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The widespread outbreak of the monkeypox virus (MPXV) recognized in 2022 poses new challenges for public healthcare systems worldwide. With more than 86,000 people infected, there is concern that MPXV may become endemic outside of its original geographical area leading to repeated human spillover infections or continue to be spread person-to-person. Fortunately, classical public health measures (e.g., isolation, contact tracing and quarantine) and vaccination have blunted the spread of the virus, but cases are continuing to be reported in 28 countries in March 2023. We describe here the vaccines and drugs available for the prevention and treatment of MPXV infections. However, although their efficacy against monkeypox (mpox) has been established in animal models, little is known about their efficacy in the current outbreak setting. The continuing opportunity for transmission raises concerns about the potential for evolution of the virus and for expansion beyond the current risk groups. The priorities for action are clear: 1) more data on the efficacy of vaccines and drugs in infected humans must be gathered; 2) global collaborations are necessary to ensure that government authorities work with the private sector in developed and low and middle income countries (LMICs) to provide the availability of treatments and vaccines, especially in historically endemic/enzootic areas; 3) diagnostic and surveillance capacity must be increased to identify areas and populations where the virus is present and may seed resurgence; 4) those at high risk of severe outcomes (e.g., immunocompromised, untreated HIV, pregnant women, and inflammatory skin conditions) must be informed of the risk of infection and be protected from community transmission of MPXV; 5) engagement with the hardest hit communities in a non-stigmatizing way is needed to increase the understanding and acceptance of public health measures; and 6) repositories of monkeypox clinical samples, including blood, fluids, tissues and lesion material must be established for researchers. This MPXV outbreak is a warning that pandemic preparedness plans need additional coordination and resources. We must prepare for continuing transmission, resurgence, and repeated spillovers of MPXV. We would like to thank Drs. Ming Fan at East Carolina University and Dara Wambach and her team at Johnson & Johnson for critically reviewing the manuscript. info:eu-repo/semantics/publishedVersion

Durinskia capensis is a kleptoplastic dinoflagellate species from high intertidal marine rock pools, which can use a variety of diatoms for photosynthesis. However, very few of the diatoms permit indefinite survival of the dinoflagellate and rbcL sequences show that D. capensis isolated from nature contains one of two closely related Nitzschia species as its kleptoplastids. In culture, without a supply of these ‘essential’ Nitzschia cells to replenish the intracellular store of diatom plastids and other organelles, D. capensis eventually loses all its kleptoplastids and dies. Inside Durinskia, diatoms do not possess frustules and so cannot be compared morphologically with free-living forms. Recently, one of the essential Nitzschia species was isolated from the type locality of D. capensis and grown in culture, allowing comparison with similar Nitzschia species, particularly N. agnita and N. kuetzingioides, examined from type material. We conclude that the ‘essential diatom’ of D. capensis differs morphologically from these and other Nitzschia species and it is therefore described as N. captiva sp. nov. Nitzschia agnita and N. kuetzingioides, on the other hand, are conspecific and N. agnita has priority. Nitzschia captiva and N. agnita are extremely similar in valve shape, dimensions, pattern and ultrastructure, but can be separated by their girdle structure. Nitzschia agnita appears to be a freshwater species, though somewhat salt-tolerant. In contrast, N. captiva, which is known principally from records of the kleptoplastids of D. capensis rather than from frustules, is so far marine. info:eu-repo/semantics/acceptedVersion

Wild adult females of a low trophic omnivore teleost species, the flathead grey mullet (Mugil cephalus), caught in the western Mediterranean were sampled. The lipid and fatty acid composition of ovaries, liver, muscle, and peritoneal fat were analysed at previtellogenesis, early-vitellogenesis —first observed at mid-summer (early August)—, late-vitellogenesis, and the post-spawning period —from mid-September to mid-October—. During ovarian development, the lipid content of muscle was low and constant (3.85%–4.92%), indicating that the muscle was not used to store lipids for gonadal growth. Although constant, lipid content in the liver was higher (18.46%–22.62%) than in the muscle, and HSI% increased during gonad development, suggesting a dynamism in the mobilization of the hepatic lipids. Total lipids in the gonads significantly increased with maturation (from 4.90% to 34.59%) in parallel with the GSI (from 0.8% to 15.5%) to decrease after spawning. Peritoneal fat was probably transitional fat that could be rapidly metabolized or transferred to other tissues but no specific function could be assigned because its presence in previtellogenic and early-vitellogenic females varied greatly. One of the main sources of lipids accumulated in the ovary was most likely diet. The total percentage of ΣMUFA, mainly 17:1 —previously not identified in high quantities in teleost vitellogenic ovaries and likely of bacterial origin— and 16:1, strongly increased in the ovaries with maturation. The 16:1 might be an important source of lipids for embryo development. High percentages of DHA, EPA, and ARA were found in the ovary during previtellogenesis available to be used during gonadal maturation. Understanding lipid and fatty acid changes in broodstock tissues can increase our knowledge of the nutritional requirements of the fish used in aquaculture breeding programs. info:eu-repo/semantics/acceptedVersion

Increasing surface air temperatures and human influences (e.g., agriculture, livestock grazing, tourism) are altering lacustrine ecosystems in the South American Andean páramo, and these influences are evident in changes in the diatom-species composition in sediment cores from the region that span the last ~ 150 years. Existing studies are limited by their short temporal scales and limited spatial extent. We analyzed two sediment cores spanning the last two millennia from the northern (Laguna Piñan) and southern (Laguna Fondococha) Andean páramo of Ecuador to provide a longer-term perspective on lake dynamics. Both lakes show shifts in the dominant diatoms through time. Fondococha diatoms shifted in dominance between two Aulacoseira species and in the planktic to benthic ratio, and these shifts are interpreted as evidence of changing lake level. The inferred shifts are corroborated by changes in sediment geochemistry. Piñan shows a directional shift in the diatom assemblage over the period of the record, from benthic diatoms tolerant of high dissolved organic carbon (DOC), low pH, and low nutrients, to an assemblage characteristic of lower DOC, higher pH, nutrients and lake levels. Shifts in Piñan’s diatoms are correlated with tephra layers in the sediment, suggesting that local volcanic deposition may have been responsible for altering the catchment and lake geochemistry. This is supported by relatively high δ13C values in organic matter associated with tephra layers, which become more negative up-section. Our study suggests that remote lakes in spatially heterogenous montane regions act as sentinels of different facets of environmental change and provide insights into Andean ecosystem responses to environmental perturbations. info:eu-repo/semantics/acceptedVersion

