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.