Amblyomma Nodosum) in an Endemic Area of Spotted Fever in Brazil Author(S): Leonardo Moerbeck, Vinicius F

Rickettsia sp. Strain NOD Infecting Ticks (Amblyomma nodosum) in an Endemic Area of Spotted Fever in Brazil Author(s): Leonardo Moerbeck, Vinicius F. Vizzoni, Stefan V. de Oliveira, Robson Cavalcante, Gerlene C. B. Coelho, Naylê F. H. Duarte, Marinete Amorim, and Gilberto S. Gazêta Source: Journal of Wildlife Diseases, 54(2):406-409. Published By: Wildlife Disease Association https://doi.org/10.7589/2017-06-137 URL: http://www.bioone.org/doi/full/10.7589/2017-06-137 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. DOI: 10.7589/2017-06-137 Journal of Wildlife Diseases, 54(2), 2018, pp. 406–409 Ó Wildlife Disease Association 2018 Rickettsia sp. Strain NOD Infecting Ticks (Amblyomma nodosum)inan Endemic Area of Spotted Fever in Brazil Leonardo Moerbeck,1,2 Vinicius F. Vizzoni,2 Stefan V. de Oliveira,3 Robson Cavalcante,4 Gerlene C. B. Coelho,5 Nayleˆ F. H. Duarte,5 Marinete Amorim,2 and Gilberto S. Gazetaˆ 1,2,6 1Programa de Pos-Graduac´ ¸aõ Stricto Sensu de Biodiversidade e Saude–Instituto´ Oswaldo Cruz/Fundaçaõ Oswaldo Cruz–Rio de Janeiro/RJ, Brazil; 2Serviço de Referenciaˆ Nacional em Vetores das Riquetsioses–Instituto Oswaldo Cruz/Fundaçaõ Oswaldo Cruz–Rio de Janeiro/RJ, Brazil; 3Secretaria de Vigilanciaˆ em Saude–Ministe´ ´rio da Saude/MS–Bras´ ´ılia/DF, Brazil; 4Secretaria Estadual de Saude´ do Estado do Ceara´, Baturite´, Brazil; 5Nucleo´ de Controle de Vetores–Coordenaçaõ de Promoçaõ e Proteçaõ aSa` ude–Secretaria´ Estadual de Saude–Ceara´ ´, Brazil; 6Corresponding author (email: [email protected]) ABSTRACT: Amblyomma nodosum ticks were col- state of Ceara´, Brazil, an endemic area of SF. lected from one collared anteater (Tamandua Tick specimens were morphologically classi- tetradactyla) in the Caatinga biome, Brazil. From one sample, we isolated a Rickettsia sp. that was fied according to Barros-Battestti et al. phylogenetically close to Rickettsia sp. strain (2006). Sixteen samples divided into 24 NOD, with 99.9, 100.0, and 99.8% identity for specimens were tested in pools or individu- gltA, htrA, and ompA genes, respectively. ally. Eleven samples were individually tested (six samples with one male each and five Amblyomma nodosum is a neotropic tick samples with one female each); two of the species commonly reported in Brazil. Usual females were partially engorged, and three hosts of immature stages are Passeriformes, were completely engorged. Five samples whereas the giant anteater (Myrmecophaga were pooled (one sample contained two flat tridactyla) and the collared anteater (Taman- females,onesamplehadtwomales,and dua tetradactyla) are the hosts of adult stages three samples had three males each). Sam- (Barros-Battestti et al. 2006). ples were submitted to DNA extraction Rickettsia sp. strain NOD (Ogrzewalska et (Aljanabi and Martinez 1997) and conven- al. 2009), Rickettsia bellii (Moerbeck et al. tional PCR targeting rickettsial genes: gltA 2016), and Rickettsia amblyommatis CS2 region (CS-78 and CS-323) and CS4 ¼ ( Rickettsia amblyommii;Karpathyetal. region (CS-239 and CS-1069), ompA 2016) are rickettsial organisms known to infect (Rr190.70F and Rr190.602R), and htrA A. nodosum. Although there are no records of (Rr1175F and Rr2608R) by using published human infestations by A. nodosum, evidence protocols (Regnery et al. 1991; Blair et al. of rickettsial infections and wide distribution 2004;Labrunaetal.2004).Onesamplewas of their host species suggest A. nodosum as a analyzed for the cox-1 gene (Folmer et al. potential vector in the enzootic cycle of 1994). All DNA fragments with expected size rickettsiae (Guglielmone et al. 2014). Moer- beck et al. (2016) reported a great diversity of were purified by using a DNA Mini Kit (Real ticks and rickettsiae species in this spotted Genomics, New Taipei City, Taiwan), and an fever (SF)–endemic area, which has a unique automated ABI 3730xl DNA analyzer (Ap- characteristic of being composed of forest plied Biosystems, Foster City, California, fragments within the Caatinga biome, a USA) was used to sequence the DNA in semiarid region of Brazil. We used our data both directions (Otto et al. 2008). Phyloge- to analyze the potential participation of A. netic analysis was performed with a concat- nodosum in the epidemic cycle of SF in the enated sequence by using neighbor-joining region. methods (MEGA 5.2, Tamura et al. 2011) In April 2013, 100 adult tick specimens (79 and the Kimura two-parameter model to males and 21 females) were collected from a estimate genetic divergency (Kimura 1980). single collared anteater from the municipality Bootstrap values were obtained from 