Introduction

Ticks are a group of hematophagous ectoparasites that are of great importance to public health due to their ability to transmit numerous pathogenic agents, such as viruses, bacteria, and protozoa (Guimarães et al. 2001; Dantas-Torres et al. 2012). These ectoparasites parasitize a wide range of hosts, and may even therefore encompass humans (Barros-Battesti et al. 2006; Palomar et al. 2012).

Coatis (Nasua nasua) can be reservoirs of numerous zoonotic agents, such as Leishmania and Trypanosoma species (Porfírio et al. 2018). Studies have shown the importance of these procyonids in the epidemiology of these microorganisms, including those transmitted by ticks, such as piroplasmids and rickettsiae (Magalhães-Matos et al. 2022; Perles et al. 2023).

Iguaçu National Park (PNI) is considered a worldwide reference for nature conservation and sustainable tourism. It comprises an area of 186,000 ha of protected and rich biodiverse Atlantic Forest, housing 12 species of amphibians, 48 species of reptiles, 158 species of mammals, 175 species of fish, 390 species of birds, and more than 800 species of invertebrates (ICMBIO, 2023). Among the species of mammals in PNI, coatis stand out, as they are commonly found in flocks and often forage for food of anthropic origin (ICMBIO, 2023). These animals are highly resistant to anthropogenic pressures and can easily adapt to modified areas, mainly because of the high availability of food (Ferreira et al. 2013). Thus, the presence of people and the flow of individuals in PNI facilitates the human approach to synanthropic animals, such as coatis (Magalhães-Matos et al. 2017) and, consequently to their ectoparasites and zoonotic pathogens.

In this context, the present study aimed to conduct molecular research on Ehrlichia spp., Anaplasma spp., Borrelia spp., and protozoans of the order Piroplasmida in ticks collected from coatis found in PNI, located in Foz do Iguaçu, state of Paraná, southern Brazil.

Material and methods

Samples

This study was conducted using DNA samples derived from ticks collected from coatis native to the Atlantic Forest in the environmental conservation area of PNI dependencies located in the municipality of Foz do Iguaçu, state of Paraná, southern Brazil. Samples of ticks were collected in September 2014 and March and April 2015 from 86 coatis.

Three collection points were chosen in the tourist area of the INP: points of access to two trails inside the forest (point I—25° 37′ 36″ S, 54° 27′ 39″ W and point II—25° 39′ 05″ S, 54° 26′ 16″ W) and the viewpoints of the falls (point III—25° 41′ 03″ S, 54° 26′ 24″ W, with a total length of approximately 1.2 km).

Sub-adult and adult ring-tailed coatis were attracted with banana, pineapple, or peanut butter bait and captured with a hand net (multifilament nylon, 60 × 120 cm) or Tomahawk traps (90 × 45 × 50 cm and 50 × 21.5 × 20 cm). After being contained, the ring-tailed coatis received a pre-anesthesia. Each animal was examined thoroughly to collect ticks, and the collected specimens were stored in RNAlater® and frozen at 20 °C until the moment of molecular analysis.

Taxonomic identification of the ticks was conducted based on morphology, using the specific dichotomous keys for ixodid ticks developed by Cooley (1946) and Kohls (1960) for Haemaphysalis nymphs, Martins et al. (2010) for Amblyomma nymphs, and Barros-Battesti et al. (2006) for adults ticks. Larvae from ticks were identified only at their genus level, since in Brazil there is no literature for specific identification. DNA extraction was performed individually from each tick by the phenol–chloroform method. Details of the methodology for collection and identification, conservation, transport, and DNA extraction from ticks are described in Magalhães-Matos et al. (2017) and Magalhaẽs-Matos et al. (2022).

A total of 553 ticks were used, including larvae of Amblyomma spp. (n=18); nymphs of Amblyomma coelebs (n=413), Amblyomma brasiliense (n=72), Haemaphysalis juxtakochi (n=5), and adults of Amblyomma ovale (n=45).

Polymerase chain reaction (PCR)

PCR assays were performed to detect the presence of DNA from potentially pathogenic bacteria and protozoa, such as Ehrlichia spp., Anaplasma spp., Borrelia spp., and protozoans of the order Piroplasmida. Specific primers were used for each agent following the original protocol for each primer. Table 1 shows the primers used along with the target genes, sizes of the amplified products, and reference protocols used. DNA from Borrelia anserina strain AL (culture), Babesia bigemina (bovine positive), Anaplasma platys (dog positive), or Ehrlichia canis (dog positive) were used as positive controls, and ultrapure water was used as a negative control.

Table 1 List of primers used for the PCR analyses in the present study
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PCR products (10 μL) were applied to a 1.5% agarose gel, separated using electrophoresis (5 V/cm), stained with ethidium bromide (0.5 μg/mL), and visualized using an ultraviolet (UV) light transilluminator.

