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

doi:10.21203/rs.3.rs-35524/v1

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

https://doi.org/10.21203/rs.3.rs-35524/v1

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published 17 Oct, 2020

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Background

Fleas are important vectors of pathogenic bacteria that pose public health concerns worldwide, including Rickettsia. Micromammals, and especially rodents, are the main flea hosts, therefore they play a fundamental role in the spread of flea-borne diseases and various species of rodent fleas 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 fleas of micromammals in areas with different human population densities in Chile.

Methods

To determine Rickettsia in fleas of micromammals, we analyzed 1,315 fleas obtained from 1,512 micromammals, which were captured in 29 locations with different human population densities in Chile. We used polymerase chain reaction (PCR) amplification of the gltA, rpoB, and ompB genes to detect Rickettsia in fleas.

Results

The prevalence of Rickettsia among flea species ranged from 0%–35.1%. Fifteen flea species were found to be Rickettsia-positive. Areas of lower human density have the highest prevalence of Rickettsia. The phylogenetic tree shows two well-differentiated clades. Rickettsia belli is positioned as basal in a clade. Another clade is subdivided into two subclades, and are related to Rickettsia of typhus group.

Conclusions

Considering that fleas are generalist parasites, and that they are frequently found and abundant in rodents, which are closely related to humans, the detection of potential emerging zoonotic pathogens in rodent fleas highlights the risk of infection to humans.

Parasitology

pathogen

bacteria

vectors

fleas

rodents

marsupials

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, fleas, 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 classified as Candidatus [4]. Recently, using new classification methods based on formal order analysis (FOA), which considers whole-genome sequencing analysis, two groups are recognized within the Rickettsia genus: the major typhus group (MTG) and major spotted fever group (MSFG). The MTG is divided into the typhus group (TG) and ancestral group (AG) and is transmitted by insects. MSFG includes the R. felis group, R. akari group, and “classical” spotted fever group that includes several species transmitted by mites and hard ticks, of which the most important are R. rickettsi and R. conorii, that cause Rocky Mountain spotted fever and Mediterranean spotted fever, respectively [4]. Since Rickettsia research has focused on species that affect humans, other species have received less attention [5]. Thus, there are several species of rickettsiae identified and are exclusively associated with arthropods. They are without known secondary hosts and associated with other organisms such as herbivorous insects, leeches, amoebas, inclusive algae, and plants, indicating that these are more common than suspected [5, 6], and that the effects they could cause in humans when contact is made are unknown.

Worldwide, micromammals, and especially rodents, are the main flea hosts. It is recognized that 74% of known flea species parasitize them; therefore, rodents play a fundamental role in the spread of flea-borne diseases, as various species of rodent fleas can also parasitize humans [7]. 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, and humans [8, 9]. In Chile, despite the great diversity of described fleas (114 species), which mainly parasitize rodents [10, 11], there is only one study that documents R. felis in cat fleas (Ctenocephalides felis) [12], and no studies have shown their presence in fleas or rodents. If this adds to the expansion of the human population invading wild areas, the chance of contacting fleas on infected rodents increases. Since, in some places, peri-urban rodents provide a link between wild rodent and human communities, humans are exposed to some zoonoses that circulate in these natural ecosystems [13, 14].

The aim of this study was to detect, characterize, and compare Rickettsia spp. from the fleas of micromammals in areas with different human population densities in Chile. The findings will provide the baseline for the future surveillance of Rickettsia spp. in Chile and inform risk assessments of human exposure to the infection.

Sample localities and micromammal-trapping procedures

A survey of micromammals and fleas was conducted in 29 localities, covering 10 regions in Chile, between − 20.2167 lat. to -53.1667 lat. (Fig. 1). It was conducted from December 2015 to January 2018, during austral summer (December to February) and austral winter (July and September). These localities were selected based on the following demographic characteristics: 1) City: urban entity that has > 5,000 inhabitants; 2) village: urban entity with a population ranging from 2,001–5,000 inhabitants, or between 1,001 and 2,000 people, and meets the economic activity requirement [15], and 3) natural area, without human settlement. Micromammals were captured using live traps baited with oats. Each locality was sampled for 2 consecutive nights. In each sampling locality, the traps were placed in four parallel lines 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 fleas was recorded (abundance); with these data, the mean infection intensity (the number of fleas collected from all species/number of infested hosts), the mean abundance of infection (the number of collected fleas from all species/number of total hosts), and prevalence (the proportion of infected hosts) were calculated. The rodents were identified based on the methods identified 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]. All procedures were approved by the Universidad de Concepción bioethics committee, Servicio Agrícola y Ganadero (SAG R.E: 8968 − 2015, 1657.2016, 73-2016, 23-2017), and Corporación Nacional Forestal (CONAF N°018-2015).

