1. Investigación

The protozoans Giardia duodenalis and Cryptosporidium spp. are common causes of gastrointestinal disease in humans and animals. While both are commonly documented in domestic animals, few studies have analysed their presence in wildlife. To assess the prevalence of both parasites in wild boar (Sus scrofa) in the Valencian Community (eastern Spain), 498 wild boar faecal samples were collected from 2018 to 2022. Cryptosporidium spp. was detected by performing a nested PCR targeting a 578 bp sequence of the small subunit ribosomal RNA gene (SSU rRNA), followed by sequencing and phylogenetic analysis. For G. duodenalis, a qPCR amplifying a fragment of 62 bp from the SSU rRNA was employed. Positive samples were genotyped for glutamate dehydrogenase and β-giardin genes. Different epidemiological factors were considered potential modulating variables in the transmission of both parasites. G. duodenalis prevalence was 1.20%, while Cryptosporidium spp. prevalence reached 21.7%. Coinfection was observed in 0.2%. Genotyping of G. duodenalis isolates only detected genotype E. Two species of Cryptosporidium spp. were identified: Cryptosporidium scrofarum and Cryptosporidium suis. The results of this study demonstrate that the exposure to Cryptosporidium spp. in wild boars is high, particularly among young individuals belonging to the Typical Mediterranean climate. Moreover, the probability of infection is dependent on both the season and the density of wild boars. On the other side, exposure to G. duodenalis seems scarce and is influenced, in turn, by the climate. Both Cryptosporidium species detected in the present study have been reported in humans. Due to wild boar increasing in number and their colonisation of urban and peri-urban areas, this could represent an inherent health risk for the human population.

Staphylococcus aureus is an opportunistic multi-host pathogen that threatens both human and animal health. Animals can act as a reservoir of S. aureus for humans, but very little is known about wild animals’ epidemiological role. Therefore, in this study, we performed a genomic characterization of S. aureus isolates from wildlife, hunters, and their auxiliary hunting animals of Eastern Spain. Of 20 different species, 242 wild animals were examined, of which 28.1% were S. aureus carriers. The common genet, the Iberian ibex, and the European hedgehog were the species with the highest S. aureus carriage. We identified 30 different sequence types (STs), including lineages associated with wild animals such as ST49 and ST581, multispecies lineages such as ST130, ST398, and ST425, and lineages commonly isolated from humans, including ST1 and ST5. The hunters and the single positive ferret shared ST5, ST398, or ST425 with wild animals. In wildlife isolates, the highest resistance levels were found for penicillin (32.8%). For virulence factors, 26.2% of them carried superantigens, while 14.8% harbored the immune evasion cluster (IEC), which indicates probable human origin. Our findings suggest that wild animals are a reservoir of clinically relevant genes and lineages that could have the potential to be transmitted to humans. These data support the notion that wildlife surveillance is necessary to better understand the epidemiology of S. aureus as a pathogen that circulates among humans, animals, and the environment.

The Iberian hare (Lepus granatensis) is a popular small game species in the Iberian Peninsula, and it has never been reported to be affected by sarcoptic mange. An adult female Iberian hare with overt skin lesions on forelimbs and ventral thorax, suggestive of sarcoptic mange, was culled in Quart de les Valls municipality in the Valencian Community, Spain, in 2019. Skin scrapings were digested in 10% KOH solutions to confirm the presence of mites. Ten Sarcoptes microsatellite markers were used to characterize the genetic structure of mites obtained from the hare, and from sympatric and allopatric wild rabbits (Oryctolagus cuniculus) and red foxes (Vulpes vulpes). A total of 56 alleles were counted across the 10 microsatellite loci. Six private alleles were found at four loci (Sarms 33, 38, 41, 45). The multivariate analysis characterized three main clusters, corresponding to mites collected on foxes originating from Catalonia, foxes from Valencia and the hare plus wild rabbits. To our knowledge, this is the first reported case of sarcoptic mange in the Iberian hare. The origin was molecularly traced back to contacts with endemically infected wild rabbits. We encourage further investigations on cross-transmissibility of S. scabiei between wild rabbits and the diverse representatives of Lepus spp.

The short-eared dog (Atelocynus microtis) is one of the least known canids of the world. Essential aspects of its ecology, activity patterns and feeding habits remain unknown. To study the composition, behavior and interactions of the western Amazonian scavenger community, a baited camera-trapping survey was performed from August to September 2018 in the surroundings of the Tiputini Biodiversity Station (Yasuní Biosphere Reserve, Ecuador). A total of 12 camera stations were set up, employing cow heads acquired from local meat processors as bait. Short-eared dogs were captured by camera-traps, and two individuals showed interest in the carrion, providing the first evidence that short-eared dogs include scavenging as part of their foraging behavior. Detection of short-eared dogs in the area was surprisingly frequent, despite the short survey period, indicating that use of baited camera-traps can be an effective tool for the study of the short-eared dog, an elusive and poorly documented species.

Prevalence and temporal evolution of the infection by the acanthocephalan Macracanthorhynchus hirudinaceus is studied in the Valencian Community (Eastern Spain), a region only recently fully colonized by the expanding native Eurasian wild boar (Sus scrofa). For 8 years, a total of 1486 wild boars were sampled in order to look for the parasite. The mean prevalence was 20.7% (95% CI, 18.6–22.8; 307/1486). We observed an increasing trend through time, both in the number of wild boars and affected districts. The prevalence of M. hirudinaceus rose in parallel to the annual capture of wild boars, and its presence has been expanding towards the East. A hotspot of M. hirudinaceus is located to the west of the study area, in Muela de Cortes Game Reserve, where 89.6% of the wild boars were positive for the infection, constituting one of the world’s highest known prevalence areas.

