Diversity of rickettsiae in ticks (Acari: Ixodidae) collected from wild vertebrates in part of the Amazon, Cerrado, and Pantanal biomes in Brazil

Abstract Ticks parasitizing 102 wild animals in the states of Mato Grosso and Goiás, Brazil were collected between 2015 and 2018. A total of 2338 ticks (865 males, 541 females, 823 nymphs, and 109 larvae) belonging to four genera (Amblyomma, Dermacentor, Haemaphysalis, and Rhipicephalus) and at least 21 species were identified. DNA extraction and a molecular survey for rickettsial agents were performed on 650 ticks. The results revealed parasitism by the following species: Rickettsia amblyommatis in Amblyomma cajennense s.s., A. cajennense s.l., Amblyomma coelebs, Amblyomma humerale, Amblyomma longirostre, Amblyomma nodosum, Amblyomma scalpturatum, Amblyomma sculptum, and Amblyomma romitii; Rickettsia parkeri in Amblyomma nodosum, Amblyomma ovale, Amblyomma scalpturatum, and Amblyomma triste; Rickettsia rhipicephali in Haemaphysalis juxtakochi; Rickettsia sp. in A. cajennense s.s., A. nodosum, and A. sculptum, and lastly, ‘Candidatus Rickettsia andeanae’ in Amblyomma parvum and Rhipicephalus microplus. This study expands the body of knowledge about tick parasitism among wild animals, including new data concerning tick-host associations, and provides information about the epidemiology of tick-borne pathogens in the Center-West region of Brazil.


Introduction
Ticks (Acari: Argasidae and Ixodidae) are ectoparasitic arthropods of numerous animal species, while humans are accidental hosts, and one of the most important arthropod vectors of infectious diseases around the world (Anderson & Magnarelli, 2008;Nava et al., 2009;Nogueira et al., 2022).Several studies have focused on ticks and their associations with wildlife in the Central-West region of Brazil (e.g., Aragão, 1936;Aragão & Fonseca, 1961;Szabó et al., 2007;Martins et al., 2011Martins et al., , 2023;;Soares et al., 2015;Witter et al., 2016;Colle et al., 2020;Serpa et al., 2021), and an understanding of aspects pertaining both to these ectoparasites and to the infectious agents they can transmit to their hosts during hematophagy is extremely important.
Rickettsiae (Rickettsiales: Rickettsiaceae) are small obligate intracellular gram-negative bacteria that infect invertebrate and vertebrate hosts worldwide with transmission related to ectoparasitic arthropods, mainly ticks (Dumler et al., 2001).Currently, the genus Rickettsia has been classified in the Spotted Fever Group (SFG), the Typhus Group (TG), the Transitional Group (TRG), the Bellii Group (BG), the Canadensis Group (CG), and several other basal groups (Weinert et al., 2009).
In Brazil, the main tick-borne disease is Brazilian Spotted Fever (BSF), caused by the bacterium Rickettsia rickettsii, which is responsible for a high mortality rate among infected humans (Oliveira et al., 2016).This zoonosis is transmitted by the ticks Amblyomma sculptum, the most important vector in Brazil (Labruna, 2009) in many parts of endemic areas in southeastern Brazil, including the states of São Paulo, Rio de Janeiro, Espírito Santo, and Minas Gerais; and Amblyomma aureolatum, a recognized vector within the metropolitan area of São Paulo municipality within the Atlantic rainforest mountain domain (Binder et al., 2021).Furthermore, another pathogenic rickettsial agent, known as Rickettsia parkeri strain Atlantic rainforest (Krawczak et al., 2016), transmitted mainly by adult Amblyomma ovale ticks (Szabó et al., 2013), has been associated with mild cases among humans in Brazil in the States of São Paulo (Spolidorio et al., 2010), Bahia (Silva et al., 2011), and Santa Catarina (Krawczak et al., 2016).Lastly, other species of the genus Rickettsia have been identified in the SFG, namely R. amblyommatis and R. rhipicephali, infecting ticks, but pathogenicity in humans is still unknown (Parola et al., 2013).
