LETTERS

5. Leung YHC, Zhang L-J, Chow C-K, JQ792166. Phylogenetic analysis was Tsang C-L, Ng C-F, Wong C-K, et conducted for sequences we identifi ed al. Poultry drinking water for avian raoultii–like infl uenza surveillance. Emerg Infect and sequences of recognized SFG Dis. 2007;13:1380–2. http://dx.doi. in rickettsial species available in org/10.3201/eid1309.070517 spp. Genbank by using the MegAlign 6. Stallknecht DE, Goekjian VH, Wilcox program (DNASTAR, Inc., Madison, BR, Poulson RL, Brown JD. Avian , Tibet, China infl uenza virus in aquatic habitats: WI, USA) and MEGA 4.0 (8). what do we need to learn? Avian Dis. To the Editor: Rickettsia raoultii Of 874 specimens, 86 were 2010;54(Suppl):461–5. http://dx.doi. is an obligate intracellular gram- D. everestianus ticks (13 male and 73 org/10.1637/8760-033109-Reg.1 female), and 788 were D. niveus ticks 7. Vong S, Ly S, Mardy S, Holl D, Buchy negative bacterium belonging to the P. Environmental contamination during spotted fever group (SFG) of the (133 male and 655 female). Samples infl uenza A virus (H5N1) outbreaks, genus Rickettsia. Genotypes RpA4, positive for gltA and ompA were Cambodia, 2006. Emerg Infect DnS14, and DnS28, originally isolated considered SFG rickettsial species. Dis. 2008;14:1303–5. http://dx.doi. Using this criterion, we found that org/10.3201/eid1408.070912 from ticks from Russia in 1999 (1), 8. World Health Organization. were designated as Rickettsia raoultii 739 tick specimens (84.6%) were Recommendations and laboratory sp. nov. on the basis of phylogenetic positive for Rickettsia spp. Of 86 D. procedures for detection of avian infl uenza analysis (2). R. raoultii has been found everestianus ticks, 85 (98.8%) were A(H5N1) virus in specimens from positive for Rickettsia spp. and of 788 suspected human cases. Revised August mainly in Dermacentor spp. ticks in 2007 [cited 2012 Jan 16]. http://www. several countries in Europe (3). It was D. niveus ticks, 654 (83.0%) were who.int/influenza/resources/documents/ detected in a Dermacentor marginatus positive. Infection rates for male and RecAIlabtestsAug07.pdf tick from the scalp of a patient with female D. niveus ticks were 87.9% 9. World Organisation for Animal Health. and 82.1%, respectively. We found Manual of diagnostics tests and vaccines tick-borne lymphadenitis in France for terrestrial animals 2010. Chapter 2.3.4: (2), which suggests that it might be a an overall prevalence of 84.6% for R. avian infl uenza [cited 2012 Jan 16]. http:// zoonotic pathogen. We determined the raoultii–like bacteria in Dermacentor www.oie.int/fi leadmin/Home/eng/Health_ prevalence of R. raoultii–like bacteria spp. in the highland regions in Tibet. standards/tahm/2.03.04_AI.pdf Nucleotide sequence identities 10. Nesbitt HJ. Rice production in Cambodia. in Dermacentor spp. in highland Manila: International Rice Research regions in Tibet. ranged from 99.2% to 100% (except Institute; 1997. Ticks from sheep (Ovis aries) for isolate WYG55, which had an near Namuco Lake (a popular tourist identity of 98.6%) for the ompA gene Address for correspondence: Philippe Buchy, destination 4,718 m above sea and from 99.2% to 99.9% (except for Institut Pasteur in Cambodia, Virology Unit, 5 level) were collected and identifi ed isolate XG86, which had an identity Monivong Blvd, PO Box 983, 12152 Phnom morphologically as D. everestianus and of 98.5%) for the ompB gene. These Penh, Cambodia; email: pbuchy@pasteur-kh. D. niveus ticks (4). Genomic DNA was results indicated that homology levels org extracted from individual specimens of most isolates were within species by using the QIAamp DNA Mini Kit thresholds (ompA ≈98.8% and ompB (QIAGEN, Hilden, Germany). All ≈99.2%) (9). Isolate WYG55 showed DNA samples were amplifi ed by using the lowest identity (98.2%) among PCRs specifi c for the citrate synthase gltA gene sequences and the lowest (gltA, 770 bp) gene (5) and the outer identity (98.6%) among ompA gene membrane protein A (ompA, 629 bp) sequences. Isolate XG86 showed gene (6). Some samples were amplifi ed lowest identity (98.5%) among ompB by using a PCR specifi c for the ompB gene sequences. These results suggest (2,479 bp) gene (7). that other Rickettsia spp. were among Randomly selected amplicons the investigated samples. for gltA (n = 27), ompA (n = 31), A BLASTn search (www. and ompB (n = 7) were cloned into ncbi.nlm.nih.gov/) for the obtained the pGEM-T Easy vector (Promega, sequences was conducted. The best Shanghai, China) and subjected to matches (highest identities) detected bidirectional sequencing (Sangon were with sequences of R. raoultii. Biotech, Shanghai, China). Sequences However, comparison of our sequences obtained were deposited in GenBank with corresponding sequences of R. under accession nos. JQ792101– raoultii in GenBank showed identity JQ792105, JQ792107, and JQ792108– ranging from 98.0% to 99.0% for

