New hard tick (Acari: Ixodidae) reports and detection of Rickettsia in ticks from Sierra Nevada de Santa Marta, Colombia

The Sierra Nevada de Santa Marta (SNSM), located in northern Colombia, is considered a geographical island with high levels of biodiversity and endemism. However, little is known about tick species and their associated microorganisms at the SNSM. In this study we sampled host-seeking ticks in areas of the town of Minca within the SNSM. We collected 47 ticks identified as Amblyomma pacae, Amblyomma longirostre, Amblyomma ovale, Amblyomma mixtum, Haemaphysalis juxtakochi, Ixodes sp. cf. Ixodes affinis and Ixodes sp. Of these ticks, we tested for Rickettsia spp. by amplifying the gltA, SCA1, and 16S rRNA genes via PCR. Rickettsia amblyommatis was detected in one pool of 3 larvae and in a female of A. pacae. Additonally, we isolated Rickettsia sp. belonging to the group of spotted fevers in larvae of A. longirostre. This study reports new findings of six species of ticks and two species of Rickettsia within the SNSM. Supplementary Information The online version contains supplementary material available at 10.1007/s10493-023-00887-z.


Introduction
Hard ticks (Acari: Ixodida: Ixodidae) are obligate blood-sucking ectoparasites that parasitize the vast majority of vertebrates (Guglielmone et al. 2014).The prevention of tickborne disease transmission to hosts depends on a thorough understanding of the presence and distribution of ticks, as well as their pathogens (Fritz 2009).This information can only be obtained through sustainable active and passive surveillance, to identify spatial and temporal acarological and epidemiological risk (Han et al.2021;Lyons et al. 2021).Without this information, it becomes difficult and inefficient to apply prevention strategies to mitigate the prevalence of ticks and tick-borne pathogens.Furthermore, without the baseline characterization of these metrics, the ability to monitor the effects of climate change or habitat modification on the risk of tick-borne diseases is lost (Johnson et al. 2022).
The Ixodidae are of great relevance for public and veterinary health, since they can cause considerable damage to their hosts such as dermatosis, anemia and even paralysis (Claveria et al. 2022).Additionally, they are vectors of a wide number of parasites including viruses, bacteria, protozoa and nematodes (Guglielmone and Robbins 2018).Infectious agents transmitted by ticks are the cause of important zoonoses that affect humans and domestic animals, among them, the best known zoonoses include Rickettsioses, Borreliosis, Babesiosis, Anaplasmosis, Ehrlichiosis, Q fever and Encephalitis (Jongejan and Uilenberg 2004;Boulanger et al. 2019).
Minca, with an altitude between 600 and 2000 masl, is considered the ecological capital of the SNSM due to its high level of biodiversity, recognized for its scientific and ecological tourism, it has a high flow of tourists, the vast majority of whom are of international origin (Mayor's Bulletin 2022a, b; WRadio Bulletin 2023).As a highly touristic destination, it could be a gateway for the exit or entry of new pathogens, enabling their establishment in the area, making it vulnerable to economic, ecological and social repercussions (Hall 2019).In general, in Colombia, eco-epidemiological tick studies are diverse, but they have been focused on areas where outbreaks have occurred, leaving a void in areas with epidemiological silence.Therefore, there is a latent need to identify and know the possible vectors and pathogens to which wild and domestic animals and humans could be exposed.The present study aims to report the presence of ticks and Rickettsia spp.collected in Minca, a touristic town located at approximately at 1000 masl, at the SNSM, Colombia.

