Ornithodoros puertoricensis (Ixodida: Argasidae) Associated With Domestic Fowl in Rural Dwellings From Córdoba Department, Caribbean Colombia

Ticks of genus Ornithodoros are nidicolous parasites associated with a wide array of vertebrates. In humans, their bites cause hypersensitivity reactions and are capable to transmit pathogens of health concern. In the department of Córdoba, Caribbean region of Colombia, the first report of an Ornithodoros soft tick was made in 1980 by Betancourt, who described the collection of Ornithodoros talaje in human dwellings. Nevertheless, current the records of O. talaje made in South America have been questioned and likely correspond to misidentifications with morphologically similar species. Between October and December of 2020, we visited rural areas of four localities from three municipalities within the department of Córdoba: Cuero Curtido and Severá (municipality of Cereté), El Espanto (municipality of Planeta Rica), and Arroyo Negro (municipality of San Carlos). Search for soft ticks was performed in 46 human domiciles and peridomiciliary areas. We searched in areas frequented by domestic animals, inspecting cracks in the walls and fowl nests. Infestation by soft ticks was found in 13% (6/46) of visited houses. Overall, 215 ticks were collected (26 larvae, 144 nymphs and 45 adults) from nests of domestic birds or in the adjacent walls. Larvae, nymphs and adults were morphologically identified as Ornithodoros puertoricensis. Molecular identification of ticks was confirmed by sequencing the tick mitochondrial 16S gene of adults, pools of nymphs and larvae. Pairwise comparisons showed a 99% of identity with O. puertoricensis from Panama. This study reports for the first time O. puertoricensis associated with domestic fowl in rural dwellings in Colombia, and expands the geographical distribution of this tick species toward the Córdoba department. Importantly, local people described exposure to tick bites while sleeping in infested houses; therefore, the transmission of soft tick-borne pathogens is now of concern in the region.

Ticks of genus Ornithodoros are nidicolous parasites associated with a wide array of vertebrates. In humans, their bites cause hypersensitivity reactions and are capable to transmit pathogens of health concern. In the department of Córdoba, Caribbean region of Colombia, the first report of an Ornithodoros soft tick was made in 1980 by Betancourt, who described the collection of Ornithodoros talaje in human dwellings. Nevertheless, current the records of O. talaje made in South America have been questioned and likely correspond to misidentifications with morphologically similar species. Between October and December of 2020, we visited rural areas of four localities from three municipalities within the department of Córdoba: Cuero Curtido and Severá (municipality of Cereté), El Espanto (municipality of Planeta Rica), and Arroyo Negro (municipality of San Carlos). Search for soft ticks was performed in 46 human domiciles and peridomiciliary areas. We searched in areas frequented by domestic animals, inspecting cracks in the walls and fowl nests. Infestation by soft ticks was found in 13% (6/46) of visited houses. Overall, 215 ticks were collected (26 larvae, 144 nymphs and 45 adults) from nests of domestic birds or in the adjacent walls. Larvae, nymphs and adults were morphologically identified as Ornithodoros puertoricensis. Molecular identification of ticks was confirmed by sequencing the tick mitochondrial 16S gene of adults, pools of nymphs and larvae. Pairwise comparisons showed a 99% of identity with O. puertoricensis from Panama. This study reports for the first time O. puertoricensis associated with domestic fowl in rural dwellings in Colombia, and expands the geographical distribution of this tick species toward the Córdoba department. Importantly, local people described exposure to tick bites while sleeping in infested houses; therefore, the transmission of soft tick-borne pathogens is now of concern in the region.
Ornithodoros puertoricensis was reported in Colombia for the first time in Ayacucho, in Cesar department, Caribbean region (20). Several years later, Betancourt (21) made the sole record of an Ornithodoros soft tick in the department of Córdoba (also in the Caribbean region). Specifically, the specimens were collected inside human dwellings at San Carlos municipality. At that time, the ticks were sent to the University of California and identified as O. talaje (21). Currently, the records of O. talaje made in South America have been questioned (16,22) and likely correspond to misidentifications with morphologically similar species of the group. Therefore, the species reported by Betancourt (21) remains to be confirmed.
Fowl are common host for soft ticks of genus Argas (23) and there is evidence that Ornithodoros spp. ticks do parasitize domestic birds in Central America (20). Although 10 species of Ornithodoros occur in Colombia, fowl have never been implicated as hosts.
We carried out a prospective study in order to confirm the presence and identity of neglected Ornithodoros in rural dwellings from the department of Córdoba, including the San Carlos municipality. Our results demonstrate that soft ticks do occur in that region of Colombia and infest fowl nests.
The three municipalities share similar environmental conditions (12-87 m of altitude, temperature between 25 and 28 • C, and average relative humidity of 81%) (24)(25)(26). Municipality of Cereté has a population of 105,815 inhabitants (26), Planeta Rica of 64,205 inhabitants (24), and San Carlos of 23,532 inhabitants (25). The four visited localities were selected by convenience criteria, considering the easiness of access, and the researchers' safety regarding public order conditions in the area.
Searches for Ornithodoros ticks were performed in 46 human domiciles and peridomiciliary areas (eight houses in locality 1, three in locality 2, eight in locality 3, and twenty-seven in locality 4). The houses were constructed with wood, "bahareque" (clay or mud with sticks or canes), and "guadua" (woody bamboo) in their walls, reinforced with cardboard. All had dirt floors and palm leaves as roofs. The presence of domestic animals such as dogs, cats, pigs, chickens, turkeys, and ducks was common in all investigated houses, and the animals circulated freely inside the houses and in the peridomiciliary area. We examined domestic animals' resting areas, chicken coops, and nests of chickens, ducks and turkeys. Around these areas, cracks in the walls and nest debris were inspected. Inspections inside the inhabitants' rooms were not allowed for privacy reasons in most of the houses.