1532 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 9, September 2012 LETTERS ompA and from 98.1% to 99.0% for transstadially and transovarially (2). of Current and Development of New ompB, which did not meet the threshold Animals bitten by infected ticks can Vaccines for Theileriosis and Babesiosis (9) for R. raoultii. We compared the acquire the pathogen and serve as of Small Ruminants (PIROVAC) Project new sequences with corresponding natural reservoirs. (KBBE-3-245145) of the European reference sequences of universally On the basis of phylogenetic Commission, Brussels, Belgium. recognized SFG group Rickettsia analysis, we found that the Rickettsia spp. in Genbank and constructed 2 spp. in ticks investigated represents a Yuefeng Wang, Zhijie Liu, phylogenetic trees (Figure). The new novel species, which can be designated Jifei Yang, Ze Chen, Jianzhi Liu, sequences were placed into separate Candidatus Rickettsia tibetani. Youquan Li, Jianxun Luo, branches, which were closely related to However, additional phylogenetic and Hong Yin R. raoultii branches. studies are needed to obtain more Author affi liations: Lanzhou Veterinary Prevalence of R. slovaca and information on the molecular biology Research Institute, Lanzhou, Gansu, China R. raoultii was 6.5% and 4.5% in of these bacteria. (Y. Wang, Z. Liu, J. Yang, Z. Chen, Y. Li, J. D. silvarum ticks in Xinjiang Uygur Luo, H. Yin); and Tibet Livestock Research Autonomous Region of China (10). Acknowledgments Institute, Lhasa, Tibet, China (J. Liu) In contrast, we found that the overall We thank Robin B. Gasser for DOI: http://dx.doi.org/10.3201/eid1809.120644 prevalence of R. raoultii–like bacteria providing comments and revising the might be ≤84.6% in D. everestianus manuscript. and D. niveus ticks in Dangxiong References County in Tibet. This study was supported by the 973 Program (2010CB530206), the Key Project 1. Rydkina E, Roux V, Rudakov N, Our fi ndings suggest that D. Gafarova M, Tarasevich I, Raoult D. New everestianus and D. niveus ticks are of Gansu Province (1002NKDA035 and Rickettsiae in ticks collected in territories potential vectors of R. raoultii–like 0801NKDA033), the National Science of the former Soviet Union. Emerg Infect Dis. 1999;5:811–4. http://dx.doi. bacteria and indicate that spread of R. Foundation of China ( 30800820, 30972182, 31072130, and 31001061), the org/10.3201/eid0506.990612 raoultii-like bacteria encompasses a 2. Mediannikov O, Matsumoto K, large area in China. In the study sites, 948 Program (2012-S04), the National Samoylenko I, Drancourt M, Roux V, yak and Tibetan sheep are the major Beef Cattle Industrial Technology System, Rydkina E, et al. Rickettsia raoultii sp. nov., a spotted fever group rickettsia domestic animals, and rodents are the Ministry of Agriculture (CARS-38), the Network for Excellence for Epizootic associated with Dermacentor ticks in major wild animals. Rodents are also Europe and Russia. Int J Syst Evol the major hosts of Dermacentor spp. Disease Diagnosis and Control (FOOD- Microbiol. 2008;58:1635–9. http://dx.doi. ticks, which can transmit R. raoultii CT-2006-016236), and the Improvement org/10.1099/ijs.0.64952-0

Figure. Unrooted phylogenetic trees inferred from comparison of A) outer membrane protein A (ompA) and B) ompB gene sequences of rickettsial species by using the neighbor-joining method. Sequences in boldface were obtained during this study. Numbers at nodes are the proportion of 100 bootstrap resamplings that support the topology shown.

