Introduction

Marsupials of the genus Didelphis are mammals geographically restricted to the American continent (Gardner 2008). In South America these animals are represented by five species divided in two groups, the D. marsupialis-group (D. marsupialis and D. aurita) and the D. albiventris-group (D. albiventris, D. pernigra, and D. imperfecta). The only Didelphis species that up to date is not found in this region is the D. virginiana, which occurs from Canada to Costa Rica (Gardner 2008). In Brazil, four species of this genus are reported, D. imperfecta (Guianan White-eared Opossum) and D. marsupialis (Common Opossum) in the Amazon rainforest, D. albiventris (White-eared Opossum) mostly in the Cerrado biome, and D. aurita (Black-eared Opossum) in the Atlantic forest (Faria and Melo 2017; Gardner 2008).

Species of the genus Didelphis are well adapted to the anthropogenic activity, being found on the roof of houses, hollows of trees and other shelters within urban centers (Jansen 2002). Due to this synanthropic behavior, the possibility of transmission of pathogens (i.e., Rickettssia spp. and Borrelia spp.) by ectoparasites to humans and domestic animals may occur (Muller et al. 2005; Cabrera et al. 2003).

It is known that ticks and fleas are commonly reported infesting Didelphis spp. (Linardi 2006; Muller et al. 2005). For instance, studies of the ectoparasite fauna of D. aurita have reported the occurrence of ticks of the genera Amblyomma and Ixodes (Luz et al. 2018, 2013; Gonzalez et al. 2017; Acosta et al. 2016; Oliveira et al. 2014; Saraiva et al. 2012; Dantas-Torres et al. 2012), and fleas of the genera Adoratopsylla, Polygenis, Leptopsylla, Xenopsylla and Ctenocephalides (Urdapilleta et al. 2019; Pinto et al. 2009; Salvador et al. 2007; Horta et al. 2007; Moraes et al. 2003). Interestingly, specimens of the genera Amblyomma and Ctenocephalides are commonly found on domestic animals (i.e., dogs, cats, horses) (Costa et al. 2017; Dantas-Torres and Otranto 2014), which may imply that these ectoparasites have major importance in the epidemiological life cycle of pathogens of medical and veterinary concern (De Sá et al. 2018; Muller et al. 2005). The aim of this study was to assess the ectoparasite fauna of D. aurita, as well as the presence of pathogens and endosymbionts in ticks and fleas.

Material and methods

Study area

The study was conducted in the municipality of Viçosa (20°45′14″S, 42°52′54″W), located in the State of Minas Gerais, Southeastern Brazil. The climate in this region is classified as Cwa (Köppen climate classification), mesothermic, with hot and rainy summers and cold and dry winters. The area is 650 m above sea level and presents an annual average temperature varying from 20 to 22 °C.

Animals and sampling

Animals were captured through Tomahawk live traps (0.45 × 0.21 × 0.21 m), which were armed and checked daily (5PM and 7AM, respectively), from January to June 2019, totaling a sampling effort of 516 trap-nights in urban environments of the study area (Fig. 1). A mix of corn flour, canned fish and banana were used as bait. After capture, the animals were mechanically restrained and classified by sex, age group (pups, subadults and adults), and marked with a small V cut at the right ear to identify recaptures (Morrant et al. 2010; Pestell and Petit 2007). Additionally, animals found dead (n = 9) on roads and streets of the study area were necropsied and fragments of spleen collected.

Fig. 1
figure 1

Points of captures of Didelphis aurita in Southeastern Brazil. Red spots are concentrated at the urban extension of the studied area. (Color figure online)

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Captured opossums were individually inspected for a period of approximately 10 min for the presence of ectoparasites. Specimens collected were stored in plastic vials (ticks in empty tubes and fleas in tubes containing 70% alcohol) for morphological identification and molecular procedures. In addition, blood samples were collected via venipuncture of the caudal ventral or jugular vein and stored at − 20 °C until molecular processing.

Laboratorial procedures

Ectoparasites were quantified, separated according to the stage and sex, and morphologically identified using dichotomy keys (Barros-Battesti et al. 2006; Aragão and Fonseca 1961; Linardi and Guimarães 2000; Bicho and Ribeiro 1998). Afterwards, 58 pools of ticks (up to 3 specimens) and 50 pools of fleas (up to 4 specimens) were formed for DNA extraction.

