Accepted Manuscript Title: Detection of rickettsiae in fleas and ticks from areas of Costa Rica with history of spotted fever group rickettsioses Author: Adriana Troyo Rolando D. Moreira-Soto Ólger Calderon-Arguedas Carlos Mata-Somarribas Jusara Ortiz-Tello Amália R.M. Barbieri Adrián Avendaño Luis E. Vargas-Castro Marcelo B. Labruna Laya Hun Lizeth Taylor PII: S1877-959X(16)30133-9 DOI: http://dx.doi.org/doi:10.1016/j.ttbdis.2016.08.009 Reference: TTBDIS 712 To appear in: Received date: 14-7-2016 Revised date: 1-8-2016 Accepted date: 22-8-2016 Please cite this article as: Troyo, Adriana, Moreira-Soto, Rolando D., Calderon- Arguedas, Ólger, Mata-Somarribas, Carlos, Ortiz-Tello, Jusara, Barbieri, Amália R.M., Avendaño, Adrián, Vargas-Castro, Luis E., Labruna, Marcelo B., Hun, Laya, Taylor, Lizeth, Detection of rickettsiae in fleas and ticks from areas of Costa Rica with history of spotted fever group rickettsioses.Ticks and Tick-borne Diseases http://dx.doi.org/10.1016/j.ttbdis.2016.08.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Detection of rickettsiae in fleas and ticks from areas of Costa Rica with history of spotted fever group rickettsioses a,* a,b a Adriana Troyo , Rolando D. Moreira-Soto , Ólger Calderon-Arguedas , Carlos Mata- a,1 a,2 c a,3 Somarribas , Jusara Ortiz-Tello , Amália R. M. Barbieri , Adrián Avendaño , Luis E. a,4 c a a,+ Vargas-Castro , Marcelo B. Labruna , Laya Hun , Lizeth Taylor a Centro de Investigación en Enfermedades Tropicales and Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. b Centro de Investigación en Estructuras Microscópicas, Universidad de Costa Rica, San José, Costa Rica. c Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, Brasil 1 Present address: Centro Nacional de Referencia de Parasitología, Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud, Cartago, Costa Rica. 2 Present address: Hospital Nacional de Geriatría y Gerontología Dr. Raúl Blanco Cervantes, San José, Costa Rica. 3 Present address: Universidad de Ciencias Médicas, San José, Costa Rica. 4 Present address: Department of Biology, University of Miami, Coral Gables, FL 33146, USA. * Corresponding author: Facultad de Microbiología, Universidad de Costa Rica. San José 11501, Costa Rica. Tel.: (506) 2511-8626. E-mail address: adriana.troyo@ucr.ac.cr + Deceased (December 2013). ABSTRACT Outbreaks of spotted fevers have been reported in Costa Rica since the 1950s, although vectors responsible for transmission to humans have not been directly identified. In this study, species of Rickettsia were detected in ectoparasites from Costa Rica, mostly from five study sites where cases of spotted fevers have been reported. Ticks and fleas were collected using drag cloths or directly from domestic and wild animals and pooled according to species, host, and location. Pools were analyzed initially by PCR to detect a fragment of Rickettsia spp. specific gltA gene, and those positive were confirmed by detection of htrA and/or ompA gene fragments. Partial sequences of the gltA gene were obtained, as well as at least one htrA and/or ompA partial sequence of each species. Rickettsia spp. were confirmed in 119 of 497 (23.9%) pools of ticks and fleas analyzed. Rickettsia rickettsii was identified in one nymph of Amblyomma mixtum and one nymph of Amblyomma varium. Other rickettsiae present were ‗Candidatus Rickettsia amblyommii‘ in A. mixtum, Amblyomma ovale, Dermacentor nitens, and Rhipicephalus sanguineus s. l.; Rickettsia bellii in Amblyomma sabanerae; Rickettsia felis in Ctenocephalides felis; and Rickettsia sp. similar to ‗Candidatus R. asemboensis‘ in C. felis, Pulex simulans, A. ovale, Rhipicephalus microplus, and A. mixtum. Results show the presence of rickettsiae in vectors that may be responsible for transmission to humans in Costa Rica, and evidence suggests exposure to rickettsial organisms in the human environment may be common. This is the first study to report R. rickettsii in A. varium and in A. mixtum in Costa Rica. Key words: Rickettsia; Ixodida; Siphonaptera; Ectoparasite; Tick-borne disease; Central America Introduction th In Latin America, the only rickettsial diseases recognized during the 20 century were Rocky Mountain spotted fever (RMSF), flea-borne typhus, and epidemic typhus caused by Rickettsia rickettsii, Rickettsia