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laticaudae from snakes on Pindai Island, New Caledonia; Emergence of 60 female Haemaphysalis longicornis, including 38 from cattle on Santo Island, Vanuatu, 10 from cattle in Port-La- africae, guerre, New Caledonia, and 12 from horses in New Cale- donia; and 7 female Rhipicephalus sanguineus from dogs Oceania in Païta, New Caledonia. Tick species were identifi ed by Carole Eldin, Oleg Mediannikov, using taxonomic keys and stored in 70% ethanol before Bernard Davoust, Olivier Cabre, Nicolas Barré, being tested. DNA was extracted from each tick with the Didier Raoult, and Philippe Parola QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) fol- lowing the manufacturer’s instructions and stored at 4°C We detected Rickettsia africae, the agent of African until it was used in PCR amplifi cations. Each sample was tick-bite fever (ATBF), by amplifi cation of fragments of gltA, tested by quantitative PCR (qPCR) in a LightCycler instru- ompA, and ompB genes from 3 specimens of Amblyomma ment (Roche Diagnostics GmbH, Mannheim, Germany) loculosum ticks collected from humans and birds in New for the presence of Rickettsia spp. DNA by using primers Caledonia. Clinicians who treat persons in this region should and Taqman probes (Eurogentec, Seraing, Belgium) that be on alert for ATBF. targeted a partial sequence of the citrate synthase (gltA) gene (6). Tick DNA samples positive by using qPCR were potted fever group (SFG) rickettsioses are caused by also subjected to standard PCR using CS2d and CS.1258n, Sobligate intracellular gram-negative of the which amplify an 1,178-bp fragment of Rickettsia gltA. genus Rickettsia and are transmitted by hematophagous An additional PCR amplifi cation was performed by using arthropods, mainly ticks. These zoonoses are important primers 190.70, 190.180, and 190.701, which amplify a emerging vector-borne infections of humans worldwide. 629–632-bp fragment of rickettsial ompA. Positive controls They share characteristic clinical features, including fever, (R. montanensis DNA) and negative controls (sterile water rash, and sometimes an inoculation eschar at the bite site, and DNA extracted from uninfected ticks from laboratory depending on the rickettsial agent that is involved (1). colonies) were included in each test. We also amplifi ed In Oceania, tick-borne rickettsioses have been reported and sequenced the 2,113–4,346-bp portion of rickettsial primarily in Australia. They include Queensland tick ompB (7). All amplifi ed products were purifi ed, directly se- (R. australis) along the east coast of Australia (2), Flinders quenced, and sequences were compared with sequences in Island (R. honei) in southeast Australia (2), the GenBank database. and variant Flinders Island spotted fever (R. honei strain Positive and negative controls gave expected results “marmionii”) in eastern Australia (2). Furthermore, the in all tests. qPCR and subsequent standard PCR for ompA DNA of at least 8 incompletely described SFG rickettsiae and gltA were positive for 3 specimens of A. loculosum: 2 have been detected in ticks, and the pathogenicity of these nymphs collected from humans on Chesterfi eld Island, New rickettsiae remains unknown (2). Additionally, R. felis, the Caledonia, and 1 female collected from birds on Walpole agent of fl ea-borne SFG , has been found in Island, New Caledonia. The 590-bp amplicons of ompA Western Australia (3), New Zealand (4), and recently in and 1,135-bp amplicons of gltA obtained from all samples New Caledonia (5). However, little is known about rick- showed 100% identity with the relevant genes of strain ettsioses in the rest of Oceania. ESF-5 of R. africae (GenBank accession no. CP001612.1). A 2,153-bp amplicon of a portion of ompB showed 99.8% The Study identity with R. africae (strain ESF-5). From February 2001 to November 2007, tick samples Multispacer typing of the detected rickettsiae was per- were obtained as part of other, ongoing studies in Oceania. formed by using the