These results demonstrate that SFG rickettsiae with public health relevance are found in ticks in central Spain as in other regions in Spain. In central Spain, the widespread distribution of tick vectors and possible wildlife hosts, the presence of persons in tick-infested recreational and hunting areas, and the transstadial and transovarial transmission of the pathogen in ticks may favor transmission to humans.Fil: Fernandez de Mera, Isabel G.. Consejo Superior de Investigaciones Cientificas. Instituto de Investigación en Recursos Cinegéticos; España;Fil: Ruiz-Fons, Francisco. Consejo Superior de Investigaciones Cientificas. Instituto de Investigación en Recursos Cinegéticos; España;Fil: de la Fuente, Gabriela. Consejo Superior de Investigaciones Cientificas. Instituto de Investigación en Recursos Cinegéticos; España;Fil: Mangold, Atilio Jose. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria, Rafaela; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Gortázar, Christian. Consejo Superior de Investigaciones Cientificas. Instituto de Investigación en Recursos Cinegéticos; España;Fil: de la Fuente, José. Oklahoma State University; Estados Unidos de América

de la Fuente, Gabriela

de la Fuente, José

Fernandez de Mera, Isabel G.

Gortázar, Christian

Mangold, Atilio Jose

Ruiz-Fons, Francisco

Emerging Infectious Diseases

PubMed

Isabel G. Fernández de Mera

Francisco Ruiz-Fons

Gabriela de la Fuente

Atilio J. Mangold

Christian Gortázar

José de la Fuente

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Spotted Fever Group Rickettsiae in Questing Ticks, Central Spain