DNA extraction and PCR amplification

DNA extraction was performed from 1,286 fleas. Each flea 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 flea’s exoskeleton was recovered and stored in 96% ethanol to later mount and identify the flea 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 amplification 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 final 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-confirmed Rickettsia DNA was amplified by the Macrogen Company (Seoul, Korea).

Table 1

Primer sequences and annealing temperatures used to detect *Rickettsia*.
Target gene, and product lenght (bp)	Annealing temperature (°C)	Primer name	Nucleotide sequence (5'–3')
gltA (401)	48 °C*	CS-78_F	GCA AGT ATC GGT GAG GAT GTA AT
		CS-323_R	GCT TCC TTA AAA TTC AAT AAA TCA GGA T
gltA (830)	48 °C*	CS-239_F	GCT CTT CTC ATC CTA TGG CTA TTA T
		CS-1069_R	CAG GGT CTT CGT GCA TTT CTT
rpoB (395)	55.5 °C	RirpoB_F	CCG ACT CAT TAC GGT CGC ATT TGT
		RirpoB_R	CCC ATC AAA GCA CGG TTA GCA TCA
ompB (862)	50 °C**	120.M59F	CCG CAG GGT TGG TAA CTG C
		120.807R	CCT TTT AGA TTA CCG CCT AA
Labruna et al. (2004); *Roux and Raoult (2000).

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 identification

After DNA extraction, each flea’s exoskeleton was recovered and mounted on glass slides using conventional procedures. The fleas were identified 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 fleas infected with Rickettsia) was calculated based on PCR results. The data were analyzed using JMP, and 95% confidence 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 fleas and types of localities. A P-value < 0.05 was considered statistically significant.

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 1,512 micromammals belonging to 18 species were captured during a trapping effort of 11,034 trap/nights. Abrothrix olivacea (n = 434), Abrothrix hirta (n = 319), Rattus (n = 250), and Oligoryzomys longicaudatus (n = 229) were the most frequently collected species. These species presented a wide distribution in Chilean territory. R. rattus was the species with the widest distribution, ranging from − 20 ° to -53 ° lat. S (present in 22 localities), followed by A. olivacea and O. longicaudatus, which were found between − 30 to -53 lat. S, in 20 and 17 localities, respectively. On the other hand, A. hirta was captured between − 34 to -45 lat. S, from 12 locations. On the contrary, other species that were restricted to a single location were Octodon bridgesi (n = 1), Abrothrix lanosus (n = 1), Loxodontomys micropus (n = 24), Phyllotis limatus (n = 2), Chelemys macronyx (n = 1), Reithrodon physodes (n = 5), Irenomys tarsalis (n = 1), Mus musculus (n = 11), and Rattus norvegicus (n = 2).

A total of 2,272 fleas 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 fleas per host and 3.2 fleas per infested host (Table 2). Excluding the species in which < 20 individuals were sampled, the micromammals that presented the highest prevalence of fleas 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 fleas of 51.4%. All of the flea species found in T. elegans corresponded to species that were also found in rodents.