Feeding represents the largest economic cost in meat production; therefore, selection to improve traits related to feed efficiency is a goal in most livestock breeding programs. Residual feed intake (RFI), that is, the difference between the actual and the expected feed intake based on animal's requirements, has been used as the selection criteria to improve feed efficiency since it was proposed by Kotch in 1963. In growing pigs, it is computed as the residual of the multiple regression model of daily feed intake (DFI), on average daily gain (ADG), backfat thickness (BFT), and metabolic body weight (MW). Recently, prediction using single-output machine learning algorithms and information from SNPs as predictor variables have been proposed for genomic selection in growing pigs, but like in other species, the prediction quality achieved for RFI has been generally poor. However, it has been suggested that it could be improved through multi-output or stacking methods. For this purpose, four strategies were implemented to predict RFI. Two of them correspond to the computation of RFI in an indirect way using the predicted values of its components obtained from (i) individual (multiple single-output strategy) or (ii) simultaneous predictions (multi-output strategy). The other two correspond to the direct prediction of RFI using (iii) the individual predictions of its components as predictor variables jointly with the genotype (stacking strategy), or (iv) using only the genotypes as predictors of RFI (single-output strategy). The single-output strategy was considered the benchmark. This research aimed to test the former three hypotheses using data recorded from 5828 growing pigs and 45,610 SNPs. For all the strategies two different learning methods were fitted: random forest (RF) and support vector regression (SVR). A nested cross-validation (CV) with an outer 10-folds CV and an inner threefold CV for hyperparameter tuning was implemented to test all strategies. This scheme was repeated using as predictor variables different subsets with an increasing number (from 200 to 3000) of the most informative SNPs identified with RF. Results showed that the highest prediction performance was achieved with 1000 SNPs, although the stability of feature selection was poor (0.13 points out of 1). For all SNP subsets, the benchmark showed the best prediction performance. Using the RF as a learner and the 1000 most informative SNPs as predictors, the mean (SD) of the 10 values obtained in the test sets were: 0.23 (0.04) for the Spearman correlation, 0.83 (0.04) for the zero–one loss, and 0.33 (0.03) for the rank distance loss. We conclude that the information on predicted components of RFI (DFI, ADG, MW, and BFT) does not contribute to improve the quality of the prediction of this trait in relation to the one obtained with the single-output strategy. info:eu-repo/semantics/publishedVersion

Background Most interactions between the host and its microbiota occur at the gut barrier, and primary colonizers are essential in the gut barrier maturation in the early life. The mother–ofspring transmission of microorganisms is the most important factor infuencing microbial colonization in mammals, and C‑section delivery (CSD) is an impor‑ tant disruptive factor of this transfer. Recently, the deregulation of symbiotic host‑microbe interactions in early life has been shown to alter the maturation of the immune system, predisposing the host to gut barrier dysfunction and infammation. The main goal of this study is to decipher the role of the early‑life gut microbiota‑barrier alterations and its links with later‑life risks of intestinal infammation in a murine model of CSD. Results The higher sensitivity to chemically induced infammation in CSD mice is related to excessive exposure to a too diverse microbiota too early in life. This early microbial stimulus has short‑term consequences on the host homeo‑ stasis. It switches the pup’s immune response to an infammatory context and alters the epithelium structure and the mucus‑producing cells, disrupting gut homeostasis. This presence of a too diverse microbiota in the very early life involves a disproportionate short‑chain fatty acids ratio and an excessive antigen exposure across the vulnerable gut barrier in the frst days of life, before the gut closure. Besides, as shown by microbiota transfer experiments, the microbiota is causal in the high sensitivity of CSD mice to chemical‑induced colitis and in most of the phenotypical parameters found altered in early life. Finally, supplementation with lactobacilli, the main bacterial group impacted by CSD in mice, reverts the higher sensitivity to infammation in ex‑germ‑free mice colonized by CSD pups’ microbiota. Conclusions Early‑life gut microbiota‑host crosstalk alterations related to CSD could be the linchpin behind the phe‑ notypic efects that lead to increased susceptibility to an induced infammation later in life in mice. Keywords C‑section delivery, Microbiota, Primary colonization, Early life, Infammation, Gut barrier, Murine model info:eu-repo/semantics/publishedVersion

Trends and ecological consequences of phosphorus (P) decline and increasing nitrogen (N) to phosphorus (N:P) ratios in rivers and estuaries are reviewed and discussed. Results suggest that re-oligotrophication is a dominant trend in rivers and estuaries of high-income countries in the last two-three decades, while in low-income countries widespread eutrophication occurs. The decline in P is well documented in hundreds of rivers of United States and the European Union, but the biotic response of rivers and estuaries besides hytoplankton decline such as trends in phytoplankton composition, changes in primary production, ecosystem shifts, cascading effects, changes in ecosystem metabolism, etc., have not been sufficiently monitored and investigated, neither the effects of N:P imbalance. N:P imbalance has significant ecological effects that need to be further investigated. There is a growing number of cases in which phytoplankton biomass have been shown to decrease due to re-oligotrophication, but the potential regime shift from phytoplankton to macrophyte dominance described in shallow lakes has been documented only in a few rivers and estuaries yet. The main reasons why regime shifts are rarely described in rivers and estuaries are, from one hand the scarcity of data on macrophyte cover trends, and from the otherhand physical factors such as peak flows or high turbidity that could prevent a general spread of submerged macrophytes as observed in shallow lakes. Moreover, re-oligotrophication effects on rivers may be different compared to lakes (e.g., lower dominance of macrophytes) or estuaries (e.g., limitation of primary production by N instead of P) or may be dependent on river/estuary type. We conclude that river and estuary re-oligotrophication effects are complex, diverse and still little known, and in some cases are equivalent to those described in shallow lakes, but the regime shift is more likely to occur in mid to high-order rivers and shallow estuaries. This work was supported by a grant from the U.S. National Science Foundation (#DBI-1639145) to the National Socio-Environmental Synthesis Center (Rivershift Project). The work was also financially supported by the Catalan Government through the funding grant ACCIÓ-Eurecat (Project AquaSCI-2022).

