Epidemiological aspects of Rickettsia parkeri in the Atlantic forest biome of Espírito Santo state, Brazil

Epidemiological aspects of Rickettsia parkeri in the Atlantic forest biome of Espírito Santo state, Brazil

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Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis

Original article

Epidemiological aspects of Rickettsia parkeri in the Atlantic forest biome of Espírito Santo state, Brazil Álvaro A. Faccini-Martíneza,b,*, Sebastián Muñoz-Lealc, Felipe S. Krawczakd, Igor C.L. Acostac, Thiago F. Martinsc, Maria Carolina A. Serpac, Amalia R.M. Barbieric, Juliana R. Tovare, Crispim Cerutti Juniora, Marcelo B. Labrunac a

Postgraduate Program in Infectious Diseases, Health Science Center, Universidade Federal do Espírito Santo, Vitória, ES, Brazil Committee of Tropical Medicine, Zoonoses and Travel Medicine, Asociación Colombiana de Infectología, Bogotá, Colombia c Department of Preventive Veterinary Medicine and Animal Health, Faculty of Veterinary Medicine, University of São Paulo, São Paulo, SP, Brazil d Setor de Medicina Veterinária Preventiva, Departamento de Medicina Veterinária, Universidade Federal de Goiás, Goiânia, GO, Brazil e Núcleo Especial de Vigilância Epidemiológica, Secretaria de Estado da Saúde do Estado do Espírito Santo, Vitória, ES, Brazil b



Keywords: Rickettsia parkeri Rickettsiosis Tick-borne diseases Epidemiology Espírito Santo Brazil

Since 2010, a new rickettsiosis caused by Rickettsia parkeri, a species included in the spotted fever group (SFG) and transmitted by the tick Amblyomma ovale, has been described in Brazil. Considering that A. ovale does occur in the Espírito Santo state (ES), the present study aimed at preliminarily describing the epidemiology of R. parkeri in the Atlantic forest of this state. Between June 2016 and September 2018, 33 villages from nine municipalities of the ES were included in a study for the molecular detection and isolation of SFG rickettsiae from adult Amblyomma ticks collected on dogs with free access to the forest. Serologic screenings against SFG rickettsiae in these animals and their owners (humans) were performed through immunofluorescence assay (IFA) using antigens of Rickettsia rickettsii and R. parkeri. Additionally, local health secretariats were informed on clinical manifestations of R. parkeri infection and told to communicate any suspected case. A total of 280 adult ticks were collected and taxonomically classified as A. ovale (n = 152), Amblyomma aureolatum (n = 127) and Amblyomma sculptum (n = 1). Overall, Rickettsia DNA was detected in 12/266 ticks. The sequencing of PCR products revealed that 0.7% (1/144) and 0.8% (1/121) of the analyzed A. ovale and A. aureolatum ticks were infected by R. parkeri strain Atlantic rainforest, respectively, and 8.3% (10/121) of the A. aureolatum ticks infected by Rickettsia bellii. Among the above PCR-positive ticks, only one isolate from one A. aureolatum tick was successfully established in the laboratory. DNA extracted from the third passage of this isolate was designated as strain M9A and molecularly characterized using primers targeting the Rickettsia gltA gene, whose sequence matched 100% the corresponding sequences of R. bellii. Seroprevalence against SFG rickettsiae in sampled dogs (n = 83) was 41% or 57%, depending on the rickettsial antigen (R. rickettsii strain Taiaçu or R. parkeri strain Atlantic rainforest, respectively). A total of 37 (45%) canine sera showed titers to R. parkeri at least fourfold higher than to R. rickettsii antigen. Among humans, 10% (4/41) of the samples reacted to at least one rickettsial antigen, with the highest endpoint titer varying from 64 to 128 for R. rickettsii and R. parkeri; no human serum showed ≥4-fold difference between the highest endpoint titers. Finally, during the study period, suspicions on cases of R. parkeri-rickettsiosis were not informed by the health secretariats. Our results confirm the presence and exposure to R. parkeri strain Atlantic rainforest, associated with two anthropophilic tick species (A. ovale and A. aureolatum) parasitizing domestic dogs with unrestrained access to forest areas. Consequently, the occurrence of R. parkeri infection in humans inhabiting the Atlantic forests of ES should not be discarded.

1. Introduction Spotted fever group (SFG) rickettsioses are tick-borne illnesses

caused by pathogenic species in the genus Rickettsia and are currently considered as emerging or re-emerging zoonotic diseases around the world (Parola et al., 2013; Kernif et al., 2016; Fang et al., 2017). Until

⁎ Corresponding author at: Postgraduate Program in Infectious Diseases, Health Science Center, Universidade Federal do Espírito Santo, Av. Marechal Campos, 1468 – Maruípe, Vitória, ES, Brazil. E-mail address: [email protected] (Á.A. Faccini-Martínez).

https://doi.org/10.1016/j.ttbdis.2019.101319 Received 31 May 2019; Received in revised form 26 September 2019; Accepted 22 October 2019 1877-959X/ © 2019 Elsevier GmbH. All rights reserved.

