1. Investigación

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.

Iberian wild goats (Capra pyrenaica, also known as Iberian ibex, Spanish ibex, and Spanish wild goat) underwent strong genetic bottlenecks during the 19th and 20th centuries due to overhunting and habitat destruction. From the 1970s to 1990s, augmentation translocations were frequently carried out to restock Iberian wild goat populations (very often with hunting purposes), but they were not systematically planned or recorded. On the other hand, recent data suggest the occurrence of hybridization events between Iberian wild goats and domestic goats (Capra hircus). Augmentation translocations and interspecific hybridization might have contributed to increase the diversity of Iberian wild goats. With the aim of investigating this issue, we have genotyped 118 Iberian wild goats from Tortosa-Beceite, Sierra Nevada, Muela de Cortes, Gredos, Batuecas, and Ordesa and Monte Perdido by using the Goat SNP50 BeadChip (Illumina). The analysis of genotypic data indicated that Iberian wild goat populations are strongly differentiated and display low diversity. Only three Iberian wild goats out from 118 show genomic signatures of mixed ancestry, a result consistent with a scenario in which past augmentation translocations have had a limited impact on the diversity of Iberian wild goats. Besides, we have detected eight Iberian wild goats from Tortosa-Beceite with signs of domestic goat introgression. Although rare, hybridization with domestic goats could become a potential threat to the genetic integrity of Iberian wild goats; hence, measures should be taken to avoid the presence of uncontrolled herds of domestic or feral goats in mountainous areas inhabited by this iconic wild ungulate.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, is considered a pathogen of animal origin that is mainly transmitted from human to human. Several animal species can be naturally or experimentally infected by SARS-CoV-2, with compelling evidence that mink is highly susceptible to SARS-CoV-2 infection. Human-to-mink infection cases have been reported and there are also suggestions that mink-to-human infection occurs. Mink infections have been reported to date only on fur farms, except for one infected freeranging wild mink near a Utah (USA) fur farm, which suggests a transmission pathway from farms to wild mink. We now report the detection of SARS-CoV-2 in 2 of 13 feral dark brown American mink (Neovison vison) trapped in the Valencian Community (Eastern Spain), during an invasive species trapping campaign. They were trapped in riverbeds in sparsely inhabited rural areas known to harbor self-sustained feral mink populations. The closest fur farm is about 20 km away. SARS-CoV-2 RNA was detected by two-step RT-PCR in these animals’ mesenteric lymph nodes and was confirmed by sequencing a 397-nucleotide amplified region of the S gene, yielding identical sequences in both animals. A molecular phylogenetic analysis was run on this sequence, which was found to correspond to the consensus SARS-CoV-2 sequence fromWuhan. Our findings appear to represent the first example of SARS-CoV-2 acquired in the wild by feral mink in self-sustained populations.

In recent decades, worldwide fish consumption has increased notably worldwide. Despite the health benefits of fish consumption, it also can suppose a risk because of fishborne diseases, including parasitic infections. Global changes are leading to the emergence of parasites in new locations and to the appearance of new sources of transmission. That is the case of the zoonotic protozoa Cryptosporidium spp., Giardia duodenalis, and Toxoplasma gondii; all of them reach aquatic environments and have been found in shellfish. Similarly, these protozoa can be present in other aquatic animals, such as fish. The present review gives an overview on these three zoonotic protozoa in order to understand their potential presence in fish and to comprehensively revise all the evidences of fish as a new potential source of Cryptosporidium spp., Giardia duodenalis, and Toxoplasma gondii transmission. All of them have been found in both marine and freshwater fishes. Until now, it has not been possible to demonstrate that fish are natural hosts for these protozoa; otherwise, they would merely act as mechanical transporters. Nevertheless, even if fish only accumulate and transport these protozoa, they could be a “new” source of infection for people.

The appearance of methicillin-resistant strains of Staphylococcus aureus (MRSA) in several animal species (including rabbits) has set o alarms for their capacity to act as reservoirs for this bacterium. This is especially important in wild animals given its epidemiological implications. The objectives of this study were to identify and characterize S. aureus, specifically MRSA, strains in wild lagomorph high-density areas. Ten hares and 353 wild rabbits from 14 towns with a high rabbit density in the Valencian region (eastern Spanish coast) were sampled. Swabs from the nasal cavity, ears, perineum and lesions (when present) were taken for microbiological studies. The detection of di erent genes and antibiotic susceptibility studies were also carried out. Of all the animals, 41.3% were positive for S. aureus, of which 63.3% were MRSA. Ears were the anatomical location with more S. aureus and MRSA strains. The more frequently identified MLST type was ST1945 (97.1%, 136/140). The mecA gene was found only in one sample. The rest (n = 139) carried the mecC gene and were included in CC130, except one. Penicillin resistance was detected in 28 mec-negative isolates and, in one case, bacitracin resistance. mecA isolate presented resistance to enrofloxacin and tetracycline, and 10 mecC isolates also showed bacitracin resistance. No MRSA isolate was positive for genes chp, sea, tst and PVL. Two ST1945 isolates contained IEC type E (comprising genes scn and sak). mecA-isolate was positive for blaZ. Of the 28 MSSA strains showing resistance to penicillin, 22 carried the blaZ gene. These surprising results highlight the marked presence of MRSA strains in wild rabbits in high-density areas.

Mycoplasma conjunctivae is an obligate microparasite that causes Infectious Keratoconjunctivitis (IKC) in Caprinae species. IKC is a long-recognised disease, but little attention has been paid to the mechanisms of transmission of the mycoplasma and its occurrence in locations other than the eyes. In this study, the presence of M. conjunctivae is assessed in the eyes, external ear canals (EEC), nasal cavity, and vagina of host species as well as in potential vectors, which may be involved in the transmission and persistence of infection within the host. M. conjunctivae was detected by qPCR in 7.2 % (CI 95% 4.7-11.0) of the ear swabs and 9.5 % (CI 95% 6.4-13.9) of the nasal swabs from Pyrenean chamois, Iberian ibex, domestic sheep and mouflon without statistical differences between species. Mycoplasma detection in nasal swabs was mostly associated with ocular infection (95.6%), but this was not the case for EEC (52.6%). Among the eye-positive ruminants, 27.3% were positive in ear swabs and 64.7% in nasal swabs, and the threshold cycle values of the qPCR were correlated only between eye and nasal swabs (p<0.01; r2=0.56). M. conjunctivae was detected in 1.7% - 7.1 % of Musca spp. captured during an IKC outbreak in Iberian ibex and in one out of three endemic sheep flocks. The results indicate that the transmission of M. conjunctivae may occur by direct contact with eye or nasal secretions and/or indirectly through flies. The M. conjunctivae DNA detection in EEC suggests that it can colonise the auditory tract, but the significance for its persistence within the host should be further assessed.