Molecular Evidence of Different Rickettsia Species in Villeta,Colombia

Introduction

The genus Rickettsia comprises obligate intracellular bacteria that include more than 20 pathogenic species. They are currently classified in four groups: the Spotted Fever Group (SFG) (R. rickettsii, R. conorii, R. parkeri, and others) transmitted by ticks; the Typhus Group (TG) (R. prowazekii and R. typhi) transmitted by lice and fleas, respectively; the Transitional Group (TRG) (R. australis, R. akari, and R. felis) transmitted by ticks, mites, and fleas, respectively; and the Ancestral Group (AG) with two nonpathogenic species related with ticks (R. bellii and R. canadensis) (Sahni et al. 2013). In recent decades, the diseases associated with these bacteria are considered as emerging and re-emerging diseases worldwide (Sahni et al. 2013). In Colombia, Villeta town is an endemic area for SFG rickettsioses. Thus, human cases with fatalities caused by R. rickettsii have been confirmed and high values of seropositivity have been found in the general population (Hidalgo et al. 2007). The aim of this work was to detect and identify Rickettsia spp. in ticks collected in the rural area of Villeta, Colombia.

Materials and Methods

From January of 2005 to April of 2006, 509 ticks were collected from domestic animals (cattle, horses, and dogs) and walls of houses in five rural villages of Villeta (5°0′46″N, 74°28′23″W) (Cune, El Chorrillo, La Mazata, Naranjal, and San Isidro) during a study after fatal cases of Rocky Mountain spotted fever were registered. All samples were frozen for further rickettsial detection at −80°C.

In addition, from March to April of 2012, 11 ticks were collected from the skin (only males were attached) and clothes of our research group members working in Naranjal during another epidemiological study. Last, a flea specimen found in the bed of a man living in Naranjal was also included in the molecular detection survey. Samples were kept in ethanol (70%) and subsequently classified using current taxonomic keys (Wall and Shearer 2001, Barros-Battesti et al. 2006). Number, location, and arthropod species are shown in Table 1.

DNA extraction of ticks removed from animals and walls (Table 1) was performed in pools made by the same location, source, tick species, life stage, and gender in adults (2–21 specimens) using a commercial kit (DNeasy Blood and Tissue; QIAGEN Inc., Valencia, CA) with the addition of 400 mL of guanidine; thiocyanic acid (DNAzol; Invitrogen™, Life Technologies Corp., Grand Island, NY) for tissue lysis. In the extraction process, the amount of sample was always under the maximum recommended by the manufacturer (≤25 mg). In contrast, DNA from ticks and the flea specimen collected from humans was individually extracted using a standardized technique of ammonium hydroxide (Portillo et al. 2005). PCR assays for partial gltA, htrA, 16S RNA, ompA, and ompB rickettsial genes were performed by conventional, seminested, and nested reactions, using previously standardized primers, reagents, and amplification conditions (Table 2).

Positive and negative controls (R. slovaca and R. prowazekii DNA and water, respectively) were included. The amplicons obtained were purified, directly sequenced, and compared with other available Rickettsia sequences by the BLAST tool.

Results

From 2005 to 2006, all ticks were included in 118 pools corresponding to the species: Amblyomma cajennense sensu lato (s.l.) (19 pools), Dermacentor nitens (11 pools), Rhipicephalus sanguineus s.l. (60 pools), Rhipicephalus (Boophilus) microplus (25 pools), and Amblyomma spp. (three pools). In 2012, all ticks collected over four of the researchers were further classified as A. cajennense s.l. (three nymphs, four male and four female adults). The flea was classified as Ctenocephalides felis. DNA of sufficient quality was only detected and sequenced in samples collected from humans. In the ticks analyzed, rickettsial DNA was detected in a nymph of A. cajennense s.l. from Naranjal village using ompA as the target. In this case, the nucleotide sequence was 100% identical to “Candidatus (Ca.) Rickettsia amblyommii” strain “Conduru” (acc. no. HQ231758).

For the flea sample, gltA, ompB, and 16S rRNA genes yielded positive PCR results. These sequences showed high identity (98.5%, 97.4%, and 99.2%, respectively), with R. felis as a validly published Rickettsia species (acc. no. CP000053), and showed 99.7% and 100% identity with gltA and ompB of “Candidatus Rickettsia asemboensis” (acc. nos. JN315968 and JN315972, respectively). The sequences obtained were submitted to GenBank under the accession numbers KJ433807 (tick) and KJ569090–KJ569092 (flea).

Discussion

To our knowledge, these results correspond to the first molecular detection in Colombia of “Ca. Rickettsia amblyommii” in ticks, and “Ca. Rickettssia asemboensis” in fleas. “Ca. Rickettsia amblyommii” had been previously amplified in A. cajennense from domestic animals in Panama (Bermúdez et al. 2009) and Costa Rica (Hun et al. 2011) and from vegetation in Brazil (Labruna et al. 2004a). Even though we amplified this species in only one nymph, not attached to the skin, our study extends to Colombia the geographical distribution of of “Ca. Rickettsia amblyommii”. On the other hand, the high homology of the C. felis sequences with “Ca. R. asemboensis”, agrees with recent findings in fleas collected from dogs in Ecuador (Oteo et al. 2014). Further studies are needed in Colombia regarding the characterization of SFG Rickettsia spp.