First molecular detection of Rickettsia sp . strain Atlantic rainforest in Amblyomma ovale ticks from Espírito Santo state , Brazil Primeira detecção molecular da Rickettsia sp . cepa Mata Atlântica em carrapatos Amblyomma ovale do estado do Espírito Santo , Brasil

Espírito Santo state (southeastern Brazil) is considered an endemic area for spotted fever group rickettsioses. In February 2017, we received in our laboratory seven unfed Amblyomma ovale adult ticks collected by a farmer from his clothes and body (not attached) during a working day in the rural area of Ibiraçu municipality, Espírito Santo state. By polymerase chain reaction (PCR) analyses, targeting gltA and ompA rickettsial genes, the DNA of Rickettsia was detected in 6/7 (85.7%) A. ovale. In all cases, DNA sequencing of PCR products revealed that consensus sequences of both genes were 100% identical to gltA and ompA corresponding sequences of Rickettsia sp. strain Atlantic rainforest retrieved from GenBank. This study reports the first molecular detection of Rickettsia sp. strain Atlantic rainforest in A. ovale ticks from Espírito Santo state. Our findings indicate a new Brazilian state in the southeast region at risk of human infection with this tick-borne emerging rickettsial agent.

Palavras-chave: Rickettsia sp., cepa Mata Atlântica, Amblyomma ovale, carrapatos, Espírito Santo, Brasil.Spotted fever group (SFG) rickettsioses are currently recognized as emerging or re-emerging zoonotic diseases, caused by pathogenic bacteria of the genus Rickettsia and transmitted to human beings through tick bites (PAROLA et al., 2013).In Brazil, since the 1920s, Rickettsia rickettsii has been recognized as the main etiological agent of SFG rickettsioses, associated with Amblyomma sculptum (formerly Amblyomma cajennense) and Amblyomma aureolatum ticks as vectors, causing a highly lethal illness, which has been predominant by far in the southeast Brazilian region (OLIVEIRA et al., 2016).Nevertheless, in 2010, a Brazilian case of mild eschar-associated rickettsiosis was reported from the littoral region of São Paulo state, and the molecular characterization of its etiological agent revealed another SFG agent, which was named the Rickettsia sp.strain Atlantic rainforest (SPOLIDORIO et al., 2010b).After this report, new human cases related to Rickettsia sp.Atlantic rainforest have been documented from Brazilian patients from Bahia and Santa Catarina states (SILVA et al., 2011;KRAWCZAK et al., 2016c).Research to unveil its epidemiology has identified the tick Amblyomma ovale as its main vector (KRAWCZAK et al., 2016a).The results based on experimental models and field-collected material indicate that this tick species is naturally infected with strain Atlantic rainforest in several states of the south (Rio Grande do Sul, Santa Catarina and Paraná), southeast (São Paulo and Rio de Janeiro) and northeast (Bahia and Ceará) regions of Brazil (KRAWCZAK et al., 2016b;NIERI-BASTOS et al., 2016;MOERBECK et al., 2016;LUZ et al., 2017).
Espírito Santo state (southeastern Brazil) is considered an endemic area for SFG rickettsioses (OLIVEIRA et al., 2016).Previously published reports, based on serological, immunohistochemical, and epidemiological methods, have suggested R. rickettsii as the probable etiological agent of an outbreak in the 1990s (COREY et al., 1993;SPOLIDORIO et al., 2010a).Consequently, we recently confirmed R. rickettsii as the etiological agent of fatal SFG rickettsiosis in human patients from Espírito Santo state between 2015 and 2017 (FACCINI-MARTÍNEZ et al., 2018).The aim to determine the identity of other pathogenic SFG rickettsiae circulating in Espírito Santo prompted the current study.
In February 2017, we received in our laboratory seven unfed adult ticks (stored in a plastic vial with 70% ethanol) that were collected by a farmer from his clothes and body (not attached) during a working day in the rural area of Ibiraçu municipality, Espírito Santo state (Figure 1).Ticks were identified following a taxonomic key for the Amblyomma genus (BARROS-BATTESTI et al., 2006) and tested for rickettsial DNA.For this purpose, ticks were processed individually for DNA extraction using the guanidine isothiocyanate phenol technique (SANGIONI et al., 2005).Then, the obtained material was tested by the polymerase chain reaction (PCR) technique using primers CS-78 and CS-323 targeting a 401-bp fragment of the citrate synthase gene (gltA), which is common in all representatives of the genus Rickettsia (LABRUNA et al., 2004).Samples yielding expected size amplicons were subsequently tested with primers Rr190.70p and Rr190.701n,targeting a 631-bp fragment of the rickettsial 190-kDa outer membrane protein gene (ompA) (EREMEEVA et al., 2006).The negative control tube containing ultrapure water and a positive control tube containing DNA of the Rickettsia sp.strain NOD were included in each PCR run.The obtained amplicons were purified with ExoSap (USB, Cleveland, Ohio, USA) and DNA-sequenced in an ABI automated sequencer (Applied Biosystems/Thermo Fisher Scientific, model ABI 3500 Genetic Analyzer, Foster City, California, USA) with the same primers used for PCR.The obtained sequences were assembled with Geneious R9 software and submitted to Blast analyses (NCBI, 2017) to infer the closest similarities available in GenBank.
All seven adult ticks were morphologically identified as A. ovale (4 males and 3 females).Six ticks (85.7%) were found to contain rickettsial DNA by PCR protocols targeting gltA and ompA genes.The obtained partial sequences of the gltA gene (350-bp) were identical to each other, and identical sequences of ompA gene (590-bp) were obtained for all ticks as well.Consensus sequences of both genes were 100% identical to gltA and ompA corresponding sequences of Rickettsia sp.strain Atlantic rainforest (GenBank accession numbers KJ174527 and KJ158741, respectively).The DNA sequences generated in the present study have been submitted to GenBank (accession numbers MF536974, MF536975).
The present study reports for the first time the detection and molecular characterization of the Rickettsia sp.strain Atlantic rainforest in A. ovale ticks in the Espírito Santo state.Interestingly, the ticks tested in our study were collected from the clothes and body of a farmer, and although they were not found attached to his skin, our findings indicate an eventual risk for a possible tick bite and related pathogenic Rickettsia transmission.This is because A. ovale is recognized as both an anthropophilic tick in South America (GUGLIELMONE et al., 2006) and a competent vector for Rickettsia sp.strain Atlantic rainforest (KRAWCZAK et al., 2016a).Furthermore, our data identified an additional location (Ibiraçu municipality) in the state of Espírito Santo where A. ovale was present.In a recent study on tick fauna in the same state, A. ovale was only documented for the municipalities of Santa Teresa and Santa Maria de Jetibá (ACOSTA et al., 2016).Nevertheless, it is quite likely that its distribution would be even broader in Espírito Santo since this state has an important portion of the preserved Atlantic rainforest.
Finally, our findings note a new Brazilian state within the southeast region in which the risk of human infections with Rickettsia sp.strain Atlantic rainforest, an agent of an emerging tick-borne human disease, exist.A recent study provided phylogenetic evidence that the Rickettsia sp.strain Atlantic rainforest represents a genetic variant of the human pathogen Rickettsia parkeri (PADDOCK et al., 2017).In this way, it would be important to conduct an active epidemiological search with the purpose of characterizing new eschar-associated rickettsiosis cases in Brazil.