Table 2

Micromammal species captured from 29 locations in Chile.
Family and species of micromammals	no. micromammals collected	no. micromammals with fleas	% Prevalence of fleas (95% CI)	Number of fleas collected	Mean abundance of fleas (95% CI)	Mean intensity of fleas (95% CI)
Order Rodentia
Cricetidae
Abrothrix hirta	319	191	59.9 (0.55–0.65)	643	2.0 (1.73–2.32)	3.4 (2.98–3.76)
Abrothrix lanosus	1	1	100.0	1	1.0	1.0
Abrothrix longipilis	5	4	80.0 (0.34–0.98)	9	1.8 (0.60–2.80)	2.3 (1.25–3.25)
Abrothrix olivacea	434	206	47.5 (0.43–0.52)	518	1.2 (1.03–1.37)	2.5 (2.27–2.80)
Oligoryzomys longicaudatus	229	81	35.4 (0.29–0.41)	162	0.7 (0.55–0.88)	2.0 (1.72–2.36)
Phyllotis darwini	120	49	40.8 (0.32–0.50)	133	1.1 (0.82–1.42)	2.7 (2.24–3.20)
Phyllotis limatus	2	0	0.0	0	0.0
Loxodontomys micropus	24	21	87.5 (0.69–0.96)	66	2.8 (1.96–3.79)	3.1 (2.38–4.24)
Chelemys macronyx	1	0	0.0	0	0.0	-
Reithrodon physodes	5	2	40.0 (0.05–0.85)	6	1.2 (0.00-3.20)	3.0 (1.00–3.00)
Irenomys tarsalis	1	0	0.0	0	0.0	-
Octodontidae
Octodon bridgesi	1	1	100.0	2	2.0	2.0
Octodon degus	69	54	78.3 (0.67–0.87)	387	5.6 (4.20–7.78)	7.2 (5.56–9.93)
Abrocomidae
Abrocoma bennetti	3	3	100.0	77	25.7	25.7
Muridae
Mus musculus	11	2	18.2 (0.02–0.52)	0	0.18 (0.00-0.36)	1 (0.00–0.00)
Rattus norvegicus	2	0	0.0	0	0.0
Rattus rattus	250	73	29.2 (0.27–0.35)	214	0.9 (0.64–1.14)	2.9 (2.40–3.70)
Didelphidae
Thylamys elegans	35	18	51.4 (0.34–0.69)	54	1.54 (0.83–2.97)	3.0 (1.83–5.22)
TOTAL	1,512	706	46.7 (0.44–0.49)	2,272	1.5 (1.38–1.66)	3.2 (2.99–3.59)
The total number of rodents captured for each species, number of parasitized rodents, prevalence of rodents parasitized by fleas, total number of fleas collected, mean abundance, and mean intensity are indicated.

Of all collected fleas, 1,315 flea specimens were analyzed, corresponding to 27 species from 15 genera and eight families (Table 3). The most abundant flea species were Sphinctopsylla ares (n = 211) and Neotyphloceras chilensis (n = 202; Table 4). The rodents that presented the greatest flea richness were A. olivacea (13 spp.), R. rattus (12 spp.), A. hirta (11 spp.), and O. longicaudatus (11 spp.; Table 3). Natural areas were where the largest number of flea species (n = 25) and specimens were collected (n = 784), followed by villages (species n = 18, specimen n = 349) and cities (species n = 18, specimen n = 181). Agastopsylla boxi, Ctenoparia jordani, Ctenoparia topali, Ectinorus cocyti, and Plocopsylla lewisi were exclusive to natural areas. Conversely, Xenopsylla cheopis was only found in one city (Iquique). Neotyphloceras chilensis and S. ares were the dominant species in natural areas (N. chilensis n = 119, and S. ares n = 151), and villages (N. chilensis n = 83, and S. ares n = 50), while in cities, Nosopsyllus fasciatus (n = 37), and Ctenoparia inopinata (n = 25) were the most frequently collected. Leptopsylla segnis, N. fasciatus, and X. cheopis are synanthropic rodent fleas, and were more abundant in cities than in villages and natural areas.

