Rickettsia monacensis and human disease, Spain.

We identified Rickettsia monacensis as a cause of acute tickborne rickettsiosis in 2 humans. Its pathogenic role was assessed by culture and detection of the organism in patients' blood samples. This finding increases the number of recognized human rickettsial pathogens and expands the known geographic distribution of Mediterranean spotted fever-like cases.

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Tickborne rickettsioses are produced by spotted fever group (SFG) rickettsiae and cause an expanding spectrum of clinical signs. Rickettsia conorii is the etiologic agent of Mediterranean spored fever (MSF) and is transmitted by Rhipicephalus sanguineus. Rickettsia helvetica, a widespread species, is carried by Ixodes ricinus (1). Recently, other SFG rickettsiae have been found in I. ricinus from Spain (2), Slovakia (3), and northeastern Italy (4), as well as in I. nipponensis from Japan (5). Subsequently, a new rickettsia species, R. monacensis, was isolated from I. ricinus from Germany (6) and detected in Hungary (7). The pathogenicity of this species is unknown. It constitutes a new rickettsial genotype and forms a separate cluster among the SFG rickettsiae (3), close to strain Cooleyi, which was isolated from I. scapularis in Texas (8). I. ricinus is well established in areas of northern Spain (9), where MSF-like cases are increasingly reported.

Our study aim was to identify the SFG rickettsial species involved in MSF-like rickettsioses in 2 patients in northern Spain. We report an association between R. monacensis and these rickettsioses.

The Study

Patient 1 was an 84-year-old man from La Rioja, who sought medical attention on June 19, 2003, 7 days after onset of fever (39.5[degrees]C), general discomfort, headache, and joint pain. At the time of the physical examination, he had a nonpruritic, disseminated maculopapular rash, with no inoculation eschar, of the trunk and lower extremities, including palms and soles. Other than a slightly low platelet count (82,000/[mm.sup.3]), examination findings were within normal limits. MSF was diagnosed, and serum and defibrinated blood samples were taken before a course of oral doxycycline (100 mg/12 h for 10 d) was initiated. Three days later, fever and rash were gone without sequelae. Additional serial serum samples were taken during weeks 4, 13, and 26 after onset and reserved for serologic analysis (Table).

Patient 2 was a 59-year-old woman from Basque Country, who sought medical attention on September 20, 2003, 4 days after onset of fever (38[degrees]C), headache, and an erythematous rash, with no inoculation eschar, at the site of a tick bite. The patient reported a history of tick bites, most recently 1 week before symptom onset. Blood cell counts and other blood chemistry values were normal. MSF was diagnosed, and oral doxycycline (100 mg/12 h for 10 d) was prescribed. Serial serum samples were taken the day of the visit and weeks 4 and 6 after onset and were reserved for serologic analysis (Table). Defibrinated blood was also taken 2 days after treatment was initiated. The patient recovered without sequelae.

DNA was extracted with the QIAGEN Tissue kit (IZASA S.A., Barcelona, Spain), and an ompA-nested PCR was designed. The first set of primers (Rr190.70p and Rr190.602n) have been described (10). Those used for the nested amplification were designed in this study: NompA-F (5'-AGC GAT AAT GCT GAG TAG TAG-3') and NompA-R (5'-TAT ATT TCC TAA ACC TGT ATA A-3') nucleotide positions 150-170 and 576-555, respectively, were numbered according to Regnery et al. (10). Amplification conditions were as described, except annealing temperature was 40[degrees]C for the second PCR and AmpliTaq Gold DNA Polymerase (Applied Biosystems, Branchburg, N J, USA) was used. The specificity of the method was tested against DNA obtained from Vero cells and Coxiella burnetii, and fragments of the expected sizes (532 and 427 bp) were obtained from different rickettsia species (data not shown). The amplicons obtained from blood samples were run in 1% low-melt agarose gels (Pronadisa, Barcelona, Spain), and the bands of interest were excised, purified with QIAquick Gel Extraction kit (IZASA S.A), and sequenced as described (9).