Thus, the occurrence of tick species among varied wild hosts in different biomes (Amazon, Cerrado, and Pantanal), where domestic animals and humans also inhabit, considering the importance of ticks and rickettsial diseases for public health, reinforces the need for research focused on this subject.In view of the importance of the tick-host association and research on rickettsial infection in ticks, this investigation focused on the molecular detection of rickettsiae in ticks collected from free-living or captive wild animals in the Central-West region of Brazil.

Materials and Methods
Ticks were collected between 2015 and 2018 from free-living and captive wild animals in the states of Mato Grosso (MT) in part of the Amazon, Cerrado, and Pantanal biomes, and Goiás (GO) in the Cerrado biome, both located in the Central-West region of Brazil (Figure 1).Samples were obtained from road-killed wild animals and from wild animals (n= 94) treated at the Veterinary Hospital of the Federal University of Mato Grosso (UFMT) (Cuiabá, MT), and from wild animals (n= 8) sent to the Wild Animal Screening Center (CETAS) of the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA) in Goiânia, GO (Table 1).Ticks were preserved in isopropyl alcohol for taxonomic identification and DNA extraction.Adult ticks were identified to species level, as described by Barros-Battesti et al. (2006), Martins et al. (2016), andNava et al. (2018), while Amblyomma and Haemaphysalis nymphs were identified morphologically as specified by Martins et al. (2010Martins et al. ( , 2016) ) and Nava et al. (2017), respectively.The larvae of the genus Amblyomma could not be morphologically identified to the species level because there is insufficient literature available, then the larvae were retained as Amblyomma sp. according to Vieira et al. (2004), Barros-Battesti et al. (2006), andNava et al. (2017).The larva of the genus Dermacentor was identified morphologically as described by Clifford & Anastos (1960), thus, the larva identified in the genus Dermacentor was considered to be D. nitens.Finally, adults of Rhipicephalus linnaei were identified based on Šlapeta et al. (2022).Tick specimens were deposited in the Tick Collection Coleção de Carrapatos da Universidade Federal de Mato Grosso at the Federal University of Mato Grosso, in Cuiabá, MT, under the following accession numbers: LDPP-UFMT/N.124-127; 129; 133; 135-137; 139-140; 142-144; 148-150; 155; 157-158; 160-163; 165-174; 176-179; 181; 185; 187-188; 190-195; 199-200; 203; 210; 214-217; 220; 224-227; 229; 231-239; 242; 244-247; 255-256; 260-266; 270-277; 283; 287-291; 294; 296-297 and 303-306.In the molecular screening for the detection of rickettsial agents, we attempted to choose all tick species identified, which had more than one specimen per host, including the largest number of vertebrate hosts among each order of animals sampled.Therefore, some whole ticks were subjected to DNA extraction using the guanidine isothiocyanate protocol, being placed in a 1.5-mL microtube containing 150 µL of TE buffer (Tris HCl 10 mmol/L, EDTA 1 mmol/L, pH 7.4) and homogenized by using a sterile pipette tip as described by Sangioni et al. (2005), and to amplification by a conventional polymerase chain reaction (cPCR).To this end, an initial cPCR was performed using CS-78 and CS-323 primers, which target a fragment of 401 bp (base pairs) of the citrate synthase (gltA) gene common to all Rickettsia species, according to Labruna et al. (2004).Positive samples were further tested by another cPCR protocol using Rr190.70pand Rr190.602nprimers, which amplify a ~530 bp fragment of the 190-kDa outer membrane protein gene (ompA) found only in Rickettsia of the SFG (Regnery et al., 1991).Negative (nuclease-free water) and positive controls (R. rickettsii DNA) were included in each of these reactions.a Reared from nymphs (n= 22) at the laboratory conditions which molted to the adult stage (n= 11 males, 11 females); b Reared from nymph (n= 1) at the laboratory conditions which molted to the adult stage (n= 1 male); c Reared from larvae (n= 15) at the laboratory conditions which molted to the nymph