Table 3

Flea species identified for each micromammal species collected in this study.
Family /specie of micromammals	Family of flea	Specie of flea
Cricetidae
Abrothrix hirta	Hystricopsyllidae	Chiliopsylla allophyla (Rothschild, 1915)
		Ctenoparia inopinata (Rothschild, 1909)
		Ctenoparia topali (Smit, 1963)
		Ctenoparia jordani (Smit, 1955)
	Ctenophthalmidae	Neotyphloceras crassispina (Rothschild, 1914)
		Neotyphloceras pardinasi (Sanchez and Lareschi, 2014)
		Neotyphloceras sp.
	Ceratophyllidae	Nosopsyllus fasciatus (Bosc d'Antic, 1800)
	Stephanocircidae	Sphinctopsylla ares (Rothschild, 1911)
	Rhopalopsyllidae	Tetrapsyllus amplus (Jordan and Rothschild, 1923)
		Tetrapsyllus tantillus (Jordan and Rothschild, 1923)
		Tetrapsyllus rhombus (Smit, 1955)
Abrothrix lanosus	Stephanocircidae	Sphinctopsylla ares (Rothschild, 1911)
Abrothrix longipilis	Ctenophthalmidae	Neotyphloceras chilensis (Lewis, 1976)
	Rhopalopsyllidae	Tetrapsyllus corfidii (Rothschild, 1904)
	Pulicidae	Hectopsylla sp.
Abrothrix olivacea	Hystricopsyllidae	Ctenoparia inopinata (Rothschild, 1909)
		Ctenoparia jordani (Smit, 1955)
		Ctenoparia topali (Smit, 1963)
	Ctenophthalmidae	Neotyphloceras crassispina (Rothschild, 1914)
		Neotyphloceras chilensis (Lewis, 1976)
		Neotyphloceras pardinasi (Sanchez and Lareschi, 2014)
		Agastopsylla boxi (Jordan and Rothschild, 1923)
	Ceratophyllidae	Nosopsyllus fasciatus (Bosc d'Antic, 1800)
	Stephanocircidae	Sphinctopsylla ares (Rothschild, 1911)
	Rhopalopsyllidae	Ectinorus cocyti (Rothschild, 1904)
		Tetrapsyllus amplus (Jordan and Rothschild, 1923)
		Tetrapsyllus tantillus (Jordan and Rothschild, 1923)
		Tetrapsyllus rhombus (Smit, 1955)
		Tetrapsyllus corfidii (Rothschild, 1904)
		Listronius sp.
	Pulicidae	Hectopsylla sp.
	Leptopsyllidae	Leptopsylla segnis (Schönherr, 1811)
Oligoryzomys longicaudatus	Hystricopsyllidae	Ctenoparia inopinata (Rothschild, 1909)
		Ctenoparia topali (Smit, 1963)
	Ctenophthalmidae	Neotyphloceras chilensis (Lewis, 1976)
		Neotyphloceras crassispina (Rothschild, 1914)
		Neotyphloceras pardinasi (Sánchez and Lareschi, 2014)
	Ceratophyllidae	Nosopsyllus fasciatus (Bosc d'Antic, 1800)
	Stephanocircidae	Sphinctopsylla ares (Rothschild, 1911)
	Rhopalopsyllidae	Ectinorus chilensis (Lewis, 1976)
		Tetrapsyllus amplus (Jordan and Rothschild, 1923)
		Tetrapsyllus rhombus (Smit, 1955)
	Leptopsyllidae	Leptopsylla segnis (Schönherr, 1811)
Phyllotis darwini
	Ctenophthalmidae	Neotyphloceras chilensis (Lewis, 1976)
		Neotyphloceras crassispina (Rothschild, 1914)
	Stephanocircidae	Sphinctopsylla ares (Rothschild, 1911)
	Rhopalopsyllidae	Delostichus sp.
		Delostichus phyllotis (Johnson, 1957)
		Delostichus smiti (Jameson and Fulk, 1977)
		Tetrapsyllus rhombus (Smit, 1955)
		Tetrapsyllus tantillus (Jordan and Rothschild, 1923)
	Pulicidae	Hectopsylla sp.
	Tungidae	Tunga sp.
Loxodontomys micropus	Ctenophthalmidae	Neotyphloceras sp.
	Stephanocircidae	Sphinctopsylla ares (Rothschild, 1911)
Octodontinidae
Octodon bridgesi	Rhopalopsyllidae	Delostichus phyllotis (Johnson, 1957)
		Tetrapsyllus sp.
Octodon degus	Ctenophthalmidae	Neotyphloceras sp.
		Neotyphloceras chilensis (Lewis, 1976)
	Rhopalopsyllidae	Delostichus sp.
		Delostichus coxalis (Rothschild, 1909)
		Delostichus degus (Beaucournu, Moreno and González, 2011)
		Delostichus phyllotis (Johnson, 1957)
		Delostichus smiti (Jameson and Fulk, 1977)
		Ectinorus chilensis (Lewis, 1976)
		Tetrapsylllus corfidii (Rothschild, 1904)
		Tetrapsyllus tantillus (Jordan and Rothschild, 1923)
Abrocomidae
Abrocoma bennetti	Ctenophthalmidae	Neotyphloceras sp.
		Neotyphloceras chilensis (Lewis, 1976)
	Rhopalopsyllidae	Delostichus sp.
		Delostichus coxalis (Rothschild, 1909)
		Delostichus phyllotis (Johnson, 1957)
		Delostichus smiti (Jameson and Fulk, 1977)
		Ectinorus chilensis (Lewis, 1976)
		Tetrapsyllus corfidii (Rothschild, 1904)
Muridae
Rattus rattus	Hystricopsyllidae	Ctenoparia inopinata (Rothschild, 1909)
		Ctenoparia jordani (Smit, 1955)
	Ctenophthalmidae	Neotyphloceras sp.
		Neotyphloceras chilensis (Lewis, 1976)
		Neotyphloceras pardinasi (Sanchez and Lareschi, 2014)
	Ceratophyllidae	Nosopsyllus fasciatus (Bosc d'Antic, 1800)
	Stephanocircidae	Sphinctopsylla ares (Rothschild, 1911)
		Plocopsylla sp.
		Plocopsylla wolffsohni (Rothschild, 1909)
	Rhopalopsyllidae	Delostichus coxalis (Rothschild, 1909)
		Delostichus smiti (Jameson and Fulk, 1977)
		Tetrapsyllus rhombus (Smit, 1955)
	Leptopsyllidae	Leptopsylla segnis (Schönherr, 1811)
	Pulicidae	Xenopsylla cheopis (Rothschild, 1903)
		Hectopsylla sp.
Mus musculus	Leptopsyllidae	Leptopsylla segnis (Schönherr, 1811)
Order Didelphimorphia
Didelphidae
Thylamys elegans	Stephanocircidae	Sphinctopsylla ares (Rothschild, 1911)
	Ctenophthalmidae	Neotyphloceras sp.
		Neotyphloceras chilensis (Lewis, 1976)
		Neotyphloceras crassispina (Rothschild, 1914)
	Rhopalopsyllidae	Delostichus smiti (Jameson and Fulk, 1977)
		Tetrapsyllus tantillus (Jordan and Rothschild, 1923)