1,000 of Guaramiranga (481504800 S, 3885505900 W), randomly generated trees. Sequences gener- 406 LETTERS 407 ated in this study were deposited in GenBank Barros-Battestti et al. 2006). Our report (accession no. KY660046-48). extends the distribution of this tick species All ticks were morphologically identified as into Ceara´, northeastern Brazil, and within A. nodosum. The sequence of cox-1 that we the Caatinga biome, mature stages of A. obtained was 99.4% (654/658) similar to A. nodosum parasitized T. tetradactyla,whichis nodosum voucher CAS-T028 (accession no. consistent with previous studies (Barros- KF200111). All analyzed samples were posi- Battestti et al. 2006). There is no record of tive for at least the CS2 fragment of gltA gene. human infestation by A. nodosum (Gugliel- The PCR reactions with sample LIC4363I mone et al. 2014), but it has been previously were positive for all primer sets, producing reported parasitizing a dog (Canis familiaris; amplicons of expected size. The BlastN Mazioli et al. 2012). Our results may indicate analysis (National Center for Biotechnology the possibility for epidemic and enzootic 2018) showed that gltA, htrA, and ompA cycles of rickettsiae. Immature stages of this sequences were 99.9% (1,055/1,056), 100.0% arthropod have been reported parasitizing (394/394), and 99.8% (490/491) similar to birds (Passeriformes; Ogrzewalska et al. Rickettsia sp. The NOD sequences (accession 2009). Although we did not collect birds, nos. EU567177, EU567178, and EU567180, their role as spatial dispersers of potential respectively). To improve the phylogenetic vectors of rickettsia must be considered, analysis, all gltA (1,053 base pair [bp]), htrA because that would facilitate the establish- (394 bp), and ompA (463 bp) sequences were ment and circulation of species of rickettsia concatenated (1,910 bp). The resulting tree in new areas (Ogrzewalska et al. 2009). Thus, corroborated with BlastN analysis and showed immature stages of A. nodosum may be the bacterium was closely related to Rickettsia important for increasing the dispersion of sp. NOD (Fig. 1). Rickettsia sp. NOD, whereas mature stages In our study, morphologic identification would serve to maintain this bioagent in an was corroborated by molecular analysis, con- area. firming that the tick species responsible for Amblyomma nodosum lives in preserved parasitizing of the anteater was A. nodosum. or minimally anthropically impacted areas, as In addition, the tick was infected with do its vertebrate hosts (Ogrzewalska et al. Rickettisa sp.NOD,aspreviousstudies reported (Ogrzewalska et al. 2009). This 2009). This scenario may explain the species rickettsial organism may infect diferent tick presence in a fragment of Atlantic rainforest species; however, it frequently infects A. within the Caatinga. Thus, the dispersion of nodosum, which has a wide distribution in immature stages by birds and the coloniza- Brazil (Ogrzewalska et al. 2009). Although our tion of new areas by adult specimens on study area somewhat resembled the Atlantic anteaters may contribute to the establish- rainforest biome, it actually is a semiarid ment of its enzootic cycle in forest frag- region. ments. Amblyomma nodosum has been widely Funding was provided by Coordenaçaõde reported in the Americas: Argentina, Bolivia, Aperfeiçoamento de Pessoal de Nıvel´ Supe- Brazil, Colombia, Costa Rica, Guatemala, rior for the scholarship of L.M. We thank Mexico, Nicaragua, Panama, Trinidad and Nicolau Maue´s da Serra-Freire (in memori- a Tobago, US, and Venezuela (Barros-Battestti am), Ministe´rio da Saude,´ 4 Coordenadoria et al. 2006; Mukherjee et al. 2014). In Brazil, Regional de Saude´ da Secretaria Estadual do it is reported in 11 states (Espırito´ Santo, Estado do Ceara´,Nucleo´ de Controle de Goia´s, Mato Grosso, Mato Grosso do Sul, Vetores–CoordenaçaõdePromoçaõeProte- Minas Gerais, Parana´, Rio de Janeiro, Rio çaõ aSa` ude-Secretaria´ Estadual de Saude–´ Grande do Sul, Rondonia,ˆ Santa Catarina, Ceara´, and Plataforma Genomicaˆ de Sequen- and Saõ Paulo) that encompass three biomes ciamento (RPT01A) – FundaçaõOswaldo (Atlantic Forest, Cerrado, and Pantanal; Cruz for DNA sequences. 408 JOURNAL OF WILDLIFE DISEASES, VOL. 54, NO. 2, APRIL 2018 FIGURE 1. Phylogenetic tree of concatenated spotted fever group rickettsia gltA, htrA, and ompA genes constructed by neighbor-joining method with the Kimura two-parameter evolution model. GenBank accession nos. are presented at the beginning of each rickettsial species. The numbers at nodes are the bootstrap values obtained from 1,000 resamplings. Bootstrap values below 70% are not presented.

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