stage (n= 15), thus identified at species level; d Reared from nymphs (n= 30) at the laboratory conditions which molted to the adult stage (n= 11M, 19F); e Reared from nymphs (n= 6) at the laboratory conditions which molted to the adult stage (n= 2 males, 4 females); f Reared from nymphs (n= 23) at the laboratory conditions which molted to the adult stage (n= 11 males, 11 females); g Reared from nymphs (n= 20) at the laboratory conditions which molted to the adult stage (n= 8 males); h Reared from nymphs (n= 3) at the laboratory conditions which molted to the adult stage (n= 3 females); i Reared from nymphs (n= 5) at the laboratory conditions which molted to the adult stage (n= 5 females); j Reared from nymphs (n= 2) at the laboratory conditions which molted to the adult stage (n= 2 females); l Reared from nymph (n= 1) at the laboratory conditions which molted to the adult stage (n= 1 female); m Reared from nymphs (n= 36) at the laboratory conditions which molted to the adult stage (n= 11 males, 11 females); n Reared from nymphs (n= 3) at the laboratory conditions which molted to the adult stage (n= 1 male); o Reared from nymphs (n= 91) at the laboratory conditions which molted to the adult stage (n= 26 males, 45 females).a Reared from nymphs (n= 22) at the laboratory conditions which molted to the adult stage (n= 11 males, 11 females); b Reared from nymph (n= 1) at the laboratory conditions which molted to the adult stage (n= 1 male); c Reared from larvae (n= 15) at the laboratory conditions which molted to the nymph stage (n= 15), thus identified at species level; d Reared from nymphs (n= 30) at the laboratory conditions which molted to the adult stage (n= 11M, 19F); e Reared from nymphs (n= 6) at the laboratory conditions which molted to the adult stage (n= 2 males, 4 females); f Reared from nymphs (n= 23) at the laboratory conditions which molted to the adult stage (n= 11 males, 11 females); g Reared from nymphs (n= 20) at the laboratory conditions which molted to the adult stage (n= 8 males); h Reared from nymphs (n= 3) at the laboratory conditions which molted to the adult stage (n= 3 females); i Reared from nymphs (n= 5) at the laboratory conditions which molted to the adult stage (n= 5 females); j Reared from nymphs (n= 2) at the laboratory conditions which molted to the adult stage (n= 2 females); l Reared from nymph (n= 1) at the laboratory conditions which molted to the adult stage (n= 1 female); m Reared from nymphs (n= 36) at the laboratory conditions which molted to the adult stage (n= 11 males, 11 females); n Reared from nymphs (n= 3) at the laboratory conditions which molted to the adult stage (n= 1 male); o Reared from nymphs (n= 91) at the laboratory conditions which molted to the adult stage (n= 26 males, 45 females).Rickettsia amplicons of the expected size were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Chicago, Illinois) and sent for sequencing at the company ACTGene (Porto Alegre, RS, Brazil) with the same primers used in the cPCR.To evaluate the quality of the sequences, electropherograms were verified with CLC Genomics Workbench software (Qiagen ® ).All the sequences obtained were then analyzed using the Basic Local Alignment Search Tool (BLAST; Altschul et al., 1990) to determine the closest identities with congeneric organisms available in GenBank.
Among the 650 DNA-extracted samples from ticks evaluated by cPCR targeting the gltA gene, at least 54 (8.30%) were found to contain rickettsial DNA through the gltA-cPCR (Table 2).Furthermore, 43 of these gltA-PCR positive samples yielded amplicons after the ompA-PCR assay.Overall, 30 DNA sequences were generated, involving the following tick species: A. cajennense s.s.      2.
The GenBank nucleotide sequence accession numbers for the partial sequences generated in the present study are: OP823389, OP823390, OP823391, OP823392, and OP823393 for partial sequences of the ompA gene of R. amblyommatis, OP823395 and OP823396 of R. parkeri, OP823394 of R. rhipicephali; and OP823399 for partial sequences of the gltA gene of Rickettsia sp., OP823397 of R. amblyommatis, and OP484958 of 'Ca.Rickettsia andeanae'.