Please cite this article as: Álvaro A. Faccini-Martínez, et al., Ticks and Tick-borne Diseases, https://doi.org/10.1016/j.ttbdis.2019.101319

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Fig. 1. Maps of the studied areas. (A) Map of South America showing the location of the Espírito Santo state within Brazil. (B) Visited villages and municipalities within the Espírito Santo state.

parasites and readily feed on dogs that enter the environments where these tick stages occur in nature (Guglielmone et al., 2006; Szabó et al., 2013b). Thus, the risk of transmission exists when infected ticks bite humans that enter the forest, or when unrestrained dogs return from the forest, bringing on them infected ticks that detach inside human dwellings (Szabó et al., 2013b). Meanwhile, considering that R. parkeri has been detected in A. ovale in Brazil (Sabatini et al., 2010; Barbieri et al., 2014; Vizzoni et al., 2016; Nieri-Bastos et al., 2016; Moerbeck et al., 2016; Krawczak et al., 2016b; Luz et al., 2019; Acosta et al., 2018), and in other anthropophilic ticks such as Amblyomma tigrinum (Weck et al., 2016; Dall’Agnol et al., 2018), and Amblyomma aureolatum (Medeiros et al., 2011; Barbieri et al., 2014; Dall’Agnol et al., 2018), it is quite probable that this agent could be endemic in other states in which human cases are yet-to-be-investigated. The Espírito Santo state (southeastern Brazil) is recognized as an endemic area for R. rickettsii rickettsiosis (locally known as Brazilian spotted fever) with a case-fatality rate of up to 20% (Sexton et al., 1993; Barros e Silva et al., 2014; Faccini-Martínez et al., 2018c). Moreover, the few serological studies performed in the state support exposure and circulation of SFG rickettsiae in humans (seroprevalence of 34.8%) (Spolidorio et al., 2010a), and in domestic animals (seroprevalence of 5.1–7.6% in dogs, and 20–26% in horses) (Spolidorio et al., 2010a; Vieira et al., 2018). Recently, the presence of A. ovale in the Espírito Santo state was reported for the first time, specifically at Santa Teresa, Santa Maria de Jetibá and Ibiraçu municipalities (Acosta et al., 2016, 2018). Interestingly, R. parkeri was detected in ticks from this later locality (Acosta et al., 2018). Considering the above-presented findings and that R. parkeri is recognized as an emerging tick-borne pathogen in the Americas (Nieri-Bastos et al., 2018), the present study aimed to contribute with epidemiological aspects of R. parkeri in the Atlantic

the late 20th century, Rickettsia rickettsii was recognized as the sole etiological agent of human SFG rickettsiosis in the Americas (Hidalgo et al., 2013). Nevertheless, in 2004, Rickettsia parkeri was reported as the agent of a new SFG disease in humans in the United States (Paddock et al., 2004), more than 60 years after the first isolation of this agent from ticks in that same country (Parker et al., 1939). Nowadays R. parkeri infection is recognized as the most important tick-borne rickettsiosis in Uruguay (Conti-Díaz et al., 2009; Portillo et al., 2013; Faccini-Martínez et al., 2018b), and the second most significant rickettsial infection after R. rickettsii in the United States (Biggs et al., 2016), Brazil (de Oliveira et al., 2016; Faccini-Martínez et al., 2018a) and Argentina (Romer et al., 2011, 2014; Villalba Apestegui et al., 2018). The infection with R. parkeri causes a mild febrile disease associated with a characteristic inoculation eschar and other nonspecific symptoms such as a nonpruritic maculopapular or vesiculopapular rash, myalgia, headache and regional lymphadenopathy (Spolidorio et al., 2010b; Silva et al., 2011; Romer et al., 2011; Biggs et al., 2016; Krawczak et al., 2016c; Faccini-Martínez et al., 2018a,b). In Brazil, only three confirmed cases of R. parkeri rickettsiosis have been published. One case concerning a patient from Peruíbe (southern coastal region of São Paulo state) (Spolidorio et al., 2010b); a second case from the Chapada Diamantina region in the Bahia state (Silva et al., 2011); and a third case from the Blumenau municipality in the Santa Catarina state (Krawczak et al., 2016c). Eco-epidemiological studies performed in these three Brazilian states point to an enzootic cycle in which the circulation and amplification of R. parkeri occurs in small rodents and Amblyomma ovale ticks. They would constitute the main reservoirs and vectors of this agent in the Atlantic rainforest biome, respectively (Szabó et al., 2013a; Nieri-Bastos et al., 2016; Krawczak et al., 2016a, 2018). Adults of A. ovale are anthropophilic 2

