Results and discussion
PCR and sequencing results showed that Besnoitia spp. DNA was present in four faecal samples (1.13%) analysed from 352 wild carnivores. Those positive samples corresponded to four red foxes from Castilla y León and Extremadura in western Spain. To date, this is the first finding of a B. besnoiti -like sequence from a carnivore in Europe, and from any carnivore species in a worldwide context.
To the best of our knowledge, this is the first large-scale molecular survey for Besnoitia spp. DNA in free-living carnivores ever carried out globally. The survey benefits from the inclusion of 12 different species of free-living carnivores and a national coverage, paying special attention to regions where bovine besnoitiosis is present (except in North West Spain) in conjunction with greater densities of extensive cattle production.
Serological, molecular and parasitological techniques have been used in an attempt to elucidate the role of several animal species as potential definitive host of B. besnoiti , but they failed to find it in wild and domestic carnivores, in addition to mammals, reptiles and birds (see Table 1). Several studies on felines, including the domestic cat (Felis catus ), have attempted to clarify its role as a definitive host (see Table 1). Rommel (1975) and Peteshev et al. (1974) reported inconclusive results to confirm domestic cats as definitive hosts ofB. besnoiti in experimental studies. Despite detecting oocysts in the faeces, authors could not achieve further characterization for fully confirmation of their identity as B. besnoiti. Other authors did not find B. besnoiti oocysts in the faeces of per oschallenged cats over a 3 to 20 weeks observation period (Diesing et al., 1988; Basso et al., 2011). Several serological studies have been also carried out to detect antibodies of B. besnoiti in felines (Table 1). In European felines (Millán et al., 2012) antibodies were found in eight feral cats (Felis silvestris catus ) by any of the serological techniques used (indirect fluorescent antibody test [IFAT] and by two western immunoblots [WB, one with tachyzoite and the other with bradyzoite antigen]), but no individual showed positivity/contact by IFAT and one of the WBs. These animals originated from areas where no cases of bovine besnoitiosis had been detected until year 2010. The results suggested their unlikely implication in the parasite transmission. In a recent study in Namibian wildlife, antibodies have been detected in a lion, Panthera leo (Seltmann et al., 2020). On the other hand, only one study has managed to detect by molecular techniques Besnoitia spp. DNA in pond bat (Myotis dasycneme ) faeces in the Netherlands (Hornok et al., 2015). The authors hypothesized that B. besnoiti -like sequence might have originated from French cattle via bloodsucking dipterans (Stomoxys calcitrans, Tabanus spp.). In this regard, bats frequently use cattle stables for roosting, where they can prey on the mechanical vectors of B. besnoiti .
In the present survey, Besnoitia spp. DNA has been demonstrated in four individual faecal samples from red foxes from Ávila, Badajoz and Salamanca provinces (Table 2) in western Spain. All four fox-derivedBesnoitia spp. sequences were equivalent to positions 527–737 of reference sequence KX013107 (a bovine isolate of the parasite previously reported in Spain), differing from it by a single di-nucleotide site (a G/C double peak) at position 706. An additional ambiguous position (an A/G double peak) was also detected at position 711 of reference sequence KX013107 in one (GenBank accession number MW035609) of the four generated sequences. The topology of the produced phylogenetic tree clearly clustered all these sequences with other Besnoitiaspecies in large mammals (B. besnoiti , B. bennetti ,B. caprae and B. tarandi ) from European countries (Belgium, Finland, Italy, Germany, Portugal and Spain), Israel and Iran. In separate phylogenetic cluster, other species of Besnotiaspp. (B. neotomofelis , B. oryctofelisi , B. akodoni and B. darlingi ) infecting small mammals from Argentina, Brazil and USA (Figure 2) are placed. These results are in agreement with those described by Olias et al. (2011), in which ITS-1region shows the most informative nucleotide variances and phylogenetically clearly split small mammalian from large mammalianBesnoitia species. Of note, all foxes with Besnotia spp. PCR-positive faecal samples were caught within Western Spain (Figure 1), where the highest number of bovine besnoitiosis clinical cases were found in a previous survey (Nieto-Rodríguez et al., 2016).
This is the first molecular evidence of B. besnoiti in a European mesocarnivore. The red fox is present in a wide range of habitats in the Iberian Peninsula (Macdonald & Reynolds 2004) with densities of 0.7–2.5 foxes/Km2, depending on environmental conditions (Sarmento et al., 2009). In addition, this wild canid is a highly adaptable omnivorous mammal distributed across all continents on the northern hemisphere. Numerous studies on the red fox diet show it as a generalist predator, feeding mainly on prey which are abundant and easily accessible. Red foxes feed most frequently on small mammals as rodents and wild rabbits, but utilize also other food items such as carrion, birds, reptiles, amphibians, invertebrates, fruit and vegetables (Díaz-Ruiz et al., 2013).
Taking into account that in our study the faecal samples were collected from regions where beef cattle are usually raised in extensive production systems (Figure 1) and bovine besnoitiosis is widespread (Nieto-Rodríguez et al., 2016), there are three possible explanations for this interestingfindings; i) our first hypothesis is that red foxes may have a role in the transmission of the parasite as definitive host: the red fox is considered to be one of the most widespread generalist vertebrate predators in the world (Macdonald & Reynolds 2004). Therefore, predation on small mammals as rodents and wild rabbit, intermediate hosts in others species of Besnoitia spp., it could make us think that red fox might have a role as a definitive host in other species of Besnoitia, and that the sequences found represents a novel Besnoitia genotype/species closely related toB. besnoiti ; ii) our second hypothesis is that there has been consumption of carrion infected with B. besnoiti, and foxes are acting as passive carriers without developing the infection; iii) and the third and last hypothesis is that the red fox could act as an accidental or paratenic host, in which the accidental ingestion of the hypothetical B. besnoitia oocysts from the contaminated soil would be excreted or digested and we would find parasite DNA in the fox faeces.
Although we have found Besnoitia spp. DNA in red fox faeces and subsequently confirmed it by Sanger sequencing, present survey has several limitations. First, no serological analysis has been performed on these species, sampling was carried out in most cases on road- and hunter-killed animals, from accidentally found carcasses, and camera-trap surveys. Thus, fresh, good quality blood samples were unavailable for serological testing, in addition to the difficulty of finding validated techniques in wildlife for detecting this parasite (González-Barrio & Ruiz-Fons, 2019). Second, no additional parasitological techniques (e.g. floatation) were used due to the retrospective nature of this study and the insufficient amount of remaining faecal material for performing complementary techniques. Finally, identification of Besnoitiaspp. was accomplished on a single locus. Low quantity and quality of genomic DNA from faeces prevented us of conducting multilocus microsatellite analyses. However, on the other hand, fox has been the unique species out of the 12 studied in which B. besnoiti has been identified, suggesting that this species might play a role in the epidemiology of the disease.
To conclude, the low prevalence rate found here suggests that the role of the red fox in the epidemiology of bovine besnoitiosis could be of limited relevance in Spain. Additional epidemiological and experimental studies with a similar approach, may help in the search for the definitive host of this parasite, where the main hypothesis is that the definitive host is a predator or scavenger from Africa where the disease originated.