typhi, and Rickettsia prowazekii, respectively (Labruna et al., 2011). The description of new species of these intracellular ectoparasite-borne bacteria has increased in the past 25 years, and emerging human pathogens have been documented worldwide (Parola et al., 2005, 2013). There are now at least 10 more species of Rickettsia reported in Latin America, including known human pathogens (Rickettsia akari, Rickettsia felis, Rickettsia parkeri, Rickettsia massiliae, Rickettsia africae) and previously undescribed species and genotypes (Pacheco et al., 2007, 2011; Labruna et al., 2011; Miranda et al., 2012; Spolidorio et al., 2012; Troyo et al., 2014). Although infection by some of the rickettsiae present in the region may be severe or have a high fatality rate if untreated, others such as Rickettsia bellii, Rickettsia rhipicephali, and ‗Candidatus Rickettsia amblyommii‘ are considered nonpathogenic or have not been associated conclusively with disease in humans (Mediannikov et al., 2007; Labruna et al., 2011; Parola et al., 2013). In Central America, the first human cases of spotted fever were documented in Panama and Costa Rica during the 1950s (de Rodaniche and Rodaniche, 1950; Calero et al., 1952; Fuentes, 1979; Hun-Opfer, 2008). In other countries of Central America, reports of spotted fever group (SFG) rickettsiosis are more recent and include diagnosis in a traveler returning from Honduras (Chen and Wilson, 2009), and a possible outbreak that was identified in Guatemala in 2007 with at least one confirmed case (Eremeeva et al., 2012). In El Salvador and Nicaragua, cases of spotted fevers have not been officially confirmed, but there is serological evidence of infection in both countries (Peacock et al., 1971; WHO, 1993). In the case of Belize, there are no reports of human infection with rickettsiae, although the pathogenic Rickettsia sp. strain Atlantic rainforest was recently reported infecting ticks in that country (Lopes et al., 2016). The ecology of rickettsial diseases has been poorly investigated in most countries of Central America (Labruna et al., 2011), despite the evidence of possible transmission in most of the region and recurrent case reports and fatalities in Costa Rica and Panama (Estripeaut et al., 2007; Tribaldos et al., 2011; Arguello et al., 2012; De Lucas et al., 2013; Hun, 2013). In Costa Rica, R. rickettsii was identified and confirmed as the species responsible for human cases (Hun et al., 2008), although the most complete ecological studies of RMSF date from the 1980s (Fuentes et al., 1985; Fuentes, 1986). In these investigations, R. rickettsii was detected and isolated only on one occasion from an invertebrate host, the rabbit tick Haemaphysalis leporispalustris (Fuentes et al., 1985; Fuentes, 1986). Considering that information concerning vectors and reservoirs of rickettsiae in Costa Rica is limited, a project was initiated in 2008 to detect and identify species of rickettsia in ticks and fleas that may present a risk of contact and infection for the human population. Preliminary results of this study documented the presence and isolation of ‗Candidatus R. amblyommii‘ and R. felis for the first time in Costa Rica (Hun et al., 2011; Troyo et al., 2012a). This paper adds substantial evidence to complete the detection and identification of rickettsiae in different ectoparasite species, collected predominantly from domestic and peridomestic animals in areas of Costa Rica where cases of spotted fevers have historically been diagnosed or are suspected. Materials and methods Study sites The main collection sites have been described previously (Troyo et al., 2012b). Sites are located to the North and East (Caribbean) regions of Costa Rica and have reported cases of spotted fevers in the past (Campbell et al., 1978; Fuentes, 1979; Hun et al., 1991; Hun-Opfer, 2008). Specifically, 5 sites were selected in 7 districts: 1) Turrialba (954' N, 8341' W; elevation: 650 m.a.s.l.), 2) La Virgen (1023' N, 8408' W; elevation: 190 m.a.s.l.), 3) Limón (959' N, 8302' W; elevation: 5 m.a.s.l.), 4) Cahuita (944' N, 8250' W; elevation: 5 m.a.s.l.), 5) Guápiles (1013' N, 8347' W; elevation: 260 m.a.s.l.), Jiménez (1012' N, 8344' W; elevation: 230 m.a.s.l.), and Guácimo (1012' N, 8341' W; elevation: 110 m.a.s.l.). The urban