Moreno‑Salas et al. Parasites Vectors (2020) 13:523 https://doi.org/10.1186/s13071‑020‑04388‑5 Parasites & Vectors RESEARCH Open Access Molecular detection of Rickettsia in feas from micromammals in Chile Lucila Moreno‑Salas1* , Mario Espinoza‑Carniglia2 , Nicol Lizama‑Schmeisser1, Luis Gonzalo Torres‑Fuentes3, María Carolina Silva‑de La Fuente4,5, Marcela Lareschi2 and Daniel González‑Acuña4 Abstract Background: Rickettsial diseases are considered important in public health due to their dispersal capacity deter‑ mined by the particular characteristics of their reservoirs and/or vectors. Among the latter, feas play an important role, since the vast majority of species parasitize wild and invasive rodents, so their detection is relevant to be able to monitor potential emerging diseases. The aim of this study was to detect, characterize, and compare Rickettsia spp. from the feas of micromammals in areas with diferent human population densities in Chile. Methods: The presence of Rickettsia spp. was evaluated by standard polymerase chain reaction (PCR) and sequenc‑ ing in 1315 feas collected from 1512 micromammals in 29 locations, with diferent human population densities in Chile. A generalized linear model (GLM) was used to identify the variables that may explain Rickettsia prevalence in feas. Results: DNA of Rickettsia spp. was identifed in 13.2% (174 of 1315) of feas tested. Fifteen fea species were found to be Rickettsia‑positive. The prevalence of Rickettsia spp. was higher in winter, semi‑arid region and natural areas, and the infection levels in feas varied between species of fea. The prevalence of Rickettsia among fea species ranged between 0–35.1%. Areas of lower human density showed the highest prevalence of Rickettsia. The phylogenetic tree showed two well‑diferentiated clades with Rickettsia bellii positioned as basal in one clade. The second clade was subdivided into two subclades of species related to Rickettsia of the spotted fever group. Conclusions: To our knowledge, this is the frst report of the occurrence and molecular characterization of Rickett- sia spp. in 15 fea species of micromammals in Chile. In this study, feas were detected carrying Rickettsia DNA with zoonotic potential, mainly in villages and natural areas of Chile. Considering that there are diferences in the preva‑ lence of Rickettsia in feas associated with diferent factors, more investigations are needed to further understand the ecology of Rickettsia in feas and their implications for human health. Keywords: Pathogen, Bacteria, Vectors, Fleas, Rodents, Marsupials Background arthropod vector (e.g. ticks, mites, feas and lice); in some Rickettsia spp. are obligate intracellular microorganisms, cases, the latter may be afected by these bacteria [2]. Gram-negative coccobacilli, with the ability to reproduce, Tey have a worldwide distribution and are the causative both in the nucleus and in the cytoplasm of infected cells agents of serious human infections [3]. [1]. Tese bacteria have a vertebrate reservoir and an Currently, 32 species are recognized (http://www. bacte rio.net/-allna mesmr .html), and there are many *Correspondence: [email protected] strains that have not yet been characterized, while sub- 1 Universidad de Concepción, Facultad de Ciencias Naturales y species and uncultivated species are classifed as “Can- Oceanográfcas, Concepción, Chile didatus” [4]. Recently, using new classifcation methods Full list of author information is available at the end of the article based on formal order analysis (FOA), which considers © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Moreno‑Salas et al. Parasites Vectors (2020) 13:523 Page 2 of 21 whole-genome sequencing analysis, two groups are rec- Methods ognized within the genus Rickettsia: the major typhus Sample localities and micromammal‑trapping procedures group (MTG) and major spotted fever group (MSFG). A total of 1512 micromammals belonging to 18 species Te MTG is divided into the typhus group (TG) and (Table 1) were captured during a trapping efort of 11,034 ancestral group (AG) and is transmitted by insects. trap/nights from 23 localities (9 cities, 6 villages and 8 MSFG includes the R. felis group, R. akari group, and natural areas) of the 29 sampled, covering 10 administra- the “classical” spotted fever group that includes several tive regions in Chile and fve bioclimatic regions (hyper- species transmitted by mites and hard ticks, of which arid, arid, semi-arid, sub-humid and hyper-humid), the most important are R. rickettsii and R. conorii, that latitude between −20.2167 and −53.1667 (Fig. 1). It was cause Rocky Mountain spotted fever and Mediterranean conducted from December 2015 to January 2018, during spotted fever, respectively [4]. Since Rickettsia research austral summer (December to February) and austral win- has focused on species that afect humans, other species ter (July and September). Tese localities were selected have received less attention [5]. Tus, there are several based on the following demographic characteristics: (i) species of rickettsiae identifed and are exclusively asso- city, urban entity that has > 5000 inhabitants; (ii) village, ciated with arthropods. Tey are without known second- urban entity with a population ranging between 2001– ary hosts and associated with other organisms such as 5000 inhabitants, or between 1001–2000 people, where herbivorous insects, leeches, amoebas, inclusive algae, less than 50% of the population that declares having and plants, indicating that these are more common than worked, is engaged in primary activities (e.g. livestock, suspected [5, 6], and that the efects they could cause in agriculture or fshing) [18]; and (iii) natural area, without humans when contact is made are unknown. human settlement, corresponding to national park (NP; Worldwide, micromammals, and especially rodents, are unaltered areas of natural and biological diversity), and the main fea hosts. It is recognized that 74% of known national reserves (NR; areas protecting wildlife popula- fea species parasitize them; therefore, rodents play a fun- tions or natural resources). damental role in the spread of fea-borne diseases, as vari- Micromammals were captured using a Sherman trap ous species of rodent feas can also parasitize humans [7]. (23 × 7.5 × 9 cm, Sherman Co., Tallahassee, USA) and In addition to this, many rodent species are capable of wire-mesh traps (30 × 10 × 11 cm; Forma Ltd., Santiago, inhabiting wild environments and adapting to rural and Chile) baited with oats. Te associated use of both types urban environments, which could favor a continuous gra- of traps strongly reduced the likelihood of a species being dient of transmission between domestic and wild species, present but not captured. Each locality was sampled for and humans [8, 9]. In Chile, despite the great diversity two consecutive nights. In each sampling locality, the of described feas (114 species), which mainly parasitize traps were placed in four parallel lines approximately 100 rodents [10, 11], a scarce number of studies have detected m from each other, and each line was equipped with 50 Rickettsia in feas [12–15]. Tese studies have focused on traps set 10 m apart from each other. Only in cities, traps the molecular detection of pathogens in feas of domes- were used along lines with a 5–10 m inter-trap space, and tic mammals, identifying R. felis from cat and dog feas the traps were placed outside the buildings. Te rodents (Ctenocephalides felis and C. canis) in central (Metro- were removed from the traps according to standard tech- politan region) and southern Chile (Valdivia) [12–14]. niques [19], and were subsequently anesthetized with Recently, “Candidatus Rickettsia asembonensis”, “Candi- ketamine:xilazine (1:1) [20]. Flea samples from rodents datus Rickettsia senegalensis”, and R. felis, were detected were collected by hand or with forceps from the host in C. felis from cats in the Easter Island (Rapa Nui) [15]. and placed into sterile cryovials tubes with 95% ethanol. No studies have shown their presence in rodent feas. If For each rodent, the total number of extracted feas was this adds to the expansion of the human population invad- recorded (abundance); with these data, the overall mean ing wild areas, the chance of contacting feas on infected infection intensity (the number of feas collected from rodents increases. Since, in some places, peri-urban all species/number of infested hosts), the overall mean rodents provide a link between wild rodent and human abundance of infection (the number of collected feas communities, humans are exposed to some zoonotic from all species/total number of hosts), and prevalence agents that circulate in these natural ecosystems [16, 17]. (the proportion of infected hosts) were calculated. Te Te aim of this study was to detect, characterize, and micromammals were identifed following Iriarte [21]. compare Rickettsia spp. from the feas of micromam- Micromammals were released after sampling, except for mals in areas with diferent human population densities invasive rodents [black rat (Rattus rattus), Norway rat in Chile.