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Spotted fever group rickettsiae identified in Dermacentor marginatus and Ixodes ricinus ticks in Algeria
Ticks and Tick-borne Diseases 3 (2012) 379–380
Contents lists available at SciVerse ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Short communication
Spotted fever group rickettsiae identified in Dermacentor marginatus and Ixodes ricinus ticks in Algeria Tahar Kernif a,b , Dalila Messaoudene a , Soraya Ouahioune a , Philippe Parola b , Didier Raoult b , Idir Bitam a,∗ a
Service d’Ecologie et des Systèmes Vectoriels, Institut Pasteur d’Algérie, Algiers, Algeria Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198, Inserm 1095, WHO Collaborative Center for Rickettsioses and Other Arthropod Borne Bacterial Diseases, Faculté de Médecine, 27 bd Jean Moulin, 13385 Marseille cedex 5, France b
a r t i c l e
i n f o
Keywords: Ixodes ricinus ticks Dermacentor marginatus ticks Rickettsia helvetica R. monacensis R. slovaca Algeria
a b s t r a c t Our study was carried out using Ixodes ricinus ticks collected from cattle from Tizi-Ouzou and Dermacentor marginatus ticks collected from the vegetation of the Blida region, a tourist site, both regions situated in northern Algeria. The results of real-time quantitative PCR (qPCR) specific for a partial sequence of the citrate synthase gene (gltA) indicate that Rickettsia spp. were present in 11/23 (48%) and 4/9 (44%) of the examined ticks from Tizi-Ouzou and Blida, respectively. The sequences of Rickettsia helvetica and Ri. monacensis were found in I. ricinus ticks using gltA primers. In addition, Ri. slovaca was detected based on the sequences of the gltA and the outer membrane protein (OmpA) genes in D. marginatus ticks. DNA sequencing to identify the species revealed for the first time the presence of Ri. helvetica in I. ricinus ticks and Ri. slovaca in D. marginatus ticks from Algeria and confirmed the presence of Ri. monacensis. © 2012 Elsevier GmbH. All rights reserved.
Introduction Spotted fever group (SFG) Rickettsia spp. is important emerging tick-borne human infection agents that are present worldwide (Parola et al., 2005). In Algeria, several Rickettsia spp. that are pathogenic for humans have been detected in various ticks, including Rickettsia conorii conorii, the causative agent of Mediterranean spotted fever, which is transmitted by Rhipicephalus sanguineus (Mouffok et al., 2009); Ri. aeschlimannii, an emerging pathogen detected in Hyalomma marginatum marginatum; Ri. massiliae, another emerging pathogen transmitted by Rh. sanguineus and Rh. turanicus (Bitam et al., 2006); and Ri. monacensis, which is generally found in Ixodes ricinus (Dib et al., 2009). The aim of the present study was to identify rickettsial species occurring in ticks collected from a specific area of Algeria that had not been previously investigated for tick-borne rickettsiae. Materials and methods In February 2006, we used the flag technique on vegetation close to the road to capture ticks in the mountains of the Blida region (Chréa Mountain) in northern Algeria. In December 2010, ticks were collected from cattle in Bouzguene (Tizi-Ouzou department),
Algeria. These ticks were identified phenotypically by using cur˜ et al., 2004), were stored in rent taxonomic criteria (Estrada-Pena 70% ethanol, and were sent to the WHO Collaborative Center for Rickettsial Diseases and Other Arthropod-borne Bacterial Diseases in Marseille, France. DNA from these specimens and negative controls (uninfected lice maintained in colonies in our laboratory) was extracted using a QIAamp Tissue Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Rickettsial DNA was detected using a Rickettsia genus-specific real-time PCR (qPCR) with a 25-bp probe targeting the partial sequence of the citrate synthase gene (gltA) using a 7900 HT Fast instrument (Varagnol et al., 2009). The positive control was DNA from Ri. montanensis (qPCR). Ticks analyzed by qPCR were considered to be positive when the cycle threshold was less than or equal to 30. All ticks identified as positive by qPCR were confirmed by 2 different standard PCRs and sequencing for fragments of the gltA and OmpA genes. DNA sequencing reactions were performed for all samples amplified by standard PCR, as previously described (Sarih et al., 2008). Data were collected with an ABI Prism 3130xl Genetic Analyzer capillary sequencer (ABI PRISM, PE Applied Biosystems, USA). Sequences were edited and assembled using Chromas Pro 1.34 (Technelysium Pty. Ltd., Tewantin, Australia). BLAST searches were performed to identify the obtained sequences. Results
∗ Corresponding author. Tel.: +213 21 673365; fax: +213 21 673365; mobile: +213 771995693. E-mail address: [email protected] (I. Bitam). 1877-959X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2012.10.012
The ticks collected from the Blida region were divided into 2 groups: The first group contained I. ricinus and Haemaphysalis spp.