centers of Guápiles, Jiménez, and Guácimo (GP/J/GC) are less than 20 km from each other and were therefore considered to be the same study area (one site). These regions are characterized by continuous wet and warm conditions without distinct seasonality: annual rainfall is 2,500— 3,500 mm, and mean minimum and maximum temperatures are approximately 20 °C and 30 °C, respectively (CRRH, 2008). The Vector Research Laboratory (Laboratorio de Investigación en Vectores, LIVE) of the University of Costa Rica (UCR), provides the general public with a service of counseling and identification of medically important arthropods. Taking advantage of this service, additional tick and flea samples that were received by the laboratory from other areas of the country were included in the analyses. Collection and identification of ectoparasites Ectoparasites analyzed were collected or received at the LIVE, UCR, between July 2008 and March 2013. At study sites, a non-probabilistic approach was used to target households, farms, and other private properties for collection of specimens. These locations were set preferably in rural environments and in proximity to forested areas. Domestic animals were identified, and 20 to 25 live animal traps (homemade wooden box, Tomahawk® and/or Havaheart® traps) were placed one day and collected the next day (after 20-24 hours) to capture opossums, rodents, and other small mammals in the surrounding areas. Traps were placed, in most cases, for 2-3 consecutive days and at least once every year at each site during 2008-2012. Small animals trapped were measured, photographed, and liberated after inspection and collection of ticks and fleas. Identification of animals was made using general descriptions (Reid, 2000), which were confirmed by an expert mammologist. All methods for trapping and manipulating animals, as well as collecting ectoparasites in this study followed the ―Regulations about access to the biodiversity in teaching, social action, and research activities of the University of Costa Rica‖ (projects A8-127 and B1-041), the ―Law of Biodiversity 7788‖ of Costa Rica, and were approved by University of Costa Rica‘s Institutional Committee for the Use and Care of Laboratory Animals (CICUA-35-10). Fleas, ticks, and other ectoparasites were collected from their animal hosts for a period no longer than 1 man-hour per animal using combs and forceps (for example, collection would not take more than 30 minutes if two people were collecting from an animal, or no more than 15 minutes if there were 4 people). Ticks and fleas from other wild animals (reptiles, toads, sloths, raccoons) were collected during independent rescue and/or veterinary activities and were brought to the LIVE by third parties for identification and analysis. Specimens from different host species, collection sites, and specific locations (household, farm, property) were placed separately in dry glass vials (ticks), or vials with water or 70% ethanol (fleas and other ectoparasites). Specimens were kept live and at 4 ºC (< 7 days) or frozen at -20 ºC until processing for identification. When required for species identification or confirmation, some of the specimens were cleared in lactophenol and mounted in Hoyer‘s medium. Ticks (Ixodida), and fleas (Siphonaptera) were selected and identified by observing morphological characters and using identification keys (Hopkins and Rothschild, 1953; Smit, 1958; Barros-Battesti et al., 2006; Vargas, 2006). Ticks belonging to the Amblyomma cajennense species group were considered to be Amblyomma mixtum, which is considered to be the only representative species of this group in Central America (Nava et al., 2014). In addition, those identified as Rhipicephalus sanguineus were designated as ―sensu lato‖ (s. l.), since the presence of 2 species of this group has been established in Latin America (the tropical lineage is the one present in Central America) (Nava et al., 2015). Depending on the size and total number collected, specimens were grouped into pools containing usually 1 to 5 adult ticks (up to 10), 1 to 20 tick nymphs and/or larvae (up to 50), and 1 to 10 fleas from the same ectoparasite species, vertebrate host species, and location. Once prepared, all pools were conserved at –20 °C previously to PCR analyses. In