Rickettsia felis and Bartonella henselae in Fleas from Lebanon

Introduction

F leas are worldwide vectors of several important zoonoses transmitted to humans including plague (Yersinia pestis) and murine typhus (Rickettsia typhi), and they also play a role in the cycle of rural epidemic typhus (Rickettsia prowazekii) in the United States (Bitam et al. 2010).

In recent years, fleas have been associated with emerging human infections worldwide, including flea-borne spotted fever (also called cat flea typhus) caused by R. felis and the cat scratch disease (CSD) in connection with Bartonella henselae. The cat flea, Ctenocephalides felis, has been associated with both pathogens (Bitam et al. 2010).

Fleas and human flea-borne infections have been rarely studied in Lebanon (Matossian et al. 1964). In this work, we aimed to detect Rickettsia spp. and Bartonella spp. in fleas collected in this country.

Materials and Methods

From September through November 2009, 105 fleas were collected from 16 cats and 50 fleas were collected from two dogs at sites in the Naqoura and Dayr Kifa regions in southern Lebanon. They were stored in 70% ethanol, transported to France, and identified using standard taxonomic keys (Beaucournu and Launay 1990).

DNA was extracted from each specimen (Berrelha et al. 2009). Each sample was tested by real-time quantitative polymerase chain reaction (qPCR) for the presence of Rickettsia spp. DNA using primers and a Taqman probe targeting a partial sequence of the citrate synthase gene gltA as well as a primer/probe set specific for the R. felis chromosomal bioB gene (Berrelha et al. 2009). Samples that tested positive for gltA by qPCR but negative in the R. felis-specific qPCR were tested by regular PCR using primers CS409d and Rp1258n, which amplify a 396-bp fragment of rickettsial gltA, and primers 190.70 or 190.180, and 190.701, which amplify a 629- to 632-bp fragment of rickettsial ompA (Berrelha et al. 2009).

All DNA samples were also screened using Bartonella genus-specific qPCR with a Taqman probe targeting the 16S/23S rRNA gene intergenic spacer (ITS) (Varagnol et al. 2009). Samples that tested positive for Bartonella by qPCR were thereafter tested using standard PCR with primers that amplified a variable-size ITS of Bartonella spp. (Parola et al. 2003). For Bartonella species identification, DNA sequencing reactions were performed for all samples reamplified in standard Bartonella ITS PCR. Negative controls constituted DNA extracted from noninfected laboratory ticks. R. felis (qPCR) and B. elisabethae (ITS PCR) DNA samples served as positive controls. Sequences were edited, assembled, and identified as described (Berrelha et al. 2009).

Results

With the exception of one flea that was identified as C. canis, all fleas collected from cats were identified as C. felis felis. All fleas collected from dogs were identified as C. canis. All real-time qPCR- and PCR-positive and negative controls tested as expected. Rickettsia DNA was detected by qPCR in 18 (all C. felis felis) of the 155 specimens; 17 of these 18 Rickettsia-positive fleas were positive in the R. felis-specific qPCR, and 1 was negative. This one was tested by regular PCR using primers that amplified parts of the gltA and ompA genes of rickettsiae. However, after several attempts, no sequence permitting bacterial identification was obtained.

Bartonella DNA was detected by qPCR in 8 (all C. felis felis) of the 155 fleas; among them, 5 tested positive by the standard ITS PCR. Sequencing showed that the amplified 723-bp product had 100% homology with B. henselae (GenBank accession number: AJ457177.1) in three of these five fleas. The quality of the other two sequences obtained was insufficient to permit molecular identification despite several attempts. One specimen of C. felis felis was found to be coinfected with both R. felis and B. henselae.

Discussion

R. felis is an obligate intracellular Gram-negative bacterium belonging to the spotted fever group of Rickettsia (Pérez-Osorio et al. 2008). C. felis is currently the only known biological vector of R. felis, and these fleas are able to maintain stable infected progeny through transovarial transmission (Reif and Macaluso 2009). However, molecular evidence of R. felis has been found in various arthropods (Reif and Macaluso 2009). Flea-borne spotted fever caused by R. felis is to date only incompletely described (Pérez-Osorio et al. 2008). The clinical features may include fever, fatigue, headache, generalized maculopapular rash, inoculation eschar(s), and lymph nodes. Cases have been reported from Brazil, Mexico, France, Germany, Spain, Tunisia, Egypt, South Korea, Laos, and Taiwan (Reif and Macaluso 2009). Recently, a study of 134 patients conducted over 9 months in two Senegalese villages identified up to 6% of indigenous febrile nonmalaria cases as R. felis associated (Socolovschi et al. 2010).

B. henselae is a Gram-negative bacterium known as an agent of CSD. Cats have presented as a reservoir for this bacterium, with an increasing gradient of the prevalence of infection from cold climates to warm and humid climates (Chomel and Kasten 2010). Fleas appear to play an important role in the maintenance and transmission of B. henselae among cats. The possibility that B. henselae can be directly transmitted to humans by the cat flea has not been demonstrated experimentally. Transmission of B. henselae by cat fleas seems to occur mainly through infected flea feces that is inoculated by the scratching of the cat's claws (Chomel and Kasten 2010). In addition to the cat flea, new potential vectors have been suggested, including ticks (Chomel and Kasten 2010). CSD is usually characterized by persistent regional lymphadenopathy. Clinical manifestations may include low-grade fever and aching, headache, anorexia, and splenomegaly as well as various atypical manifestations (Chomel and Kasten 2010). B. henselae has been also described as an agent of bacillary angiomatosis and peliosis in highly immunocompromised individuals (Chomel and Kasten 2010).

Although we cannot address the precise prevalence or distribution of R. felis and B. henselae in Lebanon, clinicians there and elsewhere who may see patients returning from this country must now be alerted by the presence of these flea-borne agents in the country.