Ixodes tropicalis (Acari: Ixodidae) infesting a human and molecular detection of Rickettsia bellii, Colombia

Abstract Introduction: Ixodes tropicalis is a little-known tick species reported parasitizing wild rodents only in Colombia and Perú. Objective: To report a case of I. tropicalis infesting a human in the south of the metropolitan area of the Valle de Aburrá, Antioquia, Colombia, and to report the molecular detection of Rickettsia bellii in this species. Materials and methods: The tick was identified using a morphological key and sequencing of tick mitochondrial 16S rRNA. Additionally, bacterial and protozoa pathogens were evaluated using PCR for the detection of Rickettsia spp., family Anaplasmataceae, Borrelia spp., and piroplasmid. Results: We identified the tick as an I. tropicalis female according to Kohls, 1956, description and to partial 16S rRNA sequences showing a minimum of 5% divergencies compAred to Ixodes sequences. We also detected the gltA gene of R. bellii in the tick with 99.87% of identity. Conclusion: This is the first report in Colombia of a species of the Ixodes genus parasitizing a human and the first report of the detection of R. bellii in this tick species.


Ixodes tropicalis
, was described from females collected from the wild rodents Thomasomys nicefori (as Thomasomys aureus) in Valdivia (Antioquia), and from Dactylomys boliviensis in San Juan, Tambopata, Sandia (Puno, Perú) (11). Later, immature ticks determined as I. tropicalis were reported infesting another wild rodent, Nephelomys childi (as Oryzomys albigularis), in the Valle de Pichindé (Valle del Cauca), and the Pichindé virus was isolated from them (12). However, this report of I. tropicalis should be considered doubtful because its larvae and nymph have not been formally described (2). Thus, the only bona fide records of I. tropicalis correspond to those of the original description (11).
This study aims to report a case of I. tropicalis infesting a human, as well as the molecular detection of R. bellii in the south of the metropolitan area of Valle de Aburrá (Antioquia).

Materials and methods
On March 18, 2018, a 59-year-old man was gardening at his house in La Tablaza, La Estrella (Antioquia) (6º07'02''N, 75º38'14''W; 1756m). Later, he was found parasitized by a tick in the abdomen umbilical region, which was removed, placed in 96% ethanol, and sent to Universidad de Antioquia. The classification of the tick was made following the description of Kohls, 1956 (11), with a stereomicroscope (Nikon SMZ1000™, Tokyo, Japan).
For molecular studies, the tick was longitudinally bisected using sterile scalpel blades and forceps, rinsed with distilled water to remove ethanol, and crushed with a homogenization pestle. The DNA was extracted using the commercial kit PureLink Genomic DNA Mini Kit™ (Invitrogen, Germany) following the manufacturer's instructions. DNA was tested by polymerase chain reaction (PCR) targeting the tick mitochondrial 16S rRNA gene (13) and gltA and ompA genes for Rickettsia spp., 16S rRNA gene of the family Anaplasmataceae, flagellin gene of Borrelia spp., and 18S rRNA gene of piroplasmid (14)(15)(16)(17)(18).

Results
The tick (a slightly engorged specimen) was identified as a female of I. tropicalis based on the following morphological characteristics: Idiosoma suboval, length from the tip of scapulae to the posterior margin of the body (excluding capitulum) 2.15 mm, width 1.66 mm; scutum, with numerous punctations, length 1.30 mm and width 1.15 mm; elevated lateral carinas extending from the scapulae to about the mild-length of the scutum; capitulum, porose areas large and semicircular in shape, separated by about the diameter of one, cornua short and rounded, palpal segment two a little longer than segment three, auricula large and posterolaterally directed; hypostome, broken at the base; coxa I of legs with moderately long internal spur and coxae I-IV with a conspicuous external spur; spiracular plate subcircular in shape ( figure 1 A, B).
We amplified fragments of the mitochondrial 16S rRNA gene of the tick and gltA gene of Rickettsia and purified the amplicons using a PureLink Quick PCR Purification Kit TM (Invitrogen, Germany), which we sent to Macrogen (Seoul, Korea) for sequencing. We did not amplify the DNA of piroplasmid, Borrelia spp., Anaplasmataceae agents, and the ompA gene of Rickettsia. The partial sequence obtained for the 16S rRNA gene of the specimen determined as I. tropicalis (ca. 410 bp) diverged by more than 5% when compared to the remaining Ixodes sequences available at the Genbank.
The partial gltA (784 bp) sequence showed 99.87% (783/784 bp) of identity with the corresponding R. bellii sequences (GenBank accession numbers: CP000087, AY375161, U59716). The sequences generated in the study were deposited in the GenBank under the accession numbers MT158325 for the 16S rRNA gene of I. tropicalis and MT174170 for the gltA gene of R. bellii.
Regarding the detection of R. bellii in Colombia, Miranda, et al. (2014), detected it in the free-living larvae of Amblyomma sp. (38) from the northern coast of Colombia (Los Córdobas, Córdoba). In an area near Los Córdobas, R. bellii in A. ovale was detected and collected from a donkey in Necoclí (39). Besides, R. bellii has been detected in larvae of A. dissimile collected in Rhinella horribilis and Basiliscus basiliscus in the department of Magdalena (40,41). As far as we know, this is the first report of I. tropicalis infesting a human and of R. bellii in this species in Colombia, and it would broaden the panorama regarding tick species infesting humans and the exposition to rickettsial agents in the population living in the south of the metropolitan area of the Valle de Aburrá in Antioquia.
These findings demonstrate the presence of I. tropicalis as a potential parasite in humans in the south of the metropolitan area of the Valle de Aburrá Valley, as well as the report on the presence in this tick species of R. bellii, a bacteria of unknown pathogenicity in humans. Finally, it is crucial to determine other regions at risk of rickettsial agents' transmission besides those already known such as the Urabá area in Antioquia and the Villeta municipality in the department of Cundinamarca.