two pools that were of special interest, molecular confirmation of the tick species was performed by amplifying and sequencing a fragment of the mitochondrial 16S ribosomal RNA gene using primers 16S+1 and 16S-1 (460 bp product) (Black and Piesman, 1994; Mangold et al., 1998). DNA extraction, sequencing, editing, and analyses were performed in the same manner described in the section below. Detection and identification of Rickettsia spp. Ticks and fleas were washed 3 times in 0.1% iodine and 70% ethanol solution and 3 times in sterile distilled water prior to DNA extraction, to reduce external bacteria and other possible contamination. Genomic DNA was extracted from ectoparasite pools using ® QIAquick® Gel Extraction Kit (Qiagen) (2008-2010) or NucleoSpin Tissue kit (Macherey- Nagel) (2011-2013), following the manufacturers‘ instructions. DNA from each pool was analyzed by end-point polymerase chain reaction (PCR) for detection of Rickettsia-specific portions of the citrate synthase (gltA), serine protease (htrA), and outer membrane protein A (ompA) genes, as described previously (Troyo et al., 2012a). Briefly, a first PCR to detect the Rickettsia spp. gltA was performed in all pools using primers CS-78 and CS-323, which amplify a 401 bp product of a conserved region (Labruna et al., 2004). To confirm positivity and recognize SFG rickettsiae, a subsequent detection of htrA and ompA fragments was performed in pools positive for the gltA using nested and semi-nested PCRs. Primers used for detection of htrA were R17-122 and R17-500 (380 bp fragment specific for Rickettsia spp.), and a subsequent nested step with TZ15 and TZ16 (SFG-specific fragment of 247 bp) or RP2 and RPID (Typhus Group-specific fragment of 286 bp) (Anderson and Tzianabos, 1989; Tzianabos et al., 1989; Massung et al., 2001). For ompA, primers used were Rr190-70 and Rr190-701, as well as Rr190-70 and Rr190-602 for a semi-nested PCR (632 and 532 bp fragments, respectively, for SFG Rickettsia) (Regnery et al., 1991; Roux et al., 1996). Pools were considered positive for Rickettsia spp. when the expected fragments for at least two of the three genes analyzed were detected by PCR. In addition, a minimum infection rate (MIR) was calculated for each species of ectoparasite [(number of positive pools/total number of ticks or fleas tested) × 100]. To identify Rickettsia species, amplicons of pools that tested positive for gltA were sequenced. PCR products were purified using BigDye® XTerminator™ Purification Kit, TM sequenced with BigDye Terminator V 3.1, and visualized in the Genetic Analyzer 3130 (Applied Biosystems/HITACHI). Positive amplicons obtained in 2012 and 2013 were purified with Exonuclease I and FastAP (Thermo Fisher Scientific Inc.) and sequenced in Macrogen, Inc. (Seoul, South Korea). All sequences were edited and assembled using DNA Baser (DNA Sequence Assembler v4, 2013, Heracle BioSoft, www.DnaBaser.com), and compared by BLAST against the NCBI database to search for similar sequences. In addition, ompA fragments (primers Rr190-70 and Rr190-701) or ompB fragments (primers 120-M59f/120-807; 856 bp product) (Roux and Raoult, 2000) were sequenced and analyzed in a similar manner to confirm the identity in at least one pool of each species of Rickettsia that was detected. Results A total 4 588 ticks and fleas were obtained from different sources: cattle, horses, domestic dogs, cats, humans, rats, opossums, wood turtles, snakes, toads, sloths, raccoons, and vegetation/environment (Table 1). Ten species of ticks and 3 species of fleas were identified. In addition, several larvae and nymphs were only identified morphologically as Amblyomma spp. Overall, 497 pools of ectoparasites were analyzed by PCR. The distribution of pools containing DNA of Rickettsia spp. and minimum infection rates (MIR), according to ectoparasite species and study site, are presented in Table 2. Detection of at least two of the three Rickettsia spp. gene fragments was possible in 119 (23.9%) pools. A high prevalence of Rickettsia spp. was observed in Amblyomma pools, especially in A. mixtum from horses (67.7% of A. mixtum pools; MIR: 9.9%), Amblyomma ovale from dogs (15.4% of A. ovale pools; MIR: 9.3%), and Amblyomma sabanerae from wood turtles (66.7% of A. sabanerae pools; MIR: 11.1%). However, only 