Sequencing

The material for sequencing was purified from 5 μL of the PCR product of the positive samples and treated with Exo-Sap-IT (GE Healthcare), following the manufacturer’s protocol. The fragments were sequenced in both directions using an automated genetic analyzer (ABI 3730 DNA Analyzer, Thermo Fisher Scientific). The obtained sequences were aligned using the DNA Baser® program and subjected to a homology search with other sequences deposited in GenBank using the BLASTn tool.

Phylogenetic analyses

For all phylogenetic analyses, the sequences obtained in our study were aligned with those in databases using the MUSCLE tool (Edgar 2004) in the Seaview4 program (Gouy et al. 2010). Phylogenetic relationships were estimated using phylogenetic inference with the maximum likelihood (ML) method, which was implemented using PhyML (Guindon and Gascuel 2003) under a sequence evolution model chosen after hierarchical testing of alternative models based on the Bayesian information criterion in MEGA version 7 (Kumar et al. 2016). Statistical support for clades was assessed using a heuristic search with 1000 bootstrap replicates. Phylogenetic relationships were visualized using FigTree v.1.4 software (Rambaut 2012).

Results

Of the total number of ticks analyzed using PCR (553), 1.99% (11/553) amplified DNA from at least one of the tested groups (Anaplasma spp., Borrelia spp., or Piroplasmida), and all samples were negative for Ehrlichia spp. The frequencies of the positive tick species are listed in Table 2.

Table 2 Tick species and frequency of positive samples for Anaplasma spp., Borrelia spp., and agents of the Order Piroplasmida, by tick species
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The four samples positive for Anaplasma spp. in A. coelebs nymphs were identical and had a similarity of 94.7% (447/472) and 98.09% (820/836) with the 23S and 16S ribosomal genes of Anaplasma marginale cepa Florida (CP001079), respectively. The partial sequences of the flaB and 16S rRNA genes of Borrelia spp. present in ticks of the species Amblyomma sp. larvae, A. coelebs nymphs, and A. ovale adult female exhibited little difference (99.65 to 100% identity) and an identity 87.7% (556/634) and 99.1% (500/505), respectively, with spirochetes from the relapsing fever group (RFG) (MG944997 and KT364340, respectively). Finally, the partial sequences of the piroplasmid 18S rRNA gene in two ticks of the A. coelebs specie showed 97.60% (447/458) similarity with Theileria cervi (MW008518).

Samples positive for Borrelia spp. were not amplified in the PCR assays for the hpt and glpQ genes, and two of them did not amplify for the 16SrRNA gene. This was likely due to the sensitivity of the primers used and/or the fact that the samples did not have sufficient DNA concentration for amplification.

GenBank accession numbers for the partial sequences obtained in the present study are as follows: MT018000 (Borrelia sp. strain AC129, flaB), MT018001 (Borrelia sp. strain AC425, flaB), MT018002 (Borrelia sp. strain AO17, flaB), MT018003 (Borrelia sp. strain AC444, flaB), MT018004 (Borrelia spp. strain AC549, flaB), MT019342 (Borrelia sp. AC129, 16S rRNA), MT019525 (Borrelia sp. strain AC425, 16S rRNA), MT019528 (Borrelia sp. strain AC129, 16S rRNA), MT022490 (Anaplasma sp. isolate AC240, 23S rRNA), MT019664 (Anaplasma sp. isolate AC458, 23S rRNA), MT019625 (Anaplasma sp. isolate AC401, 23S rRNA), MT019564 (Anaplasma sp. isolate AC191, 23S rRNA), MT019536 (Anaplasma sp. isolated AC191, 16S rRNA), MT019537(Anaplasma sp. isolated AC240, 16S rRNA), MT019545 (Anaplasma sp. isolated AC401, 16S rRNA), MT019560 (Anaplasma sp. isolated AC548, 16S rRNA), MT019670 (Theileria sp. isolate A. coelebs 74), and MT019669 (Theileria sp. isolate A. coelebs 262).

Phylogenetic analyses clustered the sequences detected in A. coelebs with A. marginale, Anaplasma centrale, and Anaplasma capra (16S rRNA - Bootstrap 88%), and Anaplasma sp. Paresseux78 (Figs. 1 and 2) previously detected in Bradypus tridactylus (23S rRNA - Bootstrap 99%) (Fig. 1). For the flagellin B (Fig. 4) and 16S rRNA (Fig. 3) genes for Borrelia spp., the phylogenetic analyses clustered the sequences detected in A. coelebs and A. ovale with Borrelia turcica isolated from the hard tick Hyalomma aegyptium, which infests tortoises (Testudo graeca), and others Borreliae of Reptilian (REP) group with 92% (flaB) and 100% (16SrRNA) of bootstrap (Figs. 3 and 4). Regarding 18S rRNA for piroplasmids, the phylogenetic analysis indicated that the generated sequences were close to the T. cervi sequences available in GenBank (Fig. 5).