Biomarkers are used to assess pain and analgesic drug efficacy in livestock. However, often the diagnostic sensitivity and specificity of these biomarkers for different painful conditions over time have not been described. Receiver operating characteristic (ROC) curves are graphical plots that illustrate the diagnostic ability of a test as its discrimination threshold is varied. The objective of this analysis was to use area under the curve (AUC) values derived from ROC analysis to characterize the predictive value of potential pain biomarkers at specific time points following a painful stimulus. The biomarkers included in the analysis were plasma cortisol, salivary cortisol, hair cortisol, infrared thermography (IRT), mechanical nociceptive threshold (MNT), substance P, kinematic gait analysis, and a visual analog scale for pain. A total of 7,992 biomarker outcomes collected from 7 pain studies involving pain associated with castration, dehorning, lameness, and abdominal surgery were included in the analysis. Each study consisted of 3 treatments: uncontrolled pain (tissue damage), no pain (handled controls), and analgesic use (tissue damage, administered a nonsteroidal anti-inflammatory drug). Results comparing analgesic effects to uncontrolled pain consistently yielded AUC values >0.7 (95% confidence interval: 0.40 to 0.99) for plasma cortisol (time points: 1.5, 2, 3, 4, 6, and 8 h), hair cortisol (time point: 62 d), and IRT (time point: 72 h). Results comparing analgesic effects to uncontrolled pain consistently yielded AUC values

This study evaluated the effects of different dietary inclusion levels of fermented soybean meal (FSM) as replacement for fish meal and their effects on the productive performance and intestinal condition of goldfish (Carassius auratus) produced in biofloc (BFT) system. Five isoproteic (39.5% crude protein) and isoenergetic diets (4250 kcal kg−1 of crude energy) were formulated with FSM inclusion levels of 0%, 7%, 14%, 21% and 28% (0, 11, 22, 32 and 43 fish meal replacement). A total of 400 goldfish (0.25 ± 0.02 g) were weighed and distributed in 20 glass aquariums (15 L). Fish were fed twice daily for 56 days. The diet with FSM inclusion level of 28% reduced the weight gain and the specific growth rate and increased the feed conversion of the goldfish. Diets with FSM inclusion levels of 21% and 28% increased the α-amylase activity in the fish intestine. The diet with FSM inclusion level of 21% increased total height of the intestinal villi of the goldfish. In conclusion, the inclusion of up to 21% of FSM can replace fishmeal without affecting the growth of goldfish juveniles reared in BFT system. Fish fed 21% of FSM showed evidence of improvement in the intestinal health. info:eu-repo/semantics/acceptedVersion

In the present study, we investigated the presence of four Vibrio cholerae virulence genes (ctxA, VPI, Zot and ace) in 36 Vibrio alginolyticus isolates obtained from different seawater, sediments and aquatic organisms. We tested the virulence of 13 V. alginolyticus strains against juveniles of Sparus aurata and this virulence was correlated with the presence of V. cholerae virulence genes. A positive amplification for the virulence pathogenicity island was produced by five V. alginolyticus strains and four for cholerae toxin. Some of the V. alginolyticus strains are pathogenic to aquatic animals and might have derived their virulence genes from V. cholerae. V. alginolyticus strains can be considered as a possible reservoir of V. cholerae virulence genes.

The Asian tiger mosquito Aedes albopictus is an undesirable invasive mosquito species that causes considerable nuisance through its biting behaviour, and has been proven to transmit more than 22 different viruses under laboratory conditions. Human-aided transportation, the capacity of winter diapause, and possibly global warming have contributed to the global invasion of Ae. albopictus. The species was found for the first time in the Netherlands in 2005, and since 2010 has been found introduced at many locations throughout the country. Elucidating the origin of these introduced mosquitoes could help the authorities on the planning and evaluation of the risk-based surveillance of Aedes invasive mosquitoes. This study aims to determine the genomic diversity of Ae. albopictus that is represented within and between collection sites with a database consisting of Ae. albopictus specimens from past introductions in the Netherlands, specimens from populations from other regions in the world, and data from specimens present in databases. In this study, complete mitochondrial genomes were sequenced, a recommended marker for phylogeography analysis of Ae. albopictus. Metadata is presented in a Nextstrain build containing 254 Ae. albopictus genomes up to October 2020. Overall, the phylogeny results of the Nextstrain build reveals a low mitogenomic diversity within Ae. albopictus. Genomic diversity of Ae. albopictus specimens found in the Netherlands fall within one main cluster which is hypothesised to represent the globally invasive strain of the species. Other organisations are stimulated to share data or materials for inclusion and improvement of the Nextstrain build, which can be accessed at https://nextstrain.nrcnvwa.nl/Aedes/20210728. info:eu-repo/semantics/publishedVersion

Global emergence and re-emergence of Porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus which causes a highly contagious enteric disease, have led to several studies addressing its variability. The aim of this study was to characterize the infection of weaned pigs with Swine enteric coronavirus (SeCoV) -a chimeric virus most likely originated from a recombination event between PEDV and Transmissible gastroenteritis virus, or its mutant Porcine respiratory coronavirus-, and two PEDV G1b variants, including a recently described recombinant PEDV-SeCoV (rPEDV-SeCoV), as well as to determine the degree of cross-protection achieved against the rPEDV-SeCoV. For this purpose, forty-eight 4-week-old weaned pigs were randomly allocated into four groups of 12 animals; piglets in groups B, C and D were orally inoculated with a PEDV variant (B and D) or SeCoV (C), while piglets in group A were mock inoculated and maintained as controls. At day 20 post-infection all groups were exposed to rPEDV-SeCoV; thus, group D was subjected to a homologous re-challenge, groups B and C to a heterologous re-challenge (PEDV/rPEDV-SeCoV and SeCoV/rPEDV-SeCoV, respectively) and group A was primary challenged (-/rPEDV-SeCoV). Clinical signs, viral shedding, microscopic lesions and specific humoral and cellular immune responses (IgG, IgA, neutralizing antibodies and IgA and IFN-γ-secreting cells) were monitored. After primo-infection all three viral strains induced an undistinguishable mild-to-moderate clinical disease with diarrhea as the main sign and villus shortening lesions in the small intestine. In homologous re-challenged pigs, no clinical signs or lesions were observed, and viral shedding was only detected in a single animal. This fact may be explained by the significant high level of rPEDV-SeCoV-specific neutralizing antibodies found in these pigs before the challenge. In contrast, prior exposition to a different PEDV G1b variant or SeCoV only provided partial cross-protection, allowing rPEDV-SeCoV replication and shedding in feces.

The emerging threat of ciguatera poisoning (CP) in Europe has been associated with fish captured in the Canary Islands (Spain) and Selvagens Islands (Portugal). The first are heavily populated islands where numerous scientific studies have been carried out. Conversely, the Selvagens Islands as a nature reserve with low human pressure have been rarely surveyed in terms of the marine benthic microalgae, including the epiphytic ciguatera-causing dinoflagellate species. To investigate the harmful microalgal diversity of the Selvagens Islands, a scientific cruise to these remote islands took place in September, 2018. The Gambierdiscus species composition and distribution, and the associated epiphytic dinoflagellate community, were assessed using artificial substrate devices. Gambierdiscus cells were found in all samples, reaching concentrations of up to 725 cells 100 cm–2. G. australes was the only species identified after morphological and molecular analysis of the retrieved cultures. Species identification was confirmed by molecular characterization based on the LSU D8–D10 region. Nevertheless, phylogenetic studies indicated that some strains diverged from the G. australes clade suggesting genetic differentiation. Toxicity was estimated by neuro-2a cell-based assay in four strains, ranging from 2.46–83 fg of CTX1B eq. cell–1. The epiphytic dinoflagellate community that co-occurred with Gambierdiscus comprised other toxic or potentially toxic dinoflagellates, such as Ostreopsis, Prorocentrum, Amphidinium and Coolia species. Oceanographic and meteorological data were also obtained to characterize the occurrence of Gambierdiscus. This study is the first stage in understanding the role of the Selvagens Islands in the incubation and proliferation of the ciguatera-causing dinoflagellates Gambierdiscus in the NE Atlantic. The Selvagens Islands are a ciguatera hotspot in Europe.Gambierdiscus australes was the only species observed in the Selvagens Islands.Strains diverging from the G. australes clade suggest genetic differentiation. The Selvagens Islands are a ciguatera hotspot in Europe. Gambierdiscus australes was the only species observed in the Selvagens Islands. Strains diverging from the G. australes clade suggest genetic differentiation.

