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.