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3. Spitalská E, Stefanidesova K, Kocianova Leishmania shawi, but L. panamensis seems to be E, Boldis V. Rickettsia slovaca and a subspecies or even a synonym of L. Rickettsia raoultii in Dermacentor (Viannia) guyanensis marginatus and Dermacentor reticulatus guyanensis (2). We thus classifi ed our ticks from Slovak Republic. Exp Appl Infection, Austria strain as L. guyanensis. Sequence data Acarol. 2012;57:189–7. were deposited at GenBank (accession 4. Teng KF, Jiang ZJ. Economic insect To the Editor: Infection with no. JN671917). fauna of China, Acari, Ixodidae. Beijing: Leishmania spp. was diagnosed in L. guyanesis/panamensis is found Science Press; 1991. an asymptomatic soldier during an 5. Roux V, Rydkina E, Eremeeva M, Raoult in 9 countries in Central and South D. Citrate synthase gene comparison, a explorative national cross-sectional America (3). It is a common cause new tool for phylogenetic analysis, and its serologic screening of soldiers of zoonotic cutaneous leishmaniasis application for the rickettsiae. Int J Syst volunteering for United Nations in humans. The sloths Choloepus Bacteriol. 1997;47:252–61. http://dx.doi. missions at the Military Hospital org/10.1099/00207713-47-2-252 didactylus (L. guyanensis) and C. 6. Roux V, Fournier PE, Raoult D. Vienna in 2009. Diagnosis was made hoffmanni (L. panamensis) are Differentiation of spotted fever group by using a commercial ELISA kit believed to be the principal reservoir rickettsiae by sequencing and analysis of (Ridascreen Leishmania; R-Biopharm, hosts and the sandfl y species restriction fragment length polymorphism Darmstadt, Germany). One year of PCR-amplifi ed DNA of the gene Lutzomyia umbratilis (L. guyanensis) encoding the protein rOmpA. J Clin later, the soldier was reassessed for and Lu. trapidoi (L. panamensis) to be Microbiol. 1996;34:2058–65. persisting antibodies by using the the principal vectors (3). Also, dogs 7. Blair PJ, Jiang J, Schoeler GB, Moron same ELISA and for Leishmania DNA can act as reservoirs for the L. (V.) C, Anaya E, Cespedes M, et al. in a blood sample stored in EDTA Characterization of spotted fever group guyanensis complex (4). rickettsiae in fl ea and tick specimens by using the Leishmania OligoC- The infected soldier had never from northern Peru. J Clin Microbiol. Test (Coris BioConcept, Gembloux, been to Central or South America and 2004;42:4961–7. http://dx.doi.org/10. Belgium). (The study was approved had no history of blood transfusions. 1128/JCM.42.11.4961-4967.2004 by the Research Ethics Committee of 8. Tamura K, Dudley J, Nei M, Kumar His lifetime travel history included S. MEGA4: Molecular Evolutionary the Austrian Armed Forces and written Italy, Spain, Greece, Germany, Genetics Analysis (MEGA) software informed consent was obtained from Croatia, New York City, and military version 4.0. Mol Biol Evol. 2007;24:1596– the person investigated.) Because the assignments in Kosovo. Thus, how 9. http://dx.doi.org/10.1093/molbev/msm results of both tests were positive, an 092 and where the infection had been 9. Raoult D, Fournier PE, Eremeeva M, additional PCR was performed for acquired remain open for discussion. Graves S, Kelly PJ, Oteo JA, et al. Naming identifi cation below genus level with Although sandfl ies are not of Rickettsiae and rickettsial diseases. the LITSR/L5.8S primer pair (1). To as robust as Anopheles spp., for Ann N Y Acad Sci. 2005;1063:1–12. confi rm the PCR results, we sequenced http://dx.doi.org/10.1196/annals.1355.002 example, the most plausible scenario 10. Tian ZC, Liu GY, Shen H, Xie JR, Luo J, the amplicon in both directions in 2 is that either an L. guyanensis– Tian MY. First report on the occurrence of independent setups and compared infected sandfl y or a noninfected but Rickettsia slovaca and Rickettsia raoultii the obtained 299-bp sequence with transmissive sandfl y from a disease- in Dermacentor silvarum in China. published sequences from GenBank Parasit Vectors. 2012;5:9. http://dx.doi. endemic area was transported in org/10.1186/1756-3305-5-19 by performing a multiple sequence a ship or airplane (comparable to alignment. Our sequence (strain the well-known “airport malaria” Address for correspondence: Hong Yin, EN10) showed 100% (299/299 bp) situation) to an area where the patient Lanzhou Veterinary Research Institute, Chinese identity with several strains from the had traveled. In recent years, Lu. Academy of Agricultural Sciences, Xujiaping 1, Leishmania (Viannia) guyanensis vexator has become widespread and Lanzhou, Gansu, 730046, People’s Republic of complex, including the L. guyanensis abundant in upstate New York (5). China; email: [email protected] strain MHOM/SR/87/TRUUS4 and Although, this is not a known vector the L. panamensis strains FJ948438, for L. guyanensis, its spread in New FJ948439, and FJ948446. Sequence York State shows that Lutzomyia homology to representatives of the L. spp. can rapidly adapt to new and (V.) braziliensis complex was ≈93%; to distant areas where the infection representatives of the L. (Leishmania) was previously nonendemic. Of the mexicana complex, 61%–68%; and to areas where the infected person had the L. (L.) donovani complex, 70%– traveled, at least in New York City 71%. The L. (V.) guyanensis complex and Spain, regular introduction of L. traditionally includes the species L. guyanensis by immigrants, travelers, guyanensis, L. panamensis, and L. or dogs from Central and South

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