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remnants of ticks processed by the shell vial technique, and to the ticks that resulted negative to Rickettsia detection in the initial Gimenez staining test. A PCR targeting a portion of the 16S rRNA mitochondrial tick gene using primers 16S +1 and 16S-1 was implemented as an internal control, following Mangold et al. (1998). Positive ticks were then tested for Rickettsia following the same algorithm primers and protocols described above. All PCRs were performed employing a mix of 25 μl, composed by 12.5 μl of DreamTaq Green PCR master mix (2X, Thermo Fischer Scientific Baltics UAB, Vilnius, Lithuania), 1 μl of each primer (10 pmol/μl), 8 μl of ultrapure water and 2.5 μl of template DNA. A negative control tube containing ultrapure water and a positive control tube containing DNA of Rickettsia vini strain Breclav (Nováková et al., 2016) were included in each reaction. Amplicons of the expected size were treated with ExoSap (USB, Cleveland, Ohio, USA) and 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 (Kearse et al., 2012) and submitted to BLASTn analyses (www.ncbi.nlm.nih.gov/blast) in order to infer the closest similarities to other organisms available in GenBank.

forest of Espírito Santo state using molecular, serological and cell-culture techniques. 2. Materials and methods 2.1. Study site Between June 2016 and September 2018, we visited rural areas of 33 villages located in nine municipalities (Anchieta, Cariacica, Domingos Martins, Ibiraçu, Marechal Floriano, Santa Leopoldina, Santa Maria de Jetibá, Santa Teresa, and Venda Nova do Imigrante) within the state of Espírito Santo (ES), Brazil (Fig. 1). These nine areas were chosen based on the similar vegetational cover that they share (i. e. dense Atlantic rainforest regions) (http://www.ijsn.es.gov.br) and because of the occurrence of A. ovale ticks, recently reported in three of them (Acosta et al., 2016, 2018). 2.2. Sample collection We collected ticks and blood from dogs living in rural households that gathered the following inclusion criteria: born and raised in the sampled village, absence of locomotor or neurological disabilities, unrestrained access to forest areas, and harboring adults of genus Amblyomma at the sampling moment (Pinter et al., 2016). Only adult ticks of the Amblyomma genus were collected and brought alive to the laboratory for taxonomic identification that followed Barros-Battesti et al. (2006). Blood was also collected from owners of sampled dogs. Serum was separated by centrifugation and stored at −20 °C until use. None of these humans had a history of rickettsiosis. Altitude and geographical coordinates of each sampled house were acquired using a Garmin® Etrex 10 GPS equipment.

2.4. Serological analyses Canine and human sera were tested by the indirect immunofluorescence assay (IFA) using crude antigens derived from two pathogenic SFG rickettsia isolates from Brazil (R. rickettsii strain Taiaçu and R. parkeri strain Atlantic rainforest), as previously described (Barbieri et al., 2014). Briefly, sera were diluted in 2-fold increments with phosphate-buffered saline (PBS), starting from the 1:64 dilution. Slides were incubated with fluorescein isothiocyanate-labeled rabbit anti-dog IgG, and goat anti-human IgG (Sigma, St Louis, MO, USA) for canine, and human sera, respectively. For each sample, the endpoint IgG titer reacting with each of the two rickettsial antigens was determined. An endpoint titer at least 4-fold higher for a Rickettsia species than that observed for other Rickettsia species was considered probably homologous to the first Rickettsia species or a very closely related species (Barbieri et al., 2014). In each slide, a non-reactive (negative control), and a known reactive sera (positive control) were tested at the 1:64 dilution.