The contribution of microRNAs (miRNAs) to mRNA post-transcriptional regulation has often been explored by the post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes in mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc–Göttingen minipigs. Exon–intron split analysis of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of miRNAs upregulated in fed pigs. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, exon–intron split analysis provides a valuable view of the regulation of gene expression, complementary to canonical differential expression analyses., We acknowledge the support of the Spanish Ministry of Economy and Competitivity for the Center of Excellence Severo Ochoa 2020–2023 (CEX2019-000902-S) grant awarded to the Centre for Research in Agricultural Genomics (CRAG, Bellaterra, Spain). Thanks also to the CERCA Programme of the Generalitat de Catalunya for their support. The present research work was funded by grants AGL2013-48742-C2-1-R and AGL2013-48742-C2-2-R awarded by the Spanish Ministry of Economy and Competitivity. E. Mármol-Sánchez was funded with a PhD fellowship FPU15/01733 awarded by the Spanish Ministry of Education and Culture. YRC is the recipient of a Ramon y Cajal fellowship (RYC2019-027244-I) from the Spanish Ministry of Science and Innovation.

Middle East respiratory syndrome coronavirus (MERS-CoV) is the cause of a severe respiratory disease with a high case fatality rate in humans. Since its emergence in mid-2012, 2578 laboratory-confirmed cases in 27 countries have been reported by the World Health Organization, leading to 888 known deaths due to the disease and related complications. Dromedary camels are considered the major reservoir host for this virus leading to zoonotic infection in humans. Dromedary camels, llamas, and alpacas are susceptible to MERS-CoV, developing a mild-to-moderate upper respiratory tract infection characterized by epithelial hyperplasia as well as infiltration of neutrophils, lymphocytes, and some macrophages within epithelium, lamina propria, in association with abundant viral antigen. The very mild lesions in the lower respiratory tract of these camelids correlate with absence of overt illness following MERS-CoV infection. Unfortunately, there is no approved antiviral treatment or vaccine for MERS-CoV infection in humans. Thus, there is an urgent need to develop intervention strategies in camelids, such as vaccination, to minimize virus spillover to humans. Therefore, the development of camelid models of MERS-CoV infection is key not only to assess vaccine prototypes but also to understand the biologic mechanisms by which the infection can be naturally controlled in these reservoir species. This review summarizes information on virus-induced pathological changes, pathogenesis, viral epidemiology, and control strategies in camelids, as the intermediate hosts and primary source of MERS-CoV infection in humans. info:eu-repo/semantics/acceptedVersion

Background In humans and livestock species, genome-wide association studies (GWAS) have been applied to study the association between variants distributed across the genome and a phenotype of interest. To discover genetic polymorphisms affecting the duodenum, liver, and muscle transcriptomes of 300 pigs from 3 different breeds (Duroc, Landrace, and Large White), we performed expression GWAS between 25,315,878 polymorphisms and the expression of 13,891 genes in duodenum, 12,748 genes in liver, and 11,617 genes in muscle. Results More than 9.68 × 1011 association tests were performed, yielding 14,096,080 significantly associated variants, which were grouped in 26,414 expression quantitative trait locus (eQTL) regions. Over 56% of the variants were within 1 Mb of their associated gene. In addition to the 100-kb region upstream of the transcription start site, we identified the importance of the 100-kb region downstream of the 3′UTR for gene regulation, as most of the cis-regulatory variants were located within these 2 regions. We also observed 39,874 hotspot regulatory polymorphisms associated with the expression of 10 or more genes that could modify the protein structure or the expression of a regulator gene. In addition, 2 motifs (5′-GATCCNGYGTTGCYG-3′ and a poly(A) sequence) were enriched across the 3 tissues within the neighboring sequences of the most significant single-nucleotide polymorphisms in each cis-eQTL region. Conclusions The 14 million significant associations obtained in this study are publicly available and have enabled the identification of expression-associated cis-, trans-, and hotspot regulatory variants within and across tissues, thus shedding light on the molecular mechanisms of regulatory variations that shape end-trait phenotypes. info:eu-repo/semantics/publishedVersion

Background Lactic Acid Bacteria such as Lactococcus lactis, Latilactobacillus sakei (basonym: Lactobacillus sakei) and Lactiplantibacillus plantarum (basonym: Lactobacillus plantarum) have gained importance as recombinant cell factories. Although it was believed that proteins produced in these lipopolysaccharides (LPS)-free microorganisms do not aggregate, it has been shown that L. lactis produce inclusion bodies (IBs) during the recombinant production process. These protein aggregates contain biologically active protein, which is slowly released, being a biomaterial with a broad range of applications including the obtainment of soluble protein. However, the aggregation phenomenon has not been characterized so far in L. plantarum. Thus, the current study aims to determine the formation of protein aggregates in L. plantarum and evaluate their possible applications. Results To evaluate the formation of IBs in L. plantarum, the catalytic domain of bovine metalloproteinase 9 (MMP-9cat) protein has been used as model protein, being a prone-to-aggregate (PTA) protein. The electron microscopy micrographs showed the presence of electron-dense structures in L. plantarum cytoplasm, which were further purified and analyzed. The ultrastructure of the isolated protein aggregates, which were smooth, round and with an average size of 250–300 nm, proved that L. plantarum also forms IBs under recombinant production processes of PTA proteins. Besides, the protein embedded in these aggregates was fully active and had the potential to be used as a source of soluble protein or as active nanoparticles. The activity determination of the soluble protein solubilized from these IBs using non-denaturing protocols proved that fully active protein could be obtained from these protein aggregates. Conclusions These results proved that L. plantarum forms aggregates under recombinant production conditions. These aggregates showed the same properties as IBs formed in other expression systems such as Escherichia coli or L. lactis. Thus, this places this LPS-free microorganism as an interesting alternative to produce proteins of interest for the biopharmaceutical industry, which are obtained from the IBs in an important number of cases. info:eu-repo/semantics/publishedVersion

