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Novel candidatus Rickettsia species detected in nostril tick from human, Gabon, 2014.

Ticks are hematophagous arthropods that parasitize different species of vertebrates, and they serve as intermediate hosts for infectious pathogens that can have serious implications for humans. Because of climate change and socioeconomic factors, tickborne diseases have increased in the past 3 decades, and these arthropods are second only to mosquitoes as vectors of human infectious diseases (1,2). Many ixodid tick species are found in Africa, and tickborne diseases in travelers returning from that continent have been reported worldwide (3). Among the travel-associated cases of African tick-bite fever, most occur in persons returning from travel to southern Africa with fever and systemic illness (4).

The Study

A 21-year-old female field worker from Spain visited Lope National Park in Gabon (Africa) for 13 days during January-February 2014 to observe chimpanzees and gorillas. Four days before returning to Spain, she noticed a foreign body (black spot) inside her left nostril but had no signs or symptoms of illness. After returning home, the woman sought care at the Tropical Medicine Centre at University Hospital Ramon y Cajal in Madrid, Spain, where a tick attached to the anteroinferior part of the left nasal septum (the Kiesselbach area) was extracted with forceps during rhinofibroscopy. The tick was sent to the Center of Rickettsiosis and Arthropod-Borne Diseases at Hospital San Pedro-Center of Biomedical Research of La Rioja in Logrono, Spain, for identification and molecular detection of tickborne bacteria. The tick was photographed (Figure) and identified, by morphologic features, as an Amblyomma sp. nymph, according to taxonomic keys (5).

Immature stages of Amblyomma ticks cannot be identified to the species level on the basis of morphologic features without allowing the nymph to molt. Thus, we conducted genetic analysis to identify the tick. To extract genomic DNA, we incubated the tick with ammonium hydroxide (1 mL of 25% ammonia and 19 mL of sterile water) for 20 min at 100[degrees]C and for another 20 min at 90[degrees]C. The DNA was used as template in PCR assays targeting the tick mitochondrial 16S rRNA (6), mitochondrial 12S rRNA (7), and nuclear 5.8S-28S rRNA intergenic transcribed spacer 2 (ITS2) (8). As a positive control, we used DNA extract from a tick of known identity (Haemaphysalis punctata) that was collected in La Rioja, Spain.

Subsequent detection and molecular characterization of tickborne bacteria (Rickettsia spp., Anaplasma phagocytophilum, and Borrelia spp.) were performed. To screen for the presence of rickettsiae, we used PCR assays targeting 2 fragments of the ompB gene (511 bp and 811 bp, respectively) (9,10). Four additional genetic markers were used to classify the isolated rickettsia to the species level: fragments of ompA (532 bp) (11,12), 16S rRNA (1,500 bp) (13), sca4 (623 bp) (14), and gltA (1,019 bp) (15). These markers were amplified in accordance with the taxonomic scheme for classifying rickettsiae at the genus and species levels (online Technical Appendix, reference 16, http://wwwnc.cdc.gov/EID/article/21/2/14-1048-Techapp1.pdf).

To screen for the presence of A. phagocytophilum and Borrelia spp., we performed PCR targeting the partial msp2 gene (334 bp) and Borrelia genus-specific 16S rRNA (1,350 bp) gene (online Technical Appendix, references 17,18). Each PCR included a positive control: Rickettsia slovaca strain S14ab DNA (obtained from Vero cells that had been inoculated in our facility with homogenate of an R. slovaca-infected Dermacentor marginatus tick from La Rioja Province); A. phagocytophilum strain Webster DNA (provided by D. Raoult [Unite de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Marseille, France] and J.S. Dumler [Johns Hopkins Hospital, Baltimore, MD, USA]); or Borrelia burgdorferi sensu stricto DNA (provided by V. Fingerle [German National Reference Centre for Borrelia, Oberschleissheim, Germany]). DNA-free water was included as a negative control in each set of reactions.

We used BLAST (http://www.ncbi.nlm.nih.gov/blast/ Blast.cgi) to compare sequences generated by each pair of primers with sequences in GenBank. The 16S rRNA sequence showed highest identity (91% [368/405 bp]) with the mitochondrion Amblyomma variegatum 16S rRNA gene (GenBank accession no. L34312). The 12S rRNA and ITS2 sequences reached only 89% and 91% identity, respectively, with those of A. variegatum and were closest (94.3% [298/316 bp] and 99% identity [809/817 bp], respectively) to those for an Amblyomma sp. nymph (GenBank accession nos. KC538944 and KC538941). Of interest, the 12S rRNA and ITS2 sequences also corresponded to those of a nostril tick removed from a researcher who had been visiting a national park in Uganda (online Technical Appendix, reference 19); the 16S rRNA sequence for the tick from this researcher was not in GenBank. The levels of sequence similarities that we found did not enable species determination of the tick in this study. Two previous reports about nostril ticks in humans who have visited Africa are available (online Technical Appendix, references 20,21).

