11 Flea-borne rickettsial diseases

R. felis has been identified in cats and cat fleas and is now considered to be the cause of flea-borne spotted fever (cat flea typhus). Cases of fever, rash and lymphadeopathy have been reported from Europe and the Americas. Diagnosis is based on serology and treatment with doxycycline.

Bacteria of the order Rickettsiales are Gram-negative microorganisms that grow in association with eukaryotic cells. ‘Rickettsia’ has long been used as a generic term for many small bacteria that could not be cultivated and were not otherwise identified. However, the progress in taxonomy that was made over the last 35 years with the introduction of molecular techniques resulted in the term ‘rickettsia’ applying only to arthropod-borne bacteria belonging to the genus Rickettsia within the family Rickettsiaceae, in the order Rickettsiales (Brenner et al. 1993). The Rickettsia genus is currently made of 24 recognized species, and also contains several dozens of as yet uncharacterized strains or tick amplicons (Fournier and Raoult 2007). At present the family Rickettsia is divided into three groups, namely the spotted fever group (SFG), the Typhus group, and Rickettsia bellii.

Flea-borne bacterial diseases include cat-scratch disease, plague, murine typhus and flea-borne spotted fever (FBSF) due to R. felis. The first description of a Rickettsia in a cat flea Ctenocephalides felis was in 1918 and it was named R. ctenocephali. It was rediscovered in 1990, when C. felis ticks were examined in California as potential vectors of R. typhi, the agent of murine typhus (Adams et al. 1990). A new rickettsia was observed to these ticks by electronic microscopy and named the ELB agent after the EL laboratory in Soquel, California (Adams et al. 1990). Later in 1994, the bacterium was detected by polymerase chain reaction (PCR) and genomic sequence comparison based on the 17-kDa protein gene sequencing, and was considered to be a new SFG Rickettsia named R. felis (Higgins et al. 1996). These first attempts to define the classification of R. felis have proved problematic and not confirmed since the first isolations were contaminated by R. typhi (Znazen and Raoult 2007). R. felis was definitely characterized and validated as a unique SFG Rickettsia in 2001 when culture conditions using Xenopis laevis tissue cells and mosquito cells at 28°C were established (La ScoLa et al. 2002).

R. felis is a small (0.8–2μm in length and 0.3–0.5μm in diameter) rod-shaped, Gram-negative bacillus that retains basic fuchsin when stained by the Gimenez method (Znazen and Raoult 2007). R. felis presents pili, probably involved in the attachment of the bacteria to other cells and in conjugation (Ogata et al. 2005). The genome of R. felis has been recently sequenced and a number of rickettsial genetic specificities were found. The genome of R. felis (1485 Mb) is circular and larger than other previously sequenced rickettsiae (i.e. R. prowazekii, R. conorii, R. sibirica, R. rickettsii, R. akari, and R. typhi). In R. felis were surprisingly found 2 plasmids (63 kb pRF and 39 kb pRFδ), with one of them to contain the equipment to allow conjugative plasmid transfer (Ogata et al. 2005). When R. felis was compared to other published genomes (R. prowazekii, R. typhi, R. conorii, and R. sibirica) 530 specific open-reading frames (ORFs) were found (Ogata et al. 2005). In addition, R. felis have 22 ankyrin (ank) repeats which are more than any prokaryotes sequenced so far (Ogata et al. 2005).

A high number of transposase (tnp) genes on pRF are also presented and this high occurrence of transposases suggests that pRF genes have been frequently rearranged through recombination mediated by tnp elements (Gillespie et al. 2007). Moreover, R. felis carries 14 spoT genes for its adaptation to the environment (Ogata et al. 2005). Also, 11 tetratricopeptide repeat-containing protein genes, tpr, and 5 families of toxin-antitoxin (TAT) system genes (16 toxins and 14 antitoxin genes) were found (Ogata et al. 2005). The TAT system was considered exceptional in intracellular organisms before its identification in R. felis and is associated with the increase of cell survival during nutritional stress (Ogata et al. 2005). R. felis rel BE has been demonstrated to stabilize plasmid efficiently even when it is chromosomal which may indicate its role in R. felis plasmid maintenance (Ogata et al. 2005).

R. felis is the only known species of SFG that is transmitted by fleas. Today several species of fleas have been associated with R. felis and Table 11.1 illustrates these species and the continent of their isolation. From all these species, the cat flea C. felis (Fig. 11.1) is one of the most frequent external parasites of companion animals worldwide. C. felis is generally regarded as the predominant species to find on dogs, cats and opossums. R. felis DNA have been determined in C. felis in the USA with an infection rate of 3.8% and 7.6% in Israel and up to 12% in the UK (Bauer et al. 2006; Znazen and Raoult 2007). Antibodies to R. felis have been detected in cats but current infection by PCR assay or culture have not been described (Case et al. 2006; Hawley et al. 2007). Although, data suggest that R. felis infection may be prevalent worldwide and humans can be infected after flea bites, the role of mammals, including rodents, hedgehogs, cats, and dogs, in the life cycle and circulation of R. felis is still unclear (Parola et al. 2005a).