Fig. 1
figure 1

Phylogenetic tree illustrating the relationships among Anaplasma species identified in this study (highlighted in bold). The tree is constructed based on 23S rRNA gene sequences, employing the maximum likelihood (ML) method. The numbers (>70%) presented above the branches represent bootstrap values. The scale bars correspond to an evolutionary distance of 0.03 substitutions per sequence position, and the branch labels contain GenBank accession numbers. Ehrlichia chaffensis (CP007480) and Ehrlichia muris (CP006917) were used as outgroup

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Fig. 2
figure 2

Phylogenetic tree depicting the phylogenetic relationships between Anaplasma species detected in this investigation (highlighted in bold). The tree is constructed using 16S rRNA gene sequences and the maximum likelihood (ML) method. The numbers (>70%) displayed above the branches indicate the bootstrap values. The scale bars indicate an evolutionary distance of 0.02 substitutions per sequence position, and the branch labels include GenBank accession numbers. Ehrlichia chaffensis (CP007480) and Ehrlichia muris (CP006917) were used as outgroup

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Fig. 3
figure 3

Phylogenetic tree presenting the phylogenetic relationships among the Borrelia species identified in this study (highlighted in bold). The tree is constructed based on 16S rRNA gene sequences, utilizing the maximum likelihood (ML) method. The numbers (>70%) indicated above the branches represent the bootstrap values. The scale bars correspond to an evolutionary distance of 0.02 substitutions per sequence position, and the branch labels contain GenBank accession numbers

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Fig. 4
figure 4

Phylogenetic tree demonstrating the phylogenetic relationships between the Borrelia species detected in this study (highlighted in bold). The tree is constructed using flaB gene sequences and the maximum likelihood (ML) method. The numbers (>70%) presented above the branches indicate the bootstrap values. The scale bars indicate an evolutionary distance of 0.07 substitutions per sequence position, and the branch labels include GenBank accession numbers

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Fig. 5
figure 5

Phylogenetic tree illustrating the relationships among the Theileria species identified in this study (highlighted in bold). The tree is constructed based on 18S rRNA gene sequences, employing the maximum likelihood (ML) method. The numbers (>70%) shown above the branches represent the bootstrap values. The scale bars correspond to an evolutionary distance of 0.05 substitutions per sequence position, and the branch labels contain GenBank accession numbers. Babesia divergens (LC477143) and Babesia capreoli (KX839234) were used as outgroup

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Discussion

The results of our study add to those of Magalhães-Matos et al. (2022), who detected DNA from Rickettsia spp. in ticks of coatis, reinforcing the importance of these animals as dispersers of ticks and agents transmitted by ticks in both forest and synanthropic areas of the PNI. An important finding of this study was the detection of DNA from Anaplasma sp. in the four positive samples of A. coelebs. Phylogenetic analysis revealed that they occur in the same group as A. marginale, A. centrale, and A. capra (Figs. 1 and 2).

Other studies seeking the amplification of the Anaplasmataceae 16SrRNA gene found high similarity with the findings of the present study. Benevenute et al. (2017) detected Anaplasma sp. B173 (KY391803) in a spleen fragment of a Rattus rattus in the state of Ceará-Brazil with 100% identity (467/467). When searching for Anaplasma spp. in the blood of the same group of coatis evaluated in our study, Perles et al. (2023) determined that 14.4% (7/49) were positive. The similarity of 97.78% (440/450) of the 16S rRNA gene fragment of the sequences described in coatis blood (GenBank OM811667) with those found in our study indicated that these were from two different species. However, new studies that present larger fragments and other genes are required to confirm this.

In Brazil, Anaplasma species are mostly detected in ticks of the genus Rhipicephalus, such as A. marginale, which infects cattle and has the tick Rhipicephalus microplus as its main biological vector (Vieira et al. 2019). The arthropod vectors involved in the transmission cycle of Anaplasma spp. among wild mammals in Brazil remain unknown (Sousa et al. 2017; Sousa et al. 2018). However, previous studies conducted in the Brazilian Pantanal detected DNA from Anaplasma spp. in different Amblyomma species collected from wild felines and carnivores, such as Amblyomma sculptum, Amblyomma triste, Amblyomma parvum, and A. ovale (Widmer et al. 2011; Sousa et al. 2017; Sousa et al. 2018). Our study extends the detection of Anaplasma to another tick species, A. coelebs. However, it must be considered that this positivity for Anaplasma sp. in ticks parasitizing animals may be related to the remains of the blood meal of the arthropod in the infected host (Sousa et al. 2018).

