Transmission potential of Rickettsia felis infection by Anopheles gambiae mosquitoes.

A growing number of recent reports have implicated Rickettsia felis as a human pathogen, paralleling the increasing detection of R. felis in arthropod hosts across the globe, primarily in fleas. Here Anopheles gambiae mosquitoes, the primary malarial vectors in sub-Saharan Africa, were fed with either blood meal infected with R. felis or infected cellular media administered in membrane feeding systems. In addition, a group of mosquitoes was fed on R. felis-infected BALB/c mice. The acquisition and persistence of R. felis in mosquitoes was demonstrated by quantitative PCR detection of the bacteria up to day 15 postinfection. R. felis was detected in mosquito feces up to day 14. Furthermore, R. felis was visualized by immunofluorescence in salivary glands, in and around the gut, and in the ovaries, although no vertical transmission was observed. R. felis was also found in the cotton used for sucrose feeding after the mosquitoes were fed infected blood. Natural bites from R. felis-infected An. gambiae were able to cause transient rickettsemias in mice, indicating that this mosquito species has the potential to be a vector of R. felis infection. This is particularly important given the recent report of high prevalence of R. felis infection in patients with "fever of unknown origin" in malaria-endemic areas.

Possible Role of Rickettsia felis in Acute Febrile Illness among Children in Gabon.

Rickettsia felis has been reported to be a cause of fever in sub-Saharan Africa, but this association has been poorly evaluated in Gabon. We assessed the prevalence of this bacterium among children <15 years of age in 4 areas of Gabon; the locations were in urban, semiurban, and rural areas. DNA samples from 410 febrile children and 60 afebrile children were analyzed by quantitative PCR. Overall, the prevalence of R. felis among febrile and afebrile children was 10.2% (42/410 children) and 3.3% (2/60 children), respectively. Prevalence differed among febrile children living in areas that are urban (Franceville, 1.3% [1/77]), semiurban (Koulamoutou, 2.1% [3/141]), and rural (Lastourville, 11.2% [15/134]; Fougamou, 39.7% [23/58]). Furthermore, in a rural area (Fougamou), R. felis was significantly more prevalent in febrile (39.7% [23/58]) than afebrile children (5.0% [1/20]). Additional studies are needed to better understand the pathogenic role of R. felis in this part of the world.

Horizontal transmission of Rickettsia felis between cat fleas, Ctenocephalides felis.

Rickettsia felis is a rickettsial pathogen primarily associated with the cat flea, Ctenocephalides felis. Although laboratory studies have confirmed that R. felis is maintained by transstadial and transovarial transmission in C. felis, distinct mechanisms of horizontal transmission of R. felis among cat fleas are undefined. Based on the inefficient vertical transmission of R. felis by cat fleas and the detection of R. felis in a variety of haematophagous arthropods, we hypothesize that R. felis is horizontally transmitted between cat fleas. Towards testing this hypothesis, flea transmission of R. felis via a bloodmeal was assessed weekly for 4 weeks. Rhodamine B was used to distinguish uninfected recipient and R. felis-infected donor fleas in a rickettsial horizontal transmission bioassay, and quantitative real-time PCR assay was used to measure transmission frequency; immunofluorescence assay also confirmed transmission. Female fleas acquired R. felis infection more readily than male fleas after feeding on a R. felis-infected bloodmeal for 24 h (69.3% and 43.3%, respectively) and both Rickettsia-uninfected recipient male and female fleas became infected with R. felis after cofeeding with R. felis-infected donor fleas (3.3-40.0%). Distinct bioassays were developed to further determine that R. felis was transmitted from R. felis-infected to uninfected fleas during cofeeding and copulation. Vertical transmission of R. felis by infected fleas was not demonstrated in this study. The demonstration of horizontal transmission of R. felis between cat fleas has broad implications for the ecology of R. felis rickettsiosis.

Bartonella clarridgeiae, B. henselae and Rickettsia felis in fleas from Morocco.

A total of 554 fleas were collected in the Moroccan Casablanca and Tiznit regions from domesticated animals and ruminants between August 2007 and October 2008 and were tested for the presence of Rickettsia spp. and Bartonella spp. using molecular methods. For the first time in Morocco, we found Rickettsia felis, the agent of flea-borne spotted fever in Ctenocephalides felis; B. henselae, an agent of cat scratch disease; and Bartonella clarridgeiae, a cat pathogen and potentially a human pathogen.

Dynamic gene expression in salivary glands of the cat flea during Rickettsia felis infection.

