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Human Rickettsia felis infection, Canary Islands, Spain.

We report the first cases of human infection by Rickettsia felis in the Canary Islands. Antibodies against R. felis were found in 5 adsorbed serum samples from 44 patients with clinically suspected rickettsiosis by Western blot serology. Fleas from 1 patient's dog were positive for R. felis by polymerase chain reaction.


Rickettsia felis is an intracellular bacterium (genus Rickettsia, spotted fever group [SFG]) (1,2). Its biological cycle involves the cat flea (Ctenocephalides felis) as the main vector (3). R. felis has been found in C. felis and C. canis in the Americas, Europe, Africa, Asia, and Oceania (1,3-6). Human disease caused by R. felis was unknown until 1994 (4). Since then, R. felis infection has been reported in Mexico (3 patients) (7), Germany (1 patient) (8), Brazil (2 patients) (1), and France (2 patients) (1). The clinical manifestations of the disease include high fever, rash, and elevation of liver enzymes (1,4,7). Exposure to fleas or to flea-prone animals is sometimes recorded (7,8).

On the Canary Islands (Atlantic islands of Spain), autochthonous cases of murine typhus have been reported (9). Although we suspected that some patients with a clinical picture of murine typhus actually had R. felis infection, we were not able to confirm this hypothesis. Therefore, 44 serum samples from 44 patients from the Canary Islands with suspected murine typhus were sent to the Unite des Rickettsies in Marseille, France, for specific serologic tests. Here, we describe the first 5 human infections caused by R. felis on the Canary Islands.

The Study

Forty-four patients were recruited for a prospective study of fever of intermediate duration (i.e., fever without focal symptoms lasting 7-28 days). Demographic, clinical, and laboratory data were collected for all patients. Chest radiographs and blood and urine cultures were taken. Antibodies against R. typhi were tested by direct immunofluorescence test (bioMerieux, Marcy L'Etoile, France) in the Canary Islands. Among the 44 patients, 24 showed a positive serologic result. Antibodies against other agents (Coxiella burnetii, R. conorii, Leptospira spp., Epstein-Barr virus, cytomegalovirus, HIV, and hepatitis B virus) were also tested; all were negative.

To search for evidence of infection with R. felis, all serologic results were confirmed by microimmunofluorescence (MIF) in France, as previously described (10). Systematic testing of SFG rickettsia antigens present in Europe and Africa was performed in parallel. The MIF procedure was followed by the use of Western blot and cross-adsorption studies. An immunofluorescence assay was considered positive if immunoglobulin G (IgG) titers were >1:64 or if IgM titers were >1:32. When cross-reactions were noted between the rickettsial antigens, the analysis comprised 3 steps. First, a rickettsial antigen was considered to represent the agent of infection when IgG or IgM antibody titers against this antigen were [greater than or equal to] 2 serial dilutions higher than titers of IgG or IgM antibody against other rickettsial antigens (11). Second, when the difference in titers between R. felis and other antigens was <2 dilutions, Western blot assays were performed. A rickettsial antigen was considered the agent of infection when sera reacted only against the specific protein of this antigen. Expected molecular masses of the specific proteins were [approximately equal to] 125 kDa for R. typhi and 31 kDa for R. felis. Finally, when Western blot assays were not diagnostic, cross-adsorption studies were performed, as previously described (12). Specific diagnosis criteria after cross-adsorptions studies included a Western blot assay that showed exclusive reactivity with specific proteins of a sole agent. If reactivity with the 2 tested agents was still observed, diagnosis of an indeterminate rickettsial disease was made. With this strategy, patients were classified by 3 types: R. felis infection, R. typhi infection, and indeterminate rickettsial disease.

Five fleas from the dog of 1 R. felis-infected patient were tested by polymerase chain reaction (PCR) (3). DNA was extracted and amplified with primers that targeted the citrate synthase sequence, as previously described (3). For negative controls, we used sterile water and infection-free fleas previously tested in our laboratory; both negative controls were tested after every 7 samples. Amplicons were separated by electrophoresis on 1% agarose gels and then purified by using a QIAquick PCR purification kit (Qiagen, Hilden, Germany), as described by the manufacturer. PCR products were sequenced by using the d-rhodamine terminator-cycle sequencing kit (PE Applied BioSystems, Courtabeuf, France), as described by the manufacturer. The sequences obtained were compared with those available in the GenBank DNA database by using the program Basic Local Alignment Search Tool (BLAST, version 2.0, National Center for Biotechnology Information (

A rickettsial infection was diagnosed by using MIF for 31 of the 44 patients: 5 patients with the final diagnosis of R. felis infection, 13 with the diagnosis of R. typhi infection, and 13 with the diagnosis of indeterminate rickettsioses (Table). The diagnosis of R. felis infection was based on Western blot results on adsorbed sera for all patients. All the antibodies of these patients were removed when the R. felis-adsorbed sera were analyzed with R. typhi and R. felis antigens, whereas antibodies to R. felis remained when the R. typhi-adsorbed sera were analyzed.