As one of the most relevant foodborne diseases, it is essential to know the factors related to the transmission, persistence and prevalence of Toxoplasma gondii infection. Eurasian wild boar (Sus scrofa) might play a relevant role in T.gondii's life cycle. This species is the most consumed big game animal in Spain and may act as a source of infection if the meat is eaten raw or undercooked or due to cross-contaminations. Additionally, wild boar can act as an excellent bioindicator of T.gondii circulation in the ecosystem, because its natural behaviour leads to exposure to oocysts from the soil when rooting and tissular bradyzoites when scavenging. A total of 1003 wild boar were sampled from 2010 to 2017 in Mediterranean Spain. Blood samples were tested with an indirect ELISA test giving a total of 14.1% (95% confidence interval 12.0–16.4%) positive results. The prevalence was not homogeneous in neither the animals nor the sampled districts. Significant differences were found regarding age, climatic conditions and human space occupancy. Human population aggregation, assessed by Demangeon's index, was identified as an influential factor in T.gondii infection risk. This multiple approach allows us to evaluate local risks for human and environmental contamination.

We conducted a retrospective serosurvey for antibodies against Crimean-Congo hemorrhagic fever virus in wild ungulates along the eastern Mediterranean Coast of Spain. The virus has been endemic in this region since 2010 but is mainly restricted to geographic clusters with extremely high seropositivity associated with high density of bovids.

We used anonymous questionnaires to assess the hygienic and sanitary aspects of game meat self-consumption in Eastern Spain as the first step towards a health risk assessment. The survey yielded 472 valid interviews from active hunters. The maximum possible score was 65 points (average 29 8; range 1–52). Most participants were men (95%), but women achieved significantly better scores (p = 0.003). Hunters above 65 years old scored significantly lower results than younger groups (p = 0.007). The score increased with the educational level (p = 0.046). A 92% of the collaborators consumed game meat. Veterinary inspection and freezing were irregular among the participants. Most respondents declared carrying the animals in their personal vehicles. Of the dressing process, 61% of sites were outdoors, 68% of the participants declared using specific knives, 64% used the same clothes as in the field, and 42% used disposable gloves. The most usual way to dispose of the remains was garbage containers (41%); offal abandonment in the field was 33%, and 13% fed domestic animals using the remains. We conclude that public health authorities should increase their interest in the self-consumption of game meat. Clear guidelines about domestic dressing facilities and hygienic habits should be published, these being essential when looking for synergies with hunter associations.

Fish not only harbor host-specific species/genotypes of Cryptosporidium, but also species like zoonotic C. parvum or anthroponotic C. hominis, which can pose a risk for fish consumers. This study aims to investigate fish cryptosporidiosis in an important aquaculture and fishery area of the Western Mediterranean (Comunidad Valenciana, Spain). We analyzed 404 specimens belonging to the following three groups: cultivated fish (N = 147), wild synanthropic fish (N = 147) and wild fish from extractive fisheries (N = 110). Nested PCR targeting the 18S rRNA gene, followed by sequencing and phylogenetic analysis, were performed. Positive isolates were also amplified at the actin gene locus. An overall prevalence of 4.2% was detected, with the highest prevalence in the synanthropic group (6.1%). C. molnari was identified in thirteen specimens from seven different host species. Zoonotic C. ubiquitum was detected in two European sea bass (Dicentrarchus labrax). One isolate similar to C. scophthalmi was detected in a cultivated meagre (Argyrosomus regius), and one isolate, highly divergent from all the Cryptosporidium species/genotypes described, was identified from a synanthropic round sardinella (Sardinella aurita). This study contributes to increasing the molecular data on fish cryptosporidiosis, expanding the range of known hosts for C. molnari and identifying, for the first time, zoonotic C. ubiquitum in edible marine fishes, pointing out a potential health risk.

Animals have been involved in the three known outbreaks of severe respiratory syndromes due to coronaviruses (years 2005, 2012, and 2019). The pandemic nature of the SARS-CoV-2 outbreak increases the likelihood of infection from humans of susceptible animal species that, thus, could become secondary viral hosts and even disease reservoirs. We present evidence of spillover infection of wild mustelids by reporting the presence of SARS-CoV-2 in a Eurasian river otter found near a water reservoir in the Valencian Community (Spain). We detected the virus using two different commercial RTqPCR assays on RNA extracted from the nasopharynx (swabbing) and from lung tissue and mediastinal lymph node homogenates. The corresponding samples from two additional otters from distant sites tested negative in identical assays. The diagnosis in the positive otter was confirmed by two-tube RT-PCR assay in which RNA was first retrotranscribed, and then specific regions of the spike (S), nucleocapsid (N), and ORF10 genes were separately amplified from the produced cDNA, followed by electrophoretic visualization and Sanger sequencing. The sequences of the amplified products revealed some non-synonymous changes in the N and ORF10 partial sequences, relative to the consensus sequence. These changes, identified already in human patient samples, point to human origin of the virus, although their specific combination was unique. These findings, together with our previous report of SARS-CoV-2 infection of feral American mink, highlight the need for SARS-CoV-2 surveillance of wild or feralmustelids to evaluate the risk that these animals could become SARS-CoV-2 reservoirs.