LETTERS ity with R. sibirica subsp. mongoloti- with caused by R. conorii, 8. Parola P, Inokuma H, Camicas JL, Brouqui monae when compared with those in R. rickettsii, and R. typhi (10). Most P, Raoult D. Detection and identifi cation of group rickettsiae and eh- the GenBank database. of these posterior segment manifes- rlichiae in African . Emerg Infect Dis. Infections caused by R. sibirica tations are usually asymptomatic in 2001;7:1014–7. subsp. mongolitimonae have been patients with acute Mediterranean 9. Stallings SP. Rocky Mountain spotted reported as lymphangitis-associated spotted fever (10) and can be easily fever and pregnancy: a case report and review of the literature. Obstet Gynecol rickettsiosis (4). Our case-patient had overlooked. Retinal vasculitis was re- Surv. 2001;56:37–42. the clinical symptoms reported for ported in 45%–55% of the patients, 10. Khairallah M, Ladjimi A, Chakroun M, this disease: fever, maculopapular but retinal artery occlusion secondary Messaoud R, Yahia SB, Zaouali S, et al. rash, eschar, enlarged satellite lymph to vasculitis has been described in only Posterior segment manifestations of Rick- ettsia conorii infection. Ophthalmology. nodes, and lymphangitis. Seasonal oc- 2 cases of infection with R. conorii 2004;111:529–34. currence of this disease in the spring and R. rickettsii (10) without details is common and has been reported in 9 of clinical symptoms. Because ocular Address for correspondence: Didier Bessis, of 12 cases, including the case-patient involvement could be asymptomatic Service de Dermatologie, Hôpital Saint-Eloi, reported here (2–6). A total of 75% and easily overlooked, an ophthalmic 80 Ave Augustin-Fliche, 34295 Montpellier of these R. sibirica subsp. mongoliti- evaluation should be conducted when CEDEX 5, France; email: d-bessis@chu- monae infections occurred in southern rickettsiosis is suspected. montpellier.fr France; other cases have been recently reported in Greece (5), Portugal (6), Julie Caron,* Jean-Marc Rolain,† and South Africa (7). However, the Frédéric Mura,* vector of R. sibirica subsp. mongoliti- Bernard Guillot,* Didier Raoult,† monae has not been identifi ed (7). This and Didier Bessis* has been isolated from Hy- *Université Montpellier 1, Montpellier, alomma asiaticum ticks in Inner Mon- France; and †Université de la Méditer- in golia, from H. truncatum in Niger (8), ranée, Marseilles, France , France and from H. anatolicum excavatum in Greece (5). Hyalomma spp. ticks are References To the Editor: Rickettsia felis suspected of being the vector and are 1. Fournier PE, Zhu Y, Yu X, Raoult D. Pro- belongs to the spotted fever group of widespread in Africa, southeastern posal to create subspecies of Rickettsia rickettsia. The pathogenic role of this Europe (including France), and Asia. sibirica and an amended description of intracellular in humans Rickettsiosis caused by R. rick- . Ann N Y Acad Sci. has been reported in patients from the ettsii and R. conorii during pregnancy 2006;1078:597–606. 2. Raoult D, Brouqui P, Roux V. A new United States (Texas) (1), Mexico (2), has been reported without risk for ver- spotted-fever-group rickettsiosis. Lancet. Germany (3), Brazil, and France (4). tical transmission (9). First-line anti- 1996;348:412. R. felis is widely distributed, is asso- microbial drugs used to treat rickettsi- 3. Fournier PE, Tissot-Dupont H, Gallais H, ciated with blood-sucking , al disease are cyclines and quinolones, Raoult D. Rickettsia mongolotimonae: a rare in France. Emerg Infect Dis. and has been isolated from fl eas in but they are contraindicated during 2000;6:290–2. several countries (5). pregnancy. Chloramphenicol is an 4. Fournier PE, Gouriet F, Brouqui P, Lucht To obtain new information about alternative drug for pregnant women F, Raoult D. Lymphangitis-associated the distribution of R. felis in France but it is not available in France. Mac- rickettsiosis, a new rickettsiosis caused by Rickettsia sibirica mongolotimonae: sev- and potential vectors/ reservoirs of this rolides (azithromycin, clarithromycin, en new cases and review of the literature. emerging pathogen, 550 fl eas were and josamycin) are effective against Clin Infect Dis. 2005;40:1435–44. collected from 82 dogs and 91 in 7 rickettsial disease and can be used 5. Psaroulaki A, Germanakis A, Gikas A, widely distributed locations in France safely during pregnancy. Scoulica E, Tselentis Y. Simultaneous de- tection of “Rickettsia mongolotimonae” in (Bordeaux, Toulouse, Cosnes-Cours No ocular complications were a patient and in a in Greece. J Clin sur Loire, Dijon, Moulins, Limoges, reported in the 11 previous cases of Microbiol. 