Sample collection
Ticks were collected in three places at the Minca region, located in the foothills of the SNSM through sampling in the months of February and November 2022.The SNSM is a mountainous massif that is isolated from the Andes.Its highest peak rises to 5776 masl and its total area is of approximately 17,000 km 2 (Huertas-Díaz et al. 2017).The SNSM presents variations in climate and vegetation produced by altitude (Rangel and Garzon 1995), however this region presents rapid changes in the plant formations depending on the geographic position, from dry deciduous and arid succulent shrubs to humid evergreen seasonal forest.Therefore, it has been established an altitudinal zonation with different biomes such as Lowland Xerophytic scrub forest (0-250 m), Low montane wet forest (0-600 m), Cloud Forest (700-1800 m), Andean Forest (2300-3200 m), Paramo (3300-4400 m) and Superparamo (4500 m-snow limit) (Bernal-Carlo 1991).The variety of climates and biomes that the SNSM presents, provide ecosystems with high levels of endemism and biological diversity (Camero 2002).
The first sampling site (11°08'02.8''N,74°06'07.5''W) is located at an altitude of 800 masl, the second site (11°7'1.48"N,74°5'51.78"W) is located at an altitude of 1200 masl, while the third site (11°06'42.1"N,74°05'49.5"W) is at 1296 masl (Fig. 1).All places have Cloud forests, characterized by dense, hygrophytic and sub-hygrophytic vegetation, highlighting the presence of trees, tree ferns, mosses, liverworts, lichens and bamboos, being Melastomataceae, Piperaceae, Moraceae, Bromeliaceae, Solanum, Pleurothallis, Passiflora, Polypodium, Psychotria, Bambusa, Coenogonium, Notothylas, Radula, Cladonia, Hypotrachyna and Stereocaulon the predominant families and genera (Rangel and Garzón 1995;Bernal 2016;Gradstein et al. 2016).Additionally, sampling localities have a rainfall regime of 3000 mm per year and have frequent fogs that increase humidity (Adams 1973;Rangel and Carvajal-Cogollo 2012).Tick collection was carried out by using the dragging and flagging methods, in which a 1 m 2 piece of cotton cloth was swept over the vegetation near human and animal trails (Salomon et al. 2020).At each site, the collection was carried out by four operators for 1.30 h each, in the month of February and in the month of November 2022, the distance between sites one and two is of 3.8 km and the distance between sites two and three is of 3.5 km.Ticks on the cloth were removed with fine-tipped forceps and preserved in 99% ethanol.

Taxonomic identification of ticks
Morphological identification of Amblyomma adults and nymphs was made following Martins et al. (2010) and Nava et al. (2017) and the larvae according to Barros-Battesti et al. (2005) and Barbieri et al. (2008a, b).A nymph of Haemaphysalis was identified following Nava et al. (2017).The adults of Ixodes were identified following Nava et al. (2017) and Saracho-Bottero et al. (2020), while the nymphs and larvae following Sénevet and Ripert (1967) and Durden and Keirans (1994).After identification, the specimens were discriminated by species and stage for storage.

Molecular identification of ticks
DNA extraction was performed using the MasterPure DNA purification kit (Lucigen, USA).Depending on the numbers of ticks collected per species, only a subset of the collected ticks was taken at random for the molecular analyzes.As the majority of the tick species found had not been reported for the study region, it was important to leave ticks for a possible detailed morphological description and biological collections.Adult stage ticks and nymphs were cut transversally with a sterile scalpel in the area of the festoons for the lysis process and to preserve the body structure.Larvae were processed individually or in pools (the pools consisted of a maximum of 10 larvae, depending to the number of individuals collected) and macerated with sterile pestles.All samples were Fig. 1 Map of the geographic location of the sampling sites at Minca, Sierra Nevada de Santa Marta, Colombia incubated at 37 °C overnight.DNA quality was verified through 1% agarose gel electrophoresis, using RedGel (Biotum).Subsequently, the mitochondrial gene cytochrome c oxidase subunit I (cox1) and the 16S rDNA gene, also mitochondrial, were amplified by conventional PCR.For the cox1 amplification, we used the primers LCO1490 (5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3') and HCO2198 (5'-TAA ACT TCA GGG TGA CCA AAA ATC A-3') described by Folmer et al. (1994), generating a 658 bp fragment.A second pair of primers, ArF2 (GCICCIGAY ATR GCITTYCCICG) and ArR5 (CCIGTI-YTIGCIGGIGCI ATYAC) described by Gibson et al. (2014) was also used.For the 16S rDNA, we used the primers 16S + 1 (5'-CCG GTC TGA ACT CAG ATC AAGT-3') and 16S-1 (5'-GCT CAA TGA TTT TTA AAT TGC TGT -3') described by Mangold et al. (1998), obtaining a 460 bp fragment.

