Pathogen screening in the red fox (Vulpes Vulpes) from Lithuania

INTRODUCTION

The red fox (Vulpes vulpes) is the most widely distributed of all wild canids, with a natural range from the deserts to the Arctic tundra (Schipper et al., 2008; Edwards et al., 2012). The red fox is adapted to different environments and can easily survive in urban areas (Teacher et al., 2011; Scott et al., 2014). Living in close proximity to people may pose a risk in the case of the transmission of zoonoses and veterinary diseases (Truyen et al., 1998; Hodžić et al., 2015; Koneval et al., 2017; Víchová et al., 2018). Determining the impact of wildlife for pathogen transmission is important for epidemiological studies.

In the past, red foxes were most commonly associated with the epidemiological cycle of rabies (Chautan et al., 2000; Vos, 2003; Zienius et al., 2007). Also, several studies showed that red foxes are a reservoir for zoonotic parasites, such as Echinococcus multilocularis, Trichinella spp., and Toxocara canis (Saeed et al., 2006; Bružinskaitė-Schmidhalter et al., 2012; Franssen et al., 2014; Karamon et al., 2018). Recent studies have revealed that in Europe, red foxes are infected with vector-borne pathogens (Hodžić et al., 2015; Koneval et al., 2017; Hodžić et al., 2018).

A long-term rabies persistence period in the red fox populations was reported in Lithuania (Zienius et al., 2007). Other studies investigated zoonotic helminths of red foxes (Bružinskaitė-Schmidhalter et al., 2012; Janulaitis et al., 2014). However, the real role of the red foxes as a source of different pathogens is unclear. Therefore, the principal aim of this study was to screen free-ranging red foxes from Lithuania for the presence of different vector-borne pathogens.

MATERIALS AND METHODS

Collection of samples

A total of 31 red foxes from three districts of Lithuania were included in the present study (Fig. 1). From 2016 to 2018, carcases of red foxes were collected in collaboration with hunters. The data on sex, the area of origin, and the hunting date were recorded for each individual red fox (Table 1). During necropsy, spleen samples were collected and frozen at –20°C until DNA extraction.

DNA extraction, PCR amplification

DNA was isolated using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Lithuania) according to the manufacturer’s instructions and stored at –20°C for further analyses.

All DNA samples were screened for the presence of Anaplasma spp., Bartonella spp., Rickettsia spp., Borrellia spp., and Babesia spp. using multiplex real time-PCR assay. Primer sequences and target gene used in this study are presented in Table 2. RT-PCR reactions were done in total volume of 15 μl consisting of 100 ng of extracted DNA, (1x) SensiMix™ II Probe No-ROX (Bioline), 1 μM of each primer, and 0.5 μM of each probe. Cycling reactions started with an initial activation step of 95°C for 10 min followed by 45 cycles of 95°C for 20 s, 60°C for 60 s (for Anaplasma, Borellia, and Babesia) and 50°C for 60 s (for Bartonella ant Rickettsia), and 72°C for 20 s. Cycling reactions were carried out using a Rotor Gene 6000 (Corbett Research, Australia).

Mycoplasma spp. and Dirofilaria spp. were detected using conventional PCR method. For Mycoplasma spp. were amplified 16S RNA region using 322s and 938as primers according Varanat et al. (2011). For filarial screening, pan-filarial primers (DIDR-F1, DIDR-R1) were used that amplify fragments of different length of the internal transcribed spacer region 2 (ITS2) of the ribosomal DNA from six different filarioid species (Dirofilaria repens, D. immitis, Acanthocheilonema reconditum, A. dracunculoides, Brugia pahangi and B. malayi). The PCR were conducted as described by Rishniw et al. (2006). All amplification products were electrophoresed on a 1.5% agarose gel and visualized under UV light after staining with ethidium bromide.

Table 2. Primers used for the amplification of DNA of different pathogens

Statistical analysis

The prevalence of different pathogen infection analysis was performed using Mixrosoft excel software. The InteractiveVenn tool was used to create the Venn diagram and calculate coinfections (Heberle et al., 2015).

RESULTS AND DISCUSSION

In general, vector-borne pathogens were detected in 83.9% (26/31) of red foxes. Five different pathogens were detected: Anaplasma spp., Bartonella spp., Rickettsia spp., Borrellia spp., and Babesia spp. (Table 3).

The most prevalent pathogen in red foxes from Lithuania was Babesia spp. (20/31, 64.5%). This pathogen was detected in all studied areas (Table 3). Females (60.0%; 12/20) were more infected with Babesia spp. than males (40.0%; 8/20). A number of studies reported that red foxes were infected with such Babesia spp. as B. canis, B. venatorum, B. vulpes (synonyms: B. microti, B. cf. microti, B. annae) (Karbowiak et al., 2010; Cardoso et al., 2013; Duscher et al., 2014; Najm et al., 2014; Farkas et al., 2015; Hodžić et al., 2015; Koneval et al., 2017; Hodžić et al., 2018; Baneth et al., 2019). Ticks are the main vector of this blood parasite. Several studies show the presence of Babesia spp. in ticks (Ixodes hexagonus, I. ricinus) collected from red foxes (Najm et al., 2014; Checa et al., 2018).