2005;43:3558–9. and Aix-en-Provence). Specimens rickettsiosis caused by R. subsp. mon- 6. de Sousa R, Barata C, Vitorino L, Santos- were collected by combing, recorded, golitimonae. However, ocular lesions, Silva M, Carrapato C, Torgal J, et al. Rick- ettsia sibirica isolation from a patient and and stored at –20°C. Samples were including optic disk staining, white detection in ticks, Portugal. Emerg Infect shipped on dry ice to the entomologic retinal lesions, retinal hemorrhages, Dis. 2006;12:1103–8. laboratory of the Institute of Compara- multiple hypofl uorescent choroidal 7. Pretorius AM, Birtles RJ. Rickettsia mon- tive Tropical Medicine and Parasitol- dots, mild vitritis, and retinal vascu- golotimonae infection in South Africa. Emerg Infect Dis. 2004;10:125–6. ogy in Munich, Germany, and species litis, have been described in patients identifi cation was performed by using

684 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 4, April 2008 LETTERS light microscopy and following the 0.5 μL (5 U/mL) of Taq polymerase (Moulins) to 37% (Bordeaux) for cats determination key of Hopkins and (Roche). Conditions for the gltA and (Table). Of 550 fl eas, 96 were positive Rothschild (6). Because infestation ompA PCRs were as described by Ber- for both genes (gltA and ompA) and levels varied (1–150 fl eas/animal), we tolotti et al. (8). Negative and positive 3 of 5 species of fl eas were infected: randomly analyzed 1–8 fl eas (mean controls were included in all PCRs. 10 with C. canis, 85 with C. felis, 3.4) from each host animal. All PCR products were separated by and 1 with A. erinacei. All sequences We homogenized fl eas individually electrophoresis on 1.5% agarose gels matched gltA and ompA genes from R. in 80 μL of phosphate-buffered saline by at 100 V for 60 min and examined un- felis (similarity 99%–100%). using 5-mm steel beads in a RETSCH der UV light. For both genes, positive Our investigation provides new Tissue Lyser Mixer Mill 300 (QIA- samples were purifi ed by using the information about distribution of R. GEN, Hilden, Germany). A total of 100 QIAquick PCR Purifi cation Kit (QIA- felis and widespread fl ea infection μL of ATL buffer and 20 μL of protei- GEN) and sent for sequencing to the with R. felis in France. A total of 88% nase K (QIAGEN) were added, and the MWG Biotech Company (Martinried, of infected fl eas were C. felis, but we homogenate was incubated at 56°C in Germany). Sequences were compared found infected C. canis in Bordeaux a thermomixer (Eppendorf, Hamburg, with those of previously characterized and Toulouse and infected A. erinacei Germany) until the tissues were lysed. rickettsia in GenBank by using basic in Limoges. We report the presence in DNA was extracted from each fl ea by local alignment search tool (BLAST) France of R. felis in C. canis and A. using a QIAamp DNA Mini Kit (QIA- (www.ncbi.nlm.nih.gov) analysis. erinacei in France. R. felis in dog fl eas GEN) according to the manufacturer’s Five species of fl eas were identi- in Uruguay and in hedgehog fl eas in instructions (tissue protocol) and stored fi ed: Ctenocephalides felis (500, 224 Algeria has been reported (9,10). Our at –20°C until used in a PCR. from dogs and 276 from cats), C. fi ndings indicate that these 2 fl ea spe- PCR amplifi cation of rickettsial canis (37 from dogs), Pulex irritans cies may be vectors of human R. felis DNA was performed by using previ- (11 from dogs), Spilopsyllus cuniculi rickettsiosis in France. ously described oligonucleotide prim- (1 from a ), and Archaeopsylla eri- er pairs Rp CS.877p/Rp CS.1258n nacei (1 from a cat). Five dogs had This study was supported by Merial targeting the citrate synthase (gltA) mixed populations of fl eas; 3 of these (Lyon, France). gene and, for the positive samples, had P. irritans and C. felis, and 2 had Jeremie Gilles,*1 Rr 190.70p/Rr 190.602n targeting the C. felis and C. canis. One cat had P. Frank Thomas Just,* outer membrane protein A (ompA) irritans and C. felis, and another cat Cornelia Silaghi,* gene (7). Amplifi cation was conduct- had S. cuniculi and C. felis. A total of Ingrid Pradel,* Heidi Lengauer,† ed in 50-μL volumes that contained 5 52 (19%) of the 272 fl eas from dogs Klaus Hellmann,† μL of DNA, 30 μL of distilled water, and 44 (16%) of the 278 fl eas from and Kurt Pfi ster* 10 μL of 5× Taq buffer (Roche, Man- cats were positive for both the gltA *Ludwig-Maximilians-University, Munich, Ger- nheim, Germany), 3 μL of 25 mmol/L and ompA genes. Positive samples many; and †Klifovet AG, Munich, Germany

MgCl2 (Roche), 1 μL of 10 mmol/L were obtained from all locations. deoxyncleotide triphosphates (Roche), Prevalence ranged from 6% (Dijon) to 1Current affi liation: University of Kentucky, 0.25 μL of each primer (100 μM), and 43% (Toulouse) for dogs and from 3% Lexington, Kentucky, USA.