The cat flea, Ctenocephalides felis, is an arthropod vector capable of transmitting several human pathogens including Rickettsia species. Earlier studies identified Rickettsia felis in the salivary glands of the cat flea and transmission of rickettsiae during arthropod feeding. The saliva of hematophagous insects contains multiple biomolecules with anticlotting, vasodilatory and immunomodulatory activities. Notably, the exact role of salivary factors in the molecular interaction between flea-borne rickettsiae and their insect host is still largely unknown. To determine if R. felis modulates gene expression in the cat flea salivary glands, cat fleas were infected with R. felis and transcription patterns of selected salivary gland-derived factors, including antimicrobial peptides and flea-specific antigens, were assessed. Salivary glands were microdissected from infected and control cat fleas at different time points after exposure and total RNA was extracted and subjected to reverse-transcriptase quantitative PCR for gene expression analysis. During the experimental 10-day feeding period, a dynamic change in gene expression of immunity-related transcripts and salivary antigens between the two experimental groups was detected. The data indicated that defensin-2 (Cf-726), glycine-rich antimicrobial peptide (Cf-83), salivary antigens (Cf-169 and Cf-65) and deorphanized peptide (Cf-75) are flea-derived factors responsive to rickettsial infection.

Host distribution and pathogen infection of fleas (Siphonaptera) recovered from small mammals in Pennsylvania.

The number of recognized flea-borne pathogens has increased over the past decade. However, the true number of infections related to all flea-borne pathogens remains unknown. To better understand the enzootic cycle of flea-borne pathogens, fleas were sampled from small mammals trapped in central Pennsylvania. A total of 541 small mammals were trapped, with white-footed mice (Peromyscus leucopus) and southern red-backed voles (Myodes gapperi) accounting for over 94% of the captures. Only P. leucopus were positive for examined blood-borne pathogens, with 47 (18.1%) and ten (4.8%) positive for Anaplasma phagocytophilum and Babesia microti, respectively. In addition, 61 fleas were collected from small mammals and tested for pathogens. Orchopeas leucopus was the most common flea and Bartonella vinsonii subspecies arupensis, B. microti, and a Rickettsia felis-like bacterium were detected in various flea samples. To the best of our knowledge, this is the first report of B. microti DNA detected from a flea and the first report of a R. felis-like bacterium from rodent fleas in eastern North America. This study provides evidence of emerging pathogens found in fleas, but further investigation is required to resolve the ecology of flea-borne disease transmission cycles.

Domestic dogs are mammalian reservoirs for the emerging zoonosis flea-borne spotted fever, caused by Rickettsia felis.

Rickettsia felis is an obligate intracellular bacterium that is being increasingly recognized as an etiological agent of human rickettsial disease globally. The agent is transmitted through the bite of an infected vector, the cat flea, Ctenocephalides felis, however there is to date, no consensus on the pathogen's vertebrate reservoir, required for the maintenance of this agent in nature. This study for the first time, demonstrates the role of the domestic dog (Canis familiaris) as a vertebrate reservoir of R. felis. The ability of dogs to sustain prolonged periods of rickettsemia, ability to remain asymptomatically infected with normal haematological parameters and ability to act as biological vehicles for the horizontal transmission of R. felis between infected and uninfected fleas provides indication of their status as a mammalian reservoir of this emerging zoonosis.

Metagenomic analysis of human-biting cat fleas in urban northeastern United States of America reveals an emerging zoonotic pathogen.

An infestation of cat fleas in a research center led to the detection of two genotypes of Ctenocephalides felis biting humans in New Jersey, USA. The rarer flea genotype had an 83% incidence of Rickettsia asembonensis, a recently described bacterium closely related to R. felis, a known human pathogen. A metagenomics analysis developed in under a week recovered the entire R. asembonensis genome at high coverage and matched it to identical or almost identical (> 99% similarity) strains reported worldwide. Our study exposes the potential of cat fleas as vectors of human pathogens in crowded northeastern U.S, cities and suburbs where free-ranging cats are abundant. Furthermore, it demonstrates the power of metagenomics to glean large amounts of comparative data regarding both emerging vectors and their pathogens.

The genome sequence of Rickettsia felis identifies the first putative conjugative plasmid in an obligate intracellular parasite.

We sequenced the genome of Rickettsia felis, a flea-associated obligate intracellular alpha-proteobacterium causing spotted fever in humans. Besides a circular chromosome of 1,485,148 bp, R. felis exhibits the first putative conjugative plasmid identified among obligate intracellular bacteria. This plasmid is found in a short (39,263 bp) and a long (62,829 bp) form. R. felis contrasts with previously sequenced Rickettsia in terms of many other features, including a number of transposases, several chromosomal toxin-antitoxin genes, many more spoT genes, and a very large number of ankyrin- and tetratricopeptide-motif-containing genes. Host-invasion-related genes for patatin and RickA were found. Several phenotypes predicted from genome analysis were experimentally tested: conjugative pili and mating were observed, as well as beta-lactamase activity, actin-polymerization-driven mobility, and hemolytic properties. Our study demonstrates that complete genome sequencing is the fastest approach to reveal phenotypic characters of recently cultured obligate intracellular bacteria.

Cat fleas (Ctenocephalides felis) carrying Rickettsia felis and Bartonella species in Hong Kong.