380
T. Kernif et al. / Ticks and Tick-borne Diseases 3 (2012) 379–380
(not tested here, but used for other studies), and the second group contained 9 ticks identified as Dermacentor marginatus, which were tested for the presence of rickettsial DNA. A total of 4 ticks (4/9) tested positive by qPCR. These positive ticks were further screened and shown to be positive by 2 PCRs targeting the ompA and gltA genes. Analysis of the sequences obtained after these PCRs showed 100% homology with a 383-bp fragment of the Ri. slovaca gltA gene (GenBank accession number DQ821853.1) and with a 558-bp fragment of the Ri. slovaca ompA gene (GenBank accession number U43808.1). A total of 23 ticks was collected from Tizi-Ouzou; all were identified as I. ricinus. Using primers and TaqMan probes, qPCR revealed the presence of rickettsial DNA in 11/23 (48%) specimens and in the positive controls. All samples found to be positive by qPCR were screened and shown to be positive by standard PCR using gltA primers. Ri. helvetica was found in 4% (1/23) of tick specimens, and Ri. monacensis was found in 43% (10/23) of tick specimens. Sequence analysis showed 100% similarity to the gltA sequences of Ri. helvetica (accession number HM371185.1) and Ri. monacensis (accession number EU853831.1) in GenBank. Discussion Ri. slovaca is a member of the SFG rickettsiae and was first isolated from D. marginatus ticks in Slovakia in 1968. This pathogen was described as a causative agent of tick-borne lymphadenopathy (TIBOLA) in humans in 1997 (Raoult et al., 1997). Subsequently, this bacterium has been found in D. marginatus and D. reticulatus in several European countries, including France, Switzerland, Portugal, Spain, Armenia, Poland, Bulgaria, Croatia, Russia, Germany, and Morocco (Bitam and Raoult, 2009; Parola et al., 2009; Sarih et al., 2008). Here, we report for the first time the presence of R. slovaca in D. marginatus ticks from Algeria. TIBOLA (or DEBONEL for Dermacentor-borne necrosis– erythema–lymphadenopathy) has now been described for several European countries (Parola et al., 2009). In addition, Ri. slovaca has been associated with a syndrome characterized by scalp eschars and neck lymphadenopathy following tick bites, and the term ‘SENLAT’ has been proposed to collectively describe this clinical entity (Angelakis et al., 2010). Patients with similar clinical signs have been documented in France, Hungary, Spain, Bulgaria, and Italy. However, other agents, including Ri. raoultii, have been shown to cause SENLAT (Angelakis et al., 2010; Parola et al., 2009). In addition, we report here the first detection of Ri. helvetica in Algeria. This rickettsia was first isolated in Switzerland from I. ricinus in 1979 and has been identified in many European countries in I. ricinus ticks (Parola et al., 2005). In North Africa, it has been detected in I. ricinus ticks in Morocco and Tunisia (Sarih et al., 2008; Sfar et al., 2008). Ri. helvetica was considered to be a nonpathogenic rickettsia for approximately 20 years after its discovery. In 2000, seroconversion for Ri. helvetica was described in a patient in France with a nonspecific febrile illness (Fournier et al., 2000). Although several reports have suggested the association of Ri. helvetica with various syndromes in humans, a clear demonstration of its pathogenic role is still pending. Ri. monacensis was described and isolated from I. ricinus ticks collected in the English Garden in Munich, Germany, in 1998 (Simser et al., 2002). Furthermore, 2 human cases of infection due to Ri. monacensis have been documented in Spain (Jado et al., 2007).