Table. Prevalence of Rickettsia felis in fleas from dogs and cats, France* Locality Animal No. animals No. fleas species No. (%) gltA+ ompA+ Aix-en-Provence Dog 6 20 Ctenocephalis felis,† C. canis,† 6 (30) Pulex irritans Bordeaux Dog 14 67 C. felis,† C. canis, P. irritans 8(12) Cat 11 38 C. felis† 14 (37) Cosnes-Cours sur Loire Dog 15 44 C. felis,† C. canis 7(16) Cat 17 50 C. felis†3(6) Dijon Dog 6 18 C. felis,† C. canis 1(17) Cat 1 3 C. felis† 1 (33) Limoges Dog 15 45 C. felis† 7 (16) Cat 21 61 C. felis,† Archaeopsylla erinacei† 11 (18) Moulins Dog 12 36 C. felis,† C. canis 5(14) Cat 22 65 C. felis,† Spilopsyllus cuniculi 2(3) Toulouse Dog 14 42 C. felis,† C. canis†18(43) Cat 19 61 C. felis†13(21) *gltA, citrate synthase A; ompA, outer membrane protein A. †Species positive for gltA and ompA.

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References Novel Nonstructural this strain in comparison with those of other NSP4 gene sequences available 1. Schriefer ME, Sacci JB Jr, Dumler JS, Protein 4 Genetic Bullen MG, Azad AF. Identifi cation of a in the GenBank database. novel rickettsial infection in a patient di- Group in Rotavirus The full-length of NSP4 gene was agnosed with murine . J Clin Mi- of Porcine Origin amplifi ed by NSP4–1a and NSP4–2b crobiol. 1994;32:949–54. primer pairs (8). The PCR amplicon 2. Zavala-Velasquez JE, Ruiz-Sosa JA, Sanchez-Elias RA, Becerra-Carmona G, To the Editor: Infection with was sequenced in both directions by Walker DH. Rickettsia felis in Yucatan. group A rotavirus is the main cause using the BigDye Terminator Cycle Lancet. 2000;356:1079–80. of acute gastroenteritis in infants and Sequencing kit (PerkinElmer-Ap- 3. Richter J, Fournier P, Petridou J, Hauss- young children worldwide and in plied Biosystems, Inc., Foster City, inger D, Raoult D. Rickettsia felis infec- tion acquired in Europe and documented young animals of many species, in- CA, USA) on an automated sequenc- by polymerase chain reaction. Emerg In- cluding piglets. In recent years, sev- er (ABI 3100; PerkinElmer-Applied fect Dis. 2002;8:207–8. eral epidemiologic studies designed Biosystems, Inc.). The sequence of 4. Rolain J-M, Franc M, Davoust B, Raoult to monitor the appearance of novel CMP034 was compared with those D. Molecular detection of quintana, B. koehlerae, B. henselae, B. or atypical rotavirus antigenic types of reference strains available in the clarridgeiae, Rickettsia felis, and Wolba- have provided evidence for the in- National Center for Biotechnology chia pipientis in cat fl eas, France. Emerg creasing antigenic diversity of group Information GenBank database by Infect Dis. 2003;9:338–42. A rotaviruses (1–3). In addition to the using BLAST (www.ncbi.nlm.nih. 5. Jiang J, Soeatmadji DW, Henry KM, Ratiwayanto S, Bangs MJ, Richards 2 rotavirus classifi cation systems, VP7 gov/blast). The NSP4 nucleotide AL. Rickettsia felis in Xenopsylla cheo- (G) and VP4 (P) genes, the virus can sequence of the CMP034 strain was pis, Java, Indonesia. Emerg Infect Dis. also be classifi ed on the basis of the deposited in GenBank under acces- 2006;12:1281–3. nonstructural glycoprotein 4 (NSP4)– sion no. DQ534017. 