A phylogenetically informative fragment of 446 bp of gltA was also sequenced from samples by nested PCR with primers designed for this study: GLTA1F (5'-GAC GGT GAT AAA GGA ATC TTG-3') and GLTA1R (5'CAT TTC TTT CCA TTG TGC CAT C-3') for the first run, and GLTA2F (5'-CTA CGA ACT TAC CGC TAT TAG3') and GLTA2R (5'-GAC CAA AAC CCA TTA ACC TAA AC-3') for the second; nucleotide positions 279-299, 1011-989, 566-586, and 1298-1277, respectively, were numbered according to Regnery et al. (10). PCR conditions included annealing temperatures of 65[degrees]C and 50[degrees]C for the first and second runs, respectively. The rest of the parameters were identical to those used above, and samples were subjected to 35 cycles of denaturing (20 s at 95[degrees]C), annealing (30 s), and extension (2 min at 60[degrees]C), with an initial denaturing cycle of 9 min at 95[degrees]C.

Blood samples from each patient were cultured by using shell vial technique (11). Gimenez stain and PCR, performed after 7 days of incubation, confirmed the growth of a Rickettsia-like organism (strain Rp-Spl) from patient 1. The sequences of ompA and gltA of this isolate (GenBank accession nos. DQ157778 and DQ517498, respectively) were identical to those obtained from the blood samples of each patient and to that of R. monacensis (6) (GenBank accession nos. AF201329 and DQ100163). The sequences generated in this study were subjected to phylogenetic analyses as described (9) and belonged to the same clade as R. monacensis and other related strains that have been detected in I. ricinus (3,4,12) (Figure).

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In-house microimmunofluorescence assay (IFA) ([1] and references therein) that used R. monacensis, R. conorii, R. helvetica, R. akari, and R. australis as antigens was performed in serial serum samples from each patient (Table). The isolate Rp-Spl from patient 1 could not be used as antigen because of poor adaptation of this isolate to culture in Vero cell monolayers; R. monacensis slides for IFA were obtained from the Department of Entomology, University of Minnesota, Minneapolis, MN, USA. Seroconversion against the 5 rickettsia species was observed from patient 1's second serum sample (day 30 after the onset). Patient 2's first serum sample also had high titers against the 5 antigens. Although the reactivity against the 5 rickettsial antigens was similar, the titers observed were slightly higher against R. monaeensis and R. helvetica, which are phylogenetically closer to each other than to the other species tested. However, because the serologic results may only loosely implicate a given rickettsia species, isolation of R. monacensis from patient 1 and its detection by PCR for both patients confirm it as the etiologic agent.

Conclusions

We describe a new, to our knowledge, rickettsia species that caused human disease. R. monacensis was the etiologic agent of MSF-like illness in northern Spain. Strain Rp-Spl was obtained from 1 patient. Because the sequences of ompA and gltA were identical to this rickettsia species and also amplified from blood samples of each patient studied, we conclude that this rickettsia is responsible for the symptoms observed in these patients. Therefore, R. monacensis joins the list of autochthonous rickettsia species (R. conorii [13], R. slovaca [14], R. typhi [15]) confirmed as human pathogens in Spain.

We were not able to study the vectors involved; however, each patient contracted the disease in areas where L ricinus is the most prevalent tick species (9), and strains close to R. monacensis have been recently detected in I. ricinus in Spain (2, 12). Thus, I. ricinus may eventually be shown to be the vector. Studies of R. monacensis incidence in autochthonous I. ricinus specimens are in progress to evaluate the risk of its transmission to humans.

Acknowledgments

We thank Ulrike Munderloh for providing R. monacensis slides for IFA.

Grant support was provided by Fondo de Investigacion Sanitaria "Red Tematica de Investigacion Cooperativa EBATRAG (G03/057)."

Dr Jado is a microbiologist at the "Unidad de Alerta y Emergencias" and Laboratorio de Espiroquetas y Patogenos Especiales, Centro Nacional de Microbiologia, Instituto de Salud Carlos. Her research interest is bacterial zoonoses, specifically tickborne pathogens.

References

(1.) Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin Microbiol Rev. 2005;18:719-56.

(2.) Marquez FJ, Muniain MA, Soriguer RC, Izquierdo G, Rodriguez-Bano J, Borobio MV. Genotypic identification of an undescribed spotted fever group rickettsia in Ixodes ricinus from southwestern Spain. Am J Trop Med Hyg. 1998;58:570-7.

(3.) Sekeyova Z, Fournier PE, Rehacek J, Raoult D. Characterization of a new spotted fever group rickettsia detected in Ixodes ricinus (Acari: Ixodidae) collected in Slovakia. J Med Entomol. 2000;37:707-13.

(4.) Beninati T, Lo N, Noda H, Esposito F, Rizzoli A, Favia G, et al. First detection of spotted fever group rickettsiae in Ixodes ricinus from Italy. Emerg Infect Dis. 2002;8:983-6.