2.3. Isolation and molecular characterization of rickettsiae from ticks Right after the arrival at the laboratory, living ticks were initially submitted to the hemolymph test with Gimenez staining (Burgdorfer, 1970). Briefly, a leg of each tick was cut, and the drop of hemolymph that emerged from the incision was imprinted into slides and stained. The body of each tick was stored separately in sterile vials at −80 °C. Ticks that presented Rickettsia-like organisms within hemocytes after observation of stained drops of hemolymph through optical microscopy were submitted to the isolation of rickettsiae in Vero cell culture by implementing the shell vial technique, as previously described (Labruna et al., 2004b). A rickettsial isolate was considered established in cultures after at least three passages showing > 90% of infected cells, as determined by Gimenez staining (Labruna et al., 2004b). DNA extraction using the DNeasy Tissue Kit (Qiagen, Valencia, CA) was performed to a sample of infected cells obtained from the third passage. Extracted DNA was submitted to a series of conventional polymerase chain reactions (PCR) protocols using the following primers: CS-78/CS-323, which target a fragment of ∼401 base pairs (bp) of the citrate synthase gene (gltA), common to all representatives of the genus Rickettsia (Labruna et al., 2004a); primers Rr190.70p/Rr190.701n that target a ∼631-bp fragment of the ompA gene (Eremeeva et al., 2006); and primers 120-M59/120–807 targeting a ∼865-bp fragment of the ompB gene (Eremeeva et al., 2006). Samples that were negative to ompA and ompB gene PCRs were submitted to a conventional protocol using Rickettsia bellii-specific primers 5´-ATCCTGATTTGCTGAATTTTTT-3´ (forward) and 5´-TGCAATACCAGTACTGACG-3´ (reverse) that amplify a ∼338-bp fragment of the gltA gene (Szabó et al., 2013a). Finally, PCRs using primers CS-239/CS-1069 and CS-78/CS-323, which amplify two overlapping fragments (final sequence ∼1090 bp) of the rickettsial gltA gene (Labruna et al., 2004b), were applied to characterize R. belliipositive samples. DNA extraction using the guanidine isothiocyanate and phenol/ chloroform technique (Sangioni et al., 2005) was applied to the

2.5. Inquiries on human cases of Rickettsia parkeri rickettsiosis From 2017–2019, in association with Health Secretariat of Espírito Santo state, we shared diverse aspects of epidemiological surveillance, diagnosis and treatment of R. parkeri rickettsiosis in Brazil (FacciniMartínez et al., 2018a) with health authorities of the visited municipalities. This procedure aimed to raise the awareness of physicians towards this tick-borne disease. Thus, in an eventually suspected case, physicians would inform us to start an epidemiological investigation. 2.6. Ethical statement This study was approved by the Ethical Committee of Animal Use and the Ethical Committee of Human Research of the Health Science Center of the Federal University of Espírito Santo (protocol 041/2016 and protocol 2.091.157, respectively). 3. Results 3.1. Collection of ticks A total of 280 adult ticks of the genus Amblyomma were collected from dogs. After morphological identifications, A. ovale was the most abundant species (54.3%, n = 152), followed by A. aureolatum (45.3%, n = 127) and Amblyomma sculptum (0.3%, n = 1). Of the above 3

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collected ticks, 14 specimens were excluded from further analyses and deposited as voucher in the tick collection "Coleção Nacional de Carrapatos Danilo Gonçalves Saraiva" (CNC) of the Faculty of Veterinary Medicine, University of São Paulo, São Paulo, Brazil, under the following accession numbers (tick species [♂ or ♀], collection date [day-month-year], animal host, municipality, Brazilian state): CNC3561 (A. ovale [1♂, 1♀], 15-04-17, dog, Santa Teresa, ES); CNC-3562 (A. aureolatum [1♂, 1♀], 12-06-17, dog, Santa Maria de Jetibá, ES); CNC-3563 (A. aureolatum [1♀], 10-02-17, dog, Venda Nova do Imigrante, ES); CNC-3564 (A. aureolatum [1♀], A. ovale [1♂, 1♀], 0506-17, dog, Santa Leopoldina, ES); CNC-3565 (A. ovale [1♂, 1♀], 2905-17, dog, Marechal Floriano, ES); and CNC-3566 (A. aureolatum [1♂, 1♀], A. ovale [1♂, 1♀], june-17, dog, Domingos Martins, ES). Regarding altitude records, A. ovale specimens were collected at altitudes ranging from 23 to 833 m above sea level (masl), and A. aureolatum was collected at altitudes between 545 and 967 masl. These ranges indicated sympatric populations of both species occurring between 545 and 833 masl. The sole A. sculptum specimen was collected at an altitude of 248 masl in Santo Antônio village, Ibiraçu municipality (Suppl. 1). Concerning to the spatial distribution of A. ovale and A. aureolatum in the studied area, the occurrence of both species was recorded in the municipalities of Domingos Martins, Santa Leopoldina and Santa Maria de Jetibá. On the other hand, A. ovale was the only species collected in the municipalities of Anchieta, Cariacica, Ibiraçu, Marechal Floriano and Santa Teresa, while A. aureolatum was the sole species collected in Venda Nova do Imigrante municipality (Suppl. 1).

