DOI: 10.5061/dryad.ghx3ffc1z
title: Fleas of wild mammals carrying pathogenic bacteria in Argentinian Patagonia: a study based on wildlife roadkill

description: Fleas (Insecta: Siphonaptera) are recognized vectors of bacteria that
 affect human and other animal health, whose reservoirs are in the majority
 mammals. Among these, some species of the genera Rickettsia
 (Rickettsiales: Rickettsiaceae) and Bartonella (Rhizobiales:
 Bartonellaceae) are emerging and re-emerging throughout the world;
 however, their circulation across vast regions of Argentina and numerous
 animal species, particularly wild species, remains largely unknown. The
 study of wild animal roadkill provides valuable insights into parasitic
 associations and the presence of pathogenic microorganisms, allowing the
 generation of a health alert in certain ecosystems. The aim of this study
 was to describe the diversity of fleas associated with roadkilled wild
 native meso-mammals in the extreme northeast of Argentinian Patagonia and
 to detect the presence of Rickettsia and Bartonella bacteria. Five host
 species were examined, including Chaetophractus villosus Desmarest
 (Cingulata: Chlamyphoridae); Didelphis albiventris Lund (Didelphimorphia:
 Didelphidae); Lagostomus maximus Desmarest (Rodentia: Chinchillidae);
 Leopardus geoffroyi d'Orbigny & Gervais (Carnivora: Felidae)
 and Lycalopex gymnocercus Fischer (Carnivora:  Canidae). A total
 of 248 fleas were recovered, identified as Hectopsyllidae: Hectopsylla
 broscus Jordan & Rothschild and Hectopsylla cypha Jordan;
 Malacopsyllidae: Phthiropsylla agenoris Rothschild and Malacopsylla
 grossiventris Weyenbergh; Pulicidae: Ctenocephalides felis Bouché and
 Pulex irritans Linnaeus. Molecular analysis detected two flea-borne
 pathogenic bacteria: Rickettsia felis (Bouyer et al.), found in C. felis
 from D. albiventris, and Bartonella rochalimae (Eremeeva et al.), reported
 here for the first time in Argentina, detected in P. agenoris from C.
 villosus, and in P. irritans from L. maximus and L. gymnocercus. The
 results contribute to knowledge of flea diversity in Argentinian Patagonia
 and provide new information about flea-borne pathogens circulating in the
 wildlife of this region. Furthermore, this study is the first in Argentina
 to investigate ectoparasites and their associated bacteria in roadkilled
 animals, making a pioneering contribution to the field. The interesting
 findings highlight the importance of implementing and expanding road
 ecology studies, which could easily be replicated in other regions where
 information gaps on flea and flea-borne bacteria diversity still exist.
description: <strong>2.1 Study area</strong>
 The study was conducted in the extreme northeast of Río Negro
 province, Argentinian Patagonia, on the coast of the Atlantic Ocean (Fig.
 1; Supplemental Material 1). The natural ecosystem corresponds to the
 Monte Desert biome (Abraham et al. 2009). The climate is semiarid to arid,
 with high evaporation enhanced by windy conditions, and mean annual
 rainfall varies between 100 and 450 mm. The vegetation is rather uniform
 in terms of physiognomy and flora composition (Abraham et al. 2009;
 Oyarzabal et al. 2018). The area covers Provincial
 Route n°1 between two cities: Viedma (-40.177288 S; -62.9988395 W) with
 80,000 inhabitants, and Balneario El Cóndor (-41,0437078 S; -62,8288118
 W), an urban entity with less than 2,500 inhabitants, totalling 28 km.
 Between both cities, xerophytic native flora alternates with
 semi-extensive livestock (mainly cattle) and farming areas. Also, the
 progress of urbanization is evident, with the consequent loss of the
 natural ecosystem, generating a transition area where wild species,
 domestic species and humans coexist. <strong>2.2.
 Host and flea collection and identification</strong>
 The study received approval from the Secretary of Environment and
 Sustainable Development of Río Negro province (085206SAYDS/218/2015).
