Molecular Detection of Rickettsia in Fleas from Micromammals in Chile with Potential Public Health Implications

Background Fleas are important vectors of pathogenic bacteria that pose public health concerns worldwide, including Rickettsia. Micromammals, and especially rodents, are the main ea hosts, therefore they play a fundamental role in the spread of ea-borne diseases and various species of rodent eas can also parasitize humans. In addition to this, many rodent species are capable of inhabiting wild environments and adapting to rural and urban environments, which could favor a continuous gradient of transmission between domestic and wild species. The aim of this study was to detect, characterize, and compare Rickettsia spp. from the eas of micromammals in areas with different human population densities in Chile. Methods There are showed genes, GenBank accession numbers, references, similarity percent (%), ea species positive for Rickettsia, micromammal ea hosts, and locations where eas were collected.


Introduction
Rickettsia are obligate intracellular microorganisms, Gram-negative coccobacilli, with the ability to reproduce, both in the nucleus and in the cytoplasm of infected cells [1]. These bacteria have a vertebrate reservoir and an arthropod vector (e.g., ticks, mites, eas, and lice); in some cases, the latter may be affected by these bacteria [2]. They have a worldwide distribution and are the causative agents of serious human infections [3]. Currently, 32 species are recognized (http://www.bacterio.net/-allnamesmr.html), and there are many strains that have not yet been characterized, while subspecies and uncultivated species are classi ed as Candidatus [4]. Recently, using new classi cation methods based on formal order analysis (FOA), which considers whole-genome sequencing analysis, two groups are recognized approximately 100 m from each other, and each line was equipped with 50 traps set 10 m apart from each other. The rodents were removed from the traps according to standard techniques [16], and were subsequently anesthetized with ketamine:xilazine (1:1). Flea samples from rodents were collected by hand or with forceps from the host and placed into sterile cryovials tubes with 95% ethanol. For each rodent, the total number of extracted eas was recorded (abundance); with these data, the mean infection intensity (the number of eas collected from all species/number of infested hosts), the mean abundance of infection (the number of collected eas from all species/number of total hosts), and prevalence (the proportion of infected hosts) were calculated. The rodents were identi ed based on the methods identi ed by Iriarte (2007). Rodents were released after sampling, except for synanthropic rodents (Rattus, Rattus norvegicus, and Mus musculus) that were euthanized by cervical dislocation [16]. DNA extraction and PCR ampli cation DNA extraction was performed from 1,286 eas. Each ea was washed and cut between the third and fourth abdominal tergite with a scalpel. DNA was extracted using a commercial kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. The incubation time was 5 hours; following DNA extraction, the ea's exoskeleton was recovered and stored in 96% ethanol to later mount and identify the ea species.
The presence of Rickettsia was screened using three genes: citrate synthase (short fragment gltA-401 bp and long fragment gltA-830 bp) [18], outer membrane protein B (ompB) [19], and we designed a set of primers for the β-subunit of RNA polymerase (rpoB) of Rickettsia sp. (GenBank access number: AF076436; Table 1). The ampli cation conditions were as follows: 5 minutes at 95 °C, 40 cycles of 30 seconds at 95 °C, 30 seconds of annealing temperature (see Table 1), 30 seconds at 72 °C, followed by a nal extension of 5 minutes at 72 °C. The reactions were performed with Green Master Mix 2 × 12.5 µL, 5.5 µL of ultrapure nuclease-free water, 2 µL of forward primer (10 µM), 2 µL of reverse primer (10 µM), and 4 µL of DNA sample. The negative controls were carried out with ultrapure water and the positive control was genomic DNA of R. conorii (Microbiologist, Vircell, Granada, Spain). Sequence-con rmed Rickettsia DNA was ampli ed by the Macrogen Company (Seoul, Korea).

Phylogenetic and BLAST analyses
All DNA sequences were edited and aligned using the Codon Code Aligner (CodonCode Corporation, Centerville, MA, USA). All sequences of this study were compared with those available in GenBank using the BLAST program (see http://www.ncbi.nlm.nih.gov/BLAST/). A Bayesian probabilities tree was created using MrBayes 3.2 based on concatenated gltA-401 bp, gltA-830 bp, and rpoB gene fragments, using Anaplasma phagocytophilum as an outgroup. We used the GTR + G substitution model to reconstruct the tree and 10,000,000 bootstrap trials. The GenBank sequence accession numbers used to reconstruct the tree are detailed in Fig. 2.

Flea mounting and identi cation
After DNA extraction, each ea's exoskeleton was recovered and mounted on glass slides using conventional procedures. The eas were identi ed using a light microscope, taxonomic keys, and the descriptions of Johnson [20], and Sanchez and Lareschi [21]. Voucher specimens (slides) were catalogued in the Museo de Zoología at Universidad de Concepción (MZUC-UCCC, Chile).