3 pools of A. sabanerae were analyzed, and most of A. mixtum were from the Cahuita site. At all study sites, Rickettsia DNA was also frequent in pools of Ctenocephalides felis (44.9% of C. felis pools; MIR: 7.2%), most of which were collected from dogs. In contrast, rickettsiae were infrequent in pools of other common ectoparasites of the areas studied, including Rhipicephalus microplus (6.6% of R. microplus pools; MIR: 0.4%), Rhipicephalus sanguineus s. l. (1.3% of R. sanguineus s. l. pools; MIR: 0.1%), and Pulex simulans (7.1% of P. simulans pools, MIR: 1.0%) (Table 2). Sequencing of the Rickettsia spp. gltA fragment and species identification with 99.6% to 100% DNA sequence similarity was possible for 100 (84%) of the positive pools (Table 3). After several attempts, it was not possible to obtain a good quality sequence for analysis from the remaining 19 PCR-positive pools (quality value scores less than 21, noise, overlapping peaks, etc.). According to BLAST search results, sequences of gltA fragments corresponding to R. rickettsii were detected in only two pools of ticks (Table 3). In both cases, specimens were collected and brought to the laboratory by a third party. Identification of R. rickettsii was further confirmed by re-analyzing the sample and sequencing the gltA and ompA amplicons in two different laboratories (Universidad de Costa Rica and Universidade de São Paulo). The same gltA results were obtained and the fragments showed 100% (349/349 for gltA, and 534/534 for ompA) sequence similarity with R. rickettsii (CP000848). Ticks in one of the pools were identified morphologically as A. cajennense s. l., although the information of specific location and host was unavailable. An analysis of the mitochondrial 16S ribosomal RNA gene from this tick determined that the highest partial sequence homology (99.3%; 400/403) was with a sequence of A. mixtum form Texas, USA (Accession No. KM458242) (Medlin et al., 2015). The second sample that was positive for R. rickettsii was a single nymph of Amblyomma collected near the metropolitan area of San José. It was received dead and in very poor condition for morphological identification; it was collected while attached to the same person who brought it (no symptoms of disease were reported). In this case, the 16S ribosomal RNA gene fragment showed 100% (404/404) sequence similarity with Amblyomma varium also from Costa Rica (Accession No. KF702341) (Ogrzewalska et al., 2015). ‗Candidatus R. amblyommii‘ was the most common species of Rickettsia in ticks, according to similarity to several partial sequences available in GenBank, including those with accession numbers HM582435, DQ517290, and CP003334 (Table 3). This Rickettsia was detected in A. mixtum, A. ovale, D. nitens, and R. sanguineus s. l. In addition, gltA partial sequences corresponding to Rickettsia bellii (CP000087) were present in A. sabanerae tick pools from wood turtles (Table 3). Rickettsiae similar to ‗Candidatus Rickettsia asemboensis‘ (KJ569090) were identified in pools of C. felis, as was communicated preliminarily as Rickettsia sp. RF2125 (Troyo et al., 2012a). This Rickettsia was also detected in P. simulans and A. ovale collected from dogs, and in R. microplus from cows (Table 3). As was done for R. rickettsii, the presence of ‗Candidatus R. amblyommii‘, R. felis, and ‗Candidatus R. asemboensis‘ in several samples was further confirmed by sequencing the ompA or ompB gene fragment, which included at least one sample corresponding to each of these rickettsiae. In all pools analyzed, ompA and ompB partial sequences obtained had 99.8- 100% similarity with the corresponding species of Rickettsia that had been identified using the gltA fragment. Representative sequences of each Rickettsia were deposited in GenBank with accession numbers: KX544813 and KX544816 for R. rickettsii; KX544804 for R. bellii; KX544805, KX544806, KX544812, and KX544815 for ‗Candidatus R. amblyommii‘; KX544809 and KX544814 for R. felis, and KX544807, KX544808, KX544810, KX544811, and KX544817 for ‗Candidatus R. asemboensis‘. Accession numbers of the mitochondrial 16S ribosomal RNA gene fragment sequences of A. mixtum and A. varium were deposited as KX544819 and KX544818, respectively. Discussion Previous studies in Costa Rica reported the