For molecular procedures, genomic DNA of blood and spleen were extracted using a commercial kit for blood and spleen (Illustra tissue and cells genomicPrep Mini Spin Kit, GE Healthcare Life Sciences) following the manufacturer’s instructions. On the other hand, DNA extraction of ectoparasites were performed following a protocol previously described (Ramos et al. 2015).

Each DNA sample was screened for the gltA gene of Rickettsia spp., the flagellin gene of Borrelia spp., the 16S rRNA gene for Anaplasmataceae, and for the 18S gene for Babesia spp. (Table 1). Amplifications were performed using Taq Pol Master Mix Green 2 × following the manufacturer’s recommendations, 400 nmol of each primer, 5 µL of DNA sample and nuclease free water until complete 25 µL volume. DNA of Rickettsia vini (Preventive Veterinary Medicine and Animal Health Department of the University of São Paulo), Borrelia garini (Epidemiology and Public Health Department of the Federal Rural University of Rio de Janeiro), Ehrlichia canis and Babesia canis (Veterinary Medicine Department of the Federal University of Viçosa) were used as positive control, and nuclease free water as negative control.

Table 1 Primers used for molecular analysis
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All amplicons obtained were purified using PCR Purification Kit (Cellco Biotec) according to manufacturer’s recommendations. Sanger’s method was performed for sequencing amplicons in both directions (Sanger et al. 1977) in an automated sequencer AB 3500 Genetic Analyzer. DNA sequences were aligned using Mega7 (Kumar et al. 2016) and compared with sequences from GenBank using the BLAST search tool (Altschul et al. 1990).

Data analysis

Descriptive statistical analysis was performed to calculate the relative and absolute frequencies, as well as mean intensity and abundance of infestation by ticks and fleas. The normality of data was checked using the Lilliefors test. Additionally, the χ2 test with Yates correction was used to compare the occurrence of these ectoparasites according to sex and age of the animals. The significance level was set at 5%. All analyzes were carried out using the BioEstat 5.3 software.

The sites of capture of opossums were geoprocessed with the geographic information system program QGIS 3.4.12 (qgis.org). Digital maps of the Brazil, the State of Minas Gerais and the municipality of Viçosa were used as cartographic basis, and the SIRGAS2000 and the UTM coordinates system as ellipsoid of reference. The layers and points were automatically converted by the system (extension on the fly). The complement QuickMapServices was used to obtain the satellite images (Google Satellite).

Results

Fifty-eight animals were captured during the whole study, being 50% (29/58) males and 50% (29/58) females. In particular, 3.5% (2/58) were classified as pups, 46.6% (27/58) as subadults and 50% (29/58) as adults. A total of 69 ticks and 121 fleas were collected on the animals, which correspond to frequencies of infestation of 41.4% (24/58) and 60.3% (35/58), respectively (Table 2). No significant difference was observed for tick and flea infestation according to sex and age of the opossums (p > 0.05).

Table 2 Ectoparasites found in Didelphis aurita from Southeastern Brazil
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The PCR analysis detected Anaplasmataceae DNA in 34.04% (16/47) pool samples of C. felis felis, and in 66.66% (2/3) pool samples of X. cheopis. The sequence analysis of these products and comparisons in the GenBank database revealed Wolbachia pipientis with query Cover of 100% and identity of 98% as compared with BLAST results (Accession numbers: KP114101.1, KP165047.1, MF944223.1, EF121345.1, and AY026912.1). Tick, blood and spleen samples scored negative for the microorganisms assessed.

Discussion

This study assessed the occurrence of ectoparasites as well as the detection of microorganisms infecting ticks and fleas of D. aurita from Southeastern Brazil. I. loricatus was the most frequent tick species found, infesting 41.38% of the animals. In fact, most studies on ectoparasites of Didelphis spp. found the adult stage of I. loricatus to be the most predominant tick species in these marsupials (Luz et al. 2018; Gonzalez et al. 2017; Oliveira et al. 2014; Saraiva et al. 2012; Dantas-Torres et al. 2012; Muller et al. 2005; Barros-Battesti et al. 2000). In contrast with these studies, when compared with I. loricatus, A. auricularium was reported with a higher frequency on D. albiventris in a study performed in Northeastern Brazil (Lopes et al. 2018). Additionally, Acosta et al. (2016) have reported only immature stages of Amblyomma spp. in D. aurita from the State of Espirito Santo, Brazil; however, few opossums of this species were inspected in that study.