Table 1 Adults of Amblyomma (with notes on rickettsial infection) collected from dogs with unrestrained access to natural environments, in rural households of Atlantic forest of Espírito Santo state, southeastern Brazil. Municipalities

Anchieta Cariacica Domingos Martins Ibiraçua Marechal Floriano Santa Leopoldina Santa Maria de Jetibá Santa Teresa Venda Nova do Imigrante Total

Ticks tested for rickettsial infection Amblyomma aureolatum

Amblyomma ovale

No. infected/ No. tested (% infection)

No. infected/ No. tested (% infection)

Rickettsia species

0/4 (0)

10/98 (10)

R. bellii

1/98 (1)

R. parkerib

0/2 (0) 0/3 (0) 0/8 (0) 1/16 (6) 0/20 (0) 0/18 (0) 0/3 (0)

Rickettsia species

R. parkerib

0/74 (0) 0/19 (0) 10/121 (8.3) 1/121 (0.8)

R. bellii R. parkeri

1/144 (0.7)

R. parkeri

a One Amblyomma sculptum tick was also collected on a dog in this municipality. b All PCR amplicons (gltA, ompA and ompB rickettsial genes) were sequenced and confirmed a 100% identity with R. parkeri strain Atlantic rainforest.

3.2. Isolations and molecular characterization of Rickettsia

were seroreactive (titer ≥64) to at least one Rickettsia species. A total of 34/83 (41%) and 47/83 (57%) dogs were seroreactive to R. rickettsii and R. parkeri, respectively (Table 2). All sera that reacted to R. rickettsii antigens also reacted to R. parkeri. A total of 37 (45%) canine sera showed titers to R. parkeri at least fourfold higher than to R. rickettsii antigen. The antibody titers in these 37 dogs were considered to have been stimulated by R. parkeri or a very closely related species. Canine IFA endpoint titers for R. parkeri varied from 64 to 8192. Among the 47 R. parkeri seroreactive dogs, 18 animals (38%) had titers ≥1024, and seven animals (15%) had titers ≥4096. Blood and serum samples were obtained from 41 dog owners, with 10% (4/41) of the samples reacting at least to one rickettsial antigen (three persons from Santa Maria de Jetibá and one person from Santa Leopoldina municipalities). The highest endpoint titers varied from 64 to 128 for R. rickettsii and R. parkeri. No human serum showed ≥4-fold difference between the highest endpoint titers, precluding any inference on a possible homologous reaction.

A total of 12 adults of the genus Amblyomma (one male of A. ovale, nine males and two females of A. aureolatum) contained Rickettsia-like organisms within their hemocytes after the hemolymph test. Attempts to isolate rickettsiae by the shell vial technique were performed with all of them; nevertheless, eleven isolates were lost during the first or second passages due to contamination with fungi or extracellular bacteria. Therefore, only one isolate (from one male of A. aureolatum tick), here designated as strain M9A, was successfully established in the laboratory and cryopreserved in our rickettsial collection. Although the majority of the attempted isolations were lost, we obtained positive results after PCR targeting Rickettsia genes in the remnants of the ticks submitted to the shell vial technique. DNA sequencing of PCR products revealed that 0.7% (1/144) and 0.8% (1/121) of the A. ovale and A. aureolatum ticks, respectively, were infected by R. parkeri strain Atlantic rainforest, since obtained gltA (350 bp), ompA (590 bp) and ompB (817 bp) partial sequences matched 100% identity with conspecific sequences available in GenBank (accession numbers MF536974, MF536975 and KU882101, respectively). On the other hand, 8.3% (10/ 121) of the A. aureolatum ticks were infected by R. bellii since gltA (1035 bp) partial sequences matched 100% identity with a sequence of R. bellii (GenBank accession number JQ906786) (Table 1). DNA extracted from the third passage of strain M9A was submitted to PCR as well, yet we obtained expected size amplicons only for gltA gene. A R. bellii-specific PCR targeting this later locus confirmed the identity for this Rickettsia species. Molecular characterization of gltA gene for the strain M9A yielded a 1035-bp haplotype 100% identical to the sequence of R. bellii strain Peruibe (GenBank accession number JQ906786). Rickettsial sequences generated in the present study were deposited in GenBank under the accession numbers MK962695, MK962696, MK962697 (gltA, ompA, and ompB from R. parkeri of A. aureolatum); MK962698, MK962699, MK962700 (gltA, ompA, ompB from R. parkeri of A. ovale), and MK962701 (gltA from R. bellii strain M9A of A. aureolatum).

