Ticks and rickettsiae from wildlife in Belize, Central America

Background The agents of spotted fevers in Latin America are Rickettsia rickettsii, R. parkeri, Rickettsia sp. strain Atlantic rainforest, and R. massiliae. In Continental Central America, R. rickettsii remains the only known pathogenic tick-borne rickettsia. In the present study, ticks were collected from wild mammals in natural areas of Belize. Besides providing new data of ticks from Belize, we investigated rickettsial infection in some of these ticks. Our results provide ticks harboring rickettsial agents for the first time in Central America. Methods Between 2010 and 2015, wild mammals were lived-trapped in the tropical broadleaf moist forests of central and southern Belize. Ticks were collected from the animals and identified to species by morphological and molecular analysis (DNA sequence of the tick mitochondrial 16S RNA gene). Some of the ticks were tested for rickettsial infection by molecular methods (DNA sequences of the rickettsial gltA and ompA genes). Results A total of 84 ticks were collected from 8 individual hosts, as follows: Amblyomma pacae from 3 Cuniculus paca; Amblyomma ovale and Amblyomma coelebs from a Nasua narica; A. ovale from an Eira Barbara; A. ovale, Amblyomma cf. oblongoguttatum, and Ixodes affinis from a Puma concolor; and A. ovale, A. coelebs, A. cf. oblongoguttatum, and I. affinis from two Panthera onca. Three rickettsial agents were detected: Rickettsia amblyommii in A. pacae, Rickettsia sp. strain Atlantic rainforest in A. ovale, and Rickettsia sp. endosymbiont in Ixodes affinis. Conclusions The present study provides unprecedented records of ticks harboring rickettsial agents in the New World. An emerging rickettsial pathogen of South America, Rickettsia sp. strain Atlantic rainforest, is reported for the first time in Central America. Besides expanding the distribution of 3 rickettsial agents in Central America, our results highlight the possible occurrence of Rickettsia sp. strain Atlantic rainforest-caused spotted fever human cases in Belize, since its possible vector, A. ovale, is recognized as one of the most important human-biting ticks in the Neotropical region.


Background
Belize is a tropical country occupying an area of only 22,800 km 2 , <5 % of continental Central America. Despite its relatively small land, Belize is recognized by its high biodiversity, especially wildlife. Among 900 tick species of the world, nearly 200 are found in the Neotropical Zoogeographic region [1,2]. The tick fauna of Belize is currently composed of 18 species, distributed in the genera Amblyomma (13 species), Ixodes (2), Rhipicephalus (2), and Dermacentor (1) [1,3].
Tick-borne rickettsiae are bacterial agents of the genus Rickettsia that cause emerging and re-emerging zoonoses worldwide [4]. Recent reviews on the occurrence of Rickettsia species in Latin America indicated that only 3 tick-borne Rickettsia species occur in Central American main land, in contrast to at least 9 rickettsial agents infecting ticks in South America [4,5]. In fact, until 2009, Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, was the only tick-borne rickettsia known to occur in Central America, with reports from Panama and Costa Rica [5]. In these two countries, R. rickettsii has been associated with three tick species, Amblyomma mixtum (reported as Amblyomma cajennense), Dermacentor nitens, and Haemaphysalis leporispalustris [6][7][8].
The current scenario of tick-borne rickettsiae in Latin America is still modest, compared to Europe, where more than 20 tick-borne agents have been reported [4]. Moreover, whereas nearly 10 tick-borne rickettsiae are agents of human illness (generally known as spotted fevers) in Europe, only 4 tick-borne rickettsiae are known to cause spotted fevers in humans in Latin America [4]. The agents of spotted fevers in Latin America are R. rickettsii, R. parkeri, Rickettsia sp. strain Atlantic rainforest, and R. massiliae [4]. In Continental Central America, R. rickettsii has remained as the only known pathogenic tickborne rickettsia.
In the present study, ticks were collected from wild mammals in natural areas of Belize. Besides providing new data of ticks from Belize, we investigated rickettsial infection in some of these ticks. Our results provide ticks harboring rickettsial agents for the first time in Central America.

Methods
Between 2010 and 2015, wild mammals were livedtrapped in the tropical broadleaf moist forests of central and southern Belize, as part of a study of wildlife movement. Ticks were collected from the animals and stored in plastic tubes containing 92 % ethanol. In the laboratory, ticks were identified to species through morphological examination following specific identification keys for Amblyomma species [15,16], and for Ixodes species [17,18]. Morphological identification was confirmed by molecular analysis. For this purpose, representative specimens of each tick species were submitted to DNA extraction using the guanidine isothiocyanate phenol technique [19] and tested by a PCR assay targeting a portion of the tick mitochondrial 16S rRNA gene, as previously described [20]. Amplicons were purified with ExoSap (USB, Cleveland, Ohio, USA) and DNAsequenced 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 sequences obtained were submitted to blast analyses (www.ncbi.nlm.nih.gov/ blast) to infer the closest similarities available in GenBank.
The mitochondrial 16S rRNA partial sequences of Ixodes ticks collected in the present study were aligned with corresponding 16S rRNA sequences of different Ixodes species available in GenBank, which included mostly those species belonging to the Ixodes ricinus species complex, as recently reported [21]. The sequence of Ixodes uriae was used as outgroup. Phylogenetic trees were inferred by the maximum parsimony methods and were performed with PAUP 4.0b10 software [22] with 1000 replicates of random-addition taxa and tree bisection and reconnection branch swapping; all positions were equally weighed.
Tick DNA samples were screened for rickettsial infection by a PCR protocol using primers CS-78 and CS-323, targeting a 401-bp fragment of the rickettsial citrate synthase gene (gltA) [23], which is relatively conserved among Rickettsia species [24]. Samples yielding visible PCR products by this PCR were further tested with primers Rr190.70p and Rr190.701n, targeting a 631-bp fragment of the rickettsial 190-kDa outer membrane protein gene (ompA) [25]. In each set of reactions, negative control tubes containing water and a positive control tube containing DNA of Rickettsia sp. strain NOD were included. Amplicons were purified and DNAsequenced as described above, and then submitted to blast analyses.