Another tick-transmitted bacteria detected in our study was Anaplasma spp. A total 48.4% (15/31) of the tested samples showed positive results. This pathogen was also detected in all the studied areas (Table 3). Infection of red foxes with A. phagocytophilum was reported from Poland (Karbowiak et al., 2009), Italy (Ebani et al., 2011), Germany (Härtwig et al., 2014), Croatia (Beck et al., 2014), Netherlands (Jahfari et al., 2014), Hungary (Tolnai et al., 2015), Romania (Dumitrache et al., 2015b), Switzerland (Hofmann-Lehmann et al., 2016), and Austria (Hodžić et al., 2018). A. ovis was reported in red foxes from Sicily (Italy) (Torina et al., 2013); A. bovis in Croatia (Beck et al., 2014); A. platys in Portugal (Cardoso et al., 2015). A. phagocytophilum has also been detected in I. ricinus ticks collected from red foxes (Dumitrache et al., 2015a; Víchová et al., 2018). Moreover, Torina et al. (2013) detected A. phagocytophilum, A. ovis, and A. marginale in fleas (Xenopsylla cheopis, Ctenocephalides canis) collected from red foxes.

Bartonella spp. are distributed in wild carnivores (Gerrikagoitia et al., 2012; Bai et al., 2016). Red foxes tested in this study were also infected with Bartonella spp. (25.8%; 8/31). These results are consistent with findings from other countries. There are some reports that infected red foxes carried B. rochalimae, B. v. berkhoffii, B. clarridgeiae (Henn et al., 2009; Kaewmongkol et al., 2011; Gerrikagoitia et al., 2012; Bai et al., 2016; Hodžić et al., 2018). Ectoparasites, such as fleas and ticks, play a role in the transmission of Bartonella species (Chomel et al., 2009). B. rochalimae was found in Pulex irritans fleas from red foxes in Spain (Márquez et al., 2009); B. henselae and B. clarridgeiae were detected in Ctenocephalides felis fleas from red foxes in Australia (Kaewmongkol et al., 2011). Bartonella spp. were found in fleas (Chaetopsylla globiceps, P. irritans, Ctenophtalmus assimilis) collected from red foxes in Slovakia (Víchová et al., 2018). The authors performed the obtained sequence analysis that showed identity or similarity to B. rochalimae and B. taylorii (Víchová et al., 2018).

Infection with Lime diseases pathogen Borrellia spp. was also found in studied red foxes from Lithuania (25.8% (8/31). Previous studies reported that red foxes were infected with B. burgdorferi sensu lato (Isogai et al., 1994; Heidrich et al., 1999; Dumitrache et al., 2015b; Lledó et al., 2016; Mysterud et al., 2019). Data on the diversity of B. burgdorferi sl in red foxes present four species B. burgdorferi sensu stricto, B. afzelii, B. lusitaniae, and B. garinii (Isogai et al., 1994; Dumitrache et al., 2015b; Sukara et al., 2019). B. burgdorferi sl has also been detected in I. ricinus and I. persulcatus ticks collected from red foxes (Isogai et al., 1994; Dumitrache et al., 2015a).

The prevalence of Rickettsia spp. infection detected in the present study is of the lowest level (9.7%; 3/31). Other authors confirmed in serological studies that foxes were exposed to R. typhi, R. slovaca, R. conorii and R. massiliae/ Bar29 (Lledó et al., 2016; Ortuño et al., 2018). R. helvetica was detected in foxes from Sweitzerland (Hofmann-Lehmann et al., 2016). Ortuño et al. (2018) reported that Rhipicephalus sanguineus complex ticks collected from red foxes were infected with R. massiliae, R. aeschlimannii, and R. slovaca (Ortuño et al., 2018). In France, arthropods collected from red foxes showed Rickettsia-positive results. Ticks (Rhipicephalus turanicus) were found to be infected with R. massiliae and fleas (Archaeopsylla erinacei) collected in the study contained R. felis (Marié et al., 2012). Also, R. felis was detected in fleas (Ctenocephalides felis) from red foxes in Sicily, Italy (Torina et al., 2013). R. massiliae DNA was detected in Rh. sanguineus ticks collected from a fox in Sardinia, Italy (Chisu et al., 2017). Moreover, Víchová et al. (2018) reported that fleas (Archaeopsylla erinacei) and ticks (Ixodes ricinus, Ixodes hexagonus, Haemaphysalis concinna) removed from red foxes in Slovakia were infected with Rickettsia spp. (Víchová et al., 2018).