3.4. Inquiries on Rickettsia parkeri rickettsiosis human cases During 2017–2019 health authorities of each municipality did not report any suspected human case of R. parkeri rickettsiosis. 4. Discussion 4.1. Ticks Amblyomma aureolatum and A. ovale are two neotropical tick species typically found in the Brazilian Atlantic rainforest biome, in which adult stages infest predominately wild carnivores such as canines and felines (Guglielmone et al., 2003). Meanwhile, in regions where human dwellings are emplaced close to forest fragments, domestic dogs with free access to this natural environment can act as alternative hosts for adults of both tick species (Guglielmone et al., 2003; Szabó et al., 2013b). Aiming to find parasitized dogs, we chose to perform this study in nine municipalities with dense Atlantic forest cover and included criteria such as unrestrained access to forest areas to maximize the collection of ticks. As expected, the majority of ticks collected on dogs

3.3. Serology Blood was obtained from 83 dogs sampled in all the localities except for Venda Nova do Imigrante municipality. Overall, ≈49% of the dogs 4

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Table 2 Seroreactivity to Rickettsia rickettsii and Rickettsia parkeri in dogs with unrestrained access to natural environments, in rural households of Atlantic forest of Espírito Santo state, southeast Brazil. Municipality


No. of tested dogs

No. seroreactive dogs to each Rickettsia species (%) R. rickettsii

R. parkeri

No. of dogs with possible homologous reaction (PAIHR in parentheses)a

Anchieta Cariacica Domingos Martins

Córrego da Prata Pau Amarelo Melgaço Baixo Soído de cima

1 3 3 1

0 3 0 0

(0) (100) (0) (0)

0 3 0 1

(0) (100) (0) (100)

— 1 (R. parkeri) — 1 (R. parkeri)


Itapiraçu Palmeiras Santo Antônio São Benedito

1 3 3 4

0 1 2 2

(0) (33) (67) (50)

0 2 3 3

(0) (67) (100) (75)

— 2 (R. parkeri) 2 (R. parkeri) 1 (R. parkeri)

Marechal Floriano

Alto Rio Fundo Costa Pereira Santa Rita

2 1 2

0 (0) 0 (0) 2 (100)

0 (0) 0 (0) 2 (100)

— — 1 (R. parkeri)

Santa Leopoldina

Bragança Luxemburgo Tirol

3 3 4

1 (33) 3 (100) 3 (75)

1 (33) 3 (100) 3 (75)

1 (R. parkeri) 1 (R. parkeri) 3 (R. parkeri)

Santa Maria de Jetibá

Alto Santa Maria Caramuru Rio Claro Rio Lamego

5 2 10 12

1 0 4 7

(20) (0) (40) (58)

2 (40) 0 (0) 7 (70) 10 (83)

1 (R. parkeri) — 7 (R. parkeri) 10 (R. parkeri)

Santa Teresa

Alto Caldeirão Alto de Santo Antônio Rio saltinho —

5 4 11 83

0 (0) 3 (75) 2 (18) 34 (41)

0 (0) 4 (100) 3 (27) 47 (57)

— 4 (R. parkeri) 2 (R. parkeri) 37 (R. parkeri)


a A homologous reaction was determined when an endpoint titer to a Rickettsia species was at least fourfold higher than those observed for the other Rickettsia species. In this case, the Rickettsia species (or a very closely related species) involved in the highest endpoint titer was considered the possible antigen involved in a homologous reaction (PAIHR).

to occur at different altitudes along their distribution within the Atlantic forest biome in four Brazilian states, any attempt to model the abiotic conditions related to each tick species must not overlook the effect of latitude.

were A. aureolatum and A. ovale. On the other hand, the low presence of A. sculptum, as shown by the collection of only one specimen, could be explained because this tick species prevails mainly in the Cerrado biome, or within degraded Atlantic forests (Martins et al., 2016), areas that were not included in this study. Based on previous records for the Espírito Santo state (Acosta et al., 2016, 2018), our results expand the geographical distribution of A. aureolatum towards Domingos Martins, Santa Leopoldina and Venda Nova do Imigrante municipalities, and into Anchieta, Cariacica, Domingos Martins, Marechal Floriano and Santa Leopoldina municipalities for A. ovale. Amblyomma aureolatum and A. ovale are both vectors of humanpathogenic rickettsiae (Szabó et al., 2013b), and both occur within the Atlantic forest biome of the Espírito Santo state (Acosta et al., 2016). Several studies have pointed out that microclimate conditions associated with the altitude would predict the distribution of these two species of ticks (Sabatini et al., 2010; Barbieri et al., 2015). For instance, Medeiros et al. (2011) and Barbieri et al. (2014) described sympatric distributions for these species at ≈ 150 masl in the Santa Catarina state (Southern Brazil). Towards the north in southeastern Brazil, A. ovale has been collected at < 100 masl and A. aureolatum over 700 masl in the state of São Paulo, depicting an allopatric distribution (Sabatini et al., 2010; Barbieri et al., 2015). Here, A. aureolatum and A. ovale were collected at altitudes ranging from 545 to 833 masl, suggesting that both species occur in sympatry between this altitudinal gap in the Espírito Santo state. Interestingly, Moerbeck et al. (2016) collected A. ovale in dogs from an Atlantic forest fragment in the Ceará state (northeastern Brazil) at > 800 masl, noting the absence of A. aureolatum. Whether ecological adaptations define that both species persist in different microclimates within the Atlantic forest biome, is an interesting hypothesis that needs further assessment. As both ticks seem