Discussion
This study revealed the presence of at least 21 tick species parasitizing wild animals in the states of Mato Grosso and Goiás, in the Central-West region of Brazil, between 2015 and 2018.The most abundant species among the collected ticks was the A. cajennense complex, followed by A. dissimile, A. nodosum, R. microplus, A. scalpturatum, A. coelebs, A. dubitatum, A. rotundatum, A. romitii, A. calcaratum, R. linnaei, A. ovale, A. triste, A. humerale, A. oblongogutattum, A. longirostre, A. parvum, A. naponense, H. juxtakochi, and D. nitens.Amphibians and reptiles are the main hosts of all the parasitic stages of A. dissimile and A. rotundatum ticks (Nava et al., 2017;Alcantara et al., 2018;Luz et al., 2018;Torres et al., 2018).This was corroborated in the present study by the discovery of lizard and snake species with terrestrial or semiaquatic habits.However, this study identified new tick-host relationships with the amphibian Rhinella diptycha and nymphs of A. rotundatum.Additionally, the current record of adult A. dissimile ticks on Eunectes notaeus and Paleosuchus palpebrosus corresponds to a new host-parasite association.With regard to adult A. humerale ticks, our records of this species parasitizing Chelonoidis denticulatus and Didelphis marsupialis represent previously described associations (Ogrzewalska et al., 2010;Soares et al., 2015;Witter et al., 2016;Colle et al., 2020).Testudines (Testudinidae) are the common hosts for adult A. humerale ticks, while an infestation of Crocodylia and Mammalia is unexpected (Guglielmone et al., 2014), as is the infestation of the reptile Phrynops geoffroanus by adult A. sculptum, which was observed here for the first time.This is an unusual discovery since A. sculptum ticks are usually found parasitizing mammals (Nava et al., 2014;Martins et al., 2016).
As for information about tick species, the largest number of ticks comprising the A. cajennense species complex, represented in Brazil by A. cajennense s.s. and A. sculptum (the vector of R. rickettsii, agent of Brazilian Spotted Fever) (Szabó et al., 2013;Nava et al., 2014;Martins et al., 2016) was expected, given the large numbers of tapirs (n=14), capybaras (n=9) and giant anteaters (n=12), usual hosts for adults and immature stages of A. sculptum (Martins et al., 2016(Martins et al., , 2023)).Birds have been recorded as hosts of all the stages of A. sculptum in Brazil (Nava et al., 2017), as reported by Luz et al. (2016) in the state of Goiás and observed in this study for A. sculptum nymphs and adult ticks infesting C. cristata in the Cerrado biome.Similarly, the large number of adult A. nodosum ticks is presumably associated with the occurrence of anteaters (Myrmecophaga tridactyla and Tamandua tetradactyla), reported to be the main hosts of adult stages of A. nodosum (Nava et al., 2017).Although A. cajennense s.s. has been observed on a wide range of domestic and wild animals, including tapirs and giant anteaters (Martins et al., 2016;Luz et al., 2020), this paper describes the first tick-host relationship with Mazama americana.
Rhipicephalus microplus is strongly associated with cattle (Nava et al., 2017;Martins et al., 2023), and all the records of parasitism on Subulo gouazoubira, Cerdocyon thous, Puma concolor, and Tapirus terrestris described herein have already been reported previously by other authors.Even though horses are the main host of D. nitens, this parasite has been found in a variety of mammals, including wild carnivores (Labruna et al., 2005b;Guglielmone et al., 2014;Nava et al., 2017).In this paper, we describe for the first time, a D. nitens larva parasitizing Chrysocyon brachyurus.Therefore, tick infestations among livestock and horses should be considered accidental findings, possibly attributable to the fact that these animals share pastures with cattle and horses (Ramos et al., 2020).
In the order Carnivora, two new tick-host associations were observed, involving the species A. cajennense s.s. and A. oblongoguttatum nymphs found on wild cats Puma concolor and Leopardus pardalis, respectively.Rhipicephalus linnaei, recently recognized as belonging to the so-called "tropical lineage" of Rhipicephalus sanguineus s.l.(Šlapeta et al., 2022), is a species of the R. sanguineus complex commonly known as the brown dog tick.It has occasionally been found on mammals of different orders, with all parasitic stages of this tick strongly associated with domestic dogs (Nava et al., 2017), and here it was found parasitizing a rodent, the capybara Hydrochoerus hydrochaeris.This is probably due to these rodents' population growth in urban and peri-urban areas, where R. linnaei is distributed throughout most of Brazil, particularly in these areas.
This study also found a new tick-host association of A. cajennense s.s.nymphs on a six-banded armadillo Euphractus sexcinctus.Although tapirs are considered the usual hosts of adults of A. coelebs, and the largest number of ticks found on one animal in our study was on a host parasitized by 14 specimens, adults and immature stages of this tick present low host specificity (Nava et al., 2017).The remaining tick-host associations described here have been previously reported.