Western blots performed with unadsorbed and adsorbed sera are represented in the Figure. Features of patients are indicated in the online appendix ( ncidod/EID/vol11no12/05-0711_app.htm). Some differences were found between groups. The interval between the beginning of clinical signs and symptoms and evaluation was significantly more prolonged in the R. felis group than others. In the R. typhi group, odynophagia, cough, and rash were more frequent. When we compared biologic data, no difference was observed between R. typhi and R. felis groups, except for milder hypertransaminasemia in the latter group. Finally, 2 PCR products were obtained and sequenced from 2 fleas. Both sequences were 100% similar to R. felis citrate synthase gene in GenBank accession no. AF210692. No fleas were positive for R. typhi. Amplification was unsuccessful in all negative controls.



In the past 10 years, application of molecular tools has resulted in discovery of several new species of pathogenic rickettsiae, including R. felis. Since then, this bacterium was cultivated, and its genome was sequenced (1,13). Its pathogenic role was recently demonstrated in patients with serologic evidence of infection in Brazil, France, and Germany (1). R. felis DNA has also been detected in sera in Texas, Mexico, Brazil, and Germany (1,4,8,14). Autochthonous human rickettsioses that occur in the Canary Islands include murine typhus; SFG infections have never been reported (9). We diagnosed 5 cases of acute R. felis infection (15). The clinical picture is globally similar to murine typhus (4). However, the R. felis infection in our study seemed to be milder, and no skin rash was observed. The incidence of R. felis infection in the Canary Islands is probably underestimated; therefore, serologic tests for R. felis should be performed in patients with prolonged fever or suspected rickettsioses.

Cross-reactions in serologic testing for R. felis are unpredictable (3). In our study, patients with R. felis infection more frequently had high antibody titers (IgM >1:32 and IgG >1:64) to R. conorii and R. typhi (2 of 5 patients) than did patients with R. typhi infection (0 of 13). On the basis of R. felis data, we conclude that patients with R. felis infection may have no cross-reactivity with other rickettsiae, cross-reactivity with SFG rickettsiae, or cross-reactivity with both SFG rickettsiae and R. typhi. Genetic support for cross-reactivity with R. conorii is plausible because most membrane proteins of SFG and R. felis are extremely close (surface cell antigen [Sca] family). Genome analysis showed that several genes were present in R. felis and R. typhi and absent for other SFG, which could explain the cross-reactivity between R. felis and R. typhi (13). Finally, if <2-fold differences in IgG/IgM titers between R. felis and other SFG and typhus group rickettsiae are observed, only Western blot and cross-adsorptions will allow a specific diagnosis once reactivity has disappeared after adsorption with R. felis antigen. By contrast, a band of [approximately equal to] 31 kDa for the R. felis antigen persists after adsorption with R. conorii and R. typhi.


We thank Kelly Johnston and Silpak Biswas for reviewing the manuscript.

This research was supported in part by a grant from the Canary Foundation of Investigation and Health.


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(2.) La Scola B, Meconi S, Fenollar F, Rolain JM, Roux V, Raoult D. Emended description of Rickettsia felis (Bouyer et al. 2001), a temperature-dependent cultured bacterium. Int J Syst Evol Microbiol. 2002;52:2035-41.

(3.) Rolain JM, Franc M, Davoust B, Raoult D. Molecular detection of Bartonella quintana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis, and Wolbachia pipientis in cat fleas, France. Emerg Infect Dis. 2003;9:338-42.

(4.) Schriefer ME, Sacci JB Jr, Dumler JS, Bullen MG, Azad AF. Identification of a novel rickettsial infection in a patient diagnosed with murine typhus. J Clin Microbiol. 1994;32:949-54.

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(11.) Raoult D, Fournier P, Fenollar F, Jensenius M, Prioe T, De Pina J, et al. Rickettsia africae, a tick-borne pathogen in travelers to Sub-Saharan Africa. N Engl J Med. 2001;344:1504-10.