Results
A total of 84 ticks were collected from 8 individual hosts, comprising 5 different tick species from 5 host species (Table 1). Four of the five tick species belonged to the genus Amblyomma, and were morphologically identified as Amblyomma ovale, Amblyomma coelebs, Amblyomma pacae, and Amblyomma cf. oblongoguttatum. This later species is provisionally treated with "cf." because its external morphology presented slight differences to A. oblongoguttatum from Brazil (type locality of A. oblongoguttatum), which will be presented in detail in another manuscript, still in progress (M.B.L., unpublished data).
Morphological identifications were corroborated by molecular analysis, as the mitochondrial 16S rRNA gene partial sequences of A. ovale, A. coelebs, and A. pacae matched ≥99 % similarity to conspecific sequences available in GenBank, while the sequences of A. cf. oblongoguttatum matched closest (≈92 % similarity) to a sequence of A. oblongoguttatum from Brazil (Table 2). Two Amblyomma larvae from the same individual host were molecularly identified as A. coelebs and A. cf. oblongoguttatum (Tables 1, 2). A total of 43 tick specimens belonged to the genus Ixodes, and were morphologically identified as Ixodes affinis. Through molecular analyses, the 16S rRNA partial sequences of 18 Ixodes specimens generated 7 different haplotypes, which were 97-99 % closest to corresponding sequences of I. affinis from southeastern United States (Table 2). In the phylogenetic analysis (Fig. 1) Voucher tick specimens collected in the present study have been deposited at the tick collection "Coleção Nacional de Carrapatos" (CNC) under the accession

Discussion
The tick species found in the present study have been previously reported in Belize [26]. Our results are in agreement with previous studies, which indicated that A. pacae is a natural parasite of C. paca [27], and that A. ovale and I. affinis are natural parasites of Carnivora hosts in the Neotropical region [28,29]. Amblyomma coelebs is primarily associated with tapirs, Tapirus spp. [30]. In fact, Tapirus bairdii is known to occur in the area where this tick was collected in the present study (data not shown). While the parasitism of immature stages of A. coelebs on P. onca has been previously reported [28], we provide the first report of A. coelebs nymph on N. narica. Furthermore, our findings of A. cf. oblongoguttatum on P. concolor and P. onca are in agreement with previous reports for A. oblongoguttatum infesting wild felids in Central America [29], and South America [27,28]. However, at this moment, we are not confident that the taxon A. oblongoguttatum represents the same tick species in these two continents, based on slightly morphological differences (data not shown) and molecular analyses ( Table 2). Because the type locality of A. oblongoguttatum is in Brazil [31], we are referring to the Belizean specimens as A. cf. oblongoguttatum. Three (33.3 %) out of 9 A. ovale ticks were found to harbor the pathogen Rickettsia sp. strain Atlantic rainforest. This rickettsial agent was recently demonstrated to cause spotted fever in humans in Brazil [32,33], where A. ovale was incriminated as a possible vector [34]. Until the present study, the occurrence of Rickettsia sp. strain Atlantic rainforest was restricted to Brazil, Colombia, and Argentina [4,35,36]. Thus, our results expand the distribution of this pathogen to Central America, where it must be considered a potential agent of human spotted fever. While previous studies showed that Rickettsia sp. strain Atlantic rainforest is phylogenetically related to R. parkeri, R. africae, and R. sibirica [32,33,35], the definitive taxonomic status of this pathogen still needs to be established.
Most of the I. affinis ticks of the present study were shown to harbor a rickettsial agent. This agent was considered to be an endosymbiont, based on previous studies on closely related Ixodes species (e.g., I. scapularis, I. pacificus, I. ricinus, I. boliviensis), which were also infected by genetically close-related rickettsial endosymbionts, usually under high infection rates [14,43]. Further studies on isolation and deeper molecular characterization are needed to elucidate the taxonomic status of the rickettsial endosymbionts of Ixodes species from Latin America.

Conclusions
The present study provides unprecedented records of ticks harboring rickettsial agents in the New World. An emerging rickettsial pathogen in South America, Rickettsia sp. strain Atlantic rainforest, is reported for the first time in Central America. Besides expanding the distribution of 3 rickettsial agents in Central America, our strain Atlantic rainforest-caused spotted fever human cases in Belize, since its possible vector, A. ovale, is recognized as one of the most important human-biting ticks in the Neotropical region [27,44].