The molecular survey indicated infection by an uncharacterized Rickettsia species belonging to the SFG in A. cajennense s.s., A. nodosum, and A. sculptum.Other studies have described uncharacterized Rickettsia in A. humerale (Colle et al., 2020) and A. nodosum (Lugarini et 2015).So, this report can indicate the possibility that other rickettsiae species not yet described should be infecting ticks from wild animals.Detection of R. rhipicephali in H. juxtakochi was expected since various studies carried out in the country have previously described this agent infecting this tick species (Labruna et al., 2005a(Labruna et al., , 2007;;Soares et al., 2015).Although its pathogenic potential for humans is still unknown, experimental infections in mice suggest that it can cause moderately severe disease (Wikswo et al., 2008;Parola et al., 2013).
Infection by R. amblyommatis in at least six tick species (A.cajennense s.s., A. cajennense s.l., A. humerale, A. longirostre, A. sculptum, A. scalpturatum, and A. oblongoguttatum) was expected, since this Rickettsia species has been reported in 34 tick species worldwide (Richardson et al., 2023).However, here we provide new data on R. amblyommatis infecting A. romitii and A. nodosum ticks.Considering the possibility of the genetic variability between the different strains of R. amblyommatis in South American (Sebastian et al., 2020), it is evident the importance of new data encompassing infection by this bacterium in two species of the genus Amblyomma, also contributing to a better understanding of ecological relationships involving ticks and agents transmitted by them in a wild environment.Despite the numerous reports of infection in ticks, the pathogenicity of R. amblyommatis to humans is still unknown.However, it is suspected that this may be a potential pathogen, in view of several serological reports of human infection, as well as a possible association with the occurrence of disease in some patients in the United States (Apperson et al., 2008;Delisle et al., 2016).Moreover, there is molecular evidence that this organism can infect dogs (Barrett et al., 2014), and cause fever and pathological signs of the disease in guinea pigs (Rivas et al., 2015).

a
Reared from nymphs (n= 22) at the laboratory conditions which molted to the adult stage (n= 11 males, 11 females); b Reared from nymph (n= 1) at the laboratory conditions which molted to the adult stage (n= 1 male); c Reared from larvae (n= 15) at the laboratory conditions which molted to the nymph stage (n= 15), thus identified at species level; d Reared from nymphs (n= 30) at the laboratory conditions which molted to the adult stage (n= 11M, 19F); e Reared from nymphs (n= 6) at the laboratory conditions which molted to the adult stage (n= 2 males, 4 females); f Reared from nymphs (n= 23) at the laboratory conditions which molted to the adult stage (n= 11 males, 11 females); g Reared from nymphs (n= 20) at the laboratory conditions which molted to the adult stage (n= 8 males); h Reared from nymphs (n= 3) at the laboratory conditions which molted to the adult stage (n= 3 females); i Reared from nymphs (n= 5) at the laboratory conditions which molted to the adult stage (n= 5 females); j Reared from nymphs (n= 2) at the laboratory conditions which molted to the adult stage (n= 2 females); l Reared from nymph (n= 1) at the laboratory conditions which molted to the adult stage (n= 1 female); m Reared from nymphs (n= 36) at the laboratory conditions which molted to the adult stage (n= 11 males, 11 females); n Reared from nymphs (n= 3) at the laboratory conditions which molted to the adult stage (n= 1 male); o Reared from nymphs (n= 91) at the laboratory conditions which molted to the adult stage (n= 26 males, 45 females).

Table 1 .
Ticks (M: male; F: female; N: nymph; L: larva) parasitizing free-living (FL) and captive (C) wild animals, collected between 2015 and 2018, in the states of Mato Grosso (MT) and Goiás (GO), in the Central-West region of Brazil.

Table 2 .
Results of molecular tests on ticks (M: adult male; F: adult female; N: nymph; L: larva) collected from free-living and captive wild animals between 2015 and 2018, in the states of Mato Grosso (MT) and Goiás (GO), in the Central-West region of Brazil.
a Results refer to minimal infection rate because PCR-positive ticks included a pool of 5 nymphs or 10 larvae.aResults refer to minimal infection rate because PCR-positive ticks included a pool of 5 nymphs or 10 larvae.
a Results refer to minimal infection rate because PCR-positive ticks included a pool of 5 nymphs or 10 larvae.