presence of R. rickettsii in rabbit ticks, H. leporispalustris, during the 1980s (Fuentes et al., 1985); however, no other rickettsiae species or confirmed tick vectors of spotted fevers had been documented in the country before the beginning of this project in 2008. In this study, R. rickettsii was detected in ticks of the genus Amblyomma. Ticks from the A. cajennense complex or identified as A. mixtum, have been implicated as possible vectors of R. rickettsii to humans in Panama (de Rodaniche, 1953; Bermúdez et al., 2016); some of the other species of this complex are also vectors of R. rickettsii in South America (Krawczak et al., 2014; Faccini-Martínez et al., 2015). Although most of the A. mixtum ticks analyzed in the present study were collected from horses in Cahuita, some of the specimens were obtained from humans, as this species complex commonly bites humans (Guglielmone et al., 2006). Unlike A. mixtum, A. varium has not been implicated in transmission of pathogenic rickettsiae and only R. bellii and ‗Candidatus R. amblyommii‘ have been detected in this tick species (Ogrzewalska et al., 2012; Lugarini et al., 2015; Ogrzewalska and Pinter, 2016). Adults of A. varium are ectoparasites of the mammal families Bradypodidae and Megalonychidae (Xenarthra), and vertebrate hosts of immature stages can include birds but have not been well characterized (Ogrzewalska and Pinter, 2016). Although confirmation is still required, a study in the State of Pará, Brazil, reported numerous immature and adult stages of A. varium parasitizing humans (Serra-Freire, 2010), as was the R. rickettsii-infected nymph analyzed in the present study. However, there are no other records of human parasitism by A. varium (Guglielmone et al., 2014). In Costa Rica, this tick species and their vertebrate hosts are common in endemic areas and were documented at sites that were investigated following reports of RMSF cases, although no rickettsiae were obtained from them at that time (Fuentes, 1986; Hun et al., 1991). Therefore, the role of adult and immature stages of A. mixtum and other Amblyomma spp. of wild animals, including A. varium, as vectors of R. rickettsii should be evaluated further. In contrast with the low infection rate of R. rickettsii, infection rate of A. mixtum with ‗Candidatus R. amblyommii‘ was high, which may be expected considering that it is a common finding in other countries of the region, including Honduras and Panamá (Novakova et al., 2015; Bermúdez et al., 2016). The infection rate in A. ovale, D. nitens, and R. sanguineus s. l. was lower, but confirms previous reports of ‗Candidatus R. amblyommii‘ in these tick species (Bermúdez et al., 2009, 2011). The presence of ‗Candidatus R. amblyommii‘ in ticks that are known to bite humans, including A. mixtum and A. ovale (Guglielmone et al., 2006), indicates that people may be at risk of exposure to this Rickettsia. Even though ‗Candidatus R. amblyommii‘ is not considered a human pathogen and has only been associated indirectly with mild disease (Apperson et al., 2008), experimental infection has produced pathology in guinea pigs and should be studied further in individuals with specific health conditions such as immunosuppression (Rivas et al., 2015). In addition, exposure may induce a partial protective immunity to R. rickettsii, as has been suggested in experimental studies (Blanton et al., 2014; Rivas et al., 2015), which could modulate disease epidemiology and severity in endemic areas of the region, such as Cahuita. Of note, A. ovale specimens analyzed in this study did not contain Rickettsia sp. strain Atlantic rainforest, which is a known human pathogen that has been detected in this tick species in Central America (Belize) and South America (Brazil and Colombia) (Barbieri et al., 2014; Londoño et al., 2014; Lopes et al., 2016). More analyses of A. ovale ticks are required, including specimens from wild animals, in order to determine if this pathogenic Rickettsia is present in Costa Rica. The infection rate of Rickettsia bellii in turtle ticks (A. sabanerae) was high in this study, although only 3 tick pools were analyzed. This species of Rickettsia was reported recently in A. sabanerae larvae on birds from Costa Rica (Ogrzewalska et al., 2015), and it was reported previously in the same