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(13.) Ogata H, Renesto P, Audic S, Robert C, Blanc G, Fournier PE, et al. The genome sequence of Rickettsia felis identifies the first putative conjugative plasmid in an obligate intracellular parasite. PLoS Biology. 2005;3:248.

(14.) Zavala-Velazquez JE, Zavala-Castro JE, Vado-Solis I, Ruiz-Sosa JA, Moron CG, Bouyer DH, et al. Identification of Ctenocephalides felis fleas as a host of Rickettsia felis, the agent of a spotted fever rickettsiosis in Yucatan, Mexico. Vector Borne Zoonotic Dis. 2002;2:69-75.

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Jose-Luis Perez-Arellano, * ([dagger]) (1) Florence Fenollar, ([double dagger]) (1) Alfonso Angel-Moreno,* ([dagger]) Margarita Bolanos, * ([dagger]) Michele Hernandez, * ([dagger]) Evora Santana, * ([dagger]) Marion Hemmersbach-Miller, * ([dagger]) Antonio-M Martin, * ([dagger]) and Didier Raoult ([double dagger])

* Hospital Universitario Insular de Las Palmas, Canary Islands, Spain; ([dagger]) Universidad de Las Palmas de Gran Canaria, Canary Islands, Spain; and ([double dagger]) Universite de la Mediterranee, Marseille, France

(1) These authors contributed equally to this article.

Address for correspondence: D. Raoult, Unite des Rickettsies, CNRS UMR 6020, Faculte de Medecine, Universite de la Mediterranee, 27 Bd Jean Moulin, 13385 Marseille CEDEX 05, France; fax: 33-4-91-38-77-72; email:

Dr Perez-Arellano is chief of the Infectious Diseases and Tropical Medicine Service and professor in the Department of Medical and Surgical Sciences, Health Sciences Faculty, University of Las Palmas de Gran Canaria. His main research interests are imported and emerging infectious diseases.
Table. Clinical, epidemiologic, and biological data between Rickettsia
felis group, R. typhi group, and indeterminate rickettsiosis group

Characteristic R. felis R. typhi

No. 5 13
Mean age, y * 45 (16) 29 (14)
Sex (M/F) 5/0 10/3
Contact with dogs or 4/5 11/13
 cats ([section])
Interval between clinical 12 (9.5-14) 9 (8.5-10.2)
 picture and evaluation,
 d ([paragraph])
Fever ([section]) 5/5 13/13
Maximal temperature 39.3 (0.8) 39.6 (0.5)
 ([degrees]C) *
Headache ([section]) 4/5 12/13
Conjunctivitis ([section]) 1/5 3/13
Arthralgia/myalgia ([section]) 4/5 6/13
Odynophagia ([section]) 0/5 6/13
Dry cough ([section]) 3/5 8/13
Nausea/vomiting ([section]) 0/5 2/13
Abdominal pain ([section]) 1/5 1/13
Rash ([section]) 0/5 9/13
Past or actual tick 1/5 2/13
 bite ([section])
Hepatomegaly ([section]) 1/5 6/13
Splenomegaly ([section]) 0/5 3/13
Anemia (hemoglobin <13 mg/dL) 0/5 3/13
Normal blood leukocyte counts 5/5 10/13 **
Normal platelet counts 4/5 11/12 ([double dagger]
 (150,000-400,000/[micro]L) [double dagger])
Normal ratio prothrombin time 4/4 10/13
Normal ESR (<10 mm/h) 1/4 11/12
Normal creatinine blood level 5/5 11/13
 (62-106 [micro]mol/L)
Normal sodium blood level 2/4 10/13
 (136-144 mmol/L)
Elevated AST (>35 IU/L) 4/5 8/13
Mean AST (U/L) 123 254
Elevated ALT (>45 IU/L) 5/5 8/13
Mean ALT (U/L) ([paragraphs]) 185 (71-374) 354 (55-1,368)
Elevated GGT (>55 IU/L) 2/5 3/13
Elevated total serum protein 0/5 0/12
 concentration (>80 g/L)
Elevated gamma globulin 2/5 5/12
 concentration (>13 g/L)