[FIGURE OMITTED]

For the strain in this study, single bands of the expected sizes for the 2 ompB rickettsial fragment genes analyzed were detected. A BLAST search revealed that these 2 sequences were genetically most similar (97.2% and 98.3% identity) to the ompB gene of Rickettsia japonica and Rickettsia heilongjiangensis, respectively (Table). The nucleotide sequence of ompA was closest (99.8% identity) to that of the ompA of Rickettsia sp. strain Davousti, and showed maximum identity (97.2%) with R. heilogjiangensis as validated species. When compared with sequences of validly published Rickettsia spp. available in GenBank, the 16S rRNA and sca4 gene sequences showed the highest identity with R. japonica (99.4%-99.6% and 98.5%, respectively). The gltA sequence shared 99.1% identity with R. japonica and R. heilogjiangensis (Table). These results are in accordance with the genetic criteria for identifying the rickettsia as Candidatus Rickettsia sp. (online Technical Appendix, reference 16).

Although reports about the circulation of Rickettsia spp. in Gabon are scarce, Rickettsia sp. strain Davousti was detected in Amblyomma tholloni ticks from African elephants in that country (online Technical Appendix, reference 22). In addition, the gltA sequence obtained in our study was 99.9% identical to the gltA sequence of Rickettsia sp. strain Davousti (Table). These findings suggest that both strains could belong to the same Rickettsia sp. No other sequences, apart from those for ompA and gltA, of Rickettsia sp. strain Davousti have been deposited in GenBank; however, on the basis of findings in the previous report (online Technical Appendix, reference 22), we propose the name Candidatus R. davousti for the strain in this study.

In addition, we did not obtain amplicons for the msp2 gene of A. phagocytophilum or 16S rRNA gene specific for Borrelia genus. For each set of PCR primers, no bands were detected on agarose gels for negative control samples.

The partial tick mitochondrial 16S rDNA, 12S rDNA, and ITS2 sequences have been deposited in GenBank under accession nos. KJ619630, KJ619636, and KJ619637, respectively. The partial ompB (2 fragment genes), ompA, 16S rRNA, sca4, and gltA sequences of the novel tick-derived rickettsia in this study have been deposited in GenBank under accession numbers KJ619632, KJ619633, KJ619631, KJ619629, KJ619634, and KJ619635.

Conclusions

We report the detection of a potentially novel Rickettsia sp. from an Amblyomma sp. nymphal tick that was removed from the nostril of a field researcher when she returned to Spain after visiting Gabon's Lope National Park; we propose the name Ca. R. davousti for this Rickettsia sp. strain.

Our findings provide further evidence of the presence of circulating Rickettsia sp. in Africa and indicate that tick bites may be a threat to persons visiting national parks in Africa. Further studies are needed to determine the prevalence of Ca. R. davousti and to establish whether this bacterium is pathogenic for humans.

Author affiliations: University Hospital Ramon y Cajal, Madrid, Spain (R. Lopez-Velez, F.F. Norman, J.A. Perez-Molina); Hospital San Pedro-Center of Biomedical Research of La Rioja, Logrono, Spain (A.M. Palomar, J.A. Oteo, A. Portillo)

DOI: http://dx.doi.org/10.3201/eid2102.141048

Acknowledgments

We thank Didier Raoult, J. Stephen Dumler, and Volker Fingerle for supplying positive control samples.

Dr. Lopez-Velez is an infectious disease and tropical medicine specialist in the National Referral Centre for Tropical Diseases, University Hospital Ramon y Cajal. He is interested in imported infectious diseases in travelers and immigrants.

References

(1.) Sonenshine DE, Lane RS, Nicholson WL. Ticks (Ixodida). In: Mullen G, Durden L, editors. Medical and veterinary entomology. New York: Academic Press; 2002. p 517-58.

(2.) Godfrey ER, Randolph SE. Economic downturn results in tick-borne disease upsurge. Parasit Vectors. 2011;4:35. http://dx.doi.org/10.1186/1756-3305-4-35

(3.) Portillo A, Oteo JA. Rickettsiosis as threat for the traveller. In: Rodriguez-Morales A, editor. Current topics in tropical medicine InTech, 2012 [cited 2014 Jun 18]. http://www. intechopen.com/books/current-topics-in-tropical-medicine/ rickettsiosis-as-threat-for-the-traveller

(4.) Mendelson M, Han PV, Vincent P, von Sonnenburg F, Cramer JP, Loutan L, et al. GeoSentinel Surveillance Network. Regional variation in travel-related illness acquired in Africa, March 1997-May 2011. Emerg Infect Dis. 2014;20:532-41. http://dx.doi. org/10.3201/eid2004.131128

(5.) Walker AR, Bouattour A, Camicas JL, Estrada-Pena A, Horak IG, Latif AA, et al. Ticks of domestic animals in Africa: a guide to identification of species. In: Edinburgh (UK): Bioscience Reports; 2003.