4.2. Rickettsial infection Our results concerning rickettsial infection in the collected ticks confirm the presence of R. parkeri strain Atlantic rainforest in A. ovale, and R. bellii in A. aureolatum from Espírito Santo state, which is in line with recently published reports (Acosta et al., 2016, 2018). Moreover, we describe the first molecular detection of R. parkeri strain Atlantic rainforest and the first rickettsial isolate (R. bellii strain M9A) from A. aureolatum ticks for the Espírito Santo state. In accordance with our results, A. ovale and A. aureolatum infected with R. parkeri strain Atlantic rainforest were previously described in a similar bio-geographical region, namely the Atlantic forest of the Santa Catarina state (Medeiros et al., 2011; Barbieri et al., 2014), where both tick species occur in sympatry (Medeiros et al., 2011). This condition, together with sharing the same vertebrate hosts (e.g., domestic dogs), may lead to horizontal transmission of this rickettsial agent via rickettsiemic hosts, since A. ovale has been confirmed as a vector for R. parkeri strain Atlantic rainforest (Krawczak et al., 2016a). This kind of transmission is likely to occur only in one way, from A. ovale to A. aureolatum, since the distribution of R. parkeri strain Atlantic rainforest has been reported only in areas with the presence A. ovale, many times without A. aureolatum (Medeiros et al., 2011; Barbieri et al., 2014). Besides, considering the anthropophilic behavior of A. aureolatum (Guglielmone et al., 2006) and its vector competence for R. rickettsii in the São Paulo Metropolitan Area (Ogrzewalska et al., 2012), this tick species could act as an alternative vector for R. parkeri strain Atlantic rainforest when 5

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based on the study of Barbieri et al. (2014) which describes a high human seroprevalence against SFG rickettsiae (47%), even in 15 inhabitants from a high R. parkeri/Amblyomma ticks infection rate region of the Santa Catarina state.

occurring in sympatry with A. ovale. In any case, this matter should be proved in future studies. Our findings of R. parkeri strain Atlantic rainforest infecting 0.7% and 0.8% of the A. ovale and A. aureolatum ticks, respectively, contrast to previous results from the Santa Catarina state, where both ticks exhibited infection rates of 3–8% (A. ovale) and 9–11% (A. aureolatum) for the same rickettsial agent (Medeiros et al., 2011; Barbieri et al., 2014). Remarkably, we also found a considerably high infection rate (8.3%) of R. bellii in A. aureolatum. Thus, it is plausible that the low R. parkeri infection rate found in the study area would be explained by the "rickettsial interference" process (Burgdorfer et al., 1981), in which R. bellii would play a significant role in inhibiting the maintenance of other pathogenic Rickettsia species (Sakai et al., 2014; Hecht et al., 2019). In this regard, some studies have shown that geographical areas where R. bellii infection rate is high coincide with a low R. parkeri infection rate in ticks (Paddock et al., 2017; Lamattina et al., 2018), and vice versa (Medeiros et al., 2011; Szabó et al., 2013a; Barbieri et al., 2014).

4.4. Inquiries on Rickettsia parkeri rickettsiosis human cases Although our overall results point the presence and circulation of the pathogen R. parkeri Atlantic rainforest in A. ovale and A. aureolatum ticks, and in infested rural dogs, the findings of low R. parkeri infection rate in ticks and low seroprevalence rates in humans suggest that the chance of R. parkeri infection in the inhabitants of these regions is also low. This fact could explain the apparent absence of suspected human cases during the study period, even with the awareness of this rickettsiosis by municipality health authorities. Either way, following the eco-epidemiological classification proposed by Pinter et al. (2016) for assessing areas of SFG-rickettsiae transmission, our study area should be classified as a "risk area with presence of Amblyomma ticks" for R. parkeri rickettsiosis. This proposal is made considering the presence of A. ovale and A. aureolatum ticks, and canine seroprevalence higher than 10%, or at least one dog with IgG titers ≥1024 by IFA test. Thus, if any human case of R. parkeri infection is diagnosed in the future, the classification of the area will change to a "transmission area" (Pinter et al., 2016). Finally, while the ecology and vector-related aspects of R. parkeri (Dall’Agnol et al., 2018; Szabó et al., 2013a, b; Krawczak et al., 2016a; Krawczak and Labruna, 2018; Nieri-Bastos et al., 2016; Weck et al., 2016) and human cases (Spolidorio et al., 2010b; Silva et al., 2011; Krawczak et al., 2016c) have already been described for Brazil, general and consolidate aware from Brazilian health authorities on the epidemiological and clinical aspects of this rickettsiosis is still incipient (Ministério da Saúde, 2017). To improve this scenario, we suggest the following issues: 1) the elaboration and publication of national guidelines on R. parkeri infection, recognizing it as a different rickettsiosis other than Brazilian spotted fever (R. rickettsii infection) (FacciniMartínez et al., 2018a); 2) the inclusion of the criterion "presence of eschar" as an item on the check-list in the "official spotted fever rickettsiosis case form"; and 3) the call for awareness of physicians on rickettsioses as a differential diagnosis of the undifferentiated febrile syndrome (Fang et al., 2017). In conclusion, our results confirm, through molecular and serological methods, the presence in the study area of the pathogen R. parkeri strain Atlantic rainforest in two anthropophilic tick species (A. ovale and A. aureolatum) and exposure of domestic dogs with unrestrained access to forest areas. Consequently, it is plausible the occurrence of R. parkeri infection in humans from the Atlantic forest of Espírito Santo state.