Characteristic Indeterminate

No. 13
Mean age, y * 40 (17)
Sex (M/F) 12/1
Contact with dogs or 11/13
 cats ([section])
Interval between clinical 9 (7.9-13.4)
 picture and evaluation,
 d ([paragraph])
Fever ([section]) 13/13
Maximal temperature 39.40
 ([degrees]C) *
Headache ([section]) 13/13
Conjunctivitis ([section]) 2/13
Arthralgia/myalgia ([section]) 5/13
Odynophagia ([section]) 0/13
Dry cough ([section]) 2/13
Nausea/vomiting ([section]) 1/13
Abdominal pain ([section]) 0/13
Rash ([section]) 6/13
Past or actual tick 1/13
 bite ([section])
Hepatomegaly ([section]) 5/13
Splenomegaly ([section]) 2/13
Anemia (hemoglobin <13 mg/dL) 1/13
Normal blood leukocyte counts 11/13 ([dagger][dagger])
Normal platelet counts 11/13 ([double dagger]
 (150,000-400,000/[micro]L) [double dagger])
Normal ratio prothrombin time 11/13
Normal ESR (<10 mm/h) 3/11
Normal creatinine blood level 10/13
 (62-106 [micro]mol/L)
Normal sodium blood level 10/12
 (136-144 mmol/L)
Elevated AST (>35 IU/L) 5/12
Mean AST (U/L) 72
Elevated ALT (>45 IU/L) 6/12
Mean ALT (U/L) ([paragraphs]) 86 (34-292)
Elevated GGT (>55 IU/L) 4/12
Elevated total serum protein 1/12
 concentration (>80 g/L)
Elevated gamma globulin 8/10
 concentration (>13 g/L)

Characteristic p value

No. --
Mean age, y * NS ([dagger])
Sex (M/F) NS ([double dagger])
Contact with dogs or NS ([double dagger])
 cats ([section])
Interval between clinical <0.05 (#)
 picture and evaluation,
 d ([paragraph])
Fever ([section]) NS ([double dagger])
Maximal temperature NS ([dagger])
 ([degrees]C) *
Headache ([section]) NS ([double dagger])
Conjunctivitis ([section]) NS ([double dagger])
Arthralgia/myalgia ([section]) NS ([double dagger])
Odynophagia ([section]) 0.01 ([double dagger])
Dry cough ([section]) 0.04 ([double dagger])
Nausea/vomiting ([section]) NS ([double dagger])
Abdominal pain ([section]) NS ([double dagger])
Rash ([section]) 0.03 ([double dagger])
Past or actual tick NS ([double dagger])
 bite ([section])
Hepatomegaly ([section]) NS ([double dagger])
Splenomegaly ([section]) NS ([double dagger])
Anemia (hemoglobin <13 mg/dL) NS
Normal blood leukocyte counts NS ([double dagger])
Normal platelet counts NS ([double dagger])
Normal ratio prothrombin time NS ([double dagger])
Normal ESR (<10 mm/h) NS ([double dagger])
Normal creatinine blood level NS ([double dagger])
 (62-106 [micro]mol/L)
Normal sodium blood level NS ([double dagger])
 (136-144 mmol/L)
Elevated AST (>35 IU/L) NS ([double dagger])
Mean AST (U/L) 0.01 ([subsection])
Elevated ALT (>45 IU/L) NS ([dagger])
Mean ALT (U/L) ([paragraphs]) <0.01 ([subsection])
Elevated GGT (>55 IU/L) NS ([double dagger])
Elevated total serum protein NS ([double dagger])
 concentration (>80 g/L)
Elevated gamma globulin NS ([double dagger])
 concentration (>13 g/L)

* Data are expressed as mean (SD). Samples are distributed normally and
have similar SD. NS, nonsignificant; ANOVA, analysis of variance test;
ESR, erythrocyte sedimentation rate, AST, aspartate aminotransferase,
ALT, alanine aminotransferase; GGT, gamma-glutamyl transpeptidase.

([dagger]) ANOVA.

([double dagger]) [chi square] test.

([section]) No. patients with these epidemiologic or clinical data/
no. patients evaluated.

([paragraph]) Data are expressed as median and 95% confidence
intervals; [greater than or equal to] 1 samples have a non-gaussian

(#) Significant differences between R. felis and R. typhi
groups (p<0.05) with Dunn test.

** Leukopenia in 1 patient (3,700/[micro]L), leukocytosis in 2 patients
(11,500/[micro]L and 16,000/[micro]L).

([dagger][dagger]) Leukopenia in 2 patients.

([double dagger][double dagger]) All patients with an abnormal platelet
count presented with thrombocytopenia in all cases.

([subsection]) ANOVA; all cases.

([paragraphs]) Data are expressed as mean (range values). Samples are
distributed normally and have similar SD.
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Title Annotation:DISPATCHES
Author:Raoult, Didier
Publication:Emerging Infectious Diseases
Geographic Code:6CANA
Date:Dec 1, 2005
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