Identifying Stress Coping Styles (SCS) in new species of interest for aquaculture has important implications for its future domestication and adaptation to captivity. Individual variability allows to select the potential positive characteristics for fish production. The main aim of this study was to identify phenotypic individual differences and characterize proactive and reactive SCS in flathead grey mullet (Mugil cephalus) juveniles by exposing fish to different stress situations and evaluating their individual and group responses to level behavioural and physiology. Juveniles were subjected to one group test (risk-taking) and five individual tests (predator, first feeding after stress, restraining, new environment and confinement). All assays were repeated twice, with a one-month interval between tests. Blood samples were taken from each individual (before and after stress) to quantify cortisol and glucose plasma concentrations. Flathead grey mullet juveniles exhibited a high inter-individual variability with two extremes of behaviours: proactive and reactive profiles that were characterized by opposed behavioural (activity time and escape attempts) and physiological (levels of cortisol and glucose) responses to stress and were consistent over time and across contexts. The flathead grey mullet juveniles showed differences in their predisposition for risk taking. Likewise, the Principal Component Analysis showed that three individual stress tests (predator, restraining and confinement tests) were reliable to characterize SCS in this fish species. This work reported for the first time the existence of stress coping styles in M. cephalus juveniles and the selection of a set of reliable behavioural tests to identify phenotypic profiles in flathead grey mullet. These results might be of interest for the aquaculture industry to improve fish welfare and health and to adjust management protocols for rearing this fish species in captivity. info:eu-repo/semantics/acceptedVersion

The fan mussel Pinna nobilis is experiencing a mass mortality event throughout the Mediterranean Sea. The survival of the remaining isolated populations and the maintenance and reproduction of individuals in captivity could determine the future of the species. This paper examines the clearance rate (CR) and oxygen consumption (VO2) of fan mussel individuals measured under 5 different temperatures (8.5, 14, 18, 23 and 28ºC). The measurements of both physiological variables revealed a threshold limit of thermal tolerance at both extreme temperatures, indicating the critical situation of the remaining populations, which are located in coastal lagoons and shallow bays where these extreme temperatures usually occur. Besides, the high clearance rates observed highlight the significant ecosystem service provided by the species in terms of water filtration, especially in confined waters with low renewal rates. Routine control of the clearance rate as an early warning system is proposed for detecting ailing individuals. Such a system could also be used to improve captivity conditions. info:eu-repo/semantics/publishedVersion

Colostrum (the first mammary gland secretion after calving) is known to contain high concentrations of nutrients as well as bioactive substances (including immunoglobulins, growth factors, and antimicrobial factors) to ensure neonatal survival. Due to its immunomodulatory, antibacterial, and antiviral activities, bovine colostrum has been used not only in calves but also in the prevention and treatment of human gastrointestinal and respiratory infections. Transition milk is the mammary secretion from the second milking to the sixth, which may contain these bioactive compounds to a lesser extent. The objective of the present study was to measure IGF-I, immunoglobulin G (IgG), and lactoferrin (LTF) concentrations in colostrum and transition milk of primiparous and multiparous cows to further assess its potential use in veterinary and nutraceutical applications. The results demonstrated that the concentrations of these three bioactive molecules decrease from the first milking to the tenth. Concentrations of IGF-I and LTF were greater in multiparous than in primiparous cows. Also, lactation number interacted with milking number in IGF-I, since primiparous cows had a smoother decline of IGF-I concentrations than multiparous ones. Overall, transition milk from the second milking showed a 46% decrease in the analysed colostrum bioactive molecules. Therefore, further studies are needed to apply this knowledge in neonate farm management practices or in developing pharmaceutical supplements from farm surpluses. info:eu-repo/semantics/publishedVersion

Novel insights were provided by contrasting the composition of wild and farmed fish gut microbiomes because the latter had essentially different environmental conditions from those in the wild. This was reflected in the gut microbiome of the wild Sparus aurata and Xyrichtys novacula studied here, which showed highly diverse microbial community structures, dominated by Proteobacteria, mostly related to an aerobic or microaerophilic metabolism, but with some common shared major species, such as Ralstonia sp. On the other hand, farmed non-fasted S. aurata individuals had a microbial structure that mirrored the microbial composition of their food source, which was most likely anaerobic, since several members of the genus Lactobacillus, probably revived from the feed and enriched in the gut, dominated the communities. The most striking observation was that after a short fasting period (86 h), farmed gilthead seabream almost lost their whole gut microbiome, and the resident community associated with the mucosa had a very much reduced diversity that was highly dominated by a single potentially aerobic species Micrococcus sp., closely related to M. flavus. The results pointed to the fact that, at least for the juvenile S. aurata studied, most of the microbes in the gut were transient and highly dependent on the feed source, and that only after fasting for at least 2 days could the resident microbiome in the intestinal mucosa be determined. Since an important role of this transient microbiome in relation to fish metabolism could not be discarded, the methodological approach needs to be well designed in order not to bias the results. The results have important implications for fish gut studies that could explain the diversity and occasional contradictory results published in relation to the stability of marine fish gut microbiomes, and might provide important information for feed formulation in the aquaculture industry. info:eu-repo/semantics/publishedVersion

This Scientific Opinion concerns the welfare of Domestic ducks (Anas platyrhynchos domesticus), Muscovy ducks (Cairina moschata domesticus) and their hybrids (Mule ducks), Domestic geese (Anser anser f. domesticus) and Japanese quail (Coturnix japonica) in relation to the rearing of breeders, birds for meat, Muscovy and Mule ducks and Domestic geese for foie gras and layer Japanese quail for egg production. The most common husbandry systems (HSs) in the European Union are described for each animal species and category. The following welfare consequences are described and assessed for each species: restriction of movement, injuries (bone lesions including fractures and dislocations, soft tissue lesions and integument damage and locomotory disorders including lameness), group stress, inability to perform comfort behaviour, inability to perform exploratory or foraging behaviour and inability to express maternal behaviour (related to prelaying and nesting behaviours). Animal-based measures relevant for the assessment of these welfare consequences were identified and described. The relevant hazards leading to the welfare consequences in the different HSs were identified. Specific factors such as space allowance (including minimum enclosure area and height) per bird, group size, floor quality, characteristics of nesting facilities and enrichment provided (including access to water to fulfil biological needs) were assessed in relation to the welfare consequences and, recommendations on how to prevent the welfare consequences were provided in a quantitative or qualitative way. info:eu-repo/semantics/publishedVersion