 Carcasses of wild native meso-mammals were then collected along Provincial
 Route n°1 and urban roads in the seaside town of Balneario El Cóndor
 between January 2020 and May 2024.  The animals were found randomly by the
 authors, dead due to vehicular collisions. Each host found was immediately
 placed in a white polyethylene bag and then transferred to a plastic
 container to prevent fleas from escaping and to facilitate their
 collection. Afterward, the mammals were taken to the
 Universidad Nacional de Río Negro laboratory, where the host species were
 identified. The native mammal species recorded were:
 <em>Chaetophractus villosus</em> (Desmarest) “Big hairy
 armadillo” (Cingulata: Chlamyphoridae); <em>Didelphis
 albiventris</em> Lund “White-eared opossum” (Didelphimorphia:
 Didelphidae); <em>Lagostomus maximus</em> (Desmarest) “Plains
 vizcacha” (Rodentia: Chinchillidae); <em>Leopardus
 geoffroyi</em> d'Orbigny & Gervais “Geoffroy's
 cat” (Carnivora:  Felidae) and <em>Lycalopex gymnocercus</em>
 (Fischer) “South American grey fox” (Carnivora: Canidae). Identification
 of the mammals was performed according to Chebez et al. (2014).
 Fleas were collected using toothbrushes and forceps, and stored
 in 96% ethyl alcohol until molecular analyses was carried out at the
 Centro de Bioinvestigaciones (CeBio). The parasitological examination and
 flea identification methods are based on the protocols outlined in Sanchez
 (2013) and Sanchez and Lareschi (2019). First, the fleas were identified
 at the genus level using a stereoscopic microscope, followed by DNA
 extraction (see section 2.3). Afterward, species identification was
 performed using the methodology described below. Fleas
 were cleared and softened in aqueous solution of potassium hydroxide 10%
 (KOH), dehydrated in an increasing series of ethanol (80-100%), further
 diaphanized in eugenol, and mounted on permanent slides with Canadian
 balsam and studied under light microscopy. Identification keys used
 included those of Hopkins and Rothschild (1953), Johnson (1957), and Smit
 (1987). <strong>2.3. DNA
 extraction</strong> For DNA extraction, one flea
 per host was randomly selected, washed, and cut between the third and
 fourth abdominal tergites using a sterile scalpel. When more than one flea
 species was present on the same host, one individual of each species was
 randomly selected and analysed separately. All materials used to handle
 the fleas, including brushes for moving the ectoparasites, Petri dishes
 for holding them, and the scalpel for making incisions, were sterilized
 between each sample. Genomic DNA extraction was performed from individual
 fleas per host, using Chelex^®^-100 (Bio-Rad Laboratories, CA, USA)
 described by Acosta et al. (2023). Following the DNA extraction, the fleas
 exoskeletons were recovered and stored in 96% ethanol and subsequently
 mounted for species identification (see section 2.2).
 <strong>2.4. PCR amplification of genes from</strong>
 <em><strong>Bartonella</strong></em>
 <strong>spp. and</strong>
 <em><strong>Rickettsia</strong></em>
 <strong>spp.</strong> The presence of
 <em>Bartonella</em> was screened using the citrate synthase
 (<em>gltA</em>) and RNA polymerase beta-subunit
 (<em>rpoB</em>) genes, while the presence of
 <em>Rickettsia</em> was screened using the citrate synthase
 (<em>gltA</em>), outer membrane protein A
 (<em>ompA</em>) and outer membrane protein B
 (<em>ompB</em>) genes (Table 1). For the amplification, the
 polymerase chain reaction (PCR) program started with an initial
 denaturation for 5 min at 95 °C, followed by 40 cycles of 95 °C for 30 s,
 gene-specific annealing °C for 30 s, and 72 °C for 30 s, and a final
 extension step at 72 °C for 5 min (Table 1). PCR reaction was set to a
 final volume of 20 μL, containing: 25-100 ng of template DNA, 1.5 mM
 MgCl<sub>2</sub>, 0.2 μM of each primer, 0.2 mM of each dNTP,
 1X reaction buffer, 0.5U of Taq Pegasus DNA polymerase and ultrapure
 sterile water to final volume. All amplifications were performed in
 conjunction with a negative (distilled water) and positive (DNA of
 <em>Bartonella henselae</em> Regnery et al., provided by
 “ANLIS Malbrán”, Buenos Aires, Argentina, and DNA of <em>Rickettsia
 parkeri</em>, provided by Instituto Nacional de Enfermedades Virales
 Humanas “Dr. Julio I. Maiztegui”, Buenos Aires, Argentina) controls. DNA
 fragment amplification was confirmed by electrophoresis on 1% w/v agarose
 gel, stained with ethidium bromide (10 mg/μL) and visualized under UV
 light. In the samples with positive PCR for the genes
 analysed, we proceeded to purification using 10U of Exonuclease I (Thermo
 Fisher Scientific) and 1U of FastAp thermosensible alkaline phosphatase
 (Thermo Fisher Scientific), incubating at 37°C for 15 min and a subsequent
 15 min at 85°C to stop the reaction. The purified samples were sequenced
 by Macrogen Co. Ltd. (South Korea). <strong>2.5.