Statistical analysis
The prevalence (percentage of eas infected with Rickettsia) was calculated based on PCR results. The data were analyzed using JMP, and 95% con dence intervals (CIs) are reported in the text. To assess the relationship between the prevalence and sample size, a Spearman correlation analysis was performed.
The chi-squared test or Fisher's exact test (if an expected cell count was < 5) was used to evaluate the differences in the prevalence of Rickettsia among species of eas and types of localities. A P-value < 0.05 was considered statistically signi cant.

Nucleotide sequence accession numbers
Rickettsia sequences generated in this study were deposited in the NCBI GenBank database under the following accession numbers: MN630893-MN630962 for gltA and MN630963-MN630997 for rpoB.
A total of 2,272 eas were collected from 13 micromammal species in 23 localities (9 cities, 6 villages, 8 natural area), with a total prevalence of 46.6% (n = 706) infected micromammals. There was a mean abundance of 1.5 eas per host and 3.2 eas per infested host ( Table 2). Excluding the species in which < 20 individuals were sampled, the micromammals that presented the highest prevalence of eas were L. micropus (87.5%) and Octodon degus (78.3%), and the lowest prevalence was found in R. rattus (29.2%). The abundance and mean intensity were higher in O. degus (Table 2). We captured only one marsupial species (Didelphimorphia), Thylamys elegans (n = 35), which had a prevalence of eas of 51.4%. All of the ea species found in T. elegans corresponded to species that were also found in rodents. The total number of rodents captured for each species, number of parasitized rodents, prevalence of rodents parasitized by eas, total number of eas collected, mean abundance, and mean intensity are indicated.

Discussion
In this study, for the rst time, we have evidence of the presence of Rickettsia DNA in 15 ea species identi ed on wild micromammals and synanthropic rodents from Chile. The eas were characterized as being highly host-opportunistic, occupying various host species [7], This is con rmed by our study, since of the 27 ea species collected, 19 occupied more than one species of micromammal. We also highlight the high ea species richness recorded in Rattus, where 10 of the 14 species identi ed in this rodent correspond to the ea species identi ed on native rodents. This rodent was mainly captured in urban areas; however, we also found it in rural and natural areas. Rickettsia-positive eas were also found in these three areas. This species could play a key role in spreading the disease from wild to urban environments [13,29]. 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 ea richness and the highest number of Rickettsia-positive eas. This species has been described to have a "random walk" type of dispersal behavior, so it can easily go from wild to domestic environments [30]. These ndings are important because these rodent species could act as "bridge hosts" and aid in the spread of the disease [29,31]. 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 eas. This rodent decreased its presence in areas with human intervention, which is consistent with the ndings reported by Monteverde and Hadora [30], 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 [31].
In this study, we found two well-differentiated clades with a high degree of support. Clade R1 is formed by sequences obtained from eas 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 [32], and which exhibits some speci city concerning its host [33]. 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 [33]. It has been classi ed as non-pathogenic for animals and humans [34], although seropositive samples have been found in dog blood in Brazil; however, the pathogenic effect is unknown [35]. 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 in ammatory reaction to necrotic scabs -typical symptomatology of other pathogenic rickettsiae [28]. Furthermore, this bacterium is capable of producing antibodies in experimental infections in opossum Didelphis aurita, but without ricketsemia [36]. These results indicate that some ea species present in wild and synanthropic micromammals could carry a new ancestral genotype of Rickettsia, just like those reported by Song et al. [37] in China from the eas of wild rodents.
The R2 clade was divided into two large groups, R2a and R2b. R2a grouped all of the sequences detected in eas as being extracted from two species of eas, 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 eas (A. hirta and A. olivacea). Conversely, R2b was formed by sequences obtained from Chiliopsylla allophyla and Ctenoparia inopinata that belong to the same family (Hystricopsylidae); both species of eas were collected in wild rodents (A. hirta and A. olivacea) from wild areas (Los Queules and Nonguén) 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) [27,28,32]. The SFG consists of > 30 species that can be found worldwide, most of them with pathogenic effects on humans [38]. 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,[39][40][41]. 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 [42], although it is associated with a greater number of ectoparasites, including eas, ticks, and mites of domestic and peridomestic animals (Ctenocephalides canis, Ctenocephalides felis, Xenopsylla cheopis, Pulex irritans, Amblyomma ovale, Rhipicephalus sanguineus, Rhipicephalus microplus, and Ornithonysus bacoti) [43][44][45][46][47]. It has also been detected in monkey blood in Malaysia [48] and in dog blood in South Africa [49]. 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 [42]. On the other hand, R. felis is an emergent, widely distributed, ea-borne human pathogen, and like R. asemboensis and R. hoogstrali, is associated with domestic and peridomestic animals and their ectoparasites. The main vector is C. felis, although mosquitoes (Anopheles gambiae) have also been detected as competent vectors [50]. 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 [43]. Although the Blast analysis shows a low percentage of similarity with R. felis (ompB 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 the presence of Rickettsia in different species of parasitic eas of wild micromammals and synanthropic rodents found in both natural and human environments. Moreover, there is evidence of at least two clades of Rickettsia associated with eas. 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.

Declarations
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