Mycoplasma is a genus of haemotropic, self replicating bacteria (Messick 2004). Most of them are responsible for a variety of diseases in humans, animals, insects, and plants (Sumithra et al., 2013). There is a lack of information about mycoplasma in wild animals. Moreover, only a few studies report the occurrence of mycoplasmas in red foxes (Kanamoto et al., 1981; Sasaki et al., 2008; Koneval et al., 2017; Millán et al., 2018). Mycoplasma spp. was not detected in any of the tested red fox spleen samples in this study. However, in a study in Slovakia, out of 300 samples of red foxes tested, Mycoplasma spp. bacteria was detected in 13 (4.3%) (Koneval et al., 2017). Also, out of 12 red foxes, only one (0.83%) was positive for M. haemocanis in Japan (Sasaki et al., 2008). A study in Spain showed 2.4% (1/41) infection of Mycoplasma spp. in red foxes (Millán et al., 2018). Considering that studies from other countries have a very low infection rate of Mycoplasma spp. in red foxes, future screening of this pathogen is required with a largest sampling site in Lithuania. Also, ectoparasites (ticks, fleas) collected from red foxes in Slovakia were tested for the presence of Mycoplasma spp. but the infection was not detected (Víchová et al., 2018).

Dirofilariasis is recognized as a zoonosis spreading across Europe (Genchi et al., 2009; Genchi et al., 2011; Simón et al., 2012). A previous study showed that D. repens is a zoonotic parasite in Lithuania (Sabūnas et al., 2019a). In Lithuania, nine human cases of D. repens during the period of 2011–2018 and the prevalence of D. repens among shelter dogs have been reported. Furthermore, recently D. immitis was found in an imported dog in Lithuania (Sabūnas et al., 2019b). Some researchers consider that free-living carnivores such as red foxes may act as a natural reservoir of zoonotic filariasis (Magi et al., 2008). Seeing that, in this study we analysed the spleen samples of red foxes from Lithuania for filarial infection in order to investigate their role as a potential wildlife reservoir of dirofilariasis. Of all tested foxes, none were positive for filarian parasites. However, D. repens infection in red foxes has been recorded in Italy (Marconcini et al., 1996; Magi et al., 2007), Slovakia (Hurníková et al., 2012), Serbia (Ćirović et al., 2014), and Romania (Ionică et al., 2017). Besides, heartworm (D. immitis) infection was found in red foxes in Italy (Marconcini et al., 1996; Magi et al., 2007), Spain (Mañas et al., 2005), Serbia (Penezić et al., 2014), Hungary (Tolnai et al., 2014), and Romania (Ionică et al., 2017). Otherwise, there are reports from other researchers that filariasis was not detected in red foxes (Hodžić et al., 2015; Härtwig et al., 2016; Hodžić et al., 2018). Considering that the distribution of filariasis in Europe is continuously spreading (Genchi et al., 2009; Genchi et al., 2011; Simón et al., 2012), future screening for filariasis in wildlife carnivores in Lithuania is required.

Majority of red foxes (77.4%; 24/31) were infected with more than one parasite species. Coinfections with two to four different pathogen species were observed (Fig. 2). Coinfection with two different pathogens were detected in 14 red foxes: one fox was infected with Babesia spp. and Borrelia spp.; one fox was infected with Babesia spp. and Rickettsia spp.; three foxes were infected with Anaplasma spp. and Borrelia spp.; three foxes were infected with Babesia spp. and Bartonella spp.; six foxes were infected with Anaplasma spp. and Babesia spp. Coinfection with three different pathogens (Babesia spp., Borrelia spp. and Bartonella spp.) was detected in one red fox. Coinfection with four different pathogens were detected in four red foxes: one fox was infected with Babesia spp., Anaplasma spp., Borrelia spp. and Rickettsia spp.; one fox was infected with Babesia spp., Anaplasma spp., Rickettsia spp. and Bartonella spp.; two foxes were infected with Babesia spp., Anaplasma spp., Borrelia spp. and Bartonella spp.

The overall presence of vector-borne pathogens in red foxes in Europe is shown in Table 4. All studies point to the importance of red foxes as a reservoir of various vector-borne pathogens. Some of them were not detected in this study. However, previous studies conducted in Lithuania showed the presence of Anaplasma spp., Babesia spp., and Dirofilarria sp. in dogs (Paulauskas et al., 2014; Sabūnas et al., 2019a; Sabūnas et al., 2019b; Tamoliūnaitė et al., 2019).

CONCLUSIONS

Our results demonstrate that vector-borne pathogens are widespread among red foxes in Lithuania. To our knowledge, this is the first report on the detection of infection with Anaplasma spp., Bartonella spp., Rickettsia spp., Borrellia spp. and Babesia spp. in red foxes from Lithuania. Further studies are needed to determine the prevalence and distribution of these vector-borne pathogens in foxes and other carnivores, and their ectoparasites. Mycoplasma spp. and filaroid parasites were not detected in red foxes in our study. Further studies of mycoplasma and filariasis in wildlife carnivores in Lithuania are required.

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Povilas Sakalauskas, Indrė Lipatova, Jana Radzijevskaja, Algimantas Paulauskas

RUDŲJŲ LAPIŲ (Vulpes vulpes) UŽSIKRĖTIMAS PATOGENAIS LIETUVOJE