tick species from El Salvador (Barbieri et al., 2012). Experimental studies indicate that R. bellii can infect guinea pigs and opossums without apparent disease (Horta et al., 2010), and exposure to R. bellii has been documented by serology in dogs (Silva Fortes et al., 2010). Although the existence of amplifying vertebrate species has not been excluded, the high prevalence of R. bellii in A. sabanerae in the region is probably the result of vertical transmission, which has been demonstrated as a very efficient transmission route in this Rickettsia (Horta et al., 2006). In fleas, R. felis was only detected in C. felis from San José and not in the specific study sites, where only rickettsiae similar to ‗Candidatus R. asemboensis‘ were identified. This Rickettsia was first reported as Rickettsia sp. genotype RF2125 from the Thailand-Myanmar border (Parola et al., 2003), but it has been more recently referred to as ‗Candidatus R. asemboensis‘, following the description of a similar Rickettsia by Jiang et al. (2013) in Asembo, Kenya. Studies have shown that DNA sequences of the gltA and other genes from both rickettsiae are very similar; hence they are probably the same species (Jiang et al., 2013; Oteo et al., 2014; Faccini-Martínez et al., 2016). At study sites, infection rate with this Rickettsia was much higher in pools of C. felis, but they were also detected in P. simulans, which commonly co-infest dogs in these areas (Troyo et al., 2012b). The lower infection rate in P. simulans, despite a large number of flea pools analyzed from the same vertebrate hosts, agrees with other studies that show absence of rickettsiae in Pulex fleas and may indicate alternative and less-frequent infection routes, such as co-feeding (Oteo et al., 2014; Brown et al., 2015). In this regard, there are few reports of DNA of R. felis or similar rickettsiae in blood of dogs, and this may be an infrequent finding in the region (Wei et al., 2014). In addition, there may be specific bacteria-flea-dog interactions that limit infection and/or vertical transmission in populations of P. simulans. An opposite effect has been reported for other bacteria like Bartonella spp., where infection rates in P. simulans are higher (fleas probably acquire bacteria from a bacteremic dog) (Rojas et al., 2015). Even though infection rates were lower than in C. felis, ‗Candidatus R. asemboensis‘ was also detected in ticks including A. ovale and R. microplus, which can also infest dogs and where they may come in close contact with infected fleas. Moreover, it is important to note that positive PCR results may indicate contamination with infected flea feces in specimens found on the same dog, or even the presence of bacteria in the animal‘s blood within the ectoparasite. Although it is possible that some of the pools may have had more than one species of Rickettsia present (the most abundant is the one evidenced after sequencing), results from this study confirm the presence of 5 species of rickettsiae in ticks and fleas in Costa Rica, many of which are common on domestic animals and may pose a threat to human health (R. rickettsii and R. felis) or may impact the epidemiology of SFG rickettsioses (i.e. ‗Candidatus R. amblyommii‘). Results indicate that rickettsiae are present in domestic and peridomestic areas, which provide an ecological context that may be suitable for their establishment and contact with humans. Therefore, awareness and recognition of rickettsial transmission in the region is necessary for adequate prevention and treatment, especially in areas where other vector-borne diseases that may challenge the differential diagnosis are common, such as dengue, chikungunya, Zika, and ehrlichiosis. This is the first report of R. rickettsii in molecularly confirmed A. mixtum in Costa Rica and the first report of R. rickettsii in A. varium. Other new associations between rickettsiae and ectoparasite species include ‗Candidatus R. asemboensis‘ in R. microplus, A. ovale, and P. simulans. In addition, the presence of R. bellii and R. rickettsii in ticks that usually parasitize wild animals suggests that these species may be important in maintaining enzootic transmission cycles of these rickettsiae, which can potentially reach humans and/or domestic animals through infected immature stages. In addition to rabbits (Fuentes, 1986), other wild animal reservoirs or amplifying hosts of R. rickettsii have not been identified in endemic areas of Central America. Hence, the role of wild animals and their ticks in the transmission cycles of rickettsiae should be evaluated more thoroughly in the region. 