This Scientific Opinion addresses a European Commission's mandate on the welfare of dairy cows as part of the Farm to Fork strategy. It includes three assessments carried out based on literature reviews and complemented by expert opinion. Assessment 1 describes the most prevalent housing systems for dairy cows in Europe: tie-stalls, cubicle housing, open-bedded systems and systems with access to an outdoor area. Per each system, the scientific opinion describes the distribution in the EU and assesses the main strengths, weaknesses and hazards potentially reducing the welfare of dairy cows. Assessment 2 addresses five welfare consequences as requested in the mandate: locomotory disorders (including lameness), mastitis, restriction of movement and resting problems, inability to perform comfort behaviour and metabolic disorders. Per each welfare consequence, a set of animal-based measures is suggested, a detailed analysis of the prevalence in different housing systems is provided, and subsequently, a comparison of the housing systems is given. Common and specific system-related hazards as well as management-related hazards and respective preventive measures are investigated. Assessment 3 includes an analysis of farm characteristics (e.g. milk yield, herd size) that could be used to classify the level of on-farm welfare. From the available scientific literature, it was not possible to derive relevant associations between available farm data and cow welfare. Therefore, an approach based on expert knowledge elicitation (EKE) was developed. The EKE resulted in the identification of five farm characteristics (more than one cow per cubicle at maximum stocking density, limited space for cows, inappropriate cubicle size, high on-farm mortality and farms with less than 2 months access to pasture). If one or more of these farm characteristics are present, it is recommended to conduct an assessment of cow welfare on the farm in question using animal-based measures for specified welfare consequences. info:eu-repo/semantics/publishedVersion

The growing interest in combining spatial and temporal patterns in nature has been fostered by the current availability of high-frequency measurements. However, we still lack a methodological framework to process and interpret spatiotemporal datasets into meaningful values, adaptable to different time windows and/or responding to different spatial structures. Here, we developed and tested a framework to evaluate spatiotemporal connectivity using two new measures: the spatiotemporal connectivity (STcon) and the spatiotemporal connectivity matrix (STconmat). To obtain these measures, we consider a set of spatially connected sites within a temporally dynamic network. These measures are calculated from a spatiotemporal matrix where spatial and temporal connections across sites are captured. These connections respond to a determined network structure, assign different values to these connections and generate different scenarios from which we obtain the spatiotemporal connectivity. We developed these measures by using a dataset of stream flow state spanning a 513-day period obtained from data loggers installed in seven temporary streams. These measures allowed us to characterise connectivity among stream reaches and relate spatiotemporal patterns with macroinvertebrate community structure and composition. Spatiotemporal connectivity differed within and among streams, with STcon and STconmat capturing different hydrological patterns. Macroinvertebrate richness and diversity were higher in more spatiotemporally connected sites. Community dissimilarity was related to STconmat showing that more spatiotemporally connected sites had similar communities for active and passive dispersers. Interestingly, both groups were related to spatiotemporal connectivity patterns for some of the analysed scenarios, highlighting the relevance of spatiotemporal connectivity in dynamic systems. As we exemplified, the proposed framework can help to disentangle and quantify spatiotemporal dynamics or be applied in the conservation of dynamic systems such as temporary streams. However, the current framework is not limited to the temporal and spatial features of temporary streams. It can be extended to other ecosystems by including different time windows and/or consider different network structures to assess spatiotemporal patterns. Such spatiotemporal measures are especially relevant in a context of global change, with the spatiotemporal dynamics of ecosystems being heavily disrupted by human activities., This research was carried out in the FEHM-Lab (Freshwater Ecology, Hydrology and Management) funded by the Agència de Gestió d'Ajuts Universitaris i de Recerca' (AGAUR) at the Generalitat de Catalunya (2017SGR1643). This work was also supported by the MECODISPER project (CTM2017-89295-P) funded by the Spanish Ministerio de Economía, Industria y Competitividad (MINECO)—Agencia Estatal de Investigación (AEI) and co-funded by the European Regional Development Fund (ERDF). David Cunillera-Montcusí and José María Fernández-Calero were also supported by the MECODISPER project. David Cunillera-Montcusí was also supported by European Union-NextGenerationEU, Ministry of Universities and Recovery, Transformation and Resilience Plan, through a call from Universitat de Girona. Miguel Cañedo-Argüelles and Núria Bonada were supported by the Serra Húnter programme of the Generalitat de Catalunya. Núria Bonada was also supported by the H2020 European Research and Innovation action Grant Agreement no. 869226 (DRYvER), and the ICREA-Academia program. Miguel Cañedo-Argüelles was also supported by a Ramón y Cajal contract funded by the Spanish Ministry of Science and Innovation (RYC2020-029829-I). This study has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 869226 (DRYvER project: www.dryver.eu). We want to also thank the two reviewers whose comments helped the current manuscript to greatly improve.

The present study aimed to investigate the effect of age, breed, and sex of broilers, as well as a probiotic or phytobiotic product on mucosal morphology, bacterial metabolites, and immune traits in the ileum of broilers. A total of 2,880 one-day-old male and female broiler chicks from two breeds (Ross308® and Cobb500®) were randomly assigned to 72 pens. Broilers were offered a wheat-soybean diet without (CO), or with either a probiotic (PO; 2.4 × 109 CFU/kg of Bacillus subtilis DSM32324 and DSM32325 and B. amyloliquefaciens DSM25840) or a phytobiotic (PY; grape extract, 165 ppm procyanidin and 585 ppm polyphenols of the diet) product. The trial was conducted with a 3 × 2 × 2 factorial arrangement of diet, breed, and sex in a completely randomized design (6 replicate-pens per treatment). At day 7, 21, and 35, one chicken per pen was slaughtered for collecting ileal tissue to evaluate of histomorphology and mRNA expression, as well as ileal digesta to measure bacterial metabolites. Data were subjected to ANOVA (the main factors; age, diet, breed, and sex) and Four-Way ANOVA (interactions) using GLM procedure. Overall, the concentration of acetate and total short chain fatty acids reached the peak and lactate decreased to its lowest on day 21, but their concentrations at day 7 and 35 were similar (p > 0.05). Spermine, spermidine, and ammonia decreased after day 7, while putrescine and cadaverine increased after day 21 (p MUC2) and claudin 5 (CLDN5) was similar; increased from day 7 to 21 and decreased afterward (p p p IFN-γ expression, while Cobb broilers had greater IL-4, IL-6 and TNF-α expression and higher total GC number (p p p > 0.05). In conclusion, aging of broilers affected ileal histomorphology, cytokine expression, and barrier integrity, as well as bacterial activity. These observed impacts could be attributed to host-microbiota interaction and the direct effects of bacterial metabolites on intestinal cells and immune system.