Phylogenetic analysis
Sequence files were edited using the Biological Sequence Alignment Editor (BioEdit v7.2.5) (Hall 1999).Subsequently, they were subjected to a similarity analysis with sequences from the NCBI database (www.ncbi.nlm.nih.gov), using the nBLAST tool (Altschul et al. 1990).As a general rule, we used a species (query sequence) percentage of 97% or greater similarity to be assigned to same species (Stackebrandt and Goebel 1994;Hebert et al. 2003;Chung et al. 2018).For the coding gene sequences, the reading frame was corrected using the Aliview software (Larsson 2014) and a data matrix was prepared with reference sequences from Genbank, which were aligned by codons and manually curated in Geneious prime (Kearse et al. 2012).Non-coding genes were aligned by nucleotides with other reference sequences and cured with Gblocks 0.91b.All data sets were aligned using the Mafft algorithm (Katoh et al. 2002).We used the PartitionFinder2 software (Lanfear et al. 2012), applying the Bayesian Inference Criterion (BIC) to find the best substitution models.Maximum Likelihood Phylogenetic analyzes were performed on IQ-TREE (Nguyen et al. 2015), using the Bootstrap algorithm with fast search and using 10,000 pseudo replicates.Also, phylogenetic trees based on Bayesian Inference were run in MrBayes (Ronquist et al. 2012), implementing 5,000,000 generations, with trees sampled every 100 generations, discarding 25% of the trees.Nodes with Bootstrap values and posterior probability greater than > 70% indicated high statistical support (Hillis and Bull 1993).

Ticks species
A total of 47 ticks (35 larvae, 4 nymphs and 8 adults) were collected and identified, distributed in three genera of the Ixodidae family: Ixodes (2 species), Amblyomma (4 species) and Haemaphysalis (1 species) (Table 1).For Ixodes, the sequence of one male, morphologically identified as Ixodes sp.cf.I. affinis (Fig. S1), presented similarly values of 96.35% for the 16S rDNA gene (KT037640) and 91.55% for the cox1 gene (KX360422) with sequences corresponding to Ixodes sp.cf.I. affinis.The analysis of Maximum Likelihood and Bayesian Inference of the 16S rDNA gene (Fig. 2), placed the sequence of Ixodes sp.cf.I. affinis within the Ixodes ricinus complex, but outside the clade made up of Ixodes fuscipes and four lineages of Ixodes sp.cf.I. affinis with reference sequences from Argentina, Panama, Colombia, the United States and Belize.Similar results were obtained using the cox1 gene sequences (Fig. 3).On the other hand, sequences from one larva and two nymphs identified as Ixodes sp.(Figs.S2, S3), presented a Blast similarity of 93.29% with sequences of Ixodes lasallei (MN727314) for the 16S gene, and of 82.35% with sequences from Ixodes dentatus (KX360374) for the cox1 gene, given that there are no sequences of the cox1 gene for I. lasallei in GenBank.In the phylogenetic analyzes, these sequences were closely related to the monophyletic clade made up of I. lasallei, Ixodes bocatorensis, Ixodes catarinensis and Ixodes spinosus, which does not indicate a clear identification of the specimens (Fig. 4).

Detection of Rickettsia
We obtained positivity for Rickettsia in A. pacae (1 female and 1 pool of three larvae) and A. longirostre (1 pool of ten larvae) (Table 1).The obtained sequences in A. pacae showed an identity of 100% for gltA (MN947702), 99.68% for SCA1 (CP015012) and 99.83-100% for 16S rRNA (CP015012) with R. amblyommatis.Similarly, the Maximum Likelihood analysis and Bayesian Inference grouped the sequences obtained with R. amblyommatis sequences (Fig. 5).In A. longirostre, the sequences obtained showed a 99% identity for gltA (MN947699), 99.83% for 16S rRNA (MK304546) with Rickettsia raoultii and a 99.35% similarity for SCA1 with R. amblyommatis (CP015012).However, in Maximum Likelihood and Bayesian Inference analyzes, this sequence forms a clade with R. amblyommatis, which is closely related to R. raoultii (Fig. 5).Since there is no clear identification for the sequence obtained from this pool, it was categorized at the genus level as Rickettsia sp.