(6.) Black WC, Piesman J. Phylogeny of hard- and soft-tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences. Proc Natl Acad Sci U S A. 1994;91:10034-8. http://dx.doi. org/10.1073/pnas.91.21.10034

(7.) Beati L, Keirans JE. Analysis of the systematic relationships among ticks of the genera Rhipicephalus and Boophilus (Acari: Ixodidae) based on mitochondrial 12S ribosomal DNA gene sequences and morphological characters. J Parasitol. 2001;87:32-48. http://dx.doi. org/10.1645/0022-3395(2001)087[0032:A0TSRA]2.0.C0;2

(8.) Zahler M, Gothe R, Rinder H. Genetic evidence against a morphologically suggestive conspecificity of Dermacentor reticulatus and Dermacentor marginatus (Acari: Ixodidae). Int J Parasitol. 1995;25:1413-9. http://dx.doi.org/10.1016/0020-7519(95)00081-X

(9.) Choi YJ, Jang WJ, Kim JY, Ryu JS, Lee SH, Park KH, et al. Spotted fever group and typhus group rickettsioses in humans, South Korea. Emerg Infect Dis. 2005;11:237-44. http://dx.doi. org/10.3201/eid1102.040603

(10.) Roux V, Raoult D. Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer-membrane protein rOmpB (ompB). Int J Syst Evol Microbiol. 2000;50:1449-55. http://dx.doi.org/10.1099/00207713-50-4-1449

(11.) Regnery RL, Spruill CL, Plikaytis BD. Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol. 1991;173:1576-89.

(12.) Roux V, Fournier PE, Raoult D. Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the gene encoding the protein r0mpA. J Clin Microbiol. 1996;34:2058-65.

(13.) Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991; 173:697-703.

(14.) Sekeyova Z, Roux V, Raoult D. Phylogeny of Rickettsia spp. inferred by comparing sequences of 'gene D', which encodes an intracytoplasmic protein. Int J Syst Evol Microbiol. 2001;51: 1353-60.

(15.) Jado I, Oteo JA, Aldamiz M, Gil H, Escudero R, Ibarra V, et al. Rickettsia monacensis and human disease, Spain. Emerg Infect Dis. 2007;13:1405-7. http://dx.doi.org/10.3201/eid1309.060186

Address for correspondence: Aranzazu Portillo, Departamento de Enfermedades Infecciosas, Hospital San Pedro-CIBIR, Edificio CIBIR, C/Piqueras 98-3a planta; 26006 Logrono (La Rioja), Spain; email: aportillo@riojasalud.es
Table. Maximum identities of rickettsial sequences obtained from an
Amblyomma sp. tick from Gabon with validated Rickettsia spp. published
in GenBank *

                              % identity with Rickettsia spp.
                                 (basepairs) ([dagger])

Gene sequence,                                   R. heilongjiangensis,
GenBank accession        R. japonica, GenBank    GenBank accession no.
 no.                    accession no. AP011533          CP002912

ompB, KJ619633              97.2 (442/455)           96.9 (441/455)
ompB, KJ619632              96.9 (746/770)           98.3 (742/755)
ompA, KJ619631              96.1 (472/491)           97.2 (477/491)
16S rRNA, KJ619629            99.4-99.6                99.3-99.6
                           (1369-1373/1378)         (1368-1372/1378)
sca4, KJ619634              98.5 (509/517)           98.3 (508/517)
gltA. KJ619635              99.1 (903/911)           99.1 (903/911)

                        % identity with Rickettsia
                        spp. (basepairs) ([dagger])

Gene sequence,           Rickettsia sp. Davousti,
GenBank accession         GenBank accession nos.
 no.                          DQ402516-402517

ompB, KJ619633                      NA
ompB, KJ619632                      NA
ompA, KJ619631                99.8 (490/491)
16S rRNA, KJ619629                  NA

sca4, KJ619634                      NA
gltA. KJ619635                99.9 (691/692)

* The tick was removed in 2014 from the nostril of a woman who
returned home to Spain after visiting Lope National Park in Gabon.
NA, not available.

([dagger]) Percentages of identity with sequences of Rickettsia sp.
Davousti have been also included when available due to the high level
of similarity with our sequences.
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Article Details
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Author:Lopez-Velez, Rogelio; Palomar, Ana M.; Oteo, Jose A.; Norman, Francesca F.; Perez-Molina, Jose A.; P
Publication:Emerging Infectious Diseases
Article Type:Clinical report
Geographic Code:6GABO
Date:Feb 1, 2015
Words:2123
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