Fleas are commonly recorded on stray as well as domestic dogs and cats in Hong Kong. Fleas can be a major cause of pruritus in dogs and cats and also vectors of potentially zoonotic bacteria in the genera Rickettsia and Bartonella. Morphological examination of 174 fleas from dogs and cats living in Hong Kong revealed only cat fleas (Ctenocephalides felis). Cytochrome c oxidase subunit 1 gene (cox1) genotyping of 20 randomly selected specimens, revealed three cox1 haplotypes (HK-h1 to HK-h3). The most common haplotype was HK-h1 with 17 specimens (17/20, 85%). HK-h1 was identical to cox1 sequences of fleas in Thailand and Fiji. HK-h1 and HK-h2 form a distinct cat flea cox1 clade previously recognized as the Clade 3. HK-h3 forms a new Clade 6. A multiplex Bartonella and Rickettsia real-time PCR of DNA from 20 C. felis found Bartonella and Rickettsia DNA in three (15%) and ten (50%) C. felis, respectively. DNA sequencing confirmed the presence of R. felis, B. clarridgeiae and Bartonella henselae. This is the first reported study of that kind in Hong Kong, and further work is required to expand the survey of companion animals in the geographical region. The sampling of fleas on domestic cats and dogs in Hong Kong revealed them to be exclusively infested by the cat flea and to be harbouring pathogens of zoonotic potential.

Rickettsia felis, from culture to genome sequencing.

Rickettsia felis has been recently cultured in XTC2 cells. This allows production of enough bacteria to create a genomic bank and to sequence it. The chromosome of R. felis is longer than that of previously sequenced rickettsiae and it possess 2 plasmids. Microscopically, this bacterium exhibits two forms of pili: one resembles a conjugative pilus and another forms hair-like projections that may play a role in pathogenicity. R. felis also exhibits several copies of ankyrin-repeat genes and tetratricopeptide encoding gene that are specifically linked to pathogenic host-associated bacteria. It also contains toxin-antitoxin system encoding genes that are extremely rare in intracellular bacteria and may be linked to plasmid maintenance.

Prevalence of Rickettsia and Bartonella species in Spanish cats and their fleas.

The aim of this study was to determine the prevalence of Bartonella henselae, Rickettsia felis, and Rickettsia typhi in fleas and companion cats (serum and claws) and to assess their presence as a function of host, host habitat, and level of parasitism. Eighty-nine serum and claw samples and 90 flea pools were collected. Cat sera were assayed by IFA for Bartonella henselae and Rickettssia species IgG antibodies. Conventional PCRs were performed on DNA extracted from nails and fleas collected from cats. A large portion (55.8%) of the feline population sampled was exposed to at least one of the three tested vector-borne pathogens. Seroreactivity to B. henselae was found in 50% of the feline studied population, and to R. felis in 16.3%. R. typhi antibodies were not found in any cat. No Bartonella sp. DNA was amplified from the claws. Flea samples from 41 cats (46%) showed molecular evidence for at least one pathogen; our study demonstrated a prevalence rate of 43.3 % of Rickettsia sp and 4.4% of Bartonella sp. in the studied flea population. None of the risk factors studied (cat's features, host habitat, and level of parasitation) was associated with either the serology or the PCR results for Bartonella sp. and Rickettsia sp.. Flea-associated infectious agents are common in cats and fleas and support the recommendation that stringent flea control should be maintained on cats.

A Case of Rickettsia felis Infection Imported From Nepal.

Rickettsia felis is an emerging spotted fever group pathogen that may be responsible for potentially life-threatening infections. A cosmopolitan distribution has been postulated though most human cases were observed in Africa and the Americas. We report an imported case from Nepal that occurred in an Italian tourist who presented with a 1-week history of fever, headache, nausea, vomiting, and a mild maculopapular rash 14 days after return.

Pathogen carriage by the cat flea Ctenocephalides felis (Bouché) in the United Kingdom.

The carriage of Bartonella, Rickettsia felis and haemoplasma species was investigated in cat fleas (Ctenocephalides felis) collected from 121 cats and dogs in the United Kingdom. DNA extracted from fleas was analysed using genus and species-specific PCR and amplicons were characterised using DNA sequencing. Fifty percent of flea samples were PCR positive for at least one pathogen. Twenty one percent were positive for R. felis, 17% for Bartonella henselae, 40% for haemoplasma species and 20% were infected with more than one of the pathogen species studied. It is clear from the results in this study that companion cats and dogs are commonly infested with Ct. felis carrying bacterial pathogens of significance to human and animal health. These findings raise the possibility that Ct. felis found on dogs and cats are a potential source of infection with such pathogens for humans.

Vertical transmission of Rickettsia felis in the cat flea (Ctenocephalides felis Bouché).

Rickettsia felis can be maintained in cat fleas by vertical transmission for up to 12 generations without the benefit of an infected host. Horizontal transmission or the acquisition of R. felis by fleas feeding on cats or artificially infected meals were not demonstrated in this study. Horizontal transmission of R. felis by the ingestion of feces or eggs by flea larvae was not detected. We also tested for and found no evidence to support horizontal transmission by contact among positive fleas and negative fleas.