In Algeria, Ri. monacensis was found in 35 I. ricinus ticks (59.3%) collected from cattle in the El Traf region of northeastern Algeria in 2009 (Dib et al., 2009); the presence of this rickettsia in North Africa was first documented when it was detected in I. ricinus ticks in Morocco and Tunisia (Sarih et al., 2008; Sfar et al., 2008). In our study, we confirmed the presence of Ri. monacensis in ticks from Tizi-Ouzou, approximately 120 km east from Algiers, the capital of Algeria. In conclusion, the results of this study confirmed that Ri. slovaca, Ri. helvetica, and Ri. monacensis are present in Algeria. The mountains of Chréa (Blida, Algeria) are increasingly visited by tourists, and there is a risk of being bitten by D. marginatus ticks carrying Ri. slovaca in this area. Additional studies are needed to determine the epidemiological and clinical relevance of different rickettsioses in these 2 regions. Clinicians should consider rickettsiosis due to these rickettsia species when diagnosing rickettsiosis cases in patients who have been bitten by ticks in Algeria. Acknowledgments We are grateful to IRD jeune équipe d’Algérie (associated with the IRD 198, Marseille) for their technical assistance. References Angelakis, E., Pulcini, C., Waton, J., Imbert, P., Socolovschi, C., Edouard, S., Dellamonica, P., Raoult, D., 2010. Scalp eschar and neck lymphadenopathy caused by Bartonella henselae after tick bite. Clin. Infect. Dis. 50, 549–551. Bitam, I., Parola, P., Matsumoto, K., Rolain, J.M., Baziz, B., Boubidi, S.C., Harrat, Z., Belkaid, M., Raoult, D., 2006. First molecular detection of R. conorii, R. aeschlimannii, and R. massiliae in ticks from Algeria. Ann. N. Y. Acad. Sci. 1078, 368–372. Bitam, I., Raoult, D., 2009. Other tick-borne diseases in Europe. Curr. Probl. Dermatol. 37, 130–154. Dib, L., Bitam, I., Bensouilah, M., Parola, P., Raoult, D., 2009. First description of Rickettsia monacensis in Ixodes ricinus in Algeria. Clin. Microbiol. Infect. 15 (Suppl. 2), 261–262. ˜ A., Bouattour, A., Camicas, J.-L., Walker, A.R., 2004. Ticks of DomesEstrada-Pena, tic Animals in the Mediterranean Region. A Guide to Identification of Species. University of Zaragoza, Spain, 131 pp. Fournier, P.E., Grunnenberger, F., Jaulhac, B., Gastinger, G., Raoult, D., 2000. Evidence of Rickettsia helvetica infection in humans, eastern France. Emerg. Infect. Dis. 6, 389–392. Jado, I., Oteo, J.A., Aldámiz, M., Gil, H., Escudero, R., Ibarra, sV., Portu, J., Portillo, A., Lezaun, M.J., Garcia-Amil, C., Rodríguez-Moreno, I., Anda, P., 2007. Rickettsia monacensis and human disease, Spain. Emerg. Infect. Dis. 13, 1405–1407. Mouffok, N., Parola, P., Abdennour, D., Aouati, A., Razik, F., Benabdellah, A., Raoult, D., 2009. Mediterranean spotted fever in Algerian children. Clin. Microbiol. Infect. 15 (Suppl. 2), 290–291. Parola, P., Paddock, C.D., Raoult, D., 2005. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin. Microbiol. Rev. 18, 719–756. Parola, P., Rovery, C., Rolain, J.M., Brouqui, P., Davoust, B., Raoult, D., 2009. Rickettsia slovaca and R. raoultii in tick-borne rickettsioses. Emerg. Infect. Dis. 15, 1105–1108. Raoult, D., Berbis, P., Roux, V., Xu, W., Maurin, M., 1997. A new tick-transmitted disease due to Rickettsia slovaca. Lancet 350, 112–113. Sarih, M., Socolovschi, C., Boudebouch, N., Hassar, M., Raoult, D., Parola, P., 2008. Spotted fever group rickettsiae in ticks, Morocco. Emerg. Infect. Dis. 14, 1067–1073. Sfar, N., M’ghirbi, Y., Letaief, A., Parola, P., Bouattour, A., Raoult, D., 2008. First report of Rickettsia monacensis and Rickettsia helvetica from Tunisia. Ann. Trop. Med. Parasitol. 102, 561–564. Simser, J.A., Palmer, A.T., Fingerle, V., Wilske, B., Kurtti, T.J., Munderloh, U.G., 2002. Rickettsia monacensis sp. nov., a spotted fever group Rickettsia, from ticks (Ixodes ricinus) collected in a European city park. Appl. Environ. Microbiol. 68, 4559–4566. Varagnol, M., Parola, P., Jouan, R., Beaucournu, J.C., Rolain, J.M., Raoult, D., 2009. First detection of Rickettsia felis and Bartonella clarridgeiae in fleas from Laos. Clin. Microbiol. Infect. 15 (Suppl. 2), 334–335.