dksA-xerC, mppA-purC, and rpmE- A total of 92 ticks were collected: 14 Amblyomma locu- tRNA-fMet intergenic spacers (8). The sequence of the dk- losum (13 nymphs, 1 female), including 2 from humans sA-xerC intergenic spacer was found to be 242 bp instead on Chesterfi eld Island, New Caledonia, and 12 from birds of the 177 bp described for the ESF-5 strain of R. africae on Walpole Island, New Caledonia; 9 female A. breviscu- (GenBank accession no. DQ008280), due to the 65-bp re- tatum from swine on Santo Island, Vanuatu; 2 female A. peat of portion 1–65. A single-nucleotide polymorphism was also found. The rpmE-tRNA-fMet (GenBank acces- Author affi liations: Université de la Méditerranée, Marseille, France sion no. DQ008246) spacer showed 100% identity with the (C. Eldin, O. Mediannikov, B. Davoust, D. Raoult, P. Parola); French rpmE-tRNA-fMet spacer of R. africae. The mppA-purC am- Army Health Service, Marseille (B. Davoust, O. Cabre); and Institut plifi cation attempt was negative. Whether this intergenic Agronomique néo-Calédonien, Païta, New Caledonia (N. Barré) spacer has been lost or substantially modifi ed remains un- DOI: 10.3201/eid1701.101081 known. Finally, qPCR with primers 1267F/1267R specifi c

100 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 1, January 2011 Rickettsia africae, Oceania for a sequence of the R. africae plasmid (pRA) produced Further investigations are necessary to evaluate the preva- positive results in 3 ticks infected by R. africae (9). lence and distribution of R. africae in New Caledonia and in other islands of Oceania, as well as its interactions with Conclusions A. loculosum ticks. However, clinicians in Oceania and By using molecular criteria (10), we identifi ed R. af- around the world should be aware of the presence of R. ricae in an unexpected place and in unexpected tick spe- africae in New Caledonia, an island increasingly visited by cies. Indeed, R. africae is the typical agent of African tourists from all over the world, and in other islands where tick-bite fever. This SFG rickettsia is prevalent mostly in A. loculosum ticks are prevalent. sub-Saharan Africa, where vectors are ticks of the genus Amblyomma (mainly A. hebraeum and A. variegatum) (11). Dr Eldin is a resident specialist in infectious diseases and trop- The rates of R. africae infection in ticks in ATBF-endemic ical medicine at the University Hospital of Marseille. Her research areas are typically high and may reach 100% (11). These interests include medical entomology and tick-borne rickettsioses. ticks have also been shown to act as reservoirs for R. afri- cae (12). ATBF occurs 5 to 10 days after the bite of a tick infected with R. africae (1). Clinical features include an References inoculation eschar, frequently more than one, predominant 1. Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the on the lower limbs; fever; regional lymphadenopathy; and world: emerging diseases challenging old concepts. Clin Microbiol a rash that is sometimes vesicular (1). Rev. 2005;18:719–56. DOI: 10.1128/CMR.18.4.719-756.2005 In our study, 2 of 3 amplifi ed and sequenced genes 2. Graves S, Stenos J. Rickettsioses in Australia. Ann N Y Acad Sci. 2009;1166:151–5. DOI: 10.1111/j.1749-6632.2009.04530.x showed 100% identity with R. africae, and a third gene 3. Schloderer D, Owen H, Clark P, Stenos J, Fenwick SG. Rickettsia (ompB) showed a degree of difference less than that needed felis in fl eas, Western Australia. Emerg Infect Dis. 2006;12:841–3. to determine a new species (10). Thus, our fi ndings dem- 4. Kelly PJ, Meads N, Theobald A, Fournier PE, Raoult D. Rickettsia onstrate the existence of a variant R. africae, despite the felis, henselae, and B.clarridgeiae, New Zealand. Emerg Infect Dis. 2004;10:967–8. previously identifi ed clonality of its strains in Africa (9) 5. Mediannikov O, Cabre O, Qu F, Socolovschi C, Davoust B, Marié and the West Indies, where it was carried by A. variegatum JL, et al. and Bartonella clarridgeiae in fl eas from ticks from western Africa in the 18th century (1). New Caledonia. Vector Borne Zoonotic Dis. Epub 2010 Jun 23. New Caledonia is a French territory (249,000 inhabit- 6. Rolain JM, Sthul L, Maurin M, Raoult D. Evaluation of antibiotic 2 susceptibilities of three rickettsial species including Rickettsia felis ants; 18,575.5 km of land area) located in the subregion by a quantitative PCR DNA assay. Antimicrob Agents Chemother. of Melanesia in the southwest Pacifi c. Chesterfi eld Island 2002;46:2747–51. DOI: 10.1128/AAC.46.9.2747-2751.2002 and Walpole Island belong to the archipelago and are un- 7. Roux V, Raoult D. Phylogenetic analysis of members of the genus inhabited coral sand cays colonized by marine birds. We Rickettsia using the gene encoding the outer-membrane protein rOmpB (ompB). Int J Syst Evol Microbiol. 2000;50:1449–55. detected R. africae in A. loculosum ticks in Oceania. This 8. Fournier PE, Zhu Y, Ogata H, Raoult D. Use of highly variable tick is known to infest marine birds that are distributed on intergenic spacer sequences for multispacer typing of Rickettsia numerous tropical islands of the southern oceans (13). A. conorii strains. J Clin Microbiol. 2004;42:5757–66. DOI: 10.1128/ loculosum ticks have been found in the Indian Ocean (Tan- JCM.42.12.5757-5766.2004 9. Fournier PE, El Karkouri K, Leroy Q, Robert C, Giumelli B, Ren- zania, Seychelles Islands, Mauritius, Cocos [Keeling] Is- esto P, et al. Analysis of Rickettsia africae genome reveals that viru- land group, Madagascar), on the Coral Sea Islands (Queen- lence acquisition in Rickettsia species may be explained by genome sland, numerous islands and reefs), near New Caledonia reduction. BMC Genomics. 2009;10:166. DOI: 10.1186/1471-2164- (Surprise Island), and in the Caroline Group in the Pacifi c 10-166 10. Fournier PE, Dumler JS, Greub G, Zhang J, Wu Y, Raoult D. Gene Ocean (13). Other hosts are goats (Australia) and lizards sequence-based criteria for identifi cation of new Rickettsia isolates (Seychelles) (13). Aride virus, an ungrouped arbovirus, has and description of Rickettsia heilongjiangensis sp. nov. J Clin Mi- been isolated from A. loculosum ticks collected from dead crobiol. 2003;41:5456–65. Medline DOI: 10.1128/JCM.41.12.5456- birds in the Seychelles, which suggests that this tick may be 5465.2003 11. Parola P, Inokuma H, Camicas JL, Brouqui P, Raoult D. Detection a vector of bird infections (14). A. loculosum ticks are also and identifi cation of spotted fever group Rickettsiae and Ehrlichiae known to readily feed on humans (Seychelles, Australia, in African ticks. Emerg Infect Dis. 2001;7:1014–7. DOI: 10.3201/ New Caledonia, Caroline Islands) (13). eid0706.010616 Two of the 3 specimens of A. loculosum ticks that test- 12. Socolovschi C, Huynh TP, Davoust B, Gomez J, Raoult D, Pa- rola P. Transovarial and trans-stadial transmission of Rickettsiae ed positive for rickettsiae were collected from humans who africae in Amblyomma variegatum ticks. Clin Microbiol Infect. worked with birds on Chesterfi eld Island, but those persons 2009;15(S2):317–8. only remained on the island for a short period. The dura- 13. Hoogstraal H, Wassef HY, Converse JD, Keirans JE, Clifford CM, tion of tick attachment was not known, but to the best of Feare CJ. Amblyomma loculosum (Ixodoidae): identity, marine bird and human hosts, virus infection, and distribution in the southern our knowledge, no illness developed after these tick bites. oceans. Ann Entomol Soc Am. 1976;69:3–14.

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14. Converse JD, Hoogstraal H, Moussa MI, Kaiser MN, Casals J, Feare Address for correspondence: Philippe Parola, Unité de Recherche en CJ. Aride virus, a new ungrouped arbovirus infecting Amblyomma Maladies Infectieuses et Tropicales Emergentes (URMITE), UMR CNRS- loculosum ticks from roseate terns in the Seychelles. Arch Virol. IRD 6236-198, Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille 1976;50:237–40. DOI: 10.1007/BF01320577 CEDEX 5, France; email: [email protected]

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