6. Hopkins GH, Rothschild M. An illustrated catalogue 1 of the Rothschild collection of encoding gene. Sequence analyses of The complete NSP4 nucleotide fl eas (Siphonaptera) in the British Muse- the NSP4 gene indicated the presence sequence of PoRV CMP034 strain um (Natural History). With keys and short of at least 5 distinct genetic groups was 750 bp and contained a single descriptions for the identifi cation of fami- among human and animal rotaviruses, long open reading frame coding for lies, genera, species and subspecies of the order. Vol. IV, Ctenophthalmidae, Dino- termed A to E (1,4,5). Among human a protein of 175 aa. Comparative psyllidae, Doratopsyllidae and Listropsyl- rotaviruses, the diversity of NSP4 analysis of the CMP034 NSP4 se- lidae. London: British Museum (Natural genes has been restricted mainly to quence with those of the 5 representa- History); 1966. genetic groups A and B; only a few tive established genetic groups (A–E) 7. Regnery RL, Spruill CL, Plikaytis BD. Genotypic identifi cation of rickettsiae and human strains possess genetic group showed the highest sequence identity, estimation of intraspecies sequence diver- C. Conversely, all 5 NSP4 genetic at 92.6% nt and 96.9% aa levels, with gence for portions of two rickettsial genes. groups (A–E) have been identifi ed in 1 PoRV strain, P21–5 (9). However, J Bacteriol. 1991;173:1576–89. rotaviruses of animal origins. To our CMP034 and P21–5 shared a low de- 8. Bertolotti L, Tomassone L, Tramuta C, Greco E, Amore G, Ambrogi C, et al. Bor- knowledge, porcine rotaviruses (PoR- gree of sequence identity with other relia lusitaniae and spotted fever group Vs) have been reported to belong only NSP4 genetic groups. The NSP4 se- rickettsia in Ixodes ricinus (Acari: Ixodi- to NSP4 genetic group B (1). quence identities of the CMPO34 and dae) in Tuscany, central Italy. J Med Ento- During an epidemiologic survey P21-5 strains ranged from 74% to mol. 2006;43:159–65. 9. Venzal JM, Perez-Martinez L, Félix ML, of PoRV from June 2000 through July 78% nt and 75%–79% aa levels with Portillo A, Blanco JR, Oteo JA. Prevalence 2001, a total of 175 fecal specimens those of genetic group A; 77%–86% nt of Rickettsia felis in Ctenocephalides felis were collected from diarrheic pig- and 79%–86% aa levels with genetic and Ctenocephalides canis from Uruguay. lets from 6 different farms in Chiang group B; 69%–73% nt and 75%–78% Ann N Y Acad Sci. 2006;1078:305–8. 10. Bitam I, Parola P, de la Cruz KD, Matsu- Mai Province, Thailand. Of these, 39 aa levels with genetic group C; 62%– moto K, Baziz B, Rolain JM, et al. First (22.3%) specimens were positive for 65% nt and 55%–60% aa levels with molecular detection of Rickettsia felis in group A rotavirus (6). A novel and un- genetic group D; and only 43%–50% fl eas from Algeria. Am J Trop Med Hyg. usual PoRV CMP034 strain was iso- nt and 29%–33% aa levels with ge- 2006;74:532–5. lated from a 7-week-old piglet during netic group E. The phylogenetic tree this survey. Molecular genetic char- confi rmed that PoRV strains CMP034 Address for correspondence: Jeremie Gilles, acterization showed that the CMP034 and P21–5 were located exclusively in Department of Entomology, University of strain carried a novel P[27] genotype a separated branch, which was distant- Kentucky, S225 Agricultural Science Center with a new lineage of G2-like rotavi- ly related to the other 5 known NSP4 North, Lexington, KY 40506, USA; email: rus genotype (7). We performed a mo- genetic groups (Figure). However, a [email protected] lecular analysis of the NSP4 gene of bootstrap support for the separation of

686 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 4, April 2008