A recent study described a possible new species named “Candidatus Anaplasma sparouinense,” suspected of an atypical case of human anaplasmosis in an Amazon rainforest region of French Guiana. The 16S rRNA sequence (GenBank ON513878) from “Ca. A. sparouinense” showed high similarity (99.8%) to the Anaplasma sequences detected in A. coelebs in our study (Duron et al. 2022). Although more data are needed, this similarity raises the possibility that our Anaplasma isolates pose a risk to human health.

DNA from Borrelia spp. was detected in ticks of the species A. ovale, A. coelebs, and Amblyomma sp. larvae. Phylogenetic analysis showed that the five isolates of Borrelia spp. detected in this study are genetically close to B. turcica and two strains that have been detected in Amblyomma variegatum collected from cattle in Côte d’Ivoire, “Candidatus Borrelia ivorensis” and “Candidatus Borrelia africana” (Ehounoud et al. 2016). They join a clade of the REP Borreliae, a new phylogenetic RGF’s subgroup, which is also vectored by ticks of the genus Amblyomma and was initially associated with reptile hosts (Takano et al. 2010). Santos et al. (2020) described a new potential species of Borrelia phylogenetically close to Borrelia spp. from Ethiopia and Côte d’Ivoire that was detected in a nymph of A. brasiliense (MN650844) and, its sequence, although shorter, is 100% identical to the Borrelia spp. described in this study.

In South America, the first Borrelia spp. detected in ticks of the genus Amblyomma was genetically closer to the species that comprise the Borrelia REP group (Pacheco et al. 2019). Since then, new strains of Borrelia have been identified in these ixodid species. In Argentina, a species of Borrelia was found to infect Amblyomma aureolatum collected from wild birds (Cicuttin et al. 2019). In Brazil, a new strain called Borrelia sp. strain Acalc110 was also recently discovered in DNA of Amblyomma calcaratum genetically close to two species of Borrelia pathogenic to humans, Borrelia miyamotoi and Borrelia venezuelensis (Araújo et al. 2022). Therefore, the present study contributes to the knowledge of Borrelia spp. in ticks of the genus Amblyomma.

Adults of A. coelebs have a parasitic preference for tapirs (Tapirus terrestris) (Guglielmone et al. 2014), and in its immature stages have been found in other mammals and birds (Ogrzewalska et al. 2010; Lopes et al. 2016). In contrast, A. ovale is more commonly recorded in wild and domestic carnivores (Guglielmone, 2003; Magalhães-Matos et al. 2017), and small rodents are also hosts for immature stages (Guglielmone, 2003). In addition, it is important to highlight the occurrence of infestation in humans by both species of ticks (Guimarães et al. 2001; Szabó et al. 2006; Garcia et al. 2015; Ito et al. 2017). However, until now, only A. ovale is considered a competent transmitter of pathogens, such as Rickettsia parkeri strain Atlantic rainforest, which causes human rickettsiosis (Szabó et al. 2013).

None of the tick samples were amplified to Babesia spp. Although we did not use specific primers, Hepatozoon procyonis appears to be a protozoan of the Order Piroplasmida frequent in Nasua nasua blood, unlike Babesia spp. (Silva et al. 2018; Perles et al. 2023). We detected DNA from Theileria sp. in two ticks of the A. coelebs specie closed to the T. cervi sequences (MW008518) detected from white-tailed deer (Odocoileus virginianus). However, the nucleotide difference in a highly conserved gene led us to believe that it is a species that has not yet been described or that at least no molecular data have been deposited in GenBank. In Brazil, DNA from Theileria spp. has been detected in several wild animals such as brown deer (Mazama gouazoubira), pampas deer (Ozotoceros bezoarticus), nine-banded armadillo (Dasypus novemcinctus), agouti (Dasyprocta sp.), paca (Cuniculus paca), and tapir (T. terrestris), including in animals with synanthropic habits such as coatis (N. nasua) (Silveira et al. 2013; Sousa et al. 2018; Gonçalves et al. 2020). It is notable that animals from the same groups also occur in PNI; however, additional investigations are necessary to obtain more specific information on other host species and to characterize the detected agent.

None of the samples amplified DNA from hemoparasites of the genus Ehrlichia. Previous studies performed in the Brazilian Pantanal have detected Ehrlichia spp. in several species of ticks of the genus Amblyomma collected from wild carnivores (Widmer et al. 2011; Melo et al. 2016; Sousa et al. 2018); however, the amplification of these samples may be related to the remains of the arthropod blood meal in the infected host (Sousa et al. 2018). Thus, there is no evidence of transmission of this pathogen in Amblyomma spp. and in coatis in Brazil to date (Gruhn et al. 2019).

Bioagents of the genera Borrelia, Anaplasma, and Theileria were found in ticks collected from coatis at the PNI in Paraná. Our study constitutes the newest report in South America of these microorganisms, which remain poorly studied. Further studies are required to clarify which species are circulating in the park and their possible hosts.