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LETTERSReferences  1. Poovorawan Y, Chongsrisawat V, Boudville I, Bock HL. Dengue virus infection: a major cause of acute hepatic liver failure in Thai children. Ann Trop Paediatr. 2006;26:17–23. http://dx.doi.org/10.1179/146532806X90565  2. Deepak NA, Patel ND. Differential diagnosis of acute liver failure in India. Ann Hepatol. 2006;5:150–6.  3 Carvalho de Macedo F, Nicol AF, Cooper LD, Yearslev M, Pires AR, Nuovo GJ. Histologic, viral and molecular correlates of dengue fever infection of the liver using highly sensitive immunohistochemistry. Diagn Mol Pathol. 2006;15:223–8. http://dx.doi.org/10.1097/01.pdm.00002 13462.60645.cd  4. Huerre MR, Lan NT, Marianneau P, Hue NB, Khun H, Hung NT, et al. Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children. Virchows Arch. 2001;438:107–15.  5. Deasy NP, Wendon J, Meire HB, Sidhu PS. The value of serial Doppler ultrasound as a predictor of clinical outcome and the need for transplantation in fulminant and severe acute liver failure. Br J Radiol. 1999;72:134–43.  6. Wachsberg RH, Bahramipour P, Sofocleous CT, Barone A. Hepatofugal flow in the portal venous system: pathophysiology, imaging findings, and diagnostic pitfalls. Radiographics. 2002;22:123–40.  7. Khongphatthanayothin A, Lertsapcharoen P, Supachokchaiwattana P, Satupan P, Thongchaiprasit K, Poovorawan Y, et al. Hepatosplanchnic circulatory dysfunction in acute hepatic infection: the case of dengue hemorrhagic fever. Shock. 2005;24:407–11. http://dx.doi.org/10.1097/01.shk.0000180981.70870.8c  8. Moriyasu F, Nishida O, Ban N, Nakamura T, Sakai M, Miyake T, et al. “Congestion index” of the portal vein. AJR Am J Roentgenol. 1986;146:735–9. http://dx.doi.org/10.2214/ajr.146.4.735  9. Sahaphong S, Riengrojpitak S, Bhamarapravati N, Chirachariyavej T. Electron microscopic study of the vascular endothelial cell in dengue hemorrhagic fever. Southeast Asian J Trop Med Public Health. 1980;11:194–204.10. Helmy A. Review article: updates in the pathogenesis and therapy of hepatic sinusoidal obstruction syndrome. Aliment Pharmacol Ther. 2006;23:11–25. http://dx.doi.org/10.1111/j.1365-2036. 2006.02742.xAddress for correspondence: Yong Poovorawan, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, 1873 Rama IV Rd., Pratumwan, Bangkok 10330, Thailand; email: yong.p@chula.ac.thSpotted Fever Group Rickettsiae in Questing Ticks, Central SpainTo the Editor: The number of spotted fever group (SFG) rickettsiae that cause diseases in humans is rap-idly increasing (1,2); infections have been described in ticks and humans in Spain (3,4). However, in Castilla-La Mancha, central Spain, where recre-ational parks and hunting estates are abundant and humans may be exposed to infected ticks, information on such infections is not available. Therefore, it is worthwhile to characterize Rick-ettsia spp. found in this area for epide-miologic studies and proper diagnosis of possible rickettsial diseases.In this study, we obtained 148 questing adult ticks, representing the most abundant species in the area: 12 Dermacentor marginatus, 26 Rhipi-cephalus bursa, 41 Rh. sanguineus, 15 Rh. turanicus, 8 Rh. pusillus, 2 Hae-maphysalis punctata, 11 Hyalomma lusitanicum, and 33 Hyalomma mar-ginatum (5). The ticks were collected from the vegetation at natural sites surveyed in Castilla-La Mancha by blanket dragging with a cotton flan-nelette during fall 2009 and spring–summer 2010 (Figure, panel A) and classified (5). Total DNA was extracted from dissected tick internal organs by us-ing the DNeasy Blood & Tissue Kit (QIAGEN, Düsseldorf, Germany) and used to analyze Rickettsia spp. DNA by PCR, cloning, and sequence analy-sis of the amplicons. At least 3 clones were sequenced for each amplicon. Genes targeted by PCR included frag-ments of adenosine triphosphate syn-thase α subunit (atpA), heat-shock protein 70 (dnaK), outer membrane protein A (ompA), outer membrane protein B (ompB), citrate synthase (gltA), 16S rRNA, recA, and initiator protein of DNA replication (dnaA) (6,7). To characterize Rickettsia spp., we compared nucleotide sequence identity to reference strains and car-ried out multilocus analysis using om-pA-ompB sequences and in silico PstI and RsaI restriction analysis of ompA sequences (7).Ticks were first screened by 16S rRNA PCR, and positive samples were analyzed for all targeted genes. The results showed that 27 (18.2%) of the 148 ticks analyzed were positive for Rickettsia spp. Of these, 11 were confirmed as R. massiliae in Rh. san-guineus, Rh. turanicus, and Rh. pusil-lus, 3 as R. raoultii in D. marginatus, 2 as R. slovaca in D. marginatus, and 2 as R. sibirica subsp. mongolitimo-nae in H. marginatum and Rh. pusillus (Figure, panel B). These species had >99% pairwise nucleotide sequence identity to reference strains R. mas-siliae MTU5 (GenBank accession no. NC_009900), R. slovaca 13-B (acces-sion no. NC_016639), and R. sibirica subsp. mongolitimonae HA-91 (ac-cession no. AHZB00000000) genome sequences for all genes analyzed, and the only R. raoultii reported sequences (accession nos. JQ792107, JQ792166, JQ792134, and NR_043755 for ompB, ompA, gltA, and 16S rRNA, respec-tively). The sequences obtained in this study were deposited in the GenBank under accession nos. KC427998–KC428040.Multilocus sequence analysis of ompA-ompB sequences (Figure, panel B) and in silico PstI and RsaI restric-tion analysis of ompA sequences also