track novel infection over the duration of the cat flea’s adult life. Through mapping the infection process in the natural host, an appreciation of the ecology of this emerging pathogen and potential points of intervention may be identified.
Source of fleas and rickettsial cultivation
Cat fleas (C. felis Bouche) purchased from Elward II (EL-Labs, Soquel, CA) were reared as previously described . The Louisiana State University (LSU) strain of R. felis was propagated in ISE6, Ixodes scapularis-derived cells, and the R. felis-infected bloodmeal preparation was carried out as described  prior to enumeration using Bac Light viability stain kit (Molecular Probes). A negative bloodmeal control was prepared from uninfected ISE6 cells in the same manner.
Newly emerged, sex-separated cat fleas were pre-fed with heat-inactivated, defibrinated bovine blood for 24 hr prior to exposure to the R. felis-infected or uninfected bloodmeal at a dilution of 8.6 × 1010 rickettsiae per ml. After 24 hr exposure, female and male fleas in each experimental group were mixed equally and maintained on defibrinated bovine blood (non heat-inactivated) with fresh blood replaced as needed for the duration of the experiment. Fleas, for the rickettsial dissemination examination, were collected at weekly intervals for 28 days after mixing the population [designated as day post-exposure (dpe)].
Genomic DNA isolation and PCR amplification
Whole fleas or individual tissues including female gut, ovary, salivary glands, and male rectal ampulla from three fleas were pooled and gDNA was extracted from the ground tissue using the DNeasy blood & tissue kit (Qiagen) according to the manufacturer’s protocol. PCR conditions for detection of the rickettsial 17-kDa antigen gene were described previously .
Flea tissues (described above) were dissected from three fleas and placed in fixative; TEM was carried out at University of Texas Medical Branch (UTMB) as described previously .
Collected fleas were placed in embedding cassettes and fixed for 24 hr in 10% normal formalin. Formalin-fixed specimens were processed and sectioned as previously described . When indicated, flea tissues were dissected and fixed with cold acetone. Rickettsiae in the formalin-fixed sections and dissected fleas tissues were detected by IFA as previously described .
Results and discussion
The current study maps the rickettsial infection process of cat fleas and establishes the baseline infection kinetics towards comparison of infection by rickettsial species and between flea species. Ultimately, Rickettsia- and flea-derived factors critical to infection of fleas and subsequent transmission of R. felis to naïve adult cat fleas can be assessed in this system.
We thank Mark Guillotte and Peter Mottram for the technical assistance and Jacqueline Macaluso for helpful comments. This research was supported by the National Institutes of Health (AI077784).
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