 Sequencing, nBLAST, and phylogenetic analysis</strong>
 The obtained sequences for the genes were analysed and manually
 edited using the BioEdit program (Hall 2004). To assign identity to each
 sequence with its respective statistical significance, it was subjected to
 a homology comparison against the GenBank nucleotide database (https://www.ncbi.nlm.nih.gov/genbank/), making use of the nBLAST algorithm (https:// blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences have been deposited in the GenBank nucleotide database (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers PP985429 - PP985431 for <em>Rickettsia</em>, and PP985432 - PP985440 for <em>Bartonella</em>. The complete set of the** gene sequences was employed for a multiple alignment performed with the ClustalW algorithm and the MEGA v.6 software (Tamura et al. 2013), together with sequences taken from the GenBank database. The resulted alignment was checked and manually corrected. Moreover, phylogenetic trees were built using the Maximun-Likelihood (ML) clustering methods, both for individual genes and for concatenated sequences. In the case of gene concatenation for both bacteria, the Farris test (Farris et al. 1994) was initially performed using Phylogenetic Analysis Using Parsimony (PAUP*) methods based on parsimony inference (Swofford and Sullivan, 2003) to establish whether these genes could be used. Subsequently, the Mesquite program (Maddison and Maddison 2021) was utilized to concatenate these sequences. The nucleotide substitution model that best fit the data was calculated using JModelTest software v2.1.4 (Darriba et al. 2012). The evolutionary history was inferred using the ML method based on the Tamura 3-parameter (I + G) model with 10,000 replicates of random-addition taxa and tree bisection and reconnection branch swapping. All positions were weighted equally.
description: # Fleas of wild mammals carrying pathogenic bacteria in Argentinian
 Patagonia: a study based on wildlife roadkill Dataset DOI:
 [10.5061/dryad.ghx3ffc1z](10.5061/dryad.ghx3ffc1z) ## Description of the
 data and file structure These data were collected to investigate the
 diversity of fleas parasitizing wild mammals in Argentinian Patagonia and
 to detect the presence of pathogenic bacteria through molecular screening.
 Fleas were collected from roadkilled mammals during field surveys, and PCR
 analyses were performed to detect *Rickettsia* spp. and Bartonella spp.
 The dataset includes host and flea species identification, collection
 locality, date, and molecular results. ### Files and variables #### File:
 Supplemental_Material_1.csv **Description:** Details of each flea sample
 analysed from roadkilled wild meso-mammals collected in Argentine
 Patagonia. The table includes sampling location, host species, flea
 species, and the presence/absence of *Rickettsia* and *Bartonella* DNA
 detected through molecular analysis. #### File:
 Supplemental_Material_2.pdf **Description:** Phylogenetic tree obtained
 with the Maximum Likelihood methodology for a 719 bp fragment *gltA* gene
 for *Rickettsia* spp. In the nodes, bootstrap values &gt; 50% are shown.
 The sequence obtained in this study is shown in bold. #### File:
 Supplemental_Material_3.pdf **Description:** Phylogenetic tree obtained
 with the Maximum Likelihood methodology for a 547 bp fragment *ompA* gene
 for *Rickettsia* spp. In the nodes, bootstrap values &gt; 50% are shown.
 The sequence obtained in this study is shown in bold. #### File:
 Supplemental_Material_4.pdf **Description:** Phylogenetic tree obtained
 with the Maximum Likelihood methodology for a 775 bp fragment *ompB* gene.
 for *Rickettsia* spp. In the nodes, bootstrap values &gt; 50% are shown.
 The sequence obtained in this study is shown in bold. #### File:
 Supplemental_Material_5.pdf **Description:** Phylogenetic tree obtained
 with the Maximum Likelihood methodology for a 181 bp fragment *gltA* gene
 for *Bartonella* spp. In the nodes, bootstrap values &gt; 50% are shown.
 The sequences obtained in this study are shown in bold. #### File:
 Supplemental_Material_6.pdf **Description:** Phylogenetic tree obtained
 with the Maximum Likelihood methodology for a 126 bp fragment *rpoB* gene
 for *Bartonella* spp. In the nodes, bootstrap values &gt; 50% are shown.
 The sequences obtained in this study are shown in bold.