confirmed the identity of the Rick-ettsia spp. identified in this study. As previously shown (7,8), multilocus analysis with ompA-ompB sequences was highly informative about the 	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	19,	No.	7,	July	2013	 1163LETTERSphylogenetic relationship between Rickettsia spp. (Figure, panel B), with similar results for maximum likeli-hood, maximum parsimony, and neigh-bor-joining methods (data not shown). Furthermore, the results suggested the tick vectors for these Rickettsia spp. in the study area (Figure, panel B) match those reported or suspected previously for these Rickettsia spp. (1–4), but for the first time, R. sibirica subsp. mon-golitimonae was identified in Hya-lomma and Rhipicephalus spp. ticks in Spain (4).These tick species are frequently found in the same area feeding on Eur-asian wild boar (Sus scrofa) and red deer (Cervus elaphus), which may act as hosts for these pathogens (5,9). To test this hypothesis, we determined the seroprevalence for SFG rickettsiae in these host species in Castilla-La Mancha. Serum samples from 235 red deer and 206 wild boar were analyzed for the presence of anti-SFG Rickett-sia antibodies by ELISA (Spotted Fe-ver Rickettsia IgG EIA Antibody Kit, Fuller Laboratories, Fullerton, CA, USA). The ELISA was adapted to test ungulate serum specimens by sub-stituting antihuman IgG-horseradish by protein G-horseradish peroxidase (Sigma-Aldrich, Madrid, Spain). Spe-cific SFG-Rickettsia antibodies were detected in 146 (70.9%) of 206 wild boar and 174 (74.0%) of 235 red deer, indicating a high seroprevalence in these species and thus the possibility that they can serve as hosts for these pathogens.These tick species also infest hu-mans, thus posing a risk for transmis-sion of rickettsiae that are pathogenic in humans (1). In fact, Castilla-La Mancha is one of the regions in Spain where a high number of SFG rickettsi-oses are reported ([10]; http://pagina.jccm.es/sanidad/salud/epidemiolo-gia/3507.pdf).In conclusion, these results dem-onstrate that SFG rickettsiae with pub-lic health relevance are found in ticks in central Spain as in other regions in Spain. In central Spain, the wide-spread distribution of tick vectors and possible wildlife hosts, the presence of persons in tick-infested recreational and hunting areas, and the transstadial and transovarial transmission of the pathogen in ticks may favor transmis-sion to humans.AcknowledgmentsWe thank M. Durán-Martínez and R. Sobrino for help with tick surveys. F. R.-F. and  I.G.F.M. are support-ed by a Juan de la Cierva contract from the Spanish Ministry for Economy and Competitiveness. Research supported by POII09-0141-8176 and European Union FP7 ANTIGONE (Anticipating the Global Onset of Novel Epidemics) project num-ber 278976.Isabel G. Fernández de Mera, Francisco Ruiz-Fons,  Gabriela de la Fuente,  Atilio J. Mangold,  Christian Gortázar,  and José de la FuenteAuthor	affiliations:	Instituto	de	Investigación	en	 Recursos	 Cinegéticos	 (IREC)–CSIC-UCLM-JCCM,	 Ciudad	 Real,	 Spain	 (I.G.	Fernández	de	Mera,	F.	Ruiz-Fons,	G.	de	la	Fuente,	C.	Gortázar,	J.	de	 la	Fuente);	Uni-versidad	 Complutense	 de	 Madrid,	 Madrid,	1164	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	19,	No.	7,	July	2013Figure.	Rickettsia	species	in	questing	ticks	collected	in	central	Spain.	A)	Study	area	with	20	collection	sites	where	ticks	were	found	(black	dots)	of	the	39	sites	surveyed	(white	and	black	dots).	B)	Multilocus	sequence	analysis	of	Rickettsia	spp.	The	evolutionary	history	was	inferred	 by	 using	 the	 neighbor-joining	method	of	ompA-ompB	 concatenated	 sequences	(total	length	=	1,189	nt).	The	optimal	tree	with	the	sum	of	branch	length	=	0.15227017	is	shown.	The	percentage	of	replicate	trees	in	which	the	associated	taxa	clustered	together	in	the	bootstrap	test	(1,000	replicates)	is	shown	next	to	the	branches.	The	tree	is	drawn	to	 scale,	 with	 branch	 lengths	 in	 the	 same	 units	 as	 those	 of	 the	 evolutionary	 distances	used	 to	 infer	 the	 phylogenetic	 relationship.	 The	 evolutionary	 distances	 were	 computed	using	 the	Maximum	Composite	 Likelihood	method	 and	 are	 in	 the	 units	 of	 the	 number	of	 base	 substitutions	 per	 site.	 Codon	 positions	 included	were	 1st+2nd+3rd+noncoding.	All	 ambiguous	 positions	 were	 removed	 for	 each	 sequence	 pair.	 Evolutionary	 analyses	were	conducted	 in	MEGA5	 (www.megasofware.net).	The	number	of	 the	Rickettsia	 spp.	recognized	 in	 this	 study	 is	 shown	next	 to	 the	 tick	 spp.	 Identified	with	 them.	Clusters	of	identified	Rickettsia	spp.	are	shown.	Rc,	Rickettsia conorii	strain	Malish	7;	Ra,	R. africae strain	ESF-5;	Rr,	R. rickettsii	 strain	 Iowa;	Rs,	R. slovaca	 strain	13-B;	Rm,	R. massiliae strain	 MTU5;	 Rsm,	 R. sibirica	 subsp.	 mongolitimonae	 strain	 HA-91;	 R. raoultii	 isolate	XG86;	Rhsang,	Rhipicephalus sanguineus;	Rtur,	Rh. turanicus;	Rpus,	Rh. pusillus;	Dmar,	Dermacenter marginatus;	Hmar,	Hyalomma marginatum. Scale	bar	 indicates	number	of	nucleotide	changes	per	site.LETTERSSpain	 (I.G.	 Fernández	 de	 Mera);	 Instituto	Nacional	de	Tecnología	Agropecuaria,	San-ta	Fe,	Argentina	 (A.J.	Mangold);	and	Okla-homa	State	University,	Stillwater,	Oklahoma,	USA	(J.	de	la	Fuente).DOI:	http://dx.doi.org/10.3201/eid1907.130005References  1. Raoult D, Roux V. Rickettsioses as para-digms of new or emerging infectious diseases. Clin Microbiol Rev. 1997; 10:694–719.  