Amblyomma sculptum was detected with a very low frequency in the animals analyzed in the present study. This tick species presents great public health importance, as it is the main vector of Rickettsia rickettsii, the etiological agent of the Brazilian Spotted Fever (Parola et al. 2013). The presence of Amblyomma spp. in D. aurita captured in urban areas has a great epidemiological importance, since this opossum act as an amplifier host for R. rickettsii infection to A. sculptum ticks (Horta et al. 2009). In our study, a single animal was found parasitized by an A. sculptum nymph. Indeed, most studies reported that larvae and nymphs of Amblyomma are the most common life stages retrieved in opossums (De Sá et al. 2018; Lopes et al. 2018; Acosta et al. 2016).

Regarding the flea species found in our study, C. felis felis was predominantly identified in D. aurita, with a frequency of 60.34%. This species, known as the cat flea, has been reported with high prevalence rates in Didelphis spp. (Horta et al. 2007; Boostrom et al. 2002; Barros-Battesti and Arzua 1997). However, Salvador et al. (2007) studying ectoparasites of D. aurita in the state of Santa Catarina, Brazil, found only Adoratopsylla intermedia, Leptopsylla segnis and Xenopsylla cheopis. The authors of that study suggested that the absence of C. felis felis in the studied population was due to the lack of human habitations in the studied area, which consequently reduce the contact of the wildlife with domestic animals. In fact, it is known that C. felis felis is the most prevalent flea species in dogs and cats worldwide (Durden and Hinkle 2019). In our study, the animals were captured in urban areas, and the high frequency of C. felis felis may suggest the exchange of this ectoparasite species between companion animals and D. aurita.

The detection of X. cheopis in the present study is a relevant finding. Salvador et al. (2007) found a higher rate of D. aurita infested by this flea species in the State of Santa Catarina, Brazil, with a frequency of 15.1%. Rodents are the main hosts of X. cheopis, and this species is involved in the transmission of important zoonotic pathogens such as the bacteria Yersinia pestis and Rickettsia typhi, the causative agents of Plague and Murine Typhus, respectively (Durden and Hinkle 2019; Civen and Ngo 2008). In addition, the presence of this flea in Didelphis spp. may have important implications in public health, as it is known that these opossums are probably involved in the zoonotic cycles of pathogens such as R. typhi and R. felis (Brown and Macaluso 2016; Boostrom et al. 2002).

Interestingly, DNA of W. pipientis was detected in C. felis felis and X. cheopis. It is known that this bacterium is an endosymbiont that infects a great number of insect species worldwide, which includes various flea species (Gorham et al. 2003; Werren and Windsor 2000) such as C. felis, C. canis, Tunga penetrans, Polygenis gwyni, Orchopeas howardi, Pulex irritans, P. simulans, Echidnophaga gallinacea, Stenoponia tripectinata tripectinata, and X. cheopis (Onder et al. 2019; Cevidanes et al. 2018; Zurita et al. 2016; Heukelbach et al. 2004; Dittmar and Whiting 2004; Rolain et al. 2003; Gorham et al. 2003). In fact, Dittmar and Whiting (2004) claim that there is a potentially widespread association between these bacterial symbionts with fleas in general. In addition, W. pipientis has influence on reproduction, sex determination, speciation and behavior of arthropods, being a potential candidate to biological control of insect vectors (LePage and Bordenstein 2013).

Conclusion

This study reports the occurrence of ticks and fleas in D. aurita opossums, as well as the infection by W. pipientis in siphonaptera of the species C. felis felis and X. cheopis collected in these animals in Southeastern Brazil. Data herein obtained demonstrates the parasitism in D. aurita by some species of ectoparasites, including specimens commonly found in domestic animals (C. felis felis and A. sculptum). These findings suggest that these arthropods circulate among wildlife and urban environments, which may implicate that they have important role in the cycle of zoonotic pathogens among opossums, humans and companion animals.