Purpose Obesity during childhood has become a pandemic disease, mainly caused by a diet rich in sugars and fatty acids. Among other negative effects, these diets can induce cognitive impairment and reduce neuroplasticity. It is well known that omega-3 and probiotics have a beneficial impact on health and cognition, and we have hypothesized that a diet enriched with Bifidobacterium breve and omega-3 could potentiate neuroplasticity in prepubertal pigs on a high-fat diet. Methods Young female piglets were fed during 10 weeks with: standard diet (T1), high-fat (HF) diet (T2), HF diet including B. breve CECT8242 (T3) and HF diet including the probiotic and omega-3 fatty acids (T4). Using hippocampal sections, we analyzed by immunocytochemistry the levels of doublecortin (DCX) to study neurogenesis, and activity-regulated cytoskeleton-associated protein (Arc) as a synaptic plasticity related protein. Results No effect of T2 or T3 was observed, whereas T4 increased both DCX+ cells and Arc expression. Therefore, a diet enriched with supplements of B. breve and omega-3 increases neurogenesis and synaptic plasticity in prepubertal females on a HF diet from nine weeks of age to sexual maturity. Furthermore, the analysis of serum cholesterol and HDL indicate that neurogenesis was related to lipidic demand in piglets fed with control or HF diets, but the neurogenic effect induced by the T4 diet was exerted by mechanisms independent of this lipidic demand. Conclusion Our results show that the T4 dietary treatment is effective in potentiating neural plasticity in the dorsal hippocampus of prepubertal females on a HF diet.

Background Gut microbial composition plays an important role in numerous traits, including immune response. Integration of host genomic information with microbiome data is a natural step in the prediction of complex traits, although methods to optimize this are still largely unexplored. In this paper, we assess the impact of different modelling strategies on the predictive capacity for six porcine immunocompetence traits when both genotype and microbiota data are available. Methods We used phenotypic data on six immunity traits and the relative abundance of gut bacterial communities on 400 Duroc pigs that were genotyped for 70 k SNPs. We compared the predictive accuracy, defined as the correlation between predicted and observed phenotypes, of a wide catalogue of models: reproducing kernel Hilbert space (RKHS), Bayes C, and an ensemble method, using a range of priors and microbial clustering strategies. Combined (holobiont) models that include both genotype and microbiome data were compared with partial models that use one source of variation only. Results Overall, holobiont models performed better than partial models. Host genotype was especially relevant for predicting adaptive immunity traits (i.e., concentration of immunoglobulins M and G), whereas microbial composition was important for predicting innate immunity traits (i.e., concentration of haptoglobin and C-reactive protein and lymphocyte phagocytic capacity). None of the models was uniformly best across all traits. We observed a greater variability in predictive accuracies across models when microbiability (the variance explained by the microbiome) was high. Clustering microbial abundances did not necessarily increase predictive accuracy. Conclusions Gut microbiota information is useful for predicting immunocompetence traits, especially those related to innate immunity. Modelling microbiome abundances deserves special attention when microbiability is high. Clustering microbial data for prediction is not recommended by default.

The meagre Argyrosomus regius (Asso, 1801) is a marine fish species that has an increasing aquaculture production in Europe. Lowering the age at maturity of hatchery-produced juveniles would support meagre aquaculture by reducing time between generations in selective breeding programs and reducing industrial costs for broodstock maintenance. The aim of this work was to assess the effects of a treatment with recombinant follicle stimulating hormone (rFsh), produced in ovarian cells of Chinese hamsters, on male germ cell proliferation and apoptosis in sexually immature meagre. The rFsh-treated fish had higher gonadosomatic index, larger seminiferous tubules, more abundant luminal spermatozoa, a lower density of anti-PCNA positive single A spermatogonia, a higher density of anti-PCNA positive spermatocysts and a lower incidence of germ cell apoptosis than control groups. The present study demonstrated the effectiveness of the produced rFsh in stimulating testis development and spermatogenesis in pre-pubertal meagre. Moreover, the rFsh treatment proved to be highly efficient in removing the apoptotic block of spermatogenesis observed in juvenile meagre, allowing spermatogonial survival and progress towards meiosis. The administration of rFsh did not stimulate spermatogonial self-renewal, a process whose control still needs to be elucidated.

Given their role in lipid digestion, feed supplementation with bile salts could be an economic and sustainable solution to alterations in adiposity and intestinal inflammation generated by some strategies currently used in aquaculture. An important part of the metabolism of bile salts takes place in the intestine, where the microbiota transforms them into more toxic forms. Consequently, we aimed to evaluate the gut immune response and microbial populations in gilthead seabream (Sparus aurata) fed a diet supplemented with a blend of bile salts with proven background as a regulator of lipid metabolism and fat content. After the 90-day feeding trial, a differential modulation of the microbiota between the anterior and posterior intestine was observed. While in the anterior intestine the relative abundance of Desulfobacterota doubled, in the posterior intestine, the levels of Firmicutes increased and Proteobacteria, Actinobacteriota, and Campylobacterota were reduced when supplementing the diet with bile salts. Even so, only in the anterior intestine, there was a decrease in estimated richness (Chao1 and ACE indices) in presence of dietary bile salts. No significant differences were displayed in alpha (Shannon and Simpson indices) nor beta-diversity, showing that bile sales did not have a great impact on the intestinal microbiota. Regarding the gene expression profile in 2 h postprandial-fish, several changes were observed in the analyzed biomarkers of epithelial integrity, nutrient transport, mucus production, interleukins, cell markers, immunoglobulin production and pathogen recognition receptors. These results may indicate the development of an intestinal immune-protective status to tackle future threats. This work also suggests that this immune response is not only regulated by the presence of the dietary bile salts in the intestine, but also by the microbial populations that are in turn modulated by bile salts. After a fasting period of 2 days, the overall gene expression profile was stabilized with respect to fish fed the unsupplemented diet, indicating that the effect of bile salts was transient after short periods of fasting. On the balance, bile salts can be used as a dietary supplement to enhance S. aurata farming and production without compromising their intestinal health.

Given the hydrophilic structure of lysophospholipids (LPLs), their dietary inclusion translates into a better emulsifying capacity of the dietary components. The present study aimed to understand the mechanisms underlying the growth-promoting effect of LPL supplementation by undertaking deep analyses of the proximal intestine and liver interactomes. The Atlantic salmon (Salmo salar) was selected as the main aquaculture species model. The animals were divided into two groups: one was fed a control diet (C-diet) and the other a feed (LPL-diet) supplemented with an LPL-based digestive enhancer (0.1% AQUALYSO®, Adisseo). The LPL-diet had a positive effect on the fish by increasing the final weight by 5% and reducing total serum lipids, mainly due to a decrease in the plasma phospholipid (p < 0.05). In the intestine, the upregulated interactome suggests a more robust digestive capacity, improving vesicle-trafficking-related proteins, complex sugar hydrolysis, and lipid metabolism. In the liver, the LPL-diet promotes better nutrients, increasing several metabolic pathways. The downregulation of the responses to stress and stimuli could be related to a reduced proinflammatory state. This study on the benefits and modes of action of dietary LPLs opens a new window into fish nutrition and could be extended to other productive species. info:eu-repo/semantics/publishedVersion