2. Uchiyama T. Tropism and pathogenic-ity of rickettsiae. Front. Microbiol. 2012;3:230. http://dx.doi.org/10.3389/fmicb.2012.00230  3. Márquez FJ. Spotted fever group Rickettsia in ticks from southeastern Spain natural parks. Exp Appl Acarol. 2008;45:185–94. http://dx.doi.org/10.1007/s10493-008-9181-7  4. Aguirrebengoa K, Portillo A, Santibáñez S, Marín JJ, Montejo M, Oteo JA. Human Rickettsia sibirica mongolitimo-nae infection, Spain. Emerg Infect Dis. 2008;14:528–9. http://dx.doi.org/10.3201/eid1403.070987  5. Ruiz-Fons F, Fernández de Mera IG, Acevedo P, Höfle U, Vicente J, de la Fuente J, et al. Ticks (Acari: Ixodidae) parasitizing Iberian red deer (Cervus elaphus hispanicus) and European wild boar (Sus scrofa) from Spain: geographi-cal and temporal distribution. Vet Para-sitol. 2006;140:133–42. http://dx.doi.org/10.1016/j.vetpar.2006.03.033  6. Fernández de Mera IG, Zivkovic Z, Bolaños M, Carranza C, Pérez-Arellano JL, Gutiérrez C, et al. Rickettsia mas-siliae in the Canary Islands. Emerg In-fect Dis. 2009;15:1869–70. http://dx.doi.org/10.3201/eid1511.090681  7. Torina A, Fernández de Mera IG, Alongi A, Mangold AJ, Blanda V, Scarlata F, et al. Rickettsia conorii Indian tick typhus strain and R. slovaca in humans. Sicily. Emerg Infect Dis. 2012;18:1008–10.  8. Zhu Y, Fournier PE, Eremeeva M, Raoult D. Proposal to create subspecies of Rickettsia conorii based on multi-locus sequence typing and an emended description of Rickettsia conorii. BMC Microbiol. 2005;5:11. http://dx.doi.org/10.1186/1471-2180-5-11  9. de la Fuente J, Naranjo V, Ruiz-Fons F, Vicente J, Estrada-Peña A, Almazán C, et al. Prevalence of tick-borne patho-gens in ixodid ticks (Acari: Ixodidae) collected from European wild boar (Sus scrofa) and Iberian red deer (Cervus ela-phus hispanicus) in central Spain. Eur J Wildl Res. 2004;50:187–96. http://dx.doi.org/10.1007/s10344-004-0060-110. Bartolomé J, Lorente S, Hernández-Pérez N, Martínez-Alfaro E, Marín-Orsa A, Crespo MD. Estudio clínico-epidemi-ológico de las rickettsiosis del grupo de las fiebres exantemáticas en Albacete. Enferm Infecc Microbiol Clin. 2005;23:194–6. http://dx.doi.org/10.1157/13073143Address for correspondence: José de la Fuente, Instituto de Investigación en Recursos Cinegéticos (IREC-CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13005 Ciudad Real, Spain; email: jose_delafuente@yahoo.comNeonatal  Granulicatella  elegans  Bacteremia,  London, UKTo the Editor: Granulicatella el-egans, a bacterium found in normal hu-man oral flora, is generally associated with infective endocarditis. We discuss the identification and possible source of neonatal G. elegans bacteremia.A 29-year-old woman sought care at Northwick Park Hospital (Lon-don, UK) at 41 weeks’ gestation (first pregnancy) for spontaneous rupture of membranes and discharge of clear liquor. She had fever (37.6°C) and a heart rate of 98 beats/min; there was no evidence of fetal distress. The woman was released from the hospital.Twelve hours later, she returned because of discharge of meconium-stained liquor. Her white cell count was 18 × 109/L (reference 3–10 × 109/L), and her C-reactive protein lev-el was 277 mg/L (reference <5 mg/L). Emergency cesarean section was per-formed after a diagnosis of fetal dis-tress. A large amount of foul-smelling meconium was observed. A live male infant (3.05 kg) was delivered; Ap-gar score was normal. Blood samples were cultured for suspected sepsis, and the neonate was empirically administered intravenous benzylpeni-cillin and amikacin (6 days). He made a full clinical recovery.The mother remained gener-ally well, although she had persistent tachycardia (120 beats/min) and fever (37.6°C). She was intravenously ad-ministered amoxicillin/clavulanic acid and amikacin; over the next 2 days, her white cell count became normal, but her C-reactive protein level re-mained >400 mg/L. By postdelivery day 10, her temperature and heart rate were normal. Antimicrobial drug treatment was stopped, and she was released without further treatment. We interviewed the mother 8 months later and established that she had no dental procedures/infection or endocarditis before, during, or after pregnancy.Placental swab samples were cul-tured on Columbia horse blood agar (CBA) and chocolated CBA (both in-cubated aerobically with 5% CO2 at 37°C for 24 hours), cysteine lactose electrolyte deficient agar (incubated in air at 37°C for 24 hours), and fastidi-ous anaerobic agar with and without neomycin (incubated anaerobically at 37°C for 48 hours); all agar was from Thermo Fisher, Basingstoke, UK. On all media, the placental swab sample yielded moderate growth of tiny colo-nies, which Gram staining indicated were gram-positive coccobacilli.Culture of the neonate’s blood sample (BacTalert 3D; Becton Dick-inson, Oxford, UK) grew small, gram-variable bacilli after 17 hours of aero-bic incubation. A subculture incubated aerobically on CBA or chocolated CBA showed no bacterial growth; however, tiny colonies were seen on fastidious anaerobic agar with and without neo-mycin. Gram staining of the colonies showed gram-positive bacilli that were morphologically similar to those isolat-ed from placenta. We suspected lacto-bacilli or streptococci, but testing (API Strep and Coryne strips; bioMérieux UK Ltd, Hampshire, UK) did not con-firm this. Nutritionally variant strepto-cocci were not suspected.	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	19,	No.	7,	July	2013	 1165