Contents lists available at SciVerse ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Short communication
Spotted fever group rickettsiae identified in Dermacentor marginatus and Ixodes ricinus ticks in Algeria Tahar Kernif a,b , Dalila Messaoudene a , Soraya Ouahioune a , Philippe Parola b , Didier Raoult b , Idir Bitam a,∗ a
Service d’Ecologie et des Systèmes Vectoriels, Institut Pasteur d’Algérie, Algiers, Algeria Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198, Inserm 1095, WHO Collaborative Center for Rickettsioses and Other Arthropod Borne Bacterial Diseases, Faculté de Médecine, 27 bd Jean Moulin, 13385 Marseille cedex 5, France b
a r t i c l e
i n f o
Keywords: Ixodes ricinus ticks Dermacentor marginatus ticks Rickettsia helvetica R. monacensis R. slovaca Algeria
a b s t r a c t Our study was carried out using Ixodes ricinus ticks collected from cattle from Tizi-Ouzou and Dermacentor marginatus ticks collected from the vegetation of the Blida region, a tourist site, both regions situated in northern Algeria. The results of real-time quantitative PCR (qPCR) specific for a partial sequence of the citrate synthase gene (gltA) indicate that Rickettsia spp. were present in 11/23 (48%) and 4/9 (44%) of the examined ticks from Tizi-Ouzou and Blida, respectively. The sequences of Rickettsia helvetica and Ri. monacensis were found in I. ricinus ticks using gltA primers. In addition, Ri. slovaca was detected based on the sequences of the gltA and the outer membrane protein (OmpA) genes in D. marginatus ticks. DNA sequencing to identify the species revealed for the first time the presence of Ri. helvetica in I. ricinus ticks and Ri. slovaca in D. marginatus ticks from Algeria and confirmed the presence of Ri. monacensis. © 2012 Elsevier GmbH. All rights reserved.
Introduction Spotted fever group (SFG) Rickettsia spp. is important emerging tick-borne human infection agents that are present worldwide (Parola et al., 2005). In Algeria, several Rickettsia spp. that are pathogenic for humans have been detected in various ticks, including Rickettsia conorii conorii, the causative agent of Mediterranean spotted fever, which is transmitted by Rhipicephalus sanguineus (Mouffok et al., 2009); Ri. aeschlimannii, an emerging pathogen detected in Hyalomma marginatum marginatum; Ri. massiliae, another emerging pathogen transmitted by Rh. sanguineus and Rh. turanicus (Bitam et al., 2006); and Ri. monacensis, which is generally found in Ixodes ricinus (Dib et al., 2009). The aim of the present study was to identify rickettsial species occurring in ticks collected from a specific area of Algeria that had not been previously investigated for tick-borne rickettsiae. Materials and methods In February 2006, we used the flag technique on vegetation close to the road to capture ticks in the mountains of the Blida region (Chréa Mountain) in northern Algeria. In December 2010, ticks were collected from cattle in Bouzguene (Tizi-Ouzou department),
Algeria. These ticks were identified phenotypically by using cur˜ et al., 2004), were stored in rent taxonomic criteria (Estrada-Pena 70% ethanol, and were sent to the WHO Collaborative Center for Rickettsial Diseases and Other Arthropod-borne Bacterial Diseases in Marseille, France. DNA from these specimens and negative controls (uninfected lice maintained in colonies in our laboratory) was extracted using a QIAamp Tissue Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Rickettsial DNA was detected using a Rickettsia genus-specific real-time PCR (qPCR) with a 25-bp probe targeting the partial sequence of the citrate synthase gene (gltA) using a 7900 HT Fast instrument (Varagnol et al., 2009). The positive control was DNA from Ri. montanensis (qPCR). Ticks analyzed by qPCR were considered to be positive when the cycle threshold was less than or equal to 30. All ticks identified as positive by qPCR were confirmed by 2 different standard PCRs and sequencing for fragments of the gltA and OmpA genes. DNA sequencing reactions were performed for all samples amplified by standard PCR, as previously described (Sarih et al., 2008). Data were collected with an ABI Prism 3130xl Genetic Analyzer capillary sequencer (ABI PRISM, PE Applied Biosystems, USA). Sequences were edited and assembled using Chromas Pro 1.34 (Technelysium Pty. Ltd., Tewantin, Australia). BLAST searches were performed to identify the obtained sequences. Results
∗ Corresponding author. Tel.: +213 21 673365; fax: +213 21 673365; mobile: +213 771995693. E-mail address: [email protected] (I. Bitam). 1877-959X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2012.10.012
The ticks collected from the Blida region were divided into 2 groups: The first group contained I. ricinus and Haemaphysalis spp.