Table 4

*Rickettsia* prevalence detected on fleas for each gene used in the different flea species analyzed.
Family and species of fleas	Number of fleas analyzed		N° of fleas positive for gene fragment (% prevalence)
Family and species of fleas	Number of fleas analyzed	gltA- 401 bp	gltA- 830 bp	rpoB- 395 bp	ompB- 862 bp
Hystricopsyllidae
Chiliopsylla allophyla	7	2 (28.6)	2 (28.6)	2 (28.6)	2 (28.6)
Ctenoparia sp.	20	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Ctenoparia inopinata	85	1 (1.2)	1 (1.2)	1 (1.2)	1 (1.2)
Ctenoparia topali	2	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Ctenoparia jordani	5	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Ctenophthalmidae
Agastopsylla boxi	3	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Neotyphloceras sp.	128	40 (31.3)	7 (5.5)	10 (7.8)	0 (0.0)
Neotyphloceras crassispina	35	2 (5.7)	0 (0.0)	2 (5.7)	0 (0.0)
Neotyphloceras chilensis	202	71 (35.1)	29 (14.4)	29 (14.4)	0 (0.0)
Neotyphloceras pardinasi	43	7 (16.3)	3 (7.0)	5 (11.6)	0 (0.0)
Ceratophyllidae
Nosopsyllus fasciatus	52	7 (13.5)	1 (1.9)	2 (3.8)	0 (0.0)
Stephanocircidae
Sphinctopsylla ares	211	20 (9.5)	16 (7.6)	19 (9.0)	2 (0.9)
Plocopsylla sp.	4	2 (50.0)	0 (0.0)	0 (0.0)	0 (0.0)
Plocopsylla wolffsohni	2	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Plocopsylla lewisi	1	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Rhopalopsyllidae
Delostichus sp.	12	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Delostichus degus	22	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Delostichus coxalis	53	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Delostichus phyllotis	7	1 (14.3)	0 (0.0)	0 (0.0)	0 (0.0)
Delostichus smiti	85	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Ectinorus sp.	1	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Ectinorus cocyti	1	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Ectinorus chilensis	12	1 (8.3)	0 (0.0)	0 (0.0)	0 (0.0)
Tetrapsyllus sp.	11	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Tetrapsyllus amplus	17	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Tetrapsyllus tantillus	93	10 (10.8)	1 (1.1)	1 (1.1)	0 (0.0)
Tetrapsyllus corfidii	16	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Tetrapsyllus rhombus	74	8 (10.8)	6 (8.1)	7 (9.5)	1 (1.4)
Listronius sp.	3	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Tungidae
Tunga sp.	4	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Pulicidae
Hectopsylla sp.	30	1 (3.3)	0 (0.0)	0 (0.0)	0 (0.0)
Xenopsylla cheopis	11	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Leptopsyllidae
Leptopsylla segnis	63	1 (1.6)	0 (0.0)	0 (0.0)	0 (0.0)
TOTAL	1,315	174 (13.2)	66 (5.0)	78 (5.9)	6 (0.5)