(5.) Ishikrura M, Ando S, Shinagawa Y, Matsuura K, Hasegawa S, Nakayama T, et al. Phylogenetic analysis of spotted fever group rickettsiae based on gltA, 17-kDa, and rOmpA genes amplified by nested PCR from ticks in Japan. Microbiol Immunol. 2003;47:823-32.

(6.) Simser JA, Palmer AT, Fingerle V, Wilske B, Kurtti T J, Munderloh UG. Rickettsia monacensis sp. nov., a spotted fever group rickettsia, from ticks (Ixodes ricinus) collected in a European city park. Appl Environ Microbiol. 2002;68:4559-66.

(7.) Sreter-Lancz Z, Sreter T, Szell Z, Egyed L. Molecular evidence of Rickettsia helvetica and R. monacensis infections in Ixodes ricinus from Hungary. Ann Trop Med Parasitol. 2005;99:325-30.

(8.) Billings AN, Teltow G J, Weaver SC, Walker DH. Molecular characterization of a novel Rickettsia species from Ixodes scapularis in Texas. Emerg Infect Dis. 1998;4:305 9.

(9.) Escudero R, Barral M, Perez A, Vitutia MM, Garcia-Perez AL, Jimenez S, et al. Molecular and pathogenic characterizacion of Borrelia burgdorferi sensu lato isolates from Spain. J Clin Microbiol. 2000;38:4026-33.

(10.) Regnery RL, Spruill CL, Plikaytis BD. Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol. 1991;173:1576-89.

(11.) La Scola B, Raoult D. Diagnosis of Mediterranean spotted fever by cultivation of Rickettsia conorii from blood and skin samples using the centrifugation-shell vial technique and by detection of R. conorii in circulating endothelial cells: a 6-year follow-up. J Clin Microbiol. 1996;34:2722-7.

(12.) Fernandez-Soto P, Perez-Sanchez R, Encinas-Grandes A, Sanz RA. Detection and identification of Rickettsia helvetica and Rickettsia sp. IRS3/IRS4 in Ixodes ricinus ticks found on humans in Spain. Eur J Clin Microbiol Infect Dis. 2004;23:648-9.

(13.) Bernabeu-Wittel M, Segura-Porta F. Rickettsiosis. Enferm Infecc Microbiol Clin. 2005;23:163-72.

(14.) Oteo JA, Ibarra V, Blanco JR, Martinez de Artola V, Marquez FJ, Portillo A, et al. Dermacentor-borne necrosis erythema and lymphadenopathy: clinical and epidemiological features of a new tickborne disease. Clin Microbiol Infect. 2004; 10:327-31.

(15.) Hernandez-Cabrera M, Angel-Moreno A, Santana E, Bolanos M, Frances A, Martin-Sanchez MS, et al. Murine typhus with renal involvement in Canary Islands, Spain. Emerg Infect Dis. 2004;10:740-3.

Address for correspondence: Pedro Anda, Laboratorio de Espiroquetas y Patogenos Especiales, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, 28220-Majadahonda, Madrid, Spain; email: panda@ isciii.es

Isabel Jado, * Jose A. Oteo, ([dagger]) Mikel Aldamiz, ([double dagger]) Horacio Gil, * Raquel Escudero, * Valvanera Ibarra, ([dagger]) Joseba Portu, ([double dagger]) Aranzazu Portillo, ([dagger]) Maria J. Lezaun, ([double dagger]) Cristina Garcia-Amil, * Isabel Rodriguez-Moreno, * and Pedro Anda *

* Centro Nacional de Microbiologia, Majadahonda, Madrid, Spain; ([dagger]) Complejo San Millan-San Pedro de La Rioja, Logrono, Spain; and ([double dagger]) Hospital de Txagorritxu, Vitoria, Spain

Table. Microimmunofluorescence titers obtained with different
rickettsial antigens, 2 patients, northern Spain, 2003 *

             Week       Rickettsia      R.
Patient    ([dagger])    conorili    monacensis

1               1        <1:40         <40
                4        1,280        2,560
               13        1,280        1,280
               26        1,280        1,280

2               1         640         2,560
                4         320         1,280
                6         640         1,280

              R.           R.           R.
Patient    helvetica      akari      australis

1             <40          <40          <40
             2,560        1,280         640
             1,280        1,280        1,280
             1,280         320          320

2            2,560         640          320
             1,280         320          160
             1,280         320          160

* A 1-fold decrease in titer is considered not significant.

([dagger]) Week after symptom onset in which the samples
were extracted.