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LETTERSReferences  1. Poovorawan Y, Chongsrisawat V, Boudville I, Bock HL. Dengue virus infection: a major cause of acute hepatic liver failure in Thai children. Ann Trop Paediatr. 2006;26:17–23. http://dx.doi.org/10.1179/146532806X90565  2. Deepak NA, Patel ND. Differential diagnosis of acute liver failure in India. Ann Hepatol. 2006;5:150–6.  3 Carvalho de Macedo F, Nicol AF, Cooper LD, Yearslev M, Pires AR, Nuovo GJ. Histologic, viral and molecular correlates of dengue fever infection of the liver using highly sensitive immunohistochemistry. Diagn Mol Pathol. 2006;15:223–8. http://dx.doi.org/10.1097/01.pdm.00002 13462.60645.cd  4. Huerre MR, Lan NT, Marianneau P, Hue NB, Khun H, Hung NT, et al. Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children. Virchows Arch. 2001;438:107–15.  5. Deasy NP, Wendon J, Meire HB, Sidhu PS. The value of serial Doppler ultrasound as a predictor of clinical outcome and the need for transplantation in fulminant and severe acute liver failure. Br J Radiol. 1999;72:134–43.  6. Wachsberg RH, Bahramipour P, Sofocleous CT, Barone A. Hepatofugal flow in the portal venous system: pathophysiology, imaging findings, and diagnostic pitfalls. Radiographics. 2002;22:123–40.  7. Khongphatthanayothin A, Lertsapcharoen P, Supachokchaiwattana P, Satupan P, Thongchaiprasit K, Poovorawan Y, et al. Hepatosplanchnic circulatory dysfunction in acute hepatic infection: the case of dengue hemorrhagic fever. Shock. 2005;24:407–11. http://dx.doi.org/10.1097/01.shk.0000180981.70870.8c  8. Moriyasu F, Nishida O, Ban N, Nakamura T, Sakai M, Miyake T, et al. “Congestion index” of the portal vein. AJR Am J Roentgenol. 1986;146:735–9. http://dx.doi.org/10.2214/ajr.146.4.735  9. Sahaphong S, Riengrojpitak S, Bhamarapravati N, Chirachariyavej T. Electron microscopic study of the vascular endothelial cell in dengue hemorrhagic fever. Southeast Asian J Trop Med Public Health. 1980;11:194–204.10. Helmy A. Review article: updates in the pathogenesis and therapy of hepatic sinusoidal obstruction syndrome. Aliment Pharmacol Ther. 2006;23:11–25. http://dx.doi.org/10.1111/j.1365-2036. 2006.02742.xAddress for correspondence: Yong Poovorawan, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, 1873 Rama IV Rd., Pratumwan, Bangkok 10330, Thailand; email: yong.p@chula.ac.thSpotted Fever Group Rickettsiae in Questing Ticks, Central SpainTo the Editor: The number of spotted fever group (SFG) rickettsiae that cause diseases in humans is rap-idly increasing (1,2); infections have been described in ticks and humans in Spain (3,4). However, in Castilla-La Mancha, central Spain, where recre-ational parks and hunting estates are abundant and humans may be exposed to infected ticks, information on such infections is not available. Therefore, it is worthwhile to characterize Rick-ettsia spp. found in this area for epide-miologic studies and proper diagnosis of possible rickettsial diseases.In this study, we obtained 148 questing adult ticks, representing the most abundant species in the area: 12 Dermacentor marginatus, 26 Rhipi-cephalus bursa, 41 Rh. sanguineus, 15 Rh. turanicus, 8 Rh. pusillus, 2 Hae-maphysalis punctata, 11 Hyalomma lusitanicum, and 33 Hyalomma mar-ginatum (5). The ticks were collected from the vegetation at natural sites surveyed in Castilla-La Mancha by blanket dragging with a cotton flan-nelette during fall 2009 and spring–summer 2010 (Figure, panel A) and classified (5). Total DNA was extracted from dissected tick internal organs by us-ing the DNeasy Blood & Tissue Kit (QIAGEN, Düsseldorf, Germany) and used to analyze Rickettsia spp. DNA by PCR, cloning, and sequence analy-sis of the amplicons. At least 3 clones were sequenced for each amplicon. Genes targeted by PCR included frag-ments of adenosine triphosphate syn-thase α subunit (atpA), heat-shock protein 70 (dnaK), outer membrane protein A (ompA), outer membrane protein B (ompB), citrate synthase (gltA), 16S rRNA, recA, and initiator protein of DNA replication (dnaA) (6,7). To characterize Rickettsia spp., we compared nucleotide sequence