The constant increase of microorganisms resistant to antibiotics has been classified as a global health emergency, which is especially challenging when biofilms are formed. Herein, novel biofunctionalized gold surfaces with the antimicrobial multidomain recombinant protein JAMF1, both in the soluble form and nanostructured as nanoparticles, were developed. The interaction between His-tag termination of the protein and a nitriloacetic acid–Ni complex formed on mixed self-assembled monolayers (mixed SAMs) was exploited. The obtained antibiofilm surfaces based on the immobilization of the novel JAMF1 protein using self-assembled monolayers were characterized using a multi-technique approach including: cyclic voltammetry, X-ray photoelectron spectroscopy, atomic force microscopy and fluorescence, proving that the modification and immobilization of JAMF1 were successfully done. The antibiofilm activity against Escherichia coli and carbapenem-resistant Klebsiella pneumoniae showed that the immobilized antimicrobial proteins were able to reduce biofilm formation of both microorganisms. This strategy opens up new possibilities for controlled biomolecule immobilization for fundamental biological studies and biotechnological applications, at the interface of materials science and molecular biology. info:eu-repo/semantics/publishedVersion

Tuberculosis (TB) is a multi-host infectious disease caused by members of the Mycobacterium tuberculosis complex (MTC). In Mediterranean ecosystems, where multiple animal hosts of TB are present, identifying the role of the different species involved in the epidemiology of TB is a key point to be able to implement proper control measures. Sheep are susceptible to MTC infection but have traditionally been considered a spillover host. However, the occurrence of outbreaks involving sheep in recent years evidences the need to better understand the role of this small ruminant species in the epidemiology of the disease. Here, we aimed to determine the seroprevalence and risk factors associated with MTC seropositivity in sheep in Andalusia (southern Spain), a region with one of the highest prevalence of MTC infection in both cattle and wild ungulates. A total of 2266 sheep from 83 flocks were tested for antibodies against MTC using an in-house indirect ELISA. Anti-MTC antibodies were detected in 16 (0.7%) of the 2266 sheep (adjusted true prevalence 0.29%, 95% posterior probability interval 0.01–1.05). Seropositivity was found in 14.5% (12/83; 95%CI: 6.9–22.0) of the sheep farms analyzed. A semi-extensive management system was identified as a risk factor associated with MTC seropositivity in sheep farms (OR = 3.7; p < 0.038; 95%CI: 1.1–12.4) in the study area. To the best of the authors' knowledge, this is the first active TB surveillance study carried out to assess MTC exposure in sheep. Our results indicate MTC circulation in sheep farms in southern Spain. However, the low individual seroprevalence obtained suggests that sheep may play a limited role in the epidemiology of TB in this region. Serosurveillance programs could be a valuable tool to detect MTC circulation in sheep in risk scenarios or target farms, in order to optimize control measures on TB animal in multi-host Mediterranean ecosystems. This study was partially funded by the Spanish Ministry of Economy and Competitiveness (MINECO) research grant (AGL2013-49159-C2-2-R). This research was also supported by CIBER -Consorcio Centro de Investigación Biomédica en Red- (CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea – NextGenerationEUD. D. Jiménez-Martín holds a PhD contract granted by Own Research Plan of the University of Córdoba. Funding for open access charge: Universidad de Córdoba / CBUA. info:eu-repo/semantics/publishedVersion

Mixing, a common management strategy used to regroup pigs, has been reported to impair individual performance and affect pig welfare because of the establishment of a new social hierarchy after regrouping. In this study we aimed to determine whether mixing management (non-mixed vs. mixed) and gender (gilts vs. barrows) affect the social and non-social behavior, performance, and physiological parameters of pigs. A total of 96 growing pigs (48 barrows and 48 females) were separated into two treatments: control (CT)—pigs that were mixed once during the growing-finishing period; and social stress (SS)—pigs that were mixed thrice during the growing-finishing period. We recorded social and non-social behaviors, injury score, performance, and physiological parameters during the experimental period. Data were grouped by the period, based on each mix performed, and overall values. The statistical analysis performed considered gender and treatment. For treatment, during period–II and III, the SS group presented the highest frequency of agonistic interactions (AI), stayed longer lying laterally (LL) and sternly (LS), and explored more enrichment material (ER) than the CT group. Furthermore, SS pigs presented the highest injury score in the ear, head, and middle and posterior regions. Compared to the females, the barrows spent more time at the electronic feed station and initiated most of the agonistic interactions during period–II, and they presented a higher injury score for the ear and head regions during period–III. In conclusion, repeated regrouping significantly affected social and feeding behavior without severely altering performance and physiological parameters. Furthermore, different patterns of social and feeding behavior, agonistic interactions, and injury scores between barrows and females were observed. This study provides an understanding of the impact of mixing management and gender differences on pigs, and this knowledge can be used to improve swine productivity and welfare.

Vaccines are an essential tool for the control of viral infections in domestic animals. We generated recombinant vector herpesvirus of turkeys (vHVT) vaccines expressing computationally optimized broadly reactive antigen (COBRA) H5 of avian influenza virus (AIV) alone (vHVT-AI) or in combination with virus protein 2 (VP2) of infectious bursal disease virus (IBDV) (vHVT-IBD-AI) or fusion (F) protein of Newcastle disease virus (NDV) (vHVT-ND-AI). In vaccinated chickens, all three vHVT vaccines provided 90–100% clinical protection against three divergent clades of high pathogenicity avian influenza viruses (HPAIVs), and significantly decreased number of birds and oral viral shedding titers at 2 days post-challenge compared to shams. Four weeks after vaccination, most vaccinated birds had H5 hemagglutination inhibition antibody titers, which significantly increased post-challenge. The vHVT-IBD-AI and vHVT-ND-AI vaccines provided 100% clinical protection against IBDVs and NDV, respectively. Our findings demonstrate that multivalent HVT vector vaccines were efficacious for simultaneous control of HPAIV and other viral infections. info:eu-repo/semantics/publishedVersion

This Scientific Opinion addresses a European Commission request on the welfare of calves as part of the Farm to Fork strategy. EFSA was asked to provide a description of common husbandry systems and related welfare consequences, as well as measures to prevent or mitigate the hazards leading to them. In addition, recommendations on three specific issues were requested: welfare of calves reared for white veal (space, group housing, requirements of iron and fibre); risk of limited cow–calf contact; and animal-based measures (ABMs) to monitor on-farm welfare in slaughterhouses. The methodology developed by EFSA to address similar requests was followed. Fifteen highly relevant welfare consequences were identified, with respiratory disorders, inability to perform exploratory or foraging behaviour, gastroenteric disorders and group stress being the most frequent across husbandry systems. Recommendations to improve the welfare of calves include increasing space allowance, keeping calves in stable groups from an early age, ensuring good colostrum management and increasing the amounts of milk fed to dairy calves. In addition, calves should be provided with deformable lying surