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In order to assess the risk of SARS-CoV-2 infection, transmission and reservoir development in swine, we combined results of an experimental and two observational studies. First, intranasal and intratracheal challenge of eight pigs did not result in infection, based on clinical signs and PCR on swab and lung tissue samples. Two serum samples returned a low positive result in virus neutralization, in line with findings in other infection experiments in pigs. Next, a retrospective observational study was performed in the Netherlands in the spring of 2020. Serum samples (N =417) obtained at slaughter from 17 farms located in a region with a high human case incidence in the first wave of the pandemic. Samples were tested with protein micro array, plaque reduction neutralization test and receptor-binding-domain ELISA. None of the serum samples was positive in all three assays, although six samples from one farm returned a low positive result in PRNT (titers 40-80). Therefore we conclude that serological evidence for large scale transmission was not observed. Finally, an outbreak of respiratory disease in pigs on one farm, coinciding with recent exposure to SARS-CoV-2 infected animal caretakers, was investigated. Tonsil swabs and paired serum samples were tested. No evidence for infection with SARS-CoV-2 was found. In conclusion, Although in both the experimental and the observational study few samples returned low antibody titer results in PRNT infection with SARS-CoV-2 was not confirmed. It was concluded that sporadic infections in the field cannot be excluded, but large-scale SARS-CoV-2 transmission among pigs is unlikely. info:eu-repo/semantics/publishedVersion

The prevalence of low virulence classical swine fever virus (CSFV) strains makes viral eradication difficult in endemic countries. However, the determinants for natural CSFV attenuation and persistence in the field remain unidentified. The aim of the present study was to assess the role of the RNase activity of CSFV Erns in pathogenesis, immune response, persistent infection, and viral transmission in pigs. To this end, a functional cDNA clone pPdR-H30K-36U with an Erns lacking RNase activity was constructed based on the low virulence CSFV field isolate Pinar de Rio (PdR). Eighteen 5-day-old piglets were infected with vPdR-H30K-36U. Nine piglets were introduced as contacts. The vPdR-H30K-36U virus was attenuated in piglets compared to the parental vPdR-36U. Only RNA traces were detected in sera and body secretions and no virus was isolated from tonsils, showing that RNase inactivation may reduce CSFV persistence and transmissibility. The vPdR-H30K-36U mutant strongly activated the interferon-α (IFN-α) production in plasmacytoid dendritic cells, while in vivo, the IFN-α response was variable, from moderate to undetectable depending on the animal. This suggests a role of the CSFV Erns RNase activity in the regulation of innate immune responses. Infection with vPdR-H30K-36U resulted in higher antibody levels against the E2 and Erns glycoproteins and in enhanced neutralizing antibody responses when compared with vPdR-36U. These results pave the way toward a better understanding of viral attenuation mechanisms of CSFV in pigs. In addition, they provide novel insights relevant for the development of DIVA vaccines in combination with diagnostic assays for efficient CSF control. info:eu-repo/semantics/publishedVersion

Rotifers are widely used as a live prey for finfish larvae, although their content in minerals such as selenium and zinc is lower than in fish. In the present study, mixed microalgae (i.e., Isochrysis aff. galbana and Nannochloropsis oculata) were enriched with different amounts of Se and Zn either alone (45, 90, and 135 mg L−1 of Zn, 60, 120, and 180 mg L−1 of Se) or mixed (22.5, 45, and 90 mg Zn L−1 and 30, 60, and 120 mg Se L−1). The content of minerals in the microalgae was analysed after 1 and 3 h enrichment and their effect on the growth and mineral composition of the rotifer. Based on the results, 1 h enrichment time and 90 and 120 mg L−1 of Zn and Se, respectively, resulted in the highest amount of Se and Zn in enriched microalgae. The maximum Zn content was detected in the rotifers fed 90 mg L−1 Zn and 90 mg L−1 Zn +120 mg L−1 of Se, but the maximum Se was observed in rotifers fed with 120 and 180 mg L−1 Se. Thus, Se- and Zn-enriched rotifers can be used as a mineral delivery method to cover marine fish larvae nutritional requirements. info:eu-repo/semantics/acceptedVersion

Background The gut microbiota influences host performance playing a relevant role in homeostasis and function of the immune system. The aim of the present work was to identify microbial signatures linked to immunity traits and to characterize the contribution of host-genome and gut microbiota to the immunocompetence in healthy pigs. Results To achieve this goal, we undertook a combination of network, mixed model and microbial-wide association studies (MWAS) for 21 immunity traits and the relative abundance of gut bacterial communities in 389 pigs genotyped for 70K SNPs. The heritability (h2; proportion of phenotypic variance explained by the host genetics) and microbiability (m2; proportion of variance explained by the microbial composition) showed similar values for most of the analyzed immunity traits, except for both IgM and IgG in plasma that was dominated by the host genetics, and the haptoglobin in serum which was the trait with larger m2 (0.275) compared to h2 (0.138). Results from the MWAS suggested a polymicrobial nature of the immunocompetence in pigs and revealed associations between pigs gut microbiota composition and 15 of the analyzed traits. The lymphocytes phagocytic capacity (quantified as mean fluorescence) and the total number of monocytes in blood were the traits associated with the largest number of taxa (6 taxa). Among the associations identified by MWAS, 30% were confirmed by an information theory network approach. The strongest confirmed associations were between Fibrobacter and phagocytic capacity of lymphocytes (r = 0.37), followed by correlations between Streptococcus and the percentage of phagocytic lymphocytes (r = -0.34) and between Megasphaera and serum concentration of haptoglobin (r = 0.26). In the interaction network, Streptococcus and percentage of phagocytic lymphocytes were the keystone bacterial and immune-trait, respectively. Conclusions Overall, our findings reveal an important connection between gut microbiota composition and immunity traits in pigs, and highlight the need to consider both sources of information, host genome and microbial levels, to accurately characterize immunocompetence in pigs.

We conducted a serosurvey for Crimean-Congo hemorrhagic fever virus antibodies in various wildlife species in Catalonia, northeastern Spain. We detected high seroprevalence in southern Catalonia, close to the Ebro Delta wetland, a key stopover for birds migrating from Africa. Our findings could indicate that competent virus vectors are present in the region. info:eu-repo/semantics/publishedVersion

The measurement of glucocorticoid hormones, such as cortisol, is a technique being increasingly used as a welfare or stress indicator (Mormède et al., 2007; Palme, 2019). In response to perceived stressors, these hormones are released into the bloodstream through activation of the hypothalamic–pituitary–adrenal (HPA) axis. The release of cortisol, a principal glucocorticoid in many mammals, enables the body to cope with threatening or demanding situations. Although this adaptive response is usually beneficial for the animal, chronic activation of the HPA axis and long-term cortisol elevations can reduce growth and reproduction, compromise the immune system, and impair the individual's ability to respond to subsequent stressors (Moberg & Mench, 2000). info:eu-repo/semantics/publishedVersion

First identified in 1947, Zika virus took roughly 70 years to cause a pandemic unusually associated with virus-induced brain damage in newborns. Zika virus is transmitted by mosquitoes, mainly Aedes aegypti, and secondarily, Aedes albopictus, both colonizing a large strip encompassing tropical and temperate regions. As part of the international project ZIKAlliance initiated in 2016, 50 mosquito populations from six species collected in 12 countries were experimentally infected with different Zika viruses. Here, we show that Ae. aegypti is mainly responsible for Zika virus transmission having the highest susceptibility to viral infections. Other species play a secondary role in transmission while Culex mosquitoes are largely non-susceptible. Zika strain is expected to significantly modulate transmission efficiency with African strains being more likely to cause an outbreak. As the distribution of Ae. aegypti will doubtless expand with climate change and without new marketed vaccines, all the ingredients are in place to relive a new pandemic of Zika. Zika virus (ZIKV), the causative agent of virus-induced brain damage in newborns, is transmitted by mosquitoes, mainly Aedes aegypti, and secondarily, Aedes albopictus. Here, Obadia et al. characterize ZIKV vector competence of 50 mosquito populations from six species collected in 12 different countries to inform about epidemic risk. They find that African ZIKV strain shows higher transmission efficiency compared to American and Asian ZIKV strains and that Ae. aegypti mosquitoes have highest susceptibility to infections, while Culex mosquitoes are largely non-susceptible.

With the current expansion of vector-based research and an increasing number of facilities rearing arthropod vectors and infecting them with pathogens, common measures for containment of arthropods as well as manipulation of pathogens are becoming essential for the design and running of such research facilities to ensure safe work and reproducibility, without compromising experimental feasibility. These guidelines and comments were written by experts of the Infravec2 consortium, a Horizon 2020-funded consortium integrating the most sophisticated European infrastructures for research on arthropod vectors of human and animal diseases. They reflect current good practice across European laboratories with experience of safely handling different mosquito species and the pathogens they transmit. As such, they provide experience-based advice to assess and manage the risks to work safely with mosquitoes and the pathogens they transmit. This document can also form the basis for research with other arthropods, for example, midges, ticks or sandflies, with some modification to reflect specific requirements. info:eu-repo/semantics/publishedVersion

Dinoflagellates of the genera Gambierdiscus and Fukuyoa are known to produce several bioactive compounds including the potent neurotoxic ciguatoxins (CTXs) which are able to accumulate in fish and through the food web. When humans ingest fish contaminated with CTXs, it can result in an intoxication named ciguatera. Although not all the currently recognized species are able to produce toxins, G. australes and G. excentricus have been highlighted to be the most abundant and toxic among the species present in the Atlantic. Even though the genera Gambierdiscus and Fukuyoa are endemic to tropical areas, recently their presence was recorded in subtropical and temperate regions. In this work, the development of three molecular assays for the detection of the Gambierdiscus and Fukuyoa genera and for G. australes and G. excentricus species, based on the combination of recombinase polymerase amplification with detection via hybridization, is successfully described. Furthermore, a remarkable limit of detection of a single cell was achieved. Additionally, six different species have been used to check the ability of each primer set to give an amplified product, even in presence of potentially interfering non-target DNAs. Therefore, these developments provide a rapid and cost-effective strategy for detection of both genera and two of the most toxic species, which will undoubtedly contribute to reliable screening of samples and ciguatera risk assessment, guaranteeing seafood safety and protection of human health. info:eu-repo/semantics/acceptedVersion

Rotaviruses (RVs) are classified into nine species or groups (RVA-RVD and RVF-RVJ). RVA, RVB and RVC are well-recognized as etiological agents of enteric disease on swine farms and have been identified in all countries with a relevant pork production. Contrarily, RVH has only been identified on swine farms from Japan and more recently from Brazil, USA, South Africa and Vietnam but not yet in Europe. The occurrence of RVH was investigated in 103 Spanish pig herds. Nine farms were positive and we achieved a complete nucleotide sequences in three isolates while another RVH isolate was partially sequenced. Mean nucleotide identities with the RVH sequences available in GenBank ranged between 69.4 and 93.7 %. Phylogenetically, all genomic segments of Spanish RVH isolates clustered closely with other porcine RVH strains but were distantly related to human RVH as well as bat RVH strain. To the best of our knowledge this is the first report of RVH on swine farms in Europe including its characterization by means of complete genome sequencing. info:eu-repo/semantics/acceptedVersion

Background Livestock play an important role as reservoir of enteric pathogens and antimicrobial resistance (AMR), a health and economic concern worldwide. However, little is known regarding the transmission and maintenance of these pathogens at the wildlife-livestock interface. In this study, we assessed the occurrence, genetic diversity and AMR of Campylobacter spp. and Salmonella spp. shed by sympatric free-ranging livestock and a wild herbivore in an alpine ecosystem. Results Campylobacter spp. was isolated from 23.3 % of cattle and 7.7 % of sheep but was not isolated from horses nor Pyrenean chamois (Rupicapra pyrenaica). Campylobacter jejuni was the most frequent species. A high genetic diversity and certain host specificity of C. jejuni isolates was observed. The main AMR detected in Campylobacter isolates was to nalidixic acid (88.2 %), ciprofloxacin (82.4 %) and tetracycline (82.4 %); only 11.7 % of the isolates were pan-susceptible and 17.6 % were multi-resistant. Salmonella ser. Newport was isolated only from one Pyrenean chamois and was pan-susceptible. Conclusions Results show that free-ranging cattle and sheep are spreaders of Campylobacter as well as their AMR strains in the alpine environment. Therefore, contaminated alpine pastures or streams may constitute a source for the dissemination of AMR enteropathogens. However, apparently, alpine wild ungulates such as Pyrenean chamois play a negligible role in the epidemiology of zoonotic enteropathogens and AMR, and are not potential bioindicators of the burden of alpine environments.

Low and variable growth and survival rates (SR) of 6–10 days postfertilization zebrafish larvae are a problem. This problem seems to be linked to starter feed characteristics. This study is an attempt to find alternatives to address these requests. For this, larvae were fed fresh and lyophilized microalgae (Chlorella, Scenedesmus, and Haematococcus), egg yolk (YOLK), lyophilized Artemia nauplii (LAN), and a combination of them. The lowest SR was observed in algae-fed larvae. All died on day 11 showing an emaciated appearance, similar to starved larvae. The highest SR was observed in YOLK- and LAN-fed larvae, which also showed an elongated anterior part of the body. Negative correlations of SR with vegfaa (vascular endothelial growth factor) and morphometric traits with igf2a (insulin-like growth factor) were also found and supported by changes at the molecular level. The presence of algae in the digestive tract of the larvae and the observation of fecal droppings indicate that the algae have an appropriate size and are palatable. The increase in the digestive enzyme activity shows the larval effort to digest the algae. The fact that the algae-fed larvae died even before the larvae were kept in starvation indicates the dramatic amount of energy that the larvae spent in microalgae digestion. Although both YOLK- and LAN-fed larvae had the highest SR, LAN group started to feed on Artemia nauplii sooner. This can be linked to the delayed growth in YOLK-fed larvae and an accelerated growth in the case of LAN-fed group. LAN is an expensive feed with negative effects on water quality, whereas YOLK is a cheap and nutritionally balanced feed with fine granular texture that contributes to a larval SR similar to LAN without affecting water quality. In conclusion, microalgae cannot be considered a suitable starter food for zebrafish, whereas LAN and YOLK can be considered good starter feeds. info:eu-repo/semantics/acceptedVersion

The paraphyletic diatom genus Nitzschia comprises over 1000 morphologically distinct pennate taxa, known from the benthos and plankton of freshwater, brackish and marine environments. The principal diagnostic characters for delimitation of Nitzschia species include valve shape, the position and structure of the raphe, presence/absence and shape of the proximal raphe endings the shape of the terminal raphe fissures, areola structure, and specific morphometric features such as cell size, and stria and fibula density. In this study, we isolated 12 diatom strains into culture from samples collected at the surface or greater depths of the southeastern Adriatic Sea. Morphological analyses included LM, SEM and TEM observations, which, along with specific morphometric features, allowed us to distinguish three new Nitzschia species. These findings were congruent with the results of phylogenetic analyses performed on nuclear-encoded SSU (18S) rDNA and chloroplast-encoded rbcL and psbC genes. One of the new species (Nitzschia dalmatica sp. nov.) formed a lineage within a clade of Bacillariaceae containing members of the Nitzschia sect. Dubiae, which was sister to Psammodictyon. A second lineage was part of a novel clade that is significantly distinct from other Nitzschia species sequenced so far and includes Nitzschia adhaerens sp. nov. and N. cf. adhaerens. A further new species was found, Nitzschia inordinata sp. nov., which appeared as the sister group to the N. adhaerens clade and the conopeoid Nitzschia species in our phylogenetic trees. Our findings contribute to the overall diversity of genus Nitzschia, especially in identifying some deep branches within the Bacillariaceae, and highlight under-scoring of this genus in marine plankton.

Malaria, leishmaniasis and trypanosomiasis are arthropod-borne, parasitic diseases that constitute a major global health problem. They are generally found in developing countries, where lack of access to preventive tools and treatment hinders their management. Because these parasites share an increased demand on glucose consumption with most cancer cells, six compounds used in anti-tumoral research were selected to be tested as antiparasitic agents in in vitro models of Leishmania infantum, Trypanosoma brucei, T. cruzi, and Plasmodium falciparum: dichloroacetic acid (DCA), 3-bromopyruvic acid (3BP), 2-deoxy-D-glucose (2DG), lonidamine (LND), metformin (MET), and sirolimus (SIR). No parasite-killing activity was found in L. infantum promastigotes, whereas DCA and 3BP reduced the burden of intra-macrophagic amastigotes. For T. brucei all selected compounds, but 2DG, decreased parasite survival. DCA, 2DG, LND and MET showed parasite-killing activity in T. cruzi. Finally, anti-plasmodial activity was found for DCA, 2DG, LND, MET and SIR. These results reinforce the hypothesis that drugs with proven efficacy in the treatment of cancer by interfering with ATP production, proliferation, and survival cell strategies might be useful in treating threatening parasitic diseases and provide new opportunities for their repurposing., Graphical abstract Image 1, Highlights • Parasitic diseases are prevalent among the poorest of the poor. • Some parasitic protists degrade glucose into CO2 even aerobically making this a target. • Degrading glucose into CO2 (Warburg effect) is also characteristic for cancer cells. • Repurposing cancer glycolysis blockers may provide cost-effective treatments for the poorest.

Medicated feed is a common strategy to control the occurrence of Streptococcus suis disease in swine production, but feed additives may constitute an alternative to metaphylaxis. In a farm with post-weaning S. suis disease, the following additives were tested: lysozyme (Lys), medium chain fatty acids plus lysozyme (FA + Lys), FA plus a natural anti-inflammatory (FA + antiinf) and amoxicillin (Amox). During the course of the study, FA + antiinf and Amox groups showed lower prevalence of clinical signs compatible with S. suis disease than the rest of the groups. Piglets from the FA + antiinf group showed high diversity and richness in their nasal and faecal microbiota. Diet supplements did not have major effects on the faecal microbiota, where the genus Mitsuokella was the only differentially present in the FA + Lys group. In the nasal microbiota, piglets from FA + antiinf presented higher differential abundance of a sequence variant from Ruminococcaceae and lower abundance of an unclassified genus from Weeksellaceae. In general, we detected more significant changes in the nasal than in the feacal microbiota, and found that parity of the dams affected the microbiota composition of their offspring, with piglets born to gilts exhibiting lower richness and diversity. Our results suggest that additives could be useful to control post-weaning disease when removing antimicrobials in farms.

9 páginas., A large-scale study was carried out to determine the prevalence of antibodies againstPestivirusspecies in wild ruminants and describe their spatial variation in mainland Spain. Serum samples of 1,874 wild ruminants from different regions of this country were collected between the years 2000 and 2017. A total of 6.6% (123/1,874) animals showed antibodies againstPestivirusby both blocking ELISA (bELISA) and virus neutralization tests (VNT). The prevalence of antibodies against pestiviruses was different both among species and regions. Seroprevalence by species was 30.0% (75/250) in Southern chamois (Rupicapra pyrenaica), 7.0% (25/357) in fallow deer (Dama dama), 2.5% (10/401) in red deer (Cervus elaphus), 2.4% (8/330) in Iberian wild goat (Capra pyrenaica), 1.1% (4/369) in roe deer (Capreolus capreolus) and 0.8% (1/130) in mouflon (Ovis aries musimon), not detecting seropositivity (0/37) in Barbary sheep (Ammotragus lervia). The results confirm that exposure to pestiviruses was detected throughout mainland Spain, with significantly higher seroprevalence in Northern regions associated with the presence of Southern chamois. This indicates an endemic circulation of pestiviruses in Southern chamois and a limited circulation of these viruses in the remaining wild ruminant species during the last two decades, thus suggesting that non-chamois species are not truePestivirusreservoirs in Spain. Nonetheless, the high spatial spread of these viruses points out that new epidemic outbreaks in naive wild ruminant populations or transmission to livestock may occur, evidencing the usefulness of monitoring pestiviruses in wild ruminants, especially at the wildlife-livestock interface., The present work has benefited from the financial aid of research grants funded by MINECO (AGL2016-76358-R and CGL2017-89866-R). S. Jiménez-Ruiz holds a PhD contract from the UCLM co-supported by the European Social Fund (2018/12504).

Este artículo se encuentra disponible en la siguiente URL: https://onlinelibrary.wiley.com/doi/epdf/10.1111/tbed.13737 En este artículo de investigación también participan: Marta Cerdà Cuéllar, Teresa Ayats, Jaume Jordá, Clara Marín y Santiago Vega. This is the pre-peer reviewed version of the following article: Sevilla-Navarro, S., Cerdà-Cuéllar, M., Ayats, T., Jordá, J., Marin, C., & Vega, S. (2021). Characterization of "Salmonella Frintrop" isolated from dromedary camels (Camelus dromedarius). Transboundary and Emerging Diseases, vol. 68, i. 2 (mar.), pp. 742?746, which has been published in final form at https://doi.org/10.1111/tbed.13737. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. Este es el pre-print del siguiente artículo: Sevilla-Navarro, S., Cerdà-Cuéllar, M., Ayats, T., Jordá, J., Marin, C., & Vega, S. (2021). Characterization of "Salmonella Frintrop" isolated from dromedary camels (Camelus dromedarius). Transboundary and Emerging Diseases, vol. 68, i. 2 (mar.), pp. 742?746, que se ha publicado de forma definitiva en https://doi.org/10.1111/tbed.13737. Different studies have reported the prevalence and antibiotic resistance of Salmonella in dromedaries’ camels and its role in camelid-associated salmonellosis in humans, but little is known about the epidemiology of Campylobacter in dromedaries. Here we investigate the prevalence, genetic diversity and antibiotic resistance of Campylobacter and Salmonella in dromedary camels (Camelus dromedarius). A total of 54 individuals were sampled from two unique dromedary farms located in Tenerife (Canary Islands, Spain). Whilst all the samples were Campylobacter-negative, Salmonella prevalence was 5.5% (3/54) and the only serovar isolated was S. Frintrop. The pulsed field gel electrophoresis analysis revealed a low genetic diversity, with all isolates showing a nearly identical pulsotype (similarity > 95%). Our results indicate that dromedaries’ camels could not be a risk factor for Campylobacter human infection, but seems to be a reservoir for Salmonella transmission. Since camel ride has become one of the main touristic attractions in several countries and its popularity has considerably risen in the last years, a mandatory control, especially for zoonotic pathogens, such as Campylobacter and Salmonella should be implemented. Preprint

A retrospective evaluation of PEDV positive samples recovered in Spain before and after the re‐emergence of this coronavirus in several European countries was carried out. We described for the first time recombinant SeCoV circulating in Spain between 1993 and 2014 and its misidentification as PEDV when diagnostic assays based on the S‐protein or S‐gene of the PEDV were used. The complete S‐gene sequence of 7 Spanish SeCoV and 30 PEDV Spanish isolates was phylogenetically analysed including the S‐gene sequences of the three SeCoV and a representative selection of the PEDV strains with complete genome sequences available in the GenBank. The tree showed a common ancestor for the S‐gene of the PEDV and SeCoV, but no evolution from any known PEDV clade was shown for the SeCoV strains. Moreover, complete genome sequences were obtained from 23 PEDV strains recovered in Spanish swine farms since 2014. The phylogenetic tree showed the INDEL type genogroup of these Spanish strains, supporting the lower pathogenicity of this genogroup since no significant economic losses were reported in the affected Spanish swine farms. Four subgroups were detected among PEDV strains in Spain, closely related to the recent European strains. Moreover, eight of the most recent Spanish PEDV isolates formed a subclade together with three European strains from 2015, showing a new evolution branch with a recombinant virus. info:eu-repo/semantics/acceptedVersion

A quantitative risk assessment model was developed to estimate the annual probability of introducing bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) at the farm level through animal movements. Data from 2017 official animal movements, biosecurity questionnaires, scientific literature, and expert opinion from field veterinarians were taken into consideration for model input parameters. Purchasing or introducing cattle, rearing replacement heifers offsite, showing cattle at competitions, sharing transport vehicles with other herds, and transporting cattle in vehicles that have not been cleaned and disinfected were considered in the model. The annual probability of introducing BVDV or BoHV-1 through infected animals was very heterogeneous between farms. The median likelihoods of BVDV and BoHV-1introduction were 12 and 9%, respectively. Farms that purchased cattle from within their region (i.e., local movements) and shared transport with other farms had a higher probability for BVDV and BoHV-1 introduction. This model can be a useful tool to support decision-making on biosecurity measures that should be prioritized to reduce the probability of introduction of these 2 diseases in dairy herds This work was supported by a Grant from the Ministerio de Ciencia e Innovación of Spain (AGL2016-77269-C2-1-R and AGL2016-77269-C2-2-R). The Universidad de Nariño (Pasto, Colombia) provided funding to the first author (BBB) SI

Spotted wolffishAnarhichas minorreproduction in captivity is dependent on in vitro fertilization. However, it is often challenging to acquire sufficient fresh sperm to fertilize the eggs that are obtained. In this study, we evaluate the possibility to store spotted wolffish sperm by refrigeration. Spotted wolffish sperm has the particularity that is already motile on stripping, and currently it is not possible to immobilize and reactivate. Thus, sperm refrigeration protocols should focus in extending this motility period that usually lasts up to 2 days. In a first experiment, we evaluated the possibility that the motility period of the sperm was limited by contamination with urine. The urea concentration in the sperm obtained both by stripping (17.10 ± 1.98 mg/dL) and directly from the testis (12.59 ± 2.37 mg/dL) was similar (p > 0.05), which indicate that the sperm collection method used avoid contamination with urine. Afterwards, we tested the possibility that the sperm motility period was limited by energy stores. The ATP concentration (initial value 5.65 ± 0.86 nmol/109cells) remained stable (p = 0.099) during 30 h after sperm collection, and similar values (p = 0.329) were recorded at end of sperm storage in both diluted (3.88 ± 1.35 nmol/109cells) and undiluted samples (4.76 ± 1.08 nmol/109). This indicates that the low intracellular ATP consumption, derived from the slow sperm motility, can probably be compensated rapidly enough by mitochondrial synthesis of ATP in the spotted wolffish sperm. In both experiments, diluted sperm kept higher percentage of motile cells during the storage time.

Mammary serum amyloid A3 (M-SAA3) has shown potential in stimulating innate immunity during intramammary infections, at calving and at dryoff. In this study, we produced recombinant caprine M-SAA3 to test its ability to reduce intramammary infections with Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli, which are all common mastitis-producing pathogens. Recombinant production of M-SAA3 (followed by lipopolysaccharide removal to avoid lipopolysaccharide-nonspecific stimulation of the immune system) was successfully achieved. Mammary serum amyloid A3 stimulated the expression of IL-8 in a dose-dependent manner in primary mammary cultures. Although a direct killing effect on Staph. aureus by M-SAA3 was not detected, this acute phase protein was able to reduce Staph. aureus, Strep. uberis, and Strep. dysgalactiae infections by up to 50% and induced a reduction in E. coli counts of 67%. In general, the best concentration of caprine M-SAA3 for inhibiting infections was the lowest concentration tested (10 μg/mL), although higher concentrations (up to 160 μg/mL) increased its antimicrobial potential against some pathogens. info:eu-repo/semantics/acceptedVersion

The effect of spray‐dried porcine plasma (SDPP) on the intestinal histological organization and autochthonous microbiota composition was evaluated in Sparus aurata. Fish were fed a basal diet (51 g/kg protein, 17 g/kg fat, 20.6 MJ/kg gross energy) and a diet containing 3 g/kg SDPP for 95 days (initial body weight, BW = 9.5 ± 0.2g, mean ± SD). The inclusion of SDPP promoted growth (p < .05), being fish fed the SDPP diet 6.2% (BW = 88.2 ± 1.6 g) heavier than the control (BW = 82.7 ± 3.2 g). SDPP increased the density of intestinal goblet cells (p < .05), whereas no differences in villi height were found (p > .05) between both groups. Intestinal microbiota was dominated by Proteobacteria (>85%) and Firmicutes (5%–12%), whereas Bacteroidetes never represented more than 1.5%. γ‐Proteobacteria, and Bacilli and Clostridia were the predominant classes. The short administration of SDPP (20 days) resulted in changes in microbiota diversity and richness associated with an increase in the sequences of the genus Lactobacillus and to a decrease in the genus Vibrio, whereas these changes were reverted at 95 days. Intestinal goblet cell density was not correlated to microbiota diversity and richness changes rather than to the immunostimulatory effect of the SDPP. info:eu-repo/semantics/acceptedVersion

Background Virus neutralization test (VNT) is widely used for serological survey of classical swine fever (CSF) and efficacy evaluation of CSF vaccines. However, VNT is a time consuming procedure that requires cell culture and live virus manipulation. C-strain CSF vaccine is the most frequently used vaccine for CSF control and prevention. In this study, we presented a neutralizing monoclonal antibody (mAb) based competitive enzyme-linked immunosorbent assay (cELISA) with the emphasis on the replacement of VNT for C-strain post–vaccination monitoring. Results One monoclonal antibody (6B211) which has potent neutralizing activity against C-strain was generated. A novel cELISA was established and optimized based on the strategy that 6B211 can compete with C-strain induced neutralizing antibodies in pig serum to bind capture antigen C-strain E2. By testing C-strain VNT negative pig sera (n = 445) and C-strain VNT positive pig sera (n = 70), the 6B211 based cELISA showed 100% sensitivity (95% confidence interval: 94.87 to 100%) and 100% specificity (95% confidence interval: 100 to 100%). The C-strain antibody can be tested in pigs as early as 7 days post vaccination with the cELISA. By testing pig sera (n = 139) in parallel, the cELISA showed excellent agreement (Kappa = 0.957) with VNT. The inhibition rate of serum samples in the cELISA is highly correlated with their titers in VNT (r2 = 0.903, p Conclusions This novel cELISA demonstrated excellent agreement and high level correlation with VNT. It is a reliable tool for sero-monitoring of C-strain vaccination campaign because it is a rapid, simple, safe and cost effective assay that can be used to monitor vaccination-induced immune response at the population level.

African swine fever (ASF) is currently causing a major pandemic affecting the swine industry and protein availability from Central Europe to East and South Asia. No commercial vaccines are available, making disease control dependent on the elimination of affected animals. Here, we show that the deletion of the African swine fever virus (ASFV) E184L gene from the highly virulent ASFV Georgia 2010 (ASFV-G) isolate produces a reduction in virus virulence during the infection in swine. Of domestic pigs intramuscularly inoculated with a recombinant virus lacking the E184L gene (ASFV-G-ΔE184L), 40% experienced a significantly (5 days) delayed presentation of clinical disease and, overall, had a 60% rate of survival compared to animals inoculated with the virulent parental ASFV-G. Importantly, all animals surviving ASFV-G-ΔE184L infection developed a strong antibody response and were protected when challenged with ASFV-G. As expected, a pool of sera from ASFV-G-ΔE184L-inoculated animals lacked any detectable antibody response to peptides partially representing the E184L protein, while sera from animals inoculated with an efficacious vaccine candidate, ASFV-G-ΔMGF, strongly recognize the same set of peptides. These results support the potential use of the E184L deletion for the development of vaccines able to differentiate infected from vaccinated animals (DIVA). Therefore, it is shown here that the E184L gene is a novel ASFV determinant of virulence that can potentially be used to increase safety in preexisting vaccine candidates, as well as to provide them with DIVA capabilities. To our knowledge, E184L is the first ASFV gene product experimentally shown to be a functional DIVA antigenic marker. IMPORTANCE No commercial vaccines are available to prevent African swine fever (ASF). The ASF pandemic caused by the ASF virus Georgia 2010 (ASFV-G) strain is seriously affecting pork production in a contiguous geographical area from Central Europe to East Asia. The only effective experimental vaccines are viruses attenuated by deleting ASFV genes associated with virus virulence. Therefore, identification of such genes is of critical importance for vaccine development. Here, we report the discovery of a novel determinant of ASFV virulence, the E184L gene. Deletion of the E184L gene from the ASFV-G genome (ASFV-G-ΔE184L) produced a reduction in virus virulence, and importantly, animals surviving infection with ASFV-G-ΔE184L were protected from developing ASF after challenge with the virulent parental virus ASFV-G. Importantly, the virus protein encoded by E184L is highly immunogenic, making a virus lacking this gene a vaccine candidate that allows the differentiation of infected from vaccinated animals (DIVA). Here, we show that unlike what is observed in animals inoculated with the vaccine candidate ASFV-G-ΔMGF, ASFV-G-ΔE184L-inoculated animals do not mount a E184L-specific antibody response, indicating the feasibility of using the E184L deletion as the antigenic marker for the development of a DIVA vaccine in ASFV. info:eu-repo/semantics/acceptedVersion

Sea level rise (SLR) is threatening low-lying coastal areas such as river deltas. The Ebro river Delta (Spain) is rep-resentative of coastal systems particularly vulnerable to SLR due to significant sediment retention behind up-stream dams (up to 99%), thereby dramatically reducing the capacity for deltaic sediment accretion. Rice production is the main economic activity, covering 66% of the delta area, and is negatively affected by SLR because of flooding and soil salinization. Therefore, appropriate adaptation measures are needed to preserve rice produc-tion. We combined Geographic Information Systems and Generalized Linear Models to identify zones prone to flooding and increasing soil salinity, and to calculate the so-called sediment deficit, that is the amount of sedi-ment needed to raise the land to compensate flooding and soil salinization. We modelled SLR scenarios predicted by the IPCC Fifth Assessment Report, and analysed the economic feasibility (not the technical feasibility) of reintroducing fluvial sediments retained in the upstream river dam reservoirs into the delta plain, which can con-tribute to maintaining land elevation and rice production with SLR. To do this, the costs of the sediment reintro-duction measures and their benefits in terms of avoided loss of rice production income were evaluated with an approximate economic cost-benefit analysis. Results predicted that between 35 and 90% of the rice field area will be flooded in the best and worst SLR scenarios considered (SLR = 0.5 m and 1.8 m by 2100, respectively), with a sediment deficit of 130 and 442 million tonnes, with an associated cost of sediment reintroduction of 13 and 226 million euro . The net benefit of rice production maintenance was 24.6 and 328 euro /ha. The proposed ad-aptation measure has a positive effect on rice production and can be considered as an innovative management option for maintaining deltaic areas under SLR. (c) 2021 Elsevier B.V. All rights reserved.

The aim of this work is to propose a novel and formal approach to evaluate the direct costs of diseases caused by different pathogens as well as their economic impact on typical Mediterranean grow-out farms culturing European sea bass under different scenarios of production related to the biomass produced (farm size) and the size of the fish produced (production strategy). We employ a deterministic static model to simulate the annual income statement of those facilities to evaluate the direct costs caused by different diseases as well as, through a partial budget and sensitivity analyses, the economic impact of them. An important conclusion of this work is that the profitability and economic viability of sea bass grow-out farms suffering recurrent outbreaks of diseases caused by different pathogens depend on the farm typology (farm size) as well as the decisions taken by owners/investors about the size of the fish produced and sold in the market. Our estimations show that as the larger is the farm and the size of the fish produced, the larger are the direct cost of a disease outbreak. However, the economic impact on the net operating profit is significantly worse as smaller is the farm and smaller the fish produced. The conclusions of this research stress the need for small producers to pay attention and devote resources to prevent and treat disease outbreaks. info:eu-repo/semantics/publishedVersion

In contrast to surveys based on a few genes that often provide limited taxonomic resolution, transcriptomes provide a wealth of genomic loci that can resolve relationships among taxonomically challenging lineages. Diatoms are a diverse group of aquatic microalgae that includes important bioindicator species and many such lineages. One example is Nitzschia palea, a widespread species complex with several morphologically based taxonomic varieties, some of which are critical pollution indicators. Morphological differences among the varieties are subtle and phylogenetic studies on a few genes fail to resolve their evolutionary relationships. We conducted morphometric and transcriptome analyses of 10 Nitzschia palea strains to resolve the relationships among strains and taxonomic varieties. Nitzschia palea was resolved into three clades, one of which corresponds to a group of strains with narrow linear-lanceolate valves. The other morphological group recovered in the shape outline analysis was not monophyletic and consisted of two clades. Subsequent gene-tree concordance analyses and phylogenetic network estimations revealed patterns of incomplete lineage sorting and gene flow between intraspecific lineages. We detected reticulated evolutionary patterns among lineages with different morphologies and a resulting putative recent hybrid. Our study shows that phylogenomic analyses of many unlinked nuclear loci, complemented with morphometrics, can resolve complex evolutionary histories of recently diverged species complexes.

The feeding habits and effect of the diameter of pelleted feeds on the feeding responses of wild juvenile and adult flathead grey mullet (Mugil cephalus) in captivity were examined. Optimal pellet size for feeding was defined according to the behavioural responses and ingestion of pellets with different diameters (2, 4, 6, 8 mm) that were dropped into the tank in a random sequence. Larger pellets (6 and 8 mm) were more attractive (lower reaction time, high percentage of capture), but the small to medium-sized pellets (2 and 4 mm) were consumed the most. The optimal size was the 2- and 4- mm pellet diameter for juvenile individuals (365.50 ± 36.90 g; 28.8 ± 0.84 cm) and the 4-mm diameter pellet for adults (937.49 ± 146.54 g; 40 ± 1.12 cm). The preferred feeding area of adult mullet was also studied to estimate preference in relation to pellet characteristics such as floating or sink. Two pellet types, floating or sinking, were offered simultaneously in the water column: at the surface, mid-water column and bottom of the tank. The flathead grey mullet had a preference to feed in the mid-water column and the bottom of the tanks indicating that sinking or slow-sinking pellets would be the optimal feed type in relation to mullet feeding behaviour.

The assessment of population structure and genetic diversity is crucial for the management and conservation of threatened species. Natural and artificial barriers to dispersal (i.e., gene flow) increase populations’ differentiation and isolation by reducing genetic exchange and diversity. Freshwater ecosystems are highly fragmented because of human activities. Threatened species with small population sizes are more sensitive to habitat fragmentation effects. Here, we investigate the genetic population structure and gene flow among seven populations of Aphanius sophiae in the Kor Basin by using sequences of the complete Cyt b gene and otolith morphometry. The Cyt b gene showed low level of genetic variation, only 4.12% of the identified sites were variable, and 2.42% were parsimony informative. Overall, haplotype diversity was low to moderate and nucleotide diversity was low to extremely low. Fish populations exhibited high levels of genetic differentiation, suggesting limited gene flow among them. These differences were obtained not only among geographically distant populations, but also among neighboring localities. Genetic population structure was supported by the AMOVA analysis and by the haplotype network (only one of 21 haplotypes were shared by two localities). Otolith morphometric analysis was in agreement with genetic results, the two most distant and isolated populations were clearly separated, and genetically close populations showed less differences in morphometry. A significant pattern of isolation by distance was also detected among A. sophiae populations, with genetic distance more correlated with hydrological distance than with geographic distance. Results suggested that limited gene flow due to habitat fragmentation is an important factor contributing to genetic structuring and to the loss of genetic variation of A. sophiae populations. Aphanius sophiae population structure seems to be the result of habitat fragmentation and water pollution, but other factors such as introduced species should be considered. Given the high degree of genetic structuring, the definition of conservation groups is of particular importance for A. sophiae, which should be considered endangered according to the IUCN criteria. Conservation plans must recognize the genetic independence of populations and manage separately preventing the loss of locally adapted genotypes. info:eu-repo/semantics/acceptedVersion

EFSA received a mandate from the European Commission to assess the effectiveness of some of the control measures against diseases included in the Category A list according to Regulation (EU) 2016/429 on transmissible animal diseases (‘Animal Health Law’). This opinion belongs to a series of opinions where these control measures will be assessed, with this opinion covering the assessment of control measures for Newcastle disease (ND). In this opinion, EFSA and the AHAW Panel of experts review the effectiveness of: (i) clinical and laboratory sampling procedures, (ii) monitoring period and (iii) the minimum radius of the protection and surveillance zone, and the minimum length of time the measures should be applied in these zones. The general methodology used for this series of opinions has been published elsewhere. Several scenarios for which these control measures had to be assessed were designed and agreed prior to the start of the assessment. The monitoring period (21 days) was assessed as effective in non-vaccinated chicken and turkey flocks, although large uncertainty remains surrounding the effectiveness of this period in vaccinated galliform flocks and flocks of other bird species. It was also concluded that the protection (3 km radius) and the surveillance (10 km radius) zones contain 99% of the infections from an infectious establishment. Recommendations provided for each of the scenarios assessed aim to support the European Commission in the drafting of further pieces of legislation, as well as for plausible ad hoc requests in relation to ND. info:eu-repo/semantics/publishedVersion

Our study evaluates differences in the distribution and ecology of genetic variants within several ecologically important diatom species that are also key for Water Framework Directive monitoring of European rivers: Fistulifera saprophila (FSAP), Achnanthidium minutissimum (ADMI), Nitzschia inconspicua (NINC) and Nitzschia soratensis (NSTS). We used DADA2 to infer amplicon sequence variants (ASVs) of a short rbcL barcode in 531 environmental samples from biomonitoring campaigns in Catalonia and France. ASVs within each species showed different distribution patterns. Threshold Indicator Taxa ANalysis revealed three ecological groupings of ASVs in both ADMI and FSAP. Two of these in each species were separated by opposite responses to calcium and conductivity. Boosted regression trees additionally showed that both variables greatly influenced the occurrence of these groupings. A third grouping in FSAP was characterized by a negative response to total organic carbon and hence was better represented in waters with higher ecological status than the other FSAP ASVs, contrasting with what is generally assumed for the species. In the two Nitzschia species, our analyses confirmed earlier studies: NINC preferred higher levels of calcium and conductivity. Our findings suggest that the broad ecological tolerance of some diatom species results from overlapping preferences among genetic variants, which individually show much more restricted preferences and distributions. This work shows the importance of studying the ecological preferences of genetic variants within species complexes, now possible with DNA metabarcoding. The results will help reveal and understand biogeographical distributions and facilitate the development of more accurate biological indexes for biomonitoring programmes. info:eu-repo/semantics/acceptedVersion

14 Pág. Departamento de ​Mejora Genética Animal, Coronaviruses silently circulate in human and animal populations, causing mild to severe diseases. Therefore, livestock are important components of a "One Health" perspective aimed to control these viral infections. However, at present there is no example that considers pig genetic resources in this context. In this study, we investigated the variability of four genes (ACE2, ANPEP and DPP4 encoding for host receptors of the viral spike proteins and TMPRSS2 encoding for a host proteinase) in 23 European (19 autochthonous and three commercial breeds and one wild boar population) and two Asian Sus scrofa populations. A total of 2229 variants were identified in the four candidate genes: 26% of them were not previously described; 29 variants affected the protein sequence and might potentially interact with the infection mechanisms. The results coming from this work are a first step towards a "One Health" perspective that should consider conservation programs of pig genetic resources with twofold objectives: (i) genetic resources could be reservoirs of host gene variability useful to design selection programs to increase resistance to coronaviruses; (ii) the described variability in genes involved in coronavirus infections across many different pig populations might be part of a risk assessment including pig genetic resources., This work has received funding from the University of Bologna RFO 2016-2019 programmes, the Italian MIUR 2017 PigPhenomics project, from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634476 for the project with acronym TREASURE, from the European Open Science Cloud (EOSC) Secretariat, project “Application of animal genomics and data mining to predict and monitor novel coronavirus potential infections (VirAnimalOne)”, the EGI call for COVID-19 research projects (AnGen1H project) and from the Por Fesr Emilia-Romagna 2014-2020 (actions 1.1.4 and 1.2.2—Bando per sostenere progetti di ricerca ed innovazione per lo sviluppo di soluzioni finalizzate al contrasto dell’epidemia da COVID-19—Project LIVESTOCK-STOP-COVI). The content of this article reflects only the authors' view and the European Union Agency is not responsible for any use that may be made of the information it contains.

Streptococcus suis is one of the most important bacterial swine pathogens affecting post-weaned piglets, causing mainly meningitis, arthritis and sudden death. It not only results in severe economic losses but also raises concerns over animal welfare and antimicrobial resistance and remains an important zoonotic agent in some countries. The definition and diagnosis of S. suis-associated diseases can be complex. Should S. suis be considered a primary or secondary pathogen? The situation is further complicated when referring to respiratory disease, since the pathogen has historically been considered as a secondary pathogen within the porcine respiratory disease complex (PRDC). Is S. suis a respiratory or strictly systemic pathogen? S. suis is a normal inhabitant of the upper respiratory tract, and the presence of potentially virulent strains alone does not guarantee the appearance of clinical signs. Within this unclear context, it has been largely proposed that co-infection with some viral and bacterial pathogens can significantly influence the severity of S. suis-associated diseases and may be the key to understanding how the infection behaves in the field. In this review, we critically addressed studies reporting an epidemiological link (mixed infections or presence of more than one pathogen at the same time), as well as in vitro and in vivo studies of co-infection of S. suis with other pathogens and discussed their limitations and possibilities for improvement and proposed recommendations for future studies.

Staphylococcus pseudintermedius is the main aetiological agent of canine pyoderma. Whole genome sequencing is the most comprehensive way of obtaining relevant genomic information about micro-organisms.Oxford Nanopore technology enables quality sequencing and de novo assembly of the whole genome of S. pseudintermedius. Whole genome analysis of S. pseudintermedius may help to better understand the pathogenesis of canine pyodermas.Twenty-two strains of S. pseudintermedius isolated from the skin of five healthy dogs and 33 strains isolated from skin of 33 dogs with pyoderma were analysed. DNA was extracted and sequenced using Oxford Nanopore MinION, a new technology that delivers longer reads in a hand-held device. The pangenome was analysed and visualised with Anvi'o 6.1.Nanopore technology allowed the sequencing and de novo assembly of the genomes of 55 S. pseudintermedius strains isolated from healthy dogs and from dogs with pyoderma. The average genome size of S. pseudintermedius was 2.62 Mbp, with 48% being core genome. Pyoderma isolates contained a higher number of antimicrobial resistance genes, yet the total number of virulence factors genes did not change between isolates from healthy dogs and from dogs with pyoderma. Genomes of meticillin-resistant S. pseudintermedius (MRSP) strains were larger than those of meticillin-susceptible (MSSP) strains (2.80 Mbp versus 2.59 Mbp), as a consequence of a greater presence of antimicrobial resistance genes, phages and prophages.This technique allows much more precise and easier characterisation of canine S. pseudintermedius populations and may lead to a better understanding of the pathogenesis of canine pyodermas.Staphylococcus pseudintermedius est le principal agent étiologique de la pyodermite canine. Le séquençage du génome entier est le moyen le plus complet d'obtenir des informations génomiques pertinentes sur les micro-organismes. HYPOTHÈSE/OBJECTIFS: La technologie Oxford Nanopore permet un séquençage de qualité et un assemblage de novo de l'ensemble du génome de S. pseudintermedius. L'analyse du génome entier de S. pseudintermedius peut aider à mieux comprendre la pathogénie des pyodermites canines. MÉTHODES ET MATERIEL: Vingt-deux souches de S. pseudintermedius isolées de la peau de cinq chiens sains et 33 souches isolées de la peau de 33 chiens atteints de pyodermite ont été analysées. L'ADN a été extrait et séquencé à l'aide d'Oxford Nanopore MinION, une nouvelle technologie qui offre des lectures plus longues dans un appareil portatif. Le pangénome a été analysé et visualisé avec ANVI'O 6.1. RÉSULTATS: La technologie Nanopore a permis le séquençage et l'assemblage de novo des génomes de 55 souches de S. pseudintermedius isolées de chiens sains et de chiens atteints de pyodermite. La taille moyenne du génome de S. pseudintermedius était de 2,62 Mpb, dont 48 % étaient le génome central. Les isolats de pyodermite contenaient un nombre plus élevé de gènes de résistance aux antimicrobiens, mais le nombre total de gènes de facteurs de virulence n'a pas changé entre les isolats de chiens sains et de chiens atteints de pyodermite. Les génomes des souches de S. pseudintermedius résistantes à la méticilline (MRSP) étaient plus grands que ceux des souches sensibles à la méticilline (MSSP) (2,80 Mbp contre 2,59 Mbp), en raison d'une plus grande présence de gènes de résistance aux antimicrobiens, de phages et de prophages.Cette technique permet une caractérisation beaucoup plus précise et plus facile des populations canines de S. pseudintermedius et peut conduire à une meilleure compréhension de la pathogénie des pyodermites canines.INTRODUCCIÓN: Staphylococcus pseudintermedius es el principal agente etiológico de la pioderma canina. La secuenciación del genoma completo es la forma más completa de obtener información genómica relevante sobre microorganismos. HIPÓTESIS/OBJETIVOS: la tecnología Oxford Nanopore permite la secuenciación de calidad y el ensamblaje de novo de todo el genoma de S. pseudintermedius. El análisis del genoma completo de S. pseudintermedius puede ayudar a comprender mejor la patogenia de las piodermas caninas. MÉTODOS Y MATERIALES: Se analizaron veintidós cepas de S. pseudintermedius aisladas de la piel de cinco perros sanos y 33 cepas aisladas de la piel de 33 perros con pioderma. El DNA se extrajo y secuenció utilizando Oxford Nanopore MinION, una nueva tecnología que ofrece lecturas más largas en un dispositivo de mano. El pangenoma se analizó y visualizó con ANVI’O 6.1. RESULTADOS: la tecnología Nanopore permitió la secuenciación y el ensamblaje de novo de los genomas de 55 cepas de S. pseudintermedius aisladas de perros sanos y de perros con pioderma. El tamaño medio del genoma de S. pseudintermedius fue de 2,62 Mbp, con un 48% de genoma central. Los aislados de pioderma contenían un mayor número de genes de resistencia a los antimicrobianos, sin embargo, el número total de genes de factores de virulencia no cambió entre los aislados de perros sanos y los de perros con pioderma. Los genomas de las cepas de S. pseudintermedius resistentes a la meticilina (MRSP) fueron más grandes que los de las cepas sensibles a la meticilina (MSSP) (2,80 Mbp versus 2,59 Mbp), como consecuencia de una mayor presencia de genes de resistencia a los antimicrobianos, fagos y profagos. CONCLUSIONES E IMPORTANCIA CLÍNICA: esta técnica permite una caracterización mucho más precisa y sencilla de las poblaciones caninas de S. pseudintermedius y puede conducir a una mejor comprensión de la patogenia de las piodermas caninas.Staphylococcus pseudintermedius stellt die hauptsächliche ätiologische Ursache für die canine Pyodermie dar. Eine Gesamtgenomsequenzierung ist der umfassendste Weg, um relevante Genominformation über Mikroorganismen zu erhalten.Die Oxford Nanopore Technologie ermöglicht eine qualitative Sequenzierung und eine de novo Anordnung des Gesamtgenoms von S. pseudintermedius. Die Gesamtgenomsequenzierung von S. pseudintermedius könnte dabei helfen, die Pathogenese der caninen Pyodermie besser zu verstehen.Zweiundzwanzig Stämme von S. pseudintermedius, die von der Haut von fünf gesunden Hunden und 33 Stämme, die von 33 Hunden mit Pyodermie isoliert worden waren, wurden analysiert. DNA wurde extrahiert und mittels Oxford Nanopore MinION, einer neuen Technologie, die längere Sequenzen in einem Handgerät liefert, sequenziert. Das Pangenom wurde analysiert und mittels ANVI´O 6.1. analysiert.Die Nanopore Technologie ermöglichte eine Sequenzierung und de novo Anordnung der Genome von 55 S. pseudintermedius Stämmen, die von gesunden Hunden und von Hunden mit Pyodermie isoliert worden waren. Die durchschnittliche Genom Länge von S. pseudintermedius betrug 2,62 Mbp, wobei 48% davon das Hauptgenom ausmachte. Pyodermie Isolate beinhalteten eine größere Anzahl an antimikrobiellen Resistenzgenen; die Gesamtzahl der Virulenzfaktorgene unterschied sich allerdings nicht zwischen den Isolaten von gesunden Hunden und von Hunden mit Pyodermie. Genome von Methicillin-resistenten S. pseudintermedius (MRSP) Stämmen waren größer als jene der Methicillin-sensiblen (MSSP) Stämme (2,8 Mbp versus 2,59 Mbp), was eine Folge der vermehrten Präsenz von antimikrobiellen Resistenzgenen, Phagen und Prophagen war.Diese Technik erlaubt eine wesentlich präzisere und einfachere Charakterisierung der caninen S. pseudintermedius Populationen und könnte zu einem besseren Verstehen der Pathogenese der caninen Pyodermien führen.背景: Staphylococcus pseudintermediusは、犬の膿皮症の主な病因です。全ゲノムシーケンスは、微生物に関する関連するゲノム情報を取得するための最も包括的な方法です。 仮説/目的: Oxford Nanoporeテクノロジーにより、S。pseudintermediusの全ゲノムの高品質なシーケンスとdenovoアセンブリが可能になります。 S. pseudintermediusの全ゲノム解析は、犬の膿皮症の病因をよりよく理解するのに役立つ可能性があります。 方法と材料: 5匹の健康な犬の皮膚から分離されたS.pseudintermediusの22株と、膿皮症の33匹の犬の皮膚から分離された33株が分析されました。 DNAは、ハンドヘルドデバイスでより長い読み取りを実現する新しいテクノロジーであるOxford Nanopore MinIONを使用して抽出およびシーケンスされました。パンゲノムはANVI’O6.1で分析および視覚化されました。 結果: Nanoporeテクノロジーにより、健康な犬と膿皮症の犬から分離された55のS.pseudintermedius株のゲノムの配列決定とdenovoアセンブリが可能になりました。 S.pseudintermediusの平均ゲノムサイズは2.62Mbpで、48％がコアゲノムでした。膿皮症分離株は、より多くの抗菌薬耐性遺伝子を含んでいましたが、病原性因子遺伝子の総数は、健康な犬と膿皮症の犬からの分離株間で変化しませんでした。メチシリン耐性S.pseudintermedius（MRSP）株のゲノムは、抗菌剤耐性遺伝子、ファージ、およびプロファージの存在が大きい結果として、メチシリン感受性（MSSP）株のゲノムよりも大きかった（2.80Mbp対2.59Mbp）。 結論と臨床的重要性: この手法により、犬のS. pseudintermedius集団のより正確で簡単な特性評価が可能になり、犬の膿皮症の病因のより良い理解につながる可能性があります。.背景: 假中间型葡萄球菌是犬脓皮病的主要病原。全基因组测序是获得微生物相关基因组信息最全面的方式。 假设/目的: 纳米孔技术能够对假中间型葡萄球菌的全基因组进行质量测序和从头组装。对假中间型葡萄球菌进行全基因组分析可能有助于更好地了解犬脓皮病的发病机制。 方法和材料: 从5只健康犬皮肤中分离的22株，以及从33只脓皮病犬皮肤中分离的33株假中间型葡萄球菌，对其进行分析。使用纳米孔 MinION提取DNA并测序，这是一种手持设备，这种新技术可传递更长的测序片段。用ANVI'O 6.1分析全基因组并将其可视化。 结果: 从健康犬和脓皮病犬中分离的55株假中间型葡萄球菌，用纳米孔技术对其基因组进行测序和从头组装。假中间型葡萄球菌的平均基因组大小为2.62 Mbp，其中48%为核心基因组。脓皮病分离株含有较多数量的抗菌药物耐药基因，然而毒力因子基因的总数在健康犬和脓皮病犬的分离株之间没有变化。耐甲氧西林假中间型葡萄球菌(MRSP)菌株的基因组比甲氧西林敏感(MSSP)菌株的基因组大（2.80 Mbp与2.59 Mbp），这是由于存在更多抗菌药物耐药基因、噬菌体和前噬菌体。 结论和临床重要性: 该技术可更精确和更容易地表征犬假中间型葡萄球菌种群，并可能有助于更好地了解犬脓皮病的发病机制。.Staphylococcus pseudintermedius é o principal agente etiológico da piodermite canina. O sequenciamento completo do genoma é a forma mais aprofundada de se obter informações genômicas relevantes sobre microrganismos. HIPÓTESE/OBJETIVOS: A tecnologia de Oxford Nanopore permite o sequenciamento de boa qualidade e montagem “de novo” do genoma completo de S. pseudintermedius. A análise do genoma completo de S. pseudintermedius pode auxiliar na melhor compreensão da patogênese da piodermite canina. MÉTODOS E MATERIAIS: Vinte e duas cepas de S. pseudintermedius isoladas da pele de cinco cães saudáveis e 33 cepas isoladas da pele de 33 cães com piodermite foram analisadas. O DNA foi extraído e sequenciado utilizando Oxford Nanopore MinION, uma tecnologia nova que fornece leituras mais longas em um aparelho portátil. O pangenoma foi analisado e visualizado com ANVI’O 6.1.A tecnologia Nanopore permitiu o sequenciamento e a montagem “de novo” dos genomas de 55 cepas de S. pseudintermedius isoladas de cães saudáveis de cães com piodermite. A média do tamanho do genoma de S. pseudintermedius foi de 2,62 Mbp, sendo 48% de genoma base. Os isolados de piodermite continham um maior número de genes de resistência antimicrobiana, apesar de o número total de fatores de virulência não sofreu alteração entre os isolados de cães saudáveis e de cães com piodermite. Os genomas das cepas S. pseudintermedius resistentes à meticilina (MRSP) foram maiores que aqueles das cepas suscetíveis à meticilina (MSSP) (2,80 Mbp versus 2,59 Mbp), como consequência da maior presença de genes de resistência antimicrobiana, fagos e profagos. CONCLUSÕES E IMPORTÂNCIA CLÍNICA: A técnica testada permite uma caracterização muito mais simples e precisa das populações de S. pseudintermedius canino e pode levar ao melhor entendimento da patogênese da piodermite canina.

Simple Summary The cow dry period is a critical period presenting a high risk of contracting intramammary infections. Active molecules to boost the innate immunity of the mammary gland and increase infection resilience could be decisive for the milking performance of dairy cows in the next lactation. Metalloproteinase-9 is a protein with a relevant role in facilitating the immune function and activating the regeneration of the mammary gland. The focus of this study was to test the role of the infusion of a recombinant version of metalloproteinase 9 at cow dry off, showing, contrary to expectations, that it is not able to enhance the innate immunity nor to improve the involution and regeneration of the mammary gland. Abstract The dry period is decisive for the milking performance of dairy cows. The promptness of mammary gland involution at dry-off affects not only the productivity in the next lactation, but also the risk of new intra-mammary infections since it is closely related with the activity of the immune system. Matrix metalloproteinase-9 (MMP-9) is an enzyme present in the mammary gland and has an active role during involution by disrupting the extracellular matrix, mediating cell survival and the recruitment of immune cells. The objective of this study was to determine the potential of exogenous administration of a soluble and recombinant version of a truncated MMP-9 (rtMMP-9) to accelerate mammary involution and boost the immune system at dry-off, avoiding the use of antibiotics. Twelve Holstein cows were dried abruptly, and two quarters of each cow received an intra-mammary infusion of either soluble rtMMP-9 or a positive control based on immunostimulant inclusion bodies (IBs). The contralateral quarters were infused with saline solution as negative control. Samples of mammary secretion were collected during the week following dry-off to determine SCC, metalloproteinase activity, bovine serum albumin, lactoferrin, sodium, and potassium concentrations. The soluble form of rtMMP-9 increased endogenous metalloproteinase activity in the mammary gland compared with saline quarters but did not accelerate either the immune response or involution in comparison with control quarters. The results demonstrated that the strategy to increase the mammary gland immunocompetence by recombinant infusion of rtMMP-9 was unsuccessful.

The surveillance for West Nile virus (WNV) in Catalonia (northeastern Spain) has consistently detected flaviviruses not identified as WNV. With the aim of characterizing the flaviviruses circulating in Catalonia, serum samples from birds and horses collected between 2010 and 2019 and positive by panflavivirus competition ELISA (cELISA) were analyzed by microneutralization test (MNT) against different flaviviruses. A third of the samples tested were inconclusive by MNT, highlighting the limitations of current diagnostic techniques. Our results evidenced the widespread circulation of flaviviruses, in particular WNV, but also Usutu virus (USUV), and suggest that chicken and horses could serve as sentinels for both viruses. In several regions, WNV and USUV overlapped, but no significant geographical aggregation was observed. Bagaza virus (BAGV) was not detected in birds, while positivity to tick-borne encephalitis virus (TBEV) was sporadically detected in horses although no endemic foci were observed. So far, no human infections by WNV, USUV, or TBEV have been reported in Catalonia. However, these zoonotic flaviviruses need to be kept under surveillance, ideally within a One Health framework. info:eu-repo/semantics/publishedVersion

The use of frozen peripheral blood mononuclear cells (PBMC) is common in immunological studies. The impact of freezing PBMC has been assessed using human and mice cells, but little information is available regarding domestic animals. In the present study, the phenotype and functionality of frozen porcine PBMC were examined. In a preliminary experiment, three freezing media: fetal bovine serum plus 10% dimethyl sulfoxide, PSC cryopreservation kit, and Cryostor CS10, were compared regarding the preservation of cell viability and the response of PBMC to mitogens after thawing. After being stored one month in liquid nitrogen, cell viability was above 89% for all freezing media. The ELISPOT IFN-gamma (IFN-γ) results in response to PHA and of IgG ELISPOT in response to R848+IL-2 were similar to those obtained using fresh PBMC. In the second set of experiments, PBMC were obtained from five pigs vaccinated against Porcine reproductive and respiratory syndrome virus (PRRSV) and then frozen using Cryostor CS10. Recovered cells were phenotyped by flow cytometry using anti-CD3, CD4, CD8, and CD21 antibodies and were used to assess the PRRSV-specific responses in a proliferation experiment, an IFN-γ ELISPOT, and an IgG ELISPOT, and compared to the results obtained with fresh cells. The antigen-specific responses of frozen cells were significantly (p

In recent years, the increase in awareness of antimicrobial resistance together with the societal demand of healthier meat products have driven attention to health-related traits in livestock production. Previous studies have reported medium to high heritabilities for these traits and described genomic regions associated with them. Despite its genetic component, health- and immunity-related traits are complex and its study by association analysis with genomic markers may be missing some information. To analyse multiple phenotypes and gene-by-gene interactions, systems biology approaches, such as the association weight matrix (AWM), allows combining genome wide association study results with network inference algorithms. The present study aimed to identify gene networks, key regulators and candidate genes associated to immunocompetence in pigs by integrating multiple health-related traits, enriched for innate immune phenotypes, using the AWM approach. The co-association network analysis unveiled a network comprised of 3,636 nodes (genes) and 451,407 edges (interactions), including a total of 246 regulators. From these, five genes (ARNT2,BRMS1L,MED12L,SUPT3HandTRIM25) were selected as key regulators as they were associated with the maximum number of genes with the minimum overlapping (1,827 genes in total). The five regulators were involved in pathways related to immunity such as lymphocyte differentiation and activation, platelet activation and degranulation, megakaryocyte differentiation, FcγR-mediated phagocytosis and response to nitric oxide, among others, but also in immunometabolism. Furthermore, we identified genes co-associated with the key regulators previously reported as candidate genes (e.g.,ANGPT1, CD4, CD36, DOCK1,PDE4B, PRKCE, PTPRCandSH2B3) for immunity traits in humans and pigs, but also new candidate ones (e.g.,ACSL3,CXADR,HBB,MMP12,PTPN6,WLS) that were not previously described. The co-association analysis revealed new regulators associated with health-related traits in pigs. This approach also identified gene-by-gene interactions and candidate genes involved in pathways related to cell fate and metabolic and immune functions. Our results shed new light in the regulatory mechanisms involved in pig immunity and reinforce the use of the pig as biomedical model.

The effect of different feeding regimes on the circadian rhythms of pancreatic digestive enzyme activities was evaluated in Mugil cephalus fry weighting 0.34 ± 0.01 g. Feeding regimes (feeding ration = 3% stocked biomass) differed on the number of meals offered per day: one, two and ten meals per day (R1, R2 and R3, respectively). The number of meals per day affected somatic growth; in particular, fry from the R3 group (0.80 ± 0.01 g) grew better than their congeners from R2 (0.70 ± 0.01 g) and R1 (0.63 ± 0.01 g) groups (P < 0.05). Feeding behaviour was modulated by the feeding regime, being the maximal gut fullness values found just after meal distribution in R1 and R2 groups, whereas this trend was not observed when feed was offered continuously during light hours (R3). Fry from R1 and R2 groups showed hyperphagia as they tended to store in their gut as much as possible feed particles to be later digested due to the limited daily meals. This strategy negatively affected feed digestion due to inappropriate enzyme to substrate ratio, changes in digestive enzyme activities and chyme transit times, which ultimately impaired growth performance. Enzyme activities were modulated by the number of meals, the more frequent the meals offered, the lower enzyme activities, supporting the hypothesis that digestive function is adapted to obtain a maximum benefit of the ingested nutrients. Present results showed that feeding grey mullet fry continuously during day light hours optimized feed digestion and promoted fry growth. info:eu-repo/semantics/acceptedVersion

The results of morphological and genetic analyses of forms/species of the Coregonus lavaretus pidschian (Gmelin, 1789) complex from the Indigirka and Kolyma river basins are presented in the context of there being recent postglacial speciation events. It has been found that the studied whitefishes belong to the sparsely rakered and low lateral-line forms and have previously been described as Coregonus lavaretus pidschian n. jucagiricus Drjagin (Berg), 1932. Based on these characters, this whitefish does not differ from most Arctic whitefish populations (in particular from Coregonus lavaretus glacialis Kirillov, 1972). Analysis of variability of the ND1 gene (mtDNA) showed that whitefishes from the Indigirka and Kolyma basins belong to a distant phylogenetic lineage, which is significantly different from all previously studied whitefish lineages from the Ob, Yenisei, Lena, Anadyr, and Amur river basins. Analysis of variability of the ITS1 fragment (nDNA) showed that all studied forms/species (from Ob River to Amur River basins), including C. l. pidschian n. jucagiricus, have a tandem arrangement of two identical nucleotide fragments and very similar nucleotide composition of the ITS1 region. Based on contemporary data, this phylogenetic lineage of the C. pidschian complex could be considered a young postglacial allopatric species. info:eu-repo/semantics/acceptedVersion

The Coregonus lavaretus (Linnaeus, 1758) complex is a morphologically and genetically diverse group of whitefish. Its taxonomic structure has been controversial for almost a century. At least 25 forms of C. lavaretus have been described in Siberia, but there is still no consensus on their intraspecific structure and taxonomy. Coregonus lavaretus pidschian (Gmelin, 1789) was described as a subspecies of C. lavaretus. Recently, it was assumed that this subspecies is also a complex. The purpose of this study was to compare the distributions of pidschian-like whitefish haplotypes in two basins of large Siberian rivers, Yenisei and Lena, and to assess the gene flow between basins of these rivers, which were connected after the last glaciation. The sequence of the following mitochondrial DNA genes, 16S rRNA (partial), tRNA-Leu (full), NADH dehydrogenase subunit 1 (full), tRNA-Ile (full), and tRNA-Gln (partial), were used for the inference of intraspecific genetic structure of C. l. pidschian. Whitefish haplotypes were clustered into two groups according to their distribution between two large Siberian river basins; however, there were shared haplotypes indicating events of migration and hybridization, which could occur when Bolshoi Yenisei and Lena river systems were connected after the last glaciation (the Late Pleistocene). info:eu-repo/semantics/acceptedVersion

This survey was conducted to estimate the incidence and level of potential viral contamination in commercially collected porcine plasma. Samples of spray dried porcine plasma (SDPP) were collected over a 12- month period from eight spray drying facilities in Spain, England, Northern Ireland, Brazil, Canada, and the United States. In this survey, viral load for several porcine pathogens including SVA, TGEV, PRRSV (EU and US strains), PEDV, PCV-2, SIV, SDCoV and PPV were determined by qPCR. Regression of Ct on TCID50 of serial diluted stock solution of each virus allowed the estimate of potential viral level in SDPP and unprocessed liquid plasma (using typical solids content of commercially collected porcine plasma). In this survey SVA, TGEV or SDCoV were not detected in any of the SDPP samples. Brazil SDPP samples were free of PRRSV and PEDV. Samples of SDPP from North America primarily contained the PRRSV-US strain while the European samples contained the PRRSV-EU strain (except for one sample from each region containing a relatively low estimated level of the alternative PRRSV strain). Estimated viral level tended to be in the range from 10 TCID50 to 10 TCID50. Estimated level of SIV was the exception with a very low incidence rate but higher estimated viral load 10 TCID50. In summary, the incidence of potential viral contamination in commercially collected porcine plasma was variable and estimated virus level in samples containing viral DNA/RNA was relatively low compared with that occurring at the peak viremia during an infection for all viruses or when considering the minimal infectious dose for each of them.

Several genera of marine dinoflagellates are known to produce bioactive compounds that affect human health. Among them, Gambierdiscus and Fukuyoa stand out for their ability to produce several toxins, including the potent neurotoxic ciguatoxins (CTXs), which accumulate through the food web. Once fishes contaminated with CTXs are ingested by humans, it can result in an intoxication named ciguatera. Within the two genera, only some species are able to produce toxins, and G. australes and G. excentricus have been highlighted to be the most abundant and toxic. Although the genera Gambierdiscus and Fukuyoa are endemic to tropical areas, their presence in subtropical and temperate regions has been recently recorded. In this work, the combined use of species-specific PCR primers for G. australes and G. excentricus modified with short oligonucleotide tails allowed the development of a multiplex detection system for these two toxin-producing species. Simultaneous detection was achieved using capture probes specific for G. australes and G. excentricus immobilized on maleimide-coated magnetic beads (MBs), separately placed on the working electrodes of a dual electrode array. Additionally, a rapid DNA extraction technique based on a portable bead beater system and MBs was developed, significantly reducing the extraction time (from several hours to 30 min). The developed technique was able to detect as low as 10 cells of both Gambierdiscus species and allowed the first detection of G. excentricus in the Balearic Islands in 8 out of the 9 samples analyzed. Finally, field samples were screened for CTXs with an immunosensor, successfully reporting 13.35 ± 0.5 pg CTX1B equiv. cell−1 in one sample and traces of toxins in 3 out of the 9 samples analyzed. These developments provide rapid and cost-effective strategies for ciguatera risk assessment, with the aim of guaranteeing seafood safety. info:eu-repo/semantics/acceptedVersion

A surge in fowl adenovirus (FAdV) causing inclusion body hepatitis (IBH) outbreaks has occurred in several countries in the last two decades. In Spain, a sharp increase in case numbers in broilers and broiler breeder pullets arose since 2011, which prompted the vaccination of breeders in some regions. Our retrospective study of IBH cases in Spain from 2011 to 2021 revealed that most cases were reported in broilers (92.21%) and were caused by serotypes FAdV-8b and -11, while cases in broiler breeder pullets were caused by serotypes FAdV-2, -11, and -8b. Vertical transmission was the main route of infection, although horizontal transmission likely happened in some broiler cases. Despite the inconsistent and heterogeneous use of vaccines among regions and over time, the number of cases mirrored the use of vaccines in the country. While IBH outbreaks were recorded year-long, significantly more cases occurred during the cooler and rainier months. The geographic distribution suggested a widespread incidence of IBH and revealed the importance of a highly integrated system. Our findings contribute to a better understanding of FAdV infection dynamics under field conditions and reiterate the importance of surveillance, serological monitoring of breeders, and vaccination of breeders against circulating serotypes to protect progenies. info:eu-repo/semantics/publishedVersion

Influenza viruses represent a continuous threat to both animal and human health. The 2009 H1N1 A influenza pandemic highlighted the importance of a swine host in the adaptation of influenza viruses to humans. Nowadays, one of the most extended strategies used to control swine influenza viruses (SIVs) is the trivalent vaccine application, whose formulation contains the most frequently circulating SIV subtypes H1N1, H1N2, and H3N2. These vaccines do not provide full protection against the virus, allowing its replication, evolution, and adaptation. To better understand the main mechanisms that shape viral evolution, here, the SIV intra-host diversity was analyzed in samples collected from both vaccinated and nonvaccinated animals challenged with the H1N1 influenza A virus. Twenty-eight whole SIV genomes were obtained by next-generation sequencing, and differences in nucleotide variants between groups were established. Substitutions were allocated along all influenza genetic segments, while the most relevant nonsynonymous substitutions were allocated in the NS1 protein on samples collected from vaccinated animals, suggesting that SIV is continuously evolving despite vaccine application. Moreover, new viral variants were found in both vaccinated and nonvaccinated pigs, showing relevant substitutions in the HA, NA, and NP proteins, which may increase viral fitness under field conditions. info:eu-repo/semantics/publishedVersion

Human-made hydrocarbon-rich environments are important reservoirs of microorganisms with specific degrading abilities and pathogenic potential. In particular, black fungi are of great interest, but their presence in the environment is frequently underestimated because they are difficult to isolate. In the frame of a biodiversity study from fuel-contaminated sites involving 30 diesel car tanks and 112 fuel pump dispensers (52 diesel and 60 gasoline, respectively), a total of 181 black fungal strains were isolated. The long cold incubation (LCI) of water-suspended samples, followed by plating on Dichloran Rose Bengal Chloramphenicol Agar (DRBC), gave isolation yields up to six times (6.6) higher than those of direct plating on DRBC, and those of enrichment with a phenolic mix. The sequencing of ITS and LSU-rDNA confirmed the dominance of potentially pathogenic fungi from the family Herpotrichiellaceae and Exophiala xenobiotica. Moreover, other opportunistic species were found, including E. opportunistica, E. oligosperma, E. phaeomuriformis, and Rhinocladiella similis. The recurrent presence of E. crusticola, Knufia epidermidis, Aureobasidium melanogenum, Cladosporium spp., and Scolecobasidium spp. was also recorded. Interestingly, 12% of total isolates, corresponding to 50% of taxa found (16/32), represent new species. All the novel taxa in this study were isolated by LCI. These findings suggest that black fungal diversity in hydrocarbon-rich niches remains largely unexplored and that LCI can be an efficient tool for further investigations.

Simple Summary The alarming emergence of antimicrobial resistance (AMR) in human and veterinary medicine has activated awareness for monitoring the levels of AMR pollution in the environment and wildlife. European hedgehogs (Erinaceus europaeus) are common wild species habiting urban areas in Europe. In this study, the occurrence and distribution of extended-spectrum β-lactam (ESBL) resistant enterobacteria and AMR genes were assessed in wild European hedgehogs in Catalonia, NE Spain. The results showed that 36.8% of the animals were detected as carriers of β-lactamase/carbapenemase resistance genes, with a special occurrence of human nosocomial bacteria such as Klebsiella pneumoniae, Escherichia coli, and Citrobacter freundii. In addition, more than half of the enterobacteria presented a multidrug resistance (MDR) phenotype and 31% of the isolates had an extended XDR profile. No differences in the spatial distribution of animals with AMR genes were observed within the study region. The results of this study suggest that the close contact with human areas predispose the transmission of AMR genes to wild hedgehogs because they either inhabit and/or feed in an anthropogenic environment. In conclusion, hedgehogs could be good sentinels or bioindicators of AMR environmental pollution, especially in highly populated areas with high human activity. Abstract Wildlife has been suggested to be a good sentinel of environmental health because of its close interaction with human populations, domestic animals, and natural ecosystems. The alarming emergence of antimicrobial resistance (AMR) in human and veterinary medicine has activated/triggered the awareness of monitoring the levels of AMR pollution in wildlife. European hedgehogs (Erinaceus europaeus) are common wild species habiting urban areas in Europe. However, there are few studies conducted in hedgehogs as reservoirs of AMR bacteria or genes. The aim of this study was to assess the occurrence and distribution of ESBL, AmpC, and carbapenem-resistant enterobacteria and AMR genes in wild European hedgehogs in Catalonia, a densely populated region of NE Spain. A total of 115 hedgehogs admitted at the Wildlife Rehabilitation Center of Torreferrussa were studied. To our knowledge, this is the first description of β-lactam resistant enterobacteria in wild hedgehogs. Interestingly, 36.8% (42/114) of the animals were detected as carriers of β-lactamase/carbapenemase resistance genes. Klebsiella spp. (59.6%), and specifically K. pneumoniae (84.6%), were the bacteria with the highest proportion of resistance genes, followed by E. coli (34.6%) and C. freundii (5.8%). The most frequently detected genetic variants were blaCTX-M-15 (19.3%), blaSHV-28 (10.5%), blaCMY-1 (9.7%), blaCMY-2 (8.8%), and blaOXA-48 (1.7%). In addition, 52% (27/52) of the isolates presented a multidrug resistance (MDR) phenotype and 31% had an extended drug resistance (XDR) profile. No clustering of animals with AMR genes within the study region was shown in the spatial analysis, nor differences in the proportion of positive animals among regions, were detected. The results of this study suggest that wild European hedgehogs could be good sentinels of AMR environmental pollution, especially in areas with a high human population density, because they either inhabit and/or feed in an anthropogenic environment. In conclusion, it is crucial to raise awareness of the strong interconnection between habitats and compartments, and therefore this implies that AMR issues must be tackled under the One Health approach.

Tools are required for quick and easy preliminary evaluation of functional feeds efficiency on fisheries. The analysis of skin mucus biomarkers is a recent alternative approach providing a faster feed-back from the laboratory which is characterized by being less invasive, more rapid and with reduced costs. The effect of replacing fishmeal and fish protein hydrolysates by means of two porcine by-products, the porcine spray-dried plasma (SDPP) and pig protein hydrolysate (PPH), in compound diets (50.4% crude protein, 16.2% crude protein, 22.1 MJ/kg feed) was evaluated in juvenile meagre (Argyrosomus regius) during a two-months period. To determine the impact of these dietary replacements, growth and food performance were measured together with digestive enzymes activities and filet proximal composition. Additionally, skin mucus was collected and characterized by determining main mucus biomarkers (protein, glucose, lactate, cortisol, and antioxidant capacity) and its antibacterial properties, measured by the quick in vitro co-culture challenges. In comparison to the control group, the inclusion of PPH and SDPP, in meagre diets reduced growth (7.4–8.8% in body weight), increased feed conversion ratios (9.0–10.0%), results that were attributed to a reduction in feed intake values (24.2–33.0%) (P < 0.05). Porcine blood by-products did not modify the activity of gastric and pancreatic digestive enzymes as well as those involved in nutrient absorption (alkaline phosphatase) nor liver oxidative stress condition (P > 0.05). In contrast, a reduction in fillet lipid content associated to an increase in fillet protein levels were found in fish fed SDPP and PPH diets (P < 0.05). As compared to the control diet, the dietary replacement did not alter the levels of the skin mucus biomarkers related to stress (cortisol and antioxidant capacity) or nutritional status (soluble protein, glucose and lactate) (P > 0.05). Interestingly, regardless of the worst performance in somatic growth, meagre fed diets containing both tested porcine by-products showed a significantly improved antibacterial capacity of their skin mucus. This enhancement was more prominent for fish fed with the PPH diet, which may be attributed to a higher content of immunomodulatory bioactive compounds in PPH. Further research will be necessary to provide insights on how the inclusion of SDPP and PPH, at the expense of dietary fishmeal and fish protein hydrolysates, affects feed intake and growth performance in meagre. However, the use of skin mucus biomarkers has been demonstrated to be an excellent methodology for a preliminary characterization of the functional feeds, in particular for their prophylactic properties by the study of mucus antibacterial activity. info:eu-repo/semantics/acceptedVersion

Centro de Investigación en Sanidad Animal (CISA), Pigs are susceptible to infection with the classical bovine spongiform encephalopathy (C-BSE) agent following experimental inoculation, and PrPSc accumulation was detected in porcine tissues after the inoculation of certain scrapie and chronic wasting disease isolates. However, a robust transmission barrier has been described in this species and, although they were exposed to C-BSE agent in many European countries, no cases of natural transmissible spongiform encephalopathies (TSE) infections have been reported in pigs. Transmission of atypical scrapie to bovinized mice resulted in the emergence of C-BSE prions. Here, we conducted a study to determine if pigs are susceptible to atypical scrapie. To this end, 12, 8-9-month-old minipigs were intracerebrally inoculated with two atypical scrapie sources. Animals were euthanized between 22- and 72-months post inoculation without clinical signs of TSE. All pigs tested negative for PrPSc accumulation by enzyme immunoassay, immunohistochemistry, western blotting and bioassay in porcine PrP mice. Surprisingly, in vitro protein misfolding cyclic amplification demonstrated the presence of C-BSE prions in different brain areas from seven pigs inoculated with both atypical scrapie isolates. Our results suggest that pigs exposed to atypical scrapie prions could become a reservoir for C-BSE and corroborate that C-BSE prions emerge during interspecies passage of atypical scrapie., This work was supported by the project EFA148/16 REDPRION. The project EFA148/16 REDPRION was 65% co-financed by the European Regional Development Fund (ERDF) through the Interreg V-A Spain-France-Andorra program (POCTEFA 2014–2020). POCTEFA aims to reinforce the economic and social integration of the French–Spanish–Andorran border. Its support is focused on developing economic, social and environmental cross-border activities through joint strategies favoring sustainable territorial development. Transmission experiments in transgenic mice were founded by grant PID2019-105837RB-I00 from Spanish Ministerio de Ciencia e Innovación

This study aimed to investigate the effects of replacing fish meal (FM) with soybean protein concentrates (SPC) on the intermediary metabolism and health of Totoaba macdonaldi juveniles. Fish (initial weight 50 ± 1 g) were fed for 60 days with eight diets: a reference diet (RD) and seven experimental diets where FM was replaced gradually with 15 to 100% SPC (SPC15, SPC30, SPC45, SPC60, SPC75, SPC90, and SPC100, respectively). Hexokinase (HK), glucokinase (GK), and alanine aminotransferase (ALT) enzyme activities showed highly significant differences (p < 0.01) between fish fed RD (0% SPC) compared to fish fed the diets with 60, 75, 90, and 100% SPC. The ALT enzyme shows a highly significant (p < 0.01) decrease in activity for fish fed 75, 90, and 100% SPC inclusions compared to fish fed the RD. The aspartate aminotransferase AST/ALT ratio showed a significant increase in activity for fish fed 100% soybean compared only with fish fed the control diet. The histological organization of the liver in totoaba juveniles fed RD, SPC15, SPC30 and SPC45 diets were similar. Totoaba fed with SPC90 and SPC100 showed histological alterations in hepatic and pancreatic parenchyma. Overall, according to the findings in this study, 45% of dietary FM could be replaced by SPC without causing adverse changes in metabolism, histological organization of liver, and health of juveniles of totoaba when cultured for 60 days. However, levels greater than 60% of SPC could compromise the health status of fish. info:eu-repo/semantics/acceptedVersion