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Global surveillance of rickettsial diseases: Memorandum from a WHO meeting. Bull. World Health Organ. 71, 293–296. Table 1 Ticks and fleas collected at sites in Costa Rica and total pools analyzed for detection of Rickettsia spp. citrate synthase gene (gltA). † Ectoparasite Vertebrate host/source No. collected Stages Pools analyzed Amblyomma mixtum Horse, human, vegetation/environment 243 A,N 31 Amblyomma dissimile Boa constrictor, unidentified snake 15 A 2 Amblyomma geayi Sloth (Choloepus hoffmanni) 1 A 1 Amblyomma ovale Dog, cat, human 45 A 26 Amblyomma rotundatum Toad (Rhinella sp.) 1 A 1 Amblyomma sabanerae Wood turtle (Rhinoclemmys funerea) 20 A 3 Amblyomma sp.* Vegetation, horse, dog, human, cow, opossum (Philander opossum, 233 N,L 19 Didelphis marsupialis), spiny rat (Proechimys semispinosus) Dermacentor nitens Horse 881 A,N,L 39 Ixodes boliviensis Dog 3 A 2 Rhipicephalus microplus Cow, dog, horse, vegetation 804 A,N,L 45 Rhipicephalus sanguineus s. l. Dog, cat, vegetation/environment 678 A,N,L 75 Adoratospylla intermedia copha Opossum (D. marsupialis, P. opossum) 12 A 2 Ctenocephalides felis Dog, cat, cow, raccoon, vegetation/environment 1 051 A 167 Pulex simulans Dog 601 A 84 All species All hosts/sources 4 588 497 *One Amblyomma sp. nymph from a human was later identified molecularly as A. varium. † A: adults, N: nymphs, L: larvae. Table 2 Minimum infection rate (MIR) and proportion of pools positive for Rickettsia spp. according to ectoparasite species and study site. Ectoparasite Rickettsia spp. positive pools per site Rickettsia spp. MIR (%) Cahuita Guápiles/ La Virgen Limón Turrialba Other positive pools Jiménez/ sites Guácimo Amblyomma mixtum 12/22 - - - 1/1 8/8 21/31 (67.7%) 9.9 Amblyomma dissimile - - - - - 0/2 0/2 (0%) 0 Amblyomma geayi - - 0/1 - - - 0/1 (0%) 0 Amblyomma ovale 3/10 0/9 - - 0/1 1/6 4/26 (15.4%) 9.3 Amblyomma rotundatum - - - - 0/1 - 0/1 (0%) 0 Amblyomma sabanerae - - - - - 2/3 2/3 (66.7%) 11.1 Amblyomma sp.* 0/4 0/4 0/1 0/6 - 3/4 3/19 (15.8%) 1.4 Dermacentor nitens 2/12 0/8 1/7 0/3 1/6 0/3 4/39 (10.3%) 0.5 Ixodes boliviensis - - - - - 0/2 0/2 (0%) 0 Rhipicephalus microplus 0/2 0/12 2/9 0/1 1/11 0/10 3/45 (6.6%) 0.4 Rhipicephalus sanguineus s. l. 0/6 1/39 0/2 0/3 0/5 0/20 1/75 (1.3%) 0.1 Adoratopsylla intermedia copha - - - - 0/2 - 0/2 (0%) 0 Ctenocephalides felis 9/33 27/65 3/14 18/30 14/15 4/10 75/167 (44.9%) 7.2 Pulex simulans 2/20 1/26 1/8 1/11 1/12 0/5 6/84 (7.1%) 1.0 *One PCR-positive nymph was identified molecularly as A. varium. Table 3 Rickettsia spp. identified in pools of ticks and fleas collected in Costa Rica, according to partial sequence homologies of the gltA amplicons. No. sequences Ectoparasite Similarity (%) Length (bp) Rickettsia sp. [GenBank accession number] Vertebrate host/source obtained Amblyomma mixtum* 18 99.6-100 293-349 ‗Candidatus R. amblyommii‘ [KM652483, CP003334] Horses, cows, human, vegetation/environment 1 100 349 R. rickettsii [CP000848] Unknown Amblyomma ovale 3 100 349 ‗Candidatus R. asemboensis‘ [KJ569090] Dogs 1 100 349 ‗Candidatus R. amblyommii‘ [KM652483] Dog Amblyomma sabanerae 2 100 342, 349 R. bellii [CP000087] Wood turtle (Rhinoclemmys funerea) Amblyomma varium* 1 100 349 R. rickettsii [CP000848] Human Amblyomma sp. 1 100 349 ‗Candidatus R. amblyommii‘ [KM652483] Horse Dermacentor nitens 2 100 332, 349 ‗Candidatus R. amblyommii‘ [CP003334] Horses Rhipicephalus microplus 2 100 300, 349 ‗Candidatus R. asemboensis‘ [KJ569090] Cows Rhipicephalus sanguineus s. l. 1 100 349 ‗Candidatus R. amblyommii‘ [CP003334] Dog Ctenocephalides felis 60 99.6-100 235-349 ‗Candidatus R. asemboensis‘ [KJ569090] Dogs, cats 3 100 349 R. felis [CP000053] Dogs Pulex simulans 5 99.6-100 235-349 ‗Candidatus R. asemboensis‘ [KJ569090] Dogs * Tick identification was confirmed in pools containing R. rickettsii by molecular analysis of the mitochondrial 16S ribosomal RNA gene. 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