Tick Collection and Identification
While larvae, nymphs, adults and exuviae were collected in 70% ethanol, eggs were maintained alive, transported to the laboratory, and kept in darkness inside an incubator (25 • C, 80% relative humidity). Hatched larvae were mounted onto slides with Hoyer's medium and examined by light microscopy for morphological identification (27). A subset of adults was prepared for scanning electron microscopic examinations for greater definition of morphological characteristics. Larvae were morphologically identified following taxonomical keys (28). Morphological identification of adults and nymphs was done with original descriptions of Ornithodoros spp. (27,29).

Molecular and Phylogenetic Analyses
Morphological identification of ticks was complemented by molecular analysis. For this purpose, DNA extraction (QIAGEN DNeasy Blood & Tissue kit) was performed on individual adults, and larvae and nymphs of each locality were pooled (up to 10 larvae, up to 4 nymphs). Successful extractions were confirmed by PCR targeting the tick mitochondrial 16S gene for each sample with primers described elsewhere (30). Amplicons of expected size were Sanger-sequenced at the Molecular Genomics Core of the University of Texas Medical Branch (Galveston, TX). Obtained sequences were assembled with Geneious (31) and the consensuses compared with sequences available in GenBank using BLASTn (32).
An alignment with 33 sequences of Argasidae retrieved from GenBank was constructed in MAFFT (33). A phylogenetic analysis using the approximately maximum likelihood method was implemented in FastTree 2 (34), selecting the GTR model and five rates categories of sites. Ornithodoros brasiliensis (GU198363) and Ornithodoros rostratus (DQ295780) sequences rooted the tree.

Tick Collection
Infestation by Ornithodoros ticks was found in 12% (1/8) of the visited houses in locality 1, in 33% (1/3) of locality 2, in 25% (2/8) of locality 3, and 7% (2/27) of locality 4. Overall, 215 ticks were collected (26 larvae, 144 nymphs, 13 females, and 32 males) ( Table 1). All were found in fowl nests and on adjacent walls. Briefly, 48 ticks were collected in locality 1, of which eight were adults (males) and 40 were nymphs, all collected in a hen's nest in the kitchen of a house with a dirt floor and cardboard-reinforcedwooden walls (Figures 2C,D). In locality 2 we found exuviae during inspection of the substrate (soil/sand) of a chicken nest  adjacent to an external wall of a warehouse made of bahareque ( Figure 2B). In this wall we collected 32 ticks (four females, two males, 26 larvae and a group of eggs) ( Figure 2E). Interestingly, the owners of the warehouse recognized the ticks, named them as "Pitos, " and also referred to bites of these arthropods while sleeping. In locality 3 a total of 121 ticks [27 adults (six females, 21 males) and 94 nymphs] were collected in chicken nests made of dried-banana-leaves, found in a warehouse and a kitchen, respectively, of two houses with guadua walls, dirt floor, and palm roof ( Figure 2F). In locality 4, 14 ticks [four adults (three females and one male) and 10 nymphs] were collected in bahareque walls adjacent to chicken nests, in a warehouse and a room of two houses, respectively (Figure 2A).
Remarkably, domiciles where we found ticks were comparatively rudimental and precarious; had poor hygiene, and sanitary conditions; dirt in the most of their floors, and no record of recent pest chemical control.