4.3. Serology in dogs and humans We found that at least 49% of the dogs have been exposed to SFG rickettsiae, probably elicited by R. parkeri strain Atlantic rainforest or a very closely related species, rather than R. rickettsii, in most of the seroreactive dogs. This assumption is plausible because: 1) none canine serum reacted with R. rickettsii without reacting with R. parkeri; 2) 45% of canine sera showed titers to R. parkeri at least fourfold higher than R. rickettsii antigen; and 3) the two most prevalent Amblyomma species infesting the sampled dogs (A. ovale and A. aureolatum) were found infected with this R. parkeri strain. On the other hand, our canine seroprevalence results contrast with previous studies in the Espírito Santo state, in which less than 10% seroprevalence values were reported for canine populations among urban and rural areas (Spolidorio et al., 2010a; Vieira et al., 2018). This fact could be explained by the inclusion criteria for sampling dogs in the current study, which were not considered in the previous studies. Three of these criteria (i. e. rural area, unrestrained access to forest areas, and infestation by adult ticks of the genus Amblyomma) are directly related with seropositivity against SFG rickettsiae (Melo et al., 2011; Szabó et al., 2013a; Barbieri et al., 2014; Costa et al., 2017). At the same time, even though our results on canine seroprevalence suggest a high exposure to SFG rickettsiae (highest against R. parkeri), they are lower if compared with seroprevalence values (60–88%) observed in other Brazilian regions where R. parkeri infects humans, namely Peruíbe (São Paulo state), Chapada Diamantina region (Bahia state), and Blumenau municipality (Santa Catarina state) (Spolidorio et al., 2010b; Silva et al., 2011; Szabó et al., 2013a; Barbieri et al., 2014; Nieri-Bastos et al., 2016; Krawczak et al., 2016c). These uneven prevalence values could be explained considering the differences in the infection rate of R. parkeri in Amblyomma ticks. In our study area less than 1% of the ticks were infected, and in the above-mentioned studies 3–14% of the ticks were infected, a fact that directly raises the chances of canine exposure to infected ticks (Szabó et al., 2013a; Barbieri et al., 2014; Nieri-Bastos et al., 2016). Concerning SFG seroprevalence in the owners of the sampled dogs, we found a lower rate compared with the only one previous study from Espírito Santo state: 10% vs. 34.8%, respectively (Spolidorio et al., 2010a). Thus, in addition to our reduced number of human samples (n = 41), which could hinder the real interpretation of the exposure to SFG rickettsiae in the study area, we did not use crude antigens derived from unknown or controversially pathogenic Rickettsia species such as R. amblyommatis, R. rhipicephali and/or R. felis. The use of these antigens would contribute importantly to human seropositivity, as depicted in previous results for the State (Spolidorio et al., 2010a). Nevertheless, we also presume that low infection rates of pathogenic SFG rickettsiae in anthropophilic Amblyomma ticks in the study area may lead consequently to lower human exposure to SFG rickettsiae. This hypothesis is

Acknowledgments We thank Elisangela Vago, Rogério Ribeiro, Claudiney Biral dos Santos, Aloísio Falqueto, Marcos Antônio Correia Rodrigues, Flavia Sylvestre Janiques, Augusto Marchon Zago, Raphael L. Zanotti, and Fernanda S. Ropke for their valuable help during field work in the visited municipalities of Espírito Santo state. We are grateful to Isabella Fontana for preparing the maps of Fig. 1. Á.A.F.M. was funded in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brazil (CAPES)- Finance Code 001.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.ttbdis.2019.101319. 6

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