380
T. Kernif et al. / Ticks and Tick-borne Diseases 3 (2012) 379–380
(not tested here, but used for other studies), and the second group contained 9 ticks identified as Dermacentor marginatus, which were tested for the presence of rickettsial DNA. A total of 4 ticks (4/9) tested positive by qPCR. These positive ticks were further screened and shown to be positive by 2 PCRs targeting the ompA and gltA genes. Analysis of the sequences obtained after these PCRs showed 100% homology with a 383-bp fragment of the Ri. slovaca gltA gene (GenBank accession number DQ821853.1) and with a 558-bp fragment of the Ri. slovaca ompA gene (GenBank accession number U43808.1). A total of 23 ticks was collected from Tizi-Ouzou; all were identified as I. ricinus. Using primers and TaqMan probes, qPCR revealed the presence of rickettsial DNA in 11/23 (48%) specimens and in the positive controls. All samples found to be positive by qPCR were screened and shown to be positive by standard PCR using gltA primers. Ri. helvetica was found in 4% (1/23) of tick specimens, and Ri. monacensis was found in 43% (10/23) of tick specimens. Sequence analysis showed 100% similarity to the gltA sequences of Ri. helvetica (accession number HM371185.1) and Ri. monacensis (accession number EU853831.1) in GenBank. Discussion Ri. slovaca is a member of the SFG rickettsiae and was first isolated from D. marginatus ticks in Slovakia in 1968. This pathogen was described as a causative agent of tick-borne lymphadenopathy (TIBOLA) in humans in 1997 (Raoult et al., 1997). Subsequently, this bacterium has been found in D. marginatus and D. reticulatus in several European countries, including France, Switzerland, Portugal, Spain, Armenia, Poland, Bulgaria, Croatia, Russia, Germany, and Morocco (Bitam and Raoult, 2009; Parola et al., 2009; Sarih et al., 2008). Here, we report for the first time the presence of R. slovaca in D. marginatus ticks from Algeria. TIBOLA (or DEBONEL for Dermacentor-borne necrosis– erythema–lymphadenopathy) has now been described for several European countries (Parola et al., 2009). In addition, Ri. slovaca has been associated with a syndrome characterized by scalp eschars and neck lymphadenopathy following tick bites, and the term ‘SENLAT’ has been proposed to collectively describe this clinical entity (Angelakis et al., 2010). Patients with similar clinical signs have been documented in France, Hungary, Spain, Bulgaria, and Italy. However, other agents, including Ri. raoultii, have been shown to cause SENLAT (Angelakis et al., 2010; Parola et al., 2009). In addition, we report here the first detection of Ri. helvetica in Algeria. This rickettsia was first isolated in Switzerland from I. ricinus in 1979 and has been identified in many European countries in I. ricinus ticks (Parola et al., 2005). In North Africa, it has been detected in I. ricinus ticks in Morocco and Tunisia (Sarih et al., 2008; Sfar et al., 2008). Ri. helvetica was considered to be a nonpathogenic rickettsia for approximately 20 years after its discovery. In 2000, seroconversion for Ri. helvetica was described in a patient in France with a nonspecific febrile illness (Fournier et al., 2000). Although several reports have suggested the association of Ri. helvetica with various syndromes in humans, a clear demonstration of its pathogenic role is still pending. Ri. monacensis was described and isolated from I. ricinus ticks collected in the English Garden in Munich, Germany, in 1998 (Simser et al., 2002). Furthermore, 2 human cases of infection due to Ri. monacensis have been documented in Spain (Jado et al., 2007).