Rickettsia prevalence on fleas

Fifteen flea species were found to be Rickettsia-positive for the gltA-401 bp gene (short fragment), nine with gltA-830 bp (long fragment), ten with rpoB, and four with ompB (Table 4). Among the flea species in which > 20 individuals were analyzed, the prevalence varied between 0% and 35.1%. The Neotyphloceras species had the highest prevalence of Rickettsia (gltA-401bp = 29.4%, gltA-830bp = 9.56%, and rpoB = 11.25%; Table 4). The four genes (gltA-401 bp, gltA-830 bp, rpoB, and ompB) showed significant differences in the prevalence of detected Rickettsia (chi-squared statistic = 193.207, df = 3; P = 0.000), with the exception of gltA-830 bp and rpoB, which did not show significant differences (chi-squared statistic = 1.934, df = 1; P = 0.164). The highest prevalence (13.6%) was detected with the gltA-401 bp gene, followed by the rpoB (6.8%), gltA-830 bp (4.8%), and ompB (0.5%) genes. No association was found between the number of fleas analyzed and the prevalence of Rickettsia detected for any of the genes analyzed (rpoB: Spearman ρ = 0.4267; P = 0.12; gltA: Spearman ρ = 0.3757; P = 0.18; ompB: Spearman ρ = 0.3272; P = 0.35).

Regarding flea origins based on locality type, natural areas and villages had the highest concentrated prevalence of Rickettsia in fleas. With gltA-401 bp, the highest prevalence of Rickettsia was detected in natural areas (analyzed fleas, n = 784; Rickettsia positive, n = 126, 16.07%), followed by villages (analyzed fleas, n = 349; Rickettsia-positive, n = 39, 11.17%), and cities (analyzed fleas, n = 181; Rickettsia-positive, n = 9, 4.97%; chi-squared statistic = 17.536; P = 0.000). With gltA-830 bp, a higher prevalence was detected in villages (Rickettsia-positive, n = 32, 9.17%), followed by natural areas (Rickettsia-positive, n = 33; 4.21%), and cities (Rickettsia-positive, n = 1, 0.55%; chi-squared statistic = 21,247, df = 2; P = 0.000). A similar finding was observed with rpoB, where the highest prevalence was observed in the village (Rickettsia-positive, n = 40, 11.46%; chi-squared statistic = 28.057, df = 2; P = 0.000), and was low in cities (Rickettsia-positive, n = 3, 1.66%) and natural areas (Rickettsia-positive, n = 35, 4.46%); the latter did not differ significantly (Fisher’s exact test P = 0.382).

BLAST analysis and phylogenetic inference

We obtained 43 sequences for gltA-401 bp, 24 sequences for gltA-830, 21 for rpoB, and only two for ompB (Table 5). Only four sequences showed homology with bacteria identified at the species level: R. felis (94%, ompB) isolated from Liposcelis bostrychophila (Psocoptera: Liposcelidae) from Australia [22], Escherichia coli (100%, rpoB) isolated from humans from Myanmar [23], Candidatus Rickettsia senegalensis (gltA-830 bp) from Ctenocephalides felis in India, and R. hoogstraalii (88%, ompB) isolated from tick Carios capensis in the United States [24]. The other sequences showed similarity to Rickettsia that have not been identified at the species level and isolated from different organisms (Table 5).