Discussion
For the SNSM and the Magdalena region there were no previous records for ticks of the genera Ixodes and Haemaphysalis, despite the fact that several species of these genera have been reported in other areas in Colombia.Therefore, this is the first report in this area of   They also found differences according to geographic location and defined the I. affinis specimens as a complex made up of four lineages, leaving them in the taxonomic status of Ixodes sp.cf.I. affinis.
The male of Ixodes spp.analyzed in this study (Fig. S1) presented morphological similarity with ticks belonging to the I. affinis complex, structures such as scutal punctuations and the length of the posterior lateral denticles of the hypostome were compatible with Ixodes sp.cf.I. affinis from Argentina (Saracho-Bottero et al. 2020).In phylogenetic analyses the sequence was closely related to the I. affinis complex but unrelated from the sequences for Ixodes sp.cf.I. affinis previously detected in Colombia (Saracho-Bottero et al. 2020) and did not cluster with any of the known lineages for this complex.Therefore, further study is required to determine whether the Ixodes sp.cf.I. affinis from the SNSM corresponds to a new lineage of the I. affinis complex.
Ixodes affinis has veterinary medical importance since they are potential vectors of Borrelia burgdorferi sensu lato (Harrison et al. 2010;Maggi et al. 2010;Heller et al. 2016;Flores et al. 2020).In Colombia, there are reports indicating the presence of Borrelia species from the relapsing fever group and Lyme disease group, including B. burgdorferi sensu stricto in bats and rodents in the southwestern regions of the country (Mancilla-Agrono et al. 2022).Thus, future studies should focus in evaluating the presence of Borrelia in the Ixodes ticks of the SNSM.
One larva and two nymphs (Fig. S2, S3) were morphologically identified as Ixodes sp., as descriptions of these stages are currently lacking for many species of the genus Ixodes (Guglielmone et al. 2021).However, with the phylogenetic analysis of the 16S gene, it was possible to determine that the 3 specimens belong to the same taxa, which was grouped in the clade formed by I. bocatorensis, I. catarinensis, I. lasallei and I. spinosus.Adults of I. bocatoriensis and I. lasallei were found by Apanaskevich and Bermúdez (2017) in Colombia in the regions of Antioquia and Arauca, respectively.Although there are few records, the adults of these species have been found associated mainly with rodents of the Cunicululidae and Dasyproctidae families (Guglielmone et al. 2021).These mammals have been found in the sampled area (Díaz et al. 1986;Pineda-Guerrero et al. 2015), so future studies should be carried out to find adults of this tick species and to be able to make the corresponding morphological comparisons.It is important to highlight that it could be Ixodes venezuelensis, a morphologically similar species (Durden and Keirans 1994) and for which there is no genetic information.
Amblyomma longirostre is a Neotropical tick, with reports from Mexico to Argentina (Nava et al. 2010;Guglielmone et al. 2023).For Colombia, the presence of A. longirostre has been reported for the regions of Boyacá (Osorno-Mesa 1940), Meta (Wells et al. 1981), Arauca (Cardona-Romero et al. 2020) and Caldas (Martínez-Sánchez et al. 2021), parasitizing Coendou prehensilis (known as porcupine), Coendou sp. and birds.Adults of A. longirostre are usually found on rodents of the family Erethizontidae, while the larvae and nymphs feed on different families of birds (Guglielmone et al. 2021).A species of porcupine of the family Erethizontidae (C.prehensilis) that lives at the SNSM (Ramírez-Chaves et al. 2016), has been found with A. longirostre (Aragão 1918;Labruna et al. 2002), so it could be a common host supporting the population in this area.Regarding the epidemiological importance of A. longirostre, they are known to feed on humans (Arzua et al. 2005;Rodríguez-Peraza et al. 2014) and have shown to be positive for R. amblyommatis (Labruna et al. 2004b, c;Ogrzewalska et al. 2008;Pacheco et al. 2012) andR. belli (McIntosh et al. 2015).The Rickettsia sp.detected in this study grouped within the spotted fever group Rickettsia and was closely related to R. amblyommatis and R. raoultii, which requires further study to obtain a clear identification of this bacterium.A limitation in our study is that this positive was detected in a pool of larvae of 10 individuals, therefore the low sequence similarity may be due to multiple larvae infected with different species of Rickettsia.Thus, without extracting and testing the individual larvae we cannot discern this finding.
Amblyomma pacae is distributed from Paraguay to Mexico (Guglielmone et al. 2021), however the only reports in Colombia correspond to adults collected on Cuniculus paca (lowland paca) in the Boyaca and Meta regions, more than 50 years ago (Osorno-Mesa 1940;Wells et al. 1981).The main host is C. paca (Jones et al. 1972), a widely distributed species that is present in the SNSM (Pineda-Guerrero et al. 2015).The epidemiological importance of A. pacae is unknown, however, in this study infection by R. amblyommatis was detected, which adds to the detections of this bacteria in this tick species in Belize and Panama (Lopes et al. 2016;Bermúdez et al. 2021b).Rickettsia amblyommatis has been detected in most genera of the Ixodidae family and there are also reports of its presence in Central America, South America and in North America (Labruna and Mattar 2004;Karpathy et al. 2016;Bermúdez and Troyo 2018;Bermúdez et al. 2021a).There are previous reports for the northern part of Colombia, such as the one carried out by Quintero et al. (2017) detecting R. amblyommatis in a nymph of Amblyomma varium parasitizing a person in the rural areas of Urabá (Antioquia, Colombia) and by seropositivity with IgG antibodies for R. amblyommatis in rural workers, Equus caballus, Equus asinus and Canis familiaris.Likewise, Quintero et al. (2020) reported the infection of R. amblyommatis in two nymphs of A. patinoi collected from humans for the same geographic area.
This study reports for the first time, for Minca at the SNSM, the tick species A. pacae, A. longirostre, A. ovale, A. mixtum, H. juxtakochi, Ixodes sp.cf.I. affinis and Ixodes sp.Likewise, the presence of R. amblyommatis is reported in larvae and in a female of A. pacae and Rickettsia sp.belonging to the group of spotted fevers in A. longirostre larvae.Bioclimatic conditions can modulate or become barriers to the distribution of ticks; Clarke-Crespo et al. (2020) in their work on niche modeling in Ixodidae ticks in Mexico, showed that ticks of some genera of Amblyomma concentrate at altitudes of 1000 to 1500 masl, because these ticks have low resistance to desiccation and little tolerance to temperature changes.Likewise, Ferrell et al. (2017), only report the presence of ticks such as A. ovale and Ixodes boliviensis at elevations above 1000 masl, in their work on altitudinal gradient in ticks associated with dogs.However, species such as A. mixtum do not tolerate low temperatures, therefore, altitudinal gradients are an ecological barrier that limit their distribution (Aguilar- Domínguez et al. 2021).This correlates to the existing reports for the SNSM, as at lower elevations only species such as A. mixtum, A. dissimile and A. ovale had been reported.However, in this study, we record the presence of Ixodes tick species at elevations above 1000 masl.
In this study, site 3 presented a greater diversity, probably because it was a relic of cloud forest in a good state of conservation and a denser vegetation.On the other hand, site 2 was located in a matrix of coffee plantations and site 1 in an area with high tourist impact.These characteristics are not mentioned in the article because it was not our objective to evaluate the conservation status of the sites.The sites were chosen because they were located at altitudes above 700 masl, with entrance permits and the presence of trails that would facilitate the transit of wildlife.This study shows that the tick fauna in the SNSM is still unknown, therefore, more studies are needed to expand the information on the Ixodidae fauna, its hosts and tick-borne pathogens in this region.This information is of public and veterinary health importance because these ticks have been reported as vectors of pathogens, both of veterinary and public health concern, and because the study area (Minca, Santa Marta, Colombia), is recognized for its ecotourism, receiving thousands of national and international tourists each year.

