In this study, for the first time, we have evidence of the presence of Rickettsia DNA in 15 flea species identified on wild micromammals and synanthropic rodents from Chile. The prevalence of Rickettsia spp. infections in fleas varied between species of fleas, bioclimatic regions, seasons, and location type, and we found a higher prevalence in winter, semi-arid region and natural areas.
The fleas were characterized as being highly host-opportunistic, occupying various host species [7]. This is confirmed by our study, since of the 27 flea species collected, 19 parasitized more than one species of micromammal. We also highlight the high flea species richness recorded in R. rattus, where 10 of the 14 species identified in this rodent correspond to the flea species identified on native rodents. This rodent was mainly captured in urban areas; however, we also found it in rural and natural areas, this occurs mainly because these rodents have an omnivore diet and plasticity in their behavior, characteristics that allow them to inhabit a great diversity of environments, adapting successfully to urban, rural and wild environments. [30,31]. Rickettsia-positive fleas parasitizing R. rattus in these three areas indicate that this species could play a key role in spreading the disease from wild to urban environments [16,32]. Conversely, we also observed that wild species enter human-occupied environments since they provide shelter and food. Abrothrix olivacea was the most frequently captured wild species in urban and rural areas and had the highest flea richness and the highest number of Rickettsia-positive fleas. This species has been described to have a “random walk” type of dispersal behavior, so it can easily go from wild to domestic environments [33]. These findings are important because these rodent species could act as “bridge hosts” and aid in the spread of the disease [32,34]. On the other hand, in natural areas, the rodent species most frequently captured was A. hirta; this, like A. olivacea, had a high prevalence of Rickettsia-positive fleas. This rodent decreased its presence in areas with human intervention, which is consistent with the findings reported by Monteverde and Hadora [33], who described that this rodent preferably moves within the wild environment. Rodent populations can act as “source populations” and may be involved in the direct transmission of the pathogen to the target population [34].
The prevalence of Rickettsia spp. infections detected in our study is variable (0-35%), and associated with the identity of the flea species, season, type of locality and bioclimatic area. However, similar differences have been reported in other studies. For example, Radzijevskaja et al. report different prevalence related to the flea species analyzed (range: 0 to 43%) [35]. Also, Kuo et al. [36] carried out an extensive sampling analyzing the presence of Rickettsia in six species of fleas, reporting 0% to 12.1% of prevalence in the different species of fleas analyzed. Furthermore, flea infestations in this study were generally higher during the winter; however, this did not occur in all bioclimatic areas. Other studies have found similar results, attributing this variation to the differences in the seasonal reproductive cycles of the different species of fleas [37], which are unknown in most of the species found in this study. On the other hand, the higher prevalence of Rickettsia in fleas detected in natural areas can be explained by the greater diversity of species of micro-mammals and, therefore, of fleas. Thus, the differences in the prevalence of infection in the different species of fleas, localities, seasons and bioclimatic zones found in our study, reveals the importance of the composition of the community, both fleas, and hosts, in determining the prevalence of Rickettsia in fleas, and therefore in the risk of infection in areas with different human disturbance.
In this study, we found two well-differentiated clades with a high degree of support. Clade R1 is formed by sequences obtained from fleas of the Neotyphloceras genus, collected from rodents Phyllotis darwini, A. olivacea, O. degus, R. rattus, and the marsupial T. elegans from central-north Chile (-30° to -31° lat. S). This clade is related to R. bellii and is described as an ancestral group of Rickettsia [38], and which exhibits some specificity concerning its host [39]. This supports our results, where only bacteria detected in Neotyphloceras were found in this clade. R. belli is endosymbiont of hard (Ixodidae) and soft (Argasidae) ticks throughout the American continent [39]. It has been classified as non-pathogenic for animals and humans [40], although seropositive samples have been found in dog blood in Brazil; however, the pathogenic effect is unknown [41]. Experimentally, this bacterium grows easily in mammalian cells. In experimental inoculations in guinea pig and rabbit, it produces – depending on the inoculated dose received, from a mild inflammatory reaction to necrotic scabs – typical symptomatology of other pathogenic rickettsiae [29]. Furthermore, it is capable of producing antibodies in experimental infections in opossum Didelphis aurita, but without ricketsemia [42]. These results indicate that some flea species present in wild and synanthropic micromammals could carry a new ancestral genotype of Rickettsia, just like those reported by Song et al. [43] in China from the fleas of wild rodents.
The R2 clade was divided into two large groups, R2a and R2b. R2a grouped all of the sequences detected in fleas as being extracted from two species of fleas, S. ares (Stephanocircidae) and Tetrapsyllus rhombus (Rhopalopsyllidae), which were obtained from villages and natural environments through wide latitudinal distribution (-35° to -45° lat. S). This corresponds to the wide distribution of the hosts of infected fleas (A. hirta and A. olivacea). Conversely, R2b was formed by sequences obtained from C. allophyla and C. inopinata that belong to the same family (Hystricopsylidae); both species of fleas were collected in wild rodents (A. hirta and A. olivacea) from wild areas (Los Queules N. R. and Nonguén N. R.) in the south-central zone of Chile. These sequences are closely related to R. hoogstrali, R. asembonensis, and R. felis, all of which are members of the spotted fever group rickettsiae (SFG) [28,29,38]. The SFG consists of >30 species that can be found worldwide, most of them with pathogenic effects on humans [44]. Our analysis showed a close relationship with R. hoogstrali, a widely distributed bacterium that is still unknown for its pathogenicity in humans. This bacterium has been detected in hard ticks (Haemaphysalis punctata, Haemaphysalis sulcate, and Haemaphysalis parva), and soft ticks (Ornithodoros moubata, Carios capensis, C. sawaii, and Argas persicus) present in domestic animals, bird nests, vegetation, and human dwellings [3,45–47]. A similar situation occurs with R. asemboensis. It also has a wide distribution worldwide, having been reported in North America and South America, Asia, the Middle East, and Europe [48], although it is associated with a greater number of ectoparasites, including fleas, ticks, and mites of domestic and peridomestic animals (C. canis, C. felis, X. cheopis, Pulex irritans, Amblyomma ovale, Rhipicephalus sanguineus, Rhipicephalus microplus, and Ornithonysus bacoti) [49–53]. It has also been detected in monkey blood in Malaysia [54] and in dog blood in South Africa [55]. Although these bacteria live in parasitic arthropods close to humans and are closely associated with R. felis, there is no evidence yet of possible infection or pathogenicity [48]. On the other hand, R. felis is an emergent, widely distributed, flea-borne human pathogen, and like R. asemboensis and R. hoogstrali, is associated with domestic and peridomestic animals and their ectoparasites [56,57]. The main vector is C. felis, although mosquitoes (Anopheles gambiae) have also been detected as competent vectors [58]. Unlike R. asemboensis and R. hoogstrali, this bacterium is of known pathogenicity causing fever, fatigue, nausea, muscle aches, back pain, headaches, macular rash, joint pain, and eschar [49]. Although the Blast analysis shows a low percentage of similarity with R. felis (sca5 94%), the phylogenetic analysis shows a close relationship with Rickettsia detected in C. allophyla in south-central Chile. Until now, in Chile, only R. felis was registered in C. felis [12].
Our study reports, for the first time in Chile, the presence of Rickettsia in different species of parasitic fleas of wild micromammals and invasive rodents found in both natural and human environments. Moreover, there is evidence of at least two clades of Rickettsia associated with fleas. These data increase the knowledge of possible Rickettsia vectors/reservoirs in Chile. However, greater efforts should be made to monitor and determine the degree of pathogenicity of the detected rickettsiae.