Table 5

Similarity percentage for obtained sequences with BLAST analyses.
Rickettsia spp. (GenBank access number)	Registered host species	Similarity %	Flea species	Host species in this study	Location
gltA- 401 bp
Rickettsia sp. MEAM1 (CP016305)	Bemisia tabaci	99	Neotyphloceras chilensis	Abrothrix olivacea, Octodon degus, Phyllotis darwini, Thylamys elegans,	Las Chinchillas N.R., Canela Baja, Fray Jorge N.P.
			Neotyphloceras pardinasi	Rattus rattus	Canela Baja
			Neotyphloceras sp.	Abrothrix olivacea	Canela Baja, Fray Jorge N.P.
Rickettsia sp. Gr15 (KP675966)	Hyalomma marginatum	99	Chiliopsylla allophyla, Ctenoparia inopinata	Abrothrix hirta	Nonguén N.R. Los Queules N.R.
uncultured Rickettsia sp. (KY433588)	Tick	93	Sphinctopsylla ares	Abrothrix olivacea	Cobquecura
Rickettsia sp. (KY705378)	Amblyomma parvitarsum	100	Delosticus phyllotis	Octodon degus	La Campana N.P.
			Leptopsylla segnis	Rattus rattus	Lolol
			Neotyphloceras crassispina	Oligoryzomys longicaudatus	Laguna Torca N.R.
			Neotyphloceras pardinasi	Rattus rattus	La Campana N.P., Laguna Torca N.R.
			Neotyphloceras sp.	Abrothrix hirta, Abrothrix olivacea, Oligoryzomys longicaudatus, Thylamys elegans	Laguna Torca N.R.
			Nosopsyllus fasciatus	Rattus rattus	Lolol, Santa Cruz
			Plocopsylla sp.	Rattus rattus	La Campana N.P., Til Til
			Sphinctopsylla ares	Rattus rattus, Thylamys elegans	Laguna Torca N.R.
			Tetrapsyllus rhombus	Rattus rattus	La Campana N.P.
			Tetrapsyllus tantillus	Abrothrix olivacea, Octodon degus,	Lolol, La Campana N.P., Til Til
Rickettsia sp. (U59712)	Adalia bipunctata	97	Neotyphloceras sp.	Abrothrix hirta	Los Queules N.R.
		98	Sphinctopsylla ares	Abrothrix hirta, Abrothrix olivacea	Los Queules N.R., Cobquecura, Coyhaique N.R.
		97	Tetrapsyllus rhombus	Abrothrix hirta, Abrothrix olivacea	Los Queules N.R., Cobquecura, Coyhaique N.R.
gltA- 830 bp
Candidatus Rickettsia senegalensis (KU499847)	Ctenocephalides felis	99	Chiliopsylla allophyla	Abrothrix hirta	Nonguén N.R.
Rickettsia sp. (KY799066)	Mansonia uniformis	97	Ctenoparia inopinata	Abrothrix hirta	Los Queules N.R.
			Sphinctopsylla ares	Abrothrix olivacea	Los Queules N.R.
Rickettsia sp. (U76908)	Empoasca papayae	97	Neotyphloceras chilensis	Abrothrix olivacea, Octodon degus, Phyllotis darwini, Thylamys elegans	Canela Baja, Fray Jorge N.P.
			Neotyphloceras pardinasi	Rattus rattus	Canela Baja
			Neotyphloceras sp.	Abrothrix olivacea, Thylamys elegans	Canela Baja, Fray Jorge N.P.
Rickettsia sp. (KF646706)	Nesidiocoris tenuis	97	Neotyphloceras chilensis	Abrothrix olivacea, Phyllotis darwini	Las Chinchillas N.R.
Rickettsia sp. (AJ269522)	Adalia decempunctata	98	Sphinctopsylla ares	Abrothrix hirta Abrothrix olivacea	Los Queules N.R., Cobquecura, Coyaique N.R.
			Tetrapsyllus rhombus	Abrothrix hirta, Abrothrix olivacea,	Los Queules N.R., Cobquecura, Coyhaique N.R.
Uncultured Bartonella sp. (EF662053.1)	Ixodes scapularis	96	Tetrapsyllus tantillus	Abrothrix olivacea	Lolol
Bartonella sp. AR 15 − 3 (FN645480)	Sciurus vulgaris		Neotyphloceras pardinasi	Abrothrix hirta	Cobquecura
			Neotyphloceras chilensis	Abrothrix olivacea	Las Chinchillas N.R.
rpoB- 395 bp
Rickettsia sp. (JF966777)	Synosternus pallidus	99–100	Chiliopsylla allophyla	Abrothrix hirta	Nonguén N.R.
		97	Ctenoparia inopinata	Abrothrix hirta	Los Queules N.R.
		91	Neotyphloceras sp.	Abrothrix hirta	Los Queules N.R.
		91–95	Sphinctopsylla ares	Abrothrix hirta, Abrothrix olivacea	Los Queules N.R., Cobquecura, Coyhaique N.R.
Rickettsia sp. (CP016305)	Bemisia tabaci	98	Neotyphloceras chilensis	Abrothrix olivacea, Octodon degus, Phyllotis darwini, Thylamys elegans	Canela Baja, Fray Jorge N.P., Las Chinchillas N.R.
		93	Neotyphloceras pardinasi	Rattus rattus	Canela Baja
		98	Neotyphloceras sp.	Abrothrix olivacea	Canela Baja
Escherichia coli (AP019189)	Human	100	Neotyphloceras sp.	Abrothrix olivacea, Thylamys elegans	Fray Jorge N.P.
			Tetrapsyllus tantillus	Abrothrix olivacea	Lolol
			Nosopsyllus fasciatus	Rattus rattus	Illapel
ompB- 862 bp
Rickettsia felis (GQ385243)	Liposcelis bostrychophila	94	Chiliopsylla allophyla	Abrothrix hirta	Nonguén N.R.
			Ctenoparia inopinata	Abrothrix hirta	Los Queules N.R.
Rickettsia hoogstraalii (EF629536)	Carios capensis	88	Sphinctopsylla ares	Abrothrix olivacea	Los Queules N.R.
There are showed genes, GenBank accession numbers, references, similarity percent (%), flea species positive for Rickettsia, micromammal flea hosts, and locations where fleas were collected.