identity to reference strains and car-ried out multilocus analysis using om-pA-ompB sequences and in silico PstI and RsaI restriction analysis of ompA sequences (7).Ticks were first screened by 16S rRNA PCR, and positive samples were analyzed for all targeted genes. The results showed that 27 (18.2%) of the 148 ticks analyzed were positive for Rickettsia spp. Of these, 11 were confirmed as R. massiliae in Rh. san-guineus, Rh. turanicus, and Rh. pusil-lus, 3 as R. raoultii in D. marginatus, 2 as R. slovaca in D. marginatus, and 2 as R. sibirica subsp. mongolitimo-nae in H. marginatum and Rh. pusillus (Figure, panel B). These species had >99% pairwise nucleotide sequence identity to reference strains R. mas-siliae MTU5 (GenBank accession no. NC_009900), R. slovaca 13-B (acces-sion no. NC_016639), and R. sibirica subsp. mongolitimonae HA-91 (ac-cession no. AHZB00000000) genome sequences for all genes analyzed, and the only R. raoultii reported sequences (accession nos. JQ792107, JQ792166, JQ792134, and NR_043755 for ompB, ompA, gltA, and 16S rRNA, respec-tively). The sequences obtained in this study were deposited in the GenBank under accession nos. KC427998–KC428040.Multilocus sequence analysis of ompA-ompB sequences (Figure, panel B) and in silico PstI and RsaI restric-tion analysis of ompA sequences also confirmed the identity of the Rick-ettsia spp. identified in this study. As previously shown (7,8), multilocus analysis with ompA-ompB sequences was highly informative about the 	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	19,	No.	7,	July	2013	 1163LETTERSphylogenetic relationship between Rickettsia spp. (Figure, panel B), with similar results for maximum likeli-hood, maximum parsimony, and neigh-bor-joining methods (data not shown). Furthermore, the results suggested the tick vectors for these Rickettsia spp. in the study area (Figure, panel B) match those reported or suspected previously for these Rickettsia spp. (1–4), but for the first time, R. sibirica subsp. mon-golitimonae was identified in Hya-lomma and Rhipicephalus spp. ticks in Spain (4).These tick species are frequently found in the same area feeding on Eur-asian wild boar (Sus scrofa) and red deer (Cervus elaphus), which may act as hosts for these pathogens (5,9). To test this hypothesis, we determined the seroprevalence for SFG rickettsiae in these host species in Castilla-La Mancha. Serum samples from 235 red deer and 206 wild boar were analyzed for the presence of anti-SFG Rickett-sia antibodies by ELISA (Spotted Fe-ver Rickettsia IgG EIA Antibody Kit, Fuller Laboratories, Fullerton, CA, USA). The ELISA was adapted to test ungulate serum specimens by sub-stituting antihuman IgG-horseradish by protein G-horseradish peroxidase (Sigma-Aldrich, Madrid, Spain). Spe-cific SFG-Rickettsia antibodies were detected in 146 (70.9%) of 206 wild boar and 174 (74.0%) of 235 red deer, indicating a high seroprevalence in these species and thus the possibility that they can serve as hosts for these pathogens.These tick species also infest hu-mans, thus posing a risk for transmis-sion of rickettsiae that are pathogenic in humans (1). In fact, Castilla-La Mancha is one of the regions in Spain where a high number of SFG rickettsi-oses are reported ([10]; http://pagina.jccm.es/sanidad/salud/epidemiolo-gia/3507.pdf).In conclusion, these results dem-onstrate that SFG rickettsiae with pub-lic health relevance are found in ticks in central Spain as in other regions in Spain. In central Spain, the wide-spread distribution of tick vectors and possible wildlife hosts, the presence of persons in tick-infested recreational and hunting areas, and the transstadial and transovarial transmission of the pathogen in ticks may favor transmis-sion to humans.AcknowledgmentsWe thank M. Durán-Martínez and R. Sobrino for help with tick surveys. F. R.-F. and  I.G.F.M. are support-ed by a Juan de la Cierva contract from the Spanish Ministry for Economy and Competitiveness. Research supported by POII09-0141-8176 and European Union FP7 ANTIGONE (Anticipating the Global Onset of Novel Epidemics) project num-ber 278976.Isabel G. Fernández de Mera, Francisco Ruiz-Fons,  Gabriela de la Fuente,  Atilio J. Mangold,  Christian Gortázar,  and José de la FuenteAuthor	affiliations:	Instituto	de	Investigación	en	 Recursos	 Cinegéticos	 (IREC)–CSIC-UCLM-JCCM,	 Ciudad	 Real,	 Spain	 (I.G.	