R. felis is now known as the agent of FBSF (also called cat flea typhus) a disease with similar clinical manifestations with murine typhus (Schriefer et al. 1994). Cases of FBSF have been reported in Europe including Spain, Canary Islands, Germany, France and the UK, in Asia including Thailand and New Zealand, in Africa including Tunisia and Ethiopia, in America including Brazil and Mexico (Parola et al. 2005b). Debate over the pathogenicity of R. felis for humans was fuelled in 2000 in Mexico when three patients with fever, exanthema, headache, and central nervous system involvement were diagnosed with R. felis infection (Zavala-Velazquez et al. 2000). In 2002, FBSF was found in an adult couple in Germany with high fever (39°C), associated with marked fatigue and headache generalized maculopapular rash of 4 and 2 days. The man had enlarged, painful lymph nodes in the inguinal region and 5 days before the onset of the symptoms patients mentioned a single black, crusted, cutaneous lesion surrounded by a livid halo (on the woman’s right thigh and the man’s abdomen) (Richter et al. 2002). Recently in Spain, an adult couple, having visited a forest area two days before, presented with itching skin lesions, mainly located on flexion areas of the lower extremities, malaise, arthralgia, and pruritic papular rash over the lower extremities, abdomen, and chest. Both patients showed the same clinical picture, although fever was absent in the male. R. felis was identified by PCR (Oteo et al. 2006). R. felis can cause severe debilitating disease in some people—a 34 year old woman from rural eastern Yucatán developed central nervous system involvement with Brudzinski and Kernig signs (Zavala-Velazquez et al. 2000) and a 18 year old man presented severe case of pneumonia following R. felis infection (Zavala-Velazquez et al. 2006). To date no fatal cases have been reported (Znazen and Raoult 2007). However, more studies are needed to determine the real clinical spectrum of symptoms present in FBSF as well as the epidemiology including the role of vectors, reservoirs, hosts, and the case:fatality ratio of this disease.

The mildness of the disease, together with the non specific clinical manifestations, that may be the same in all rickettsioses, leads to the fact that the illness is difficult to recognize. Mild leucopenia, anemia, and thrombocytopenia are usually presented and hyponatremia, hypoalbuminemia, and hepatic and renal abnormalities may also occur (Znazen and Raoult 2007).

R. felis is an obligate intracellular bacterium and its culture and isolation must only be carried out in Biosafety level 3 laboratories. R. felis isolation and establishment can be obtained in XTC-2 cells at low temperature (27°C), on mosquito cell lines (C6/36 cell line) and on Ixodes scapularis-derived tick cell line (ISE6) (Pornwiroon et al. 2006; Horta et al. 2006b; Znazen and Raoult 2007). R. felis can

be detected and identified by PCR and sequencing methods targeting the citrate synthase (gltA), the 16S rRNA, the rickettsia genus-specific 17-kDa antigen, and SFG-specific outer membrane protein A (ompA) or protein B (ompB) genes (Pornwiroon et al. 2006; Znazen and Raoult 2007). Recently a quantitative real-time polymerase chain reaction (qPCR) assay, detecting outer membrane protein B genes (ompB) was proposed for the detection of the R. felis (Henry et al. 2007).

Serological tests are the most frequently used and widely available methods for the diagnosis. The Weil-Felix test was developed 90 years ago but continues to be used by many laboratories around the world. Microimmunofluorescence (MIF) is widely accepted as the reference method and is reliable but does not allow differentiation of infection among the SFG rickettsiae (Hechemy et al. 1989). One limitation of serology is the cross-reactivity that might occur between the antigens of organisms within the same genus and occasionally in different genera. R. felis harbors the antigenic profile of an SFG rickettsia and, by a neighbour-joining analysis based on MIF R. felis, was found to be related to SFG rickettsiae antigenically, clustering with R. australis, R. akari, and R. montanensis (Fang and Raoult 2003). Moreover, antisera to R. felis have low cross-reactivities with R. rickettsii, R. conorii, and R. typhi, which are the only commercially available antigens (Fang and Raoult 2003).

Western blot assays and, if needed, cross absorption studies can be used when differences in titers between several antigens are lower than two dilutions and in cases of cross-reactivity. However these techniques are time and antigen consuming (Znazen and Raoult 2007).

Fleas can also be used as epidemiological tools in order to detect the presence of a pathogen in a specific area. The fleas should be disinfected with iodinated alcohol and then rinsed with sterile water and be crushed before being inoculated onto a shell vial for culture or being processed using molecular methods (Parola et al. 1999). Arthropods which are sent to a reference centre for PCR analysis should be stored dry or frozen at −80°C before transport in dry ice (Gouriet et al. 2005).

The evaluation of antibiotic susceptibility for R. felis showed that doxycycline, fluoroquinolones, telithromycin, and rifampin are effective against the organism but trimethoprim-sulfamethoxazole, β-lactams, and erythromycin are not (Rolain et al. 2002). In fact, genome of R. felis was found to carry a gene coding to β-lactamases of class C and D, streptomycin-resistant protein, and multidrug transport-system protein (Znazen and Raoult 2007).

The conventional antibiotic regimen for SFG rickettsioses is a 7–14 day oral course of doxycycline (200 mg/day) and all reported cases of FBSF rickettsioses have been treated with doxycycline (Znazen and Raoult 2007).

Prevention efforts are aimed at reducing conditions associated with contact with the fleas of cats, dogs, rodents, and hedgehogs. To date no vaccine is available.