The phylogenetic tree shows two well-differentiated clades with 100% support in the node (Fig. 2). Clade R1 is formed by sequences obtained from Neotyphloceras fleas collected in Las Chinchillas N. R. (-31° lat S), Canela Baja (-31° lat S), and Fray Jorge N. P. (-30°lat S). R. belli (GenBank accession number: gltA DQ146481; rpoB DQ870673) is positioned on a basal branch in this group. The clade R2 is subdivided into two subclades: R2a and R2b. R2a, with 100% support in the node, is related to sequences obtained from Tetrapsyllus rhombus and S. ares collected in Los Queules N. R., Cobquecura, and Coyhaique N.R., comprising a larger area of distribution (-35° to -45° lat S) than clade R1. Subclade R2b is formed by a member of typhus group, and sequences obtained from Ctenoparia inopinata and Chiliopsylla allophyla were collected in Los Queules N. R. and Nonguén N. R. The obtained sequences were positioned closely to R. hoogstralii (gltA FJ767737, rpoB LAOB01000001) isolated from Haemaphysalis sulcata (tick) in Croatia [25] and Corvus capensis; R. asembonensis was detected in Ctenocephalides felis from Kenya (rpoB: JWSW01000001) [26], and Peru (gltA KY650697) [27] and R. felis was isolated from C. felis in Brazil (GenBank accession number: gltA JN375498, rpoB CP000053) [28].

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 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 occupied more than one species of micromammal. We also highlight the high flea species richness recorded in 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. Rickettsia-positive fleas 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 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 [30]. These findings 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 fleas. This rodent decreased its presence in areas with human intervention, which is consistent with the findings 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 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 [32], and which exhibits some specificity 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 classified 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 inflammatory 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 flea 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 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 Chiliopsylla allophyla and Ctenoparia 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 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, 39–41]. 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 fleas, 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) [43–47]. 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, flea-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 fleas 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 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.

Ethics approval and consent to participate

All procedures were approved by the Universidad de Concepción bioethics committee, Servicio Agrícola y Ganadero (SAG R.E: 8968-2015, 1657.2016, 73-2016, 23-2017), and Corporación Nacional Forestal (CONAF N°018-2015).

Consent for publication: Not applicable.

Availability of data and materials: Not applicable.

Competing interests: Not applicable.

Funding

This work was supported by the National Fund for Scientific and Technological Development [FONDECYT 11150875, 1130948 and 1170972].

Authors' contributions

Lucila Moreno Salas, Mario Espinoza-Carniglia, Nicol Lizama-Schmeisser, Luis Gonzalo Torres Fuentes, Maria Carolina Silva-de La Fuente and Daniel González-Acuña participate on rodent sampling, identifying rodent species and flea’s extraction. Lucila Moreno Salas, Nicol Lizama-Schmeisser and Marcela Lareschi made fleas identification on laboratory. Mario Espinoza-Carniglia performed DNA extraction and PCR analyses. All authors read and approved the final manuscript.

Acknowledgements

English-language editing of this manuscript was provided by Journal Prep Services.

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

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Abstract

Figures

Introduction

Materials And Methods

Sample localities and micromammal-trapping procedures

DNA extraction and PCR amplification

Phylogenetic and BLAST analyses

Flea mounting and identification

Statistical analysis

Nucleotide sequence accession numbers

Results

Rickettsia prevalence on fleas

BLAST analysis and phylogenetic inference

Discussion

Declarations

References

Supplementary Files

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Journal Publication

Version 1