Fernández	de	Mera,	F.	Ruiz-Fons,	G.	de	la	Fuente,	C.	Gortázar,	J.	de	 la	Fuente);	Uni-versidad	 Complutense	 de	 Madrid,	 Madrid,	1164	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	19,	No.	7,	July	2013Figure.	Rickettsia	species	in	questing	ticks	collected	in	central	Spain.	A)	Study	area	with	20	collection	sites	where	ticks	were	found	(black	dots)	of	the	39	sites	surveyed	(white	and	black	dots).	B)	Multilocus	sequence	analysis	of	Rickettsia	spp.	The	evolutionary	history	was	inferred	 by	 using	 the	 neighbor-joining	method	of	ompA-ompB	 concatenated	 sequences	(total	length	=	1,189	nt).	The	optimal	tree	with	the	sum	of	branch	length	=	0.15227017	is	shown.	The	percentage	of	replicate	trees	in	which	the	associated	taxa	clustered	together	in	the	bootstrap	test	(1,000	replicates)	is	shown	next	to	the	branches.	The	tree	is	drawn	to	 scale,	 with	 branch	 lengths	 in	 the	 same	 units	 as	 those	 of	 the	 evolutionary	 distances	used	 to	 infer	 the	 phylogenetic	 relationship.	 The	 evolutionary	 distances	 were	 computed	using	 the	Maximum	Composite	 Likelihood	method	 and	 are	 in	 the	 units	 of	 the	 number	of	 base	 substitutions	 per	 site.	 Codon	 positions	 included	were	 1st+2nd+3rd+noncoding.	All	 ambiguous	 positions	 were	 removed	 for	 each	 sequence	 pair.	 Evolutionary	 analyses	were	conducted	 in	MEGA5	 (www.megasofware.net).	The	number	of	 the	Rickettsia	 spp.	recognized	 in	 this	 study	 is	 shown	next	 to	 the	 tick	 spp.	 Identified	with	 them.	Clusters	of	identified	Rickettsia	spp.	are	shown.	Rc,	Rickettsia conorii	strain	Malish	7;	Ra,	R. africae strain	ESF-5;	Rr,	R. rickettsii	 strain	 Iowa;	Rs,	R. slovaca	 strain	13-B;	Rm,	R. massiliae strain	 MTU5;	 Rsm,	R. sibirica	 subsp.	mongolitimonae	 strain	 HA-91;	R. raoultii	 isolate	XG86;	Rhsang,	Rhipicephalus sanguineus;	Rtur,	Rh. turanicus;	Rpus,	Rh. pusillus;	Dmar,	Dermacenter marginatus;	Hmar,	Hyalomma marginatum. Scale	bar	 indicates	number	of	nucleotide	changes	per	site.LETTERSSpain	 (I.G.	 Fernández	 de	 Mera);	 Instituto	Nacional	de	Tecnología	Agropecuaria,	San-ta	Fe,	Argentina	 (A.J.	Mangold);	and	Okla-homa	State	University,	Stillwater,	Oklahoma,	USA	(J.	de	la	Fuente).DOI:	http://dx.doi.org/10.3201/eid1907.130005References  1. Raoult D, Roux V. Rickettsioses as para-digms of new or emerging infectious diseases. Clin Microbiol Rev. 1997; 10:694–719.  2. Uchiyama T. Tropism and pathogenic-ity of rickettsiae. Front. Microbiol. 2012;3:230. http://dx.doi.org/10.3389/fmicb.2012.00230  3. Márquez FJ. Spotted fever group Rickettsia in ticks from southeastern Spain natural parks. Exp Appl Acarol. 2008;45:185–94. http://dx.doi.org/10.1007/s10493-008-9181-7  4. Aguirrebengoa K, Portillo A, Santibáñez S, Marín JJ, Montejo M, Oteo JA. Human Rickettsia sibirica mongolitimo-nae infection, Spain. Emerg Infect Dis. 2008;14:528–9. http://dx.doi.org/10.3201/eid1403.070987  5. Ruiz-Fons F, Fernández de Mera IG, Acevedo P, Höfle U, Vicente J, de la Fuente J, et al. Ticks (Acari: Ixodidae) parasitizing Iberian red deer (Cervus elaphus hispanicus) and European wild boar (Sus scrofa) from Spain: geographi-cal and temporal distribution. Vet Para-sitol. 2006;140:133–42. http://dx.doi.org/10.1016/j.vetpar.2006.03.033  6. Fernández de Mera IG, Zivkovic Z, Bolaños M, Carranza C, Pérez-Arellano JL, Gutiérrez C, et al. Rickettsia mas-siliae in the Canary Islands. Emerg In-fect Dis. 2009;15:1869–70. http://dx.doi.org/10.3201/eid1511.090681  7. Torina A, Fernández de Mera IG, Alongi A, Mangold AJ, Blanda V, Scarlata F, et al. Rickettsia conorii Indian tick typhus strain and R. slovaca in humans. Sicily. Emerg Infect Dis. 2012;18:1008–10.  8. Zhu Y, Fournier PE, Eremeeva M, Raoult D. Proposal to create subspecies of Rickettsia conorii based on multi-locus sequence typing and an emended description of Rickettsia conorii. BMC Microbiol. 