First identification of natural infection of Rickettsia rickettsii in the Rhipicephalus sanguineus tick, in the State of Rio de Janeiro

Pesquisa Veterinária Brasileira

Rozental, Tatiana¹  Massard, Carlos L.²  Cunha, Nathalie C.³  Fonseca, Adivaldo H.³  Rezende, Jania³  Favacho, Alexsandra R.M.¹  Barreira, Jairo D.¹  Lemos, Elba R.S.¹ 

[1] FIOCruz, Rio de Janeiro, Brazil;UFRRJ;Universidade Federal Rural do Rio de Janeiro, Seropédica, Brazil

关键词: Rhipicephalus sanguineus;    Acari;    Ixodidae;    Rickettsia rickettsii;    molecular analysis;    Brazilian spotted fever.;   

DOI  :  10.1590/S0100-736X2009000200003

来源: Colegio Brasileiro de Patologia Animal-CBPA

【 摘 要 】

The Brazilian Spotted Fever (BSF) is a zoonotic disease caused by Rickettsia rickettsii and transmitted by ticks of the genus Amblyomma, more frequently, Amblyomma cajennense. The aim of this paper was to report the first molecular detection of R. rickettsii on R. sanguineus naturally infected in Rio de Janeiro, Brazil. Ticks were collected from dogs in a rural region of Resende municipality, Rio de Janeiro State, Brazil (22º30'9.46"S, 44º42'44.29"WO), where occurred five human cases of BSF in 2006. The ticks were identified under a stereoscopic microscope and separated in pools by stages, species and sex. DNA extraction was carried out using QIAamp DNA Mini Kit (QIAGEN®). The DNA was submitted to PCR amplification using 04 set of primers: Rr190.70p/Rr190.602n (OmpA, 532bp), BG1-21/BG2-20 (OmpB, 650bp), Tz15/Tz16 (17 kDa protein-encoding gene, 246bp) and RpCS.877p/RpCS.1258n (gltA, 381bp). PCR products were separated by electrophoresis on 1% agarose gels and visualized under ultraviolet light with ethidium bromide. PCR products of the expected sizes were purified by QIAquick® and sequenced by ABI PRISM®. The generated nucleotide sequences were edited with using Bioedit® software and compared with the corresponding homologous sequences available through GenBank, using Discontiguous Mega Blast (http://www.ncbi.nlm.nih.gov). It was confirmed R. rickettsii by sequencing of the material (GenBank FJ356230). The molecular characterization of R. rickettsii in the tick R. sanguineus emphasizes the role of dogs as carriers of ticks from the environment to home. Moreover, this result suggests that there is a considerable chance for active participation of R. sanguineus as one of tick species in the transmission of R. ricketsii to human being in the Brazilian territory. Index terms: Rhipicephalus sanguineus, Acari, Ixodidae, Rickettsia rickettsii, molecular analysis, Brazilian spotted fever. RESUMO A Febre Maculosa Brasileira (FMB) é uma zoonose causada por Rickettsia rickettsii e transmitida por carrapatos do gênero Amblyomma, mais freqüentemente pela espécie Amblyomma cajennense. Este trabalho tem como objetivo relatar a primeira detecção molecular de R. rickettsii em Rhipicephalus sanguineus naturalmente infectado no Rio de Janeiro, Brasil. Carrapatos foram coletados de cães, procedentes de uma região rural do município de Resende, estado do Rio de Janeiro, Brasil (22º30'9.46"S, 44º42'44.29"WO), onde ocorreram cinco casos humanos de FMB em 2006. Todos os carrapatos foram identificados segundo chave dicotômica, utilizando-se lupa estereoscópica e separados de acordo com estágio, espécie e sexo. Para a extração de DNA utilizou-se o kit comercial QIAamp DNA (QIAGEN ®). O DNA foi submetido à técnica de PCR utilizando 04 conjuntos de iniciadores para a amplificação dos genes: Rr190.70p/Rr190.602n (OmpA, 532bp), BG1-21/BG2-20 (OmpB, 650bp), Tz15/Tz16 (17 kDa gene que codifica a proteína, 246bp) e RPCs .877p/RpCS.1258n (gltA, 381bp). Os produtos da PCR foram separados por eletroforese em gel agarose 1% corados com brometo de etídio e visualizados sob luz ultravioleta e, aqueles que apresentaram bandas amplificadas foram purificados utilizando-se o kit comercial QIAquick ® e seqüenciados pelo ABI PRISM®. As seqüências nucleotídicas foram geradas usando Bioedit®, editado em software e comparados os correspondentes homólogos com as sequências disponíveis através GenBank, utilizando Discontiguous Mega Blast (http://www.ncbi.nlm.nih.gov). Confirmou-se R. rickettsii (GenBank FJ356230) no seqüenciamento de apenas um espécime, adulto de carrapato R. sanguineus. A caracterização molecular de R. rickettsii em exemplar de carrapato R. sanguineus confirma que esta espécie pode ter importante papel na transmissão de R. rickettsii para humanos no território brasileiro. Termos de indexação: Rhipicephalus sanguineus, Acari, Ixodidae, Rickettsia rickettsii, Análise molecular, Febre maculosa brasileira.    