Morphological and Genetic Identification of Specimens
Based on the examination of 10 slide mounted specimens, larvae were identified morphologically as O. puertoricensis because of the following combination of traits: dorsal plate pyriform; 17-18 pairs of dorsal setae (seven anterolateral, 6-7 posterolateral and four central); hypostome pointed, with dentition formula 3/3 in middle length (Figures 3A-C). Nymphs and adults were identified morphologically as the same species by the combination of the following characters: presence of cheeks, conical mammillae, anteromedian disk present, posteromedian file of disks merging with median disk (Figures 3D-G). Examined larvae and adults were deposited in the "Colección Parasitológica Veterinaria Julio Mario Rodríguez Peña" at the Universidad Nacional de Colombia (UNAL: CPV-UN: 2021ACAR001-003). Genetic identification of ticks was performed individually for 14 males, 10 females, nine pools of nymphs and one pool of larvae collected in the four localities ( Table 1). Two haplotypes with two polymorphisms consisting of adenine-guanine transitions were retrieved (99.5% of identity between them). Both sequences were 99.2-99.7% identical to O. puertoricensis from Panama available in GenBank (KX685710) (35). Haplotype I was found in 28 pools of ticks (14 males, nine females and five pools of nymphs) from the four localities, while haplotype II was only found in six pools of ticks (one female, four pools of nymphs and one pool of larvae) from two of the localities (El Espanto and Arroyo Negro). Tick mitochondrial 16S rDNA sequences of O. puertoricensis generated in this study were deposited in GenBank under accession numbers MZ005589 and MZ005590.
The phylogenetic analysis determined that O. puertoricensis from the Córdoba department in Colombia are closely related to a homologous species from Panama. A previous sequence of O. puertoricensis from Haiti (AF113932) branches basally to the Colombian and Panamanian ticks. Collectively, the sequences of O. puertoricensis cluster as a sister group to Ornithodoros cerradoensis (Figure 4). This topology is consistent with previous phylogenies including the O. talaje group (27).

DISCUSSION
This study reports for the first time O. puertoricensis infesting domestic fowl nests in rural dwellings in Colombia, and expands the geographical range of this tick species to the Córdoba department. However, O. puertoricensis is no stranger, since it has been reported in the northwestern region of the country. The first record of O. puertoricensis in Colombia was published by Fairchild in 1966, referring to adult specimens collected in the Ayacucho municipality, in Cesar department (20). Later, in 2009, Paternina et al. described the finding of larvae infesting dogs in the Sucre department (36), and recently, larvae and females were described infesting synanthropic rodents (i.e., Mus, Rattus) and human dwellings in Urabá region, in the Antioquia department (19,37,38). In addition, Butler and Gibbs (39) stated that O. puertoricensis occurs in the Colombian pacific region as well.
Ornithodoros puertoricensis was described based on larvae collected on rats (Rattus spp.) in Puerto Rico (40 (20,40). Dunn's descriptions are similar to our findings in that they suggest that Ornithodoros soft ticks do associate with domestic fowl. Even though in the present study we did not collect larvae feeding on animals, it is likely fowl could maintain the biological cycle of O. puertoricensis in the visited localities, since eggs, larvae, nymphs and adults were taken from their nests. Thus, our results suggest that the previous report of O. talaje made by Betancourt (21) at the same locations probably corresponds to O. puertoricensis.
Ornithodoros puertoricensis do infest human dwellings in Panama and Colombia (19,35) and mild toxicosis was reported after their bites (35). Inside houses, O. puertoricensis seems to parasitize humans during the night (35). Indeed, during our investigations, the inhabitants of infested houses recognized the ticks and reported nocturnal parasitism as well. Interestingly, the common name for O. puertoricensis in the region (i.e., "Pito"), is the same one assigned to triatomine bugs, vectors of Chagas disease in Colombia (47). To acknowledge this coincidence in common names is valuable from an epidemiological point of view and in further inquiries looking for soft ticks infesting domiciliary environments in the region. Finally, evidence that O. puertoricensis transmits human pathogens is currently lacking. However, the role as a vector should not be ruled out, since other Ornithodoros ticks are reservoirs of TBRF spirochetes of human health concern.