2005;5:11. http://dx.doi.org/10.1186/1471-2180-5-11  9. de la Fuente J, Naranjo V, Ruiz-Fons F, Vicente J, Estrada-Peña A, Almazán C, et al. Prevalence of tick-borne patho-gens in ixodid ticks (Acari: Ixodidae) collected from European wild boar (Sus scrofa) and Iberian red deer (Cervus ela-phus hispanicus) in central Spain. Eur J Wildl Res. 2004;50:187–96. http://dx.doi.org/10.1007/s10344-004-0060-110. Bartolomé J, Lorente S, Hernández-Pérez N, Martínez-Alfaro E, Marín-Orsa A, Crespo MD. Estudio clínico-epidemi-ológico de las rickettsiosis del grupo de las fiebres exantemáticas en Albacete. Enferm Infecc Microbiol Clin. 2005;23:194–6. http://dx.doi.org/10.1157/13073143Address for correspondence: José de la Fuente, Instituto de Investigación en Recursos Cinegéticos (IREC-CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13005 Ciudad Real, Spain; email: jose_delafuente@yahoo.comNeonatal  Granulicatella  elegans  Bacteremia,  London, UKTo the Editor: Granulicatella el-egans, a bacterium found in normal hu-man oral flora, is generally associated with infective endocarditis. We discuss the identification and possible source of neonatal G. elegans bacteremia.A 29-year-old woman sought care at Northwick Park Hospital (Lon-don, UK) at 41 weeks’ gestation (first pregnancy) for spontaneous rupture of membranes and discharge of clear liquor. She had fever (37.6°C) and a heart rate of 98 beats/min; there was no evidence of fetal distress. The woman was released from the hospital.Twelve hours later, she returned because of discharge of meconium-stained liquor. Her white cell count was 18 × 109/L (reference 3–10 × 109/L), and her C-reactive protein lev-el was 277 mg/L (reference <5 mg/L). Emergency cesarean section was per-formed after a diagnosis of fetal dis-tress. A large amount of foul-smelling meconium was observed. A live male infant (3.05 kg) was delivered; Ap-gar score was normal. Blood samples were cultured for suspected sepsis, and the neonate was empirically administered intravenous benzylpeni-cillin and amikacin (6 days). He made a full clinical recovery.The mother remained gener-ally well, although she had persistent tachycardia (120 beats/min) and fever (37.6°C). She was intravenously ad-ministered amoxicillin/clavulanic acid and amikacin; over the next 2 days, her white cell count became normal, but her C-reactive protein level re-mained >400 mg/L. By postdelivery day 10, her temperature and heart rate were normal. Antimicrobial drug treatment was stopped, and she was released without further treatment. We interviewed the mother 8 months later and established that she had no dental procedures/infection or endocarditis before, during, or after pregnancy.Placental swab samples were cul-tured on Columbia horse blood agar (CBA) and chocolated CBA (both in-cubated aerobically with 5% CO2 at 37°C for 24 hours), cysteine lactose electrolyte deficient agar (incubated in air at 37°C for 24 hours), and fastidi-ous anaerobic agar with and without neomycin (incubated anaerobically at 37°C for 48 hours); all agar was from Thermo Fisher, Basingstoke, UK. On all media, the placental swab sample yielded moderate growth of tiny colo-nies, which Gram staining indicated were gram-positive coccobacilli.Culture of the neonate’s blood sample (BacTalert 3D; Becton Dick-inson, Oxford, UK) grew small, gram-variable bacilli after 17 hours of aero-bic incubation. A subculture incubated aerobically on CBA or chocolated CBA showed no bacterial growth; however, tiny colonies were seen on fastidious anaerobic agar with and without neo-mycin. Gram staining of the colonies showed gram-positive bacilli that were morphologically similar to those isolat-ed from placenta. We suspected lacto-bacilli or streptococci, but testing (API Strep and Coryne strips; bioMérieux UK Ltd, Hampshire, UK) did not con-firm this. Nutritionally variant strepto-cocci were not suspected.	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	19,	No.	7,	July	2013	 1165

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Spotted Fever Group Rickettsiae in Questing Ticks, Central Spain

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