Results and discussion
PCR and sequencing results showed that Besnoitiabesnoiti -like 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 in Europe. 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 against B. besnoiti in felines (Table 1). Millán et al. (2012) found antibodies by IFAT (indirect fluorescent antibody test) in eight feral cats (Felis silvestris catus ). However, no individual tested positive by 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 two lions, Panthera leo (Seltmann et al., 2020). On the other hand, two studies have managed to detect by molecular techniques Besnoitia spp. DNA in faeces from pond bat (Myotis dasycneme ) in the Netherlands (Hornok et al., 2015) and in faecal matter from cheetahs (Acinonyx jubatus ) in Namibia (Schares et al., 2021); in the first report, authors hypothesized that B. besnoiti -like sequences 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 ofB. besnoiti . In addition, the later study (Schares et al., 2021) suggests that a so far unknown Besnoitia species closely related to B. darlingi , B. neotomofelis , B. oryctofelisi ,B. akodoni or B. jellisoni is cycling in Namibian wildlife.
In the present survey, B. besnoiti- like 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-derived Besnoitia 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 Besnoitia species in large mammals (B. bennetti ,B. caprae and B. tarandi ) but particularly with B. besnoiti , from European countries (Belgium, Finland, Italy, Germany, Portugal and Spain), Israel and Iran. The ITS1 rDNA sequence ofB. besnoiti -like from red foxes suggest a closer relationship toB. besnoiti , which infects cattle in the Old World. In a separate phylogenetic cluster and with large evolutionary divergence, other species of Besnotia genus (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-1 region shows the most informative nucleotide variances and phylogenetically clearly splits small mammalian from large mammalian Besnoitia species. Of note, all foxes withBesnotia sp. 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 the occurrence of B. besnoiti -like DNA in a European mesocarnivore gut. 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).
The prevalence rate found in red fox (2.1%; 4/187) is in agreement with worldwide reported data for the excretion of oocysts of the closely related Toxoplasmatinae parasite Toxoplasma gondii by Felidae (Hatam-Nahavandi et al., 2021). With this in mind the presence of bovine besnoitiosis could be explained in regions where the disease is endemic not only by animal trade and mechanical transmission but also by the possible high prevalence of oocysts shedding and wide abundance of foxes.
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 such interesting findings; 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 of B. darlingi , B. neotomofelis andB. oryctofelisi respectively, suggests that it could make us think that red fox might have a role as a definitive host in other species of Besnoitia. However, the sequences found in fox faeces are genetically not so closely related to small mammalianBesnoitia spp. as to B. besnoiti . In addition, other small mammalian prey of the fox may be unknown intermediate hosts of B. besnoiti , and the fox may act as a definitive host for B. 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 carrier, after the accidental ingestion of the hypothetical B. besnoitia oocysts from the contaminated soil (and foodstuff) 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, foxes were the only species among the twelve studies, where B. besnoiti -like DNA was identified, suggesting that this/such species might play a role in the epidemiology of the disease.
To conclude, Sanger sequencing analysis of four of the 129 faecal samples revealed that the presence of B. Besnoiti- like species in red fox (Vulpes vulpes ) faeces has been confirmed in Spain. Further epidemiological and experimental studies with a similar approach may help in the search for the definitive host of this parasite. In addition, future studies are needed to identify its natural intermediate host in small mammalian prey of foxes.