In Algeria, Ri. monacensis was found in 35 I. ricinus ticks (59.3%) collected from cattle in the El Traf region of northeastern Algeria in 2009 (Dib et al., 2009); the presence of this rickettsia in North Africa was first documented when it was detected in I. ricinus ticks in Morocco and Tunisia (Sarih et al., 2008; Sfar et al., 2008). In our study, we confirmed the presence of Ri. monacensis in ticks from Tizi-Ouzou, approximately 120 km east from Algiers, the capital of Algeria. In conclusion, the results of this study confirmed that Ri. slovaca, Ri. helvetica, and Ri. monacensis are present in Algeria. The mountains of Chréa (Blida, Algeria) are increasingly visited by tourists, and there is a risk of being bitten by D. marginatus ticks carrying Ri. slovaca in this area. Additional studies are needed to determine the epidemiological and clinical relevance of different rickettsioses in these 2 regions. Clinicians should consider rickettsiosis due to these rickettsia species when diagnosing rickettsiosis cases in patients who have been bitten by ticks in Algeria. Acknowledgments We are grateful to IRD jeune équipe d’Algérie (associated with the IRD 198, Marseille) for their technical assistance. References Angelakis, E., Pulcini, C., Waton, J., Imbert, P., Socolovschi, C., Edouard, S., Dellamonica, P., Raoult, D., 2010. Scalp eschar and neck lymphadenopathy caused by Bartonella henselae after tick bite. Clin. Infect. Dis. 50, 549–551. Bitam, I., Parola, P., Matsumoto, K., Rolain, J.M., Baziz, B., Boubidi, S.C., Harrat, Z., Belkaid, M., Raoult, D., 2006. First molecular detection of R. conorii, R. aeschlimannii, and R. massiliae in ticks from Algeria. Ann. N. Y. Acad. Sci. 1078, 368–372. Bitam, I., Raoult, D., 2009. Other tick-borne diseases in Europe. Curr. Probl. Dermatol. 37, 130–154. Dib, L., Bitam, I., Bensouilah, M., Parola, P., Raoult, D., 2009. First description of Rickettsia monacensis in Ixodes ricinus in Algeria. Clin. Microbiol. Infect. 15 (Suppl. 2), 261–262. ˜ A., Bouattour, A., Camicas, J.-L., Walker, A.R., 2004. Ticks of DomesEstrada-Pena, tic Animals in the Mediterranean Region. A Guide to Identification of Species. University of Zaragoza, Spain, 131 pp. Fournier, P.E., Grunnenberger, F., Jaulhac, B., Gastinger, G., Raoult, D., 2000. Evidence of Rickettsia helvetica infection in humans, eastern France. Emerg. Infect. Dis. 6, 389–392. Jado, I., Oteo, J.A., Aldámiz, M., Gil, H., Escudero, R., Ibarra, sV., Portu, J., Portillo, A., Lezaun, M.J., Garcia-Amil, C., Rodríguez-Moreno, I., Anda, P., 2007. Rickettsia monacensis and human disease, Spain. Emerg. Infect. Dis. 13, 1405–1407. Mouffok, N., Parola, P., Abdennour, D., Aouati, A., Razik, F., Benabdellah, A., Raoult, D., 2009. Mediterranean spotted fever in Algerian children. Clin. Microbiol. Infect. 15 (Suppl. 2), 290–291. Parola, P., Paddock, C.D., Raoult, D., 2005. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin. Microbiol. Rev. 18, 719–756. Parola, P., Rovery, C., Rolain, J.M., Brouqui, P., Davoust, B., Raoult, D., 2009. Rickettsia slovaca and R. raoultii in tick-borne rickettsioses. Emerg. Infect. Dis. 15, 1105–1108. Raoult, D., Berbis, P., Roux, V., Xu, W., Maurin, M., 1997. A new tick-transmitted disease due to Rickettsia slovaca. Lancet 350, 112–113. Sarih, M., Socolovschi, C., Boudebouch, N., Hassar, M., Raoult, D., Parola, P., 2008. Spotted fever group rickettsiae in ticks, Morocco. Emerg. Infect. Dis. 14, 1067–1073. Sfar, N., M’ghirbi, Y., Letaief, A., Parola, P., Bouattour, A., Raoult, D., 2008. First report of Rickettsia monacensis and Rickettsia helvetica from Tunisia. Ann. Trop. Med. Parasitol. 102, 561–564. Simser, J.A., Palmer, A.T., Fingerle, V., Wilske, B., Kurtti, T.J., Munderloh, U.G., 2002. Rickettsia monacensis sp. nov., a spotted fever group Rickettsia, from ticks (Ixodes ricinus) collected in a European city park. Appl. Environ. Microbiol. 68, 4559–4566. Varagnol, M., Parola, P., Jouan, R., Beaucournu, J.C., Rolain, J.M., Raoult, D., 2009. First detection of Rickettsia felis and Bartonella clarridgeiae in fleas from Laos. Clin. Microbiol. Infect. 15 (Suppl. 2), 334–335.