Fig. 2 Fig. 3
Fig. 2 Phylogenetic reconstruction using maximum likelihood and Bayesian inference of 16S rDNA for our sequence (yellow triangle) and sequences downloaded from GenBank of the Ixodes ricinus complex (outgroup).Numbers on nodes correspond to bootstrap values/the posterior probability.(Color figure online)

Fig. 4
Fig. 4 Phylogenetic reconstruction using maximum likelihood and Bayesian inference of 16S rDNA for our sequences (yellow triangle) and sequences downloaded from GenBank of tick species of the genus Ixodes.Numbers on nodes correspond to bootstrap values/the posterior probability.(Color figure online) the genus Ixodes (Ixodes sp.cf.I. affinis and Ixodes sp.), one species of the genus Haemaphysalis (H.juxtakochi) and two species of the genus Amblyomma (A. pacae and A. longirostre) (Acevedo-Gutiérrez et al. 2020; Uribe et al. 2020).Ixodes sp.cf.Ixodes affinis presents problems in their morphological identification, since Rodríguez-Vivas et al. (2016) found differences in the shield scores of Ixodes affinis specimens as compared to the original description by Neumann, 1899.Nadolny et al. (2016) found genetic differences in 16S rDNA sequences from specimens from Colombia, Panama, Belize and the United States, forming independent clades according to the geographic location.This was supported by Saracho-Bottero et al. (2020), when analyzing sequences of specimens from Argentina, Belize, Colombia, the United States and Panama.

Fig. 5
Fig. 5 Phylogenetic reconstruction using maximum likelihood and Bayesian inference of concatenate gltA, SCA1 and 16S rRNA genes for genus Rickettsia, including our sequences (yellow triangle) and sequences downloaded from genbank.Numbers on nodes correspond to bootstrap values/the posterior probability.(Color figure online)

Table 1
Molecular detection of Rickettsia in host-seeking Ixodidae ticks collected from Sierra Nevada de Santa Marta, Colombia.Including GenBank accession numbers of