INTRODUCTIONBrazilian spotted fever (BSF) is a life-threatening rickettsiosis caused by Rickettsia rickettsii that is transmitted to human beings by ticks of genus Amblyomma, more frequently, Amblyomma cajennense, a tick species primarily associated with horses (Dias & Martins, 1939; Oliveira et al. 2000). Although other species of Rickettsia have been described in the Brazilian territory, as Rickettsia parkeri (Horta et al. 2004, Horta et al. 2007, Labruna et al. 2007) and Rickettsia felis (Cardoso et al. 2006, Oliveira et al. 2008), only R. rickettsii has been identified in BSF human cases.Until recently, Dermancentor andersoni, D. variabilis in North American beside A. cajennense in South America were the only species of ticks associated with spotted fever rickettsioses on the American Continent (Parola et al. 2005). Nowadays, other species have been reported as A. triste (Venzal et al. 2008) in Uruguay and Rhipicephalus sanguineus in the USA (Demma et al. 2005, Wikswo et al. 2007).Rhipicephalus sanguineus, commonly known as the brown dog tick, is a three-host tick that feeds primarily on dogs and occasionally on other hosts, including human beings (Walker et al. 2000). This tick species is the main vector and reservoir of Rickettsia conorii complex that cause Mediterranean spotted fever in Europe, Africa and Asia (Parola et al. 2005).Although the role of R. sanguineus in the transmission cycle of a severe spotted fever rickettsiose to humans have been demonstrated since 1940's in Mexico (Bustamante et al. 1946, Bustamante et al. 1947), only in 2002, R. rickettsii was identified in R. sanguineus in the USA as important vector of the Rocky Mountain Spotted Fever (RMSF) in Eastern Arizona (Demma et al. 2005)Burgdorfer et al. in 1975, registered the possibility of R. sanguineus involvement in the epidemiology of RMSF, reporting that ticks of this species collected in infected dogs with Rickettsia spp. were able to transmit and cause the disease, experimentally.Recently in Brazil, human parasitism by R. sanguineus have been reported (Dantas-Torres et al. 2006, Louly et al. 2006). Till now, no data is published regarding the presence of vector-borne rickettsia in this specie of tick in a BSF endemic area.On the other hand, recently R. sanguineus has been identified as reservoir of Rickettsia felis in Brazil (Cardoso et al. 2006, Oliveira et al. 2008), although previous studies had shown evidence of infected R. sanguineus in endemic areas in Brazilian states of Minas Gerais and Rio de Janeiro (Lemos et al. 1997a, Rozental et al. 2002)The increasing number of cases of human parasitism by R. sanguineus ticks reported in the literature indicates that the interaction between human beings and R. sanguineus may be more common than it is recognized in the American continent.Usually, R. sanguineus parasitizes dogs in urban areas, but sometimes they are found in rural farming areas (Soares et al. 2006, Labruna et al. 2001). In BSF endemic area, the conglomerations of R. sanguineus create a great risk to humans whose can be attacked by infected ticks in and around their houses. This study reports the first molecular detection of R. rickettsii in R. sanguineus ticks in Rio de Janeiro State, Brazil.  MATERIALS AND METHODS The material here analyzed is part of an ongoing study that is been developing at Engenheiro Passos District (22º30'165"S, 44º42'654"WO), located in the city of Resende, Rio de Janeiro State, Brazil where in May 2006 five BSF cases with two deaths were confirmed by serology . This area is situated in the western portion of the State in Paraíba do Sul middle river valley. The native vegetation, Atlantic Forest, has been replaced by degraded areas, characterized by the presence of livestock for milk production and current reforestation with Eucalyptus spp.In June 2006, a study was conducted in this district, when ticks were collected from dogs and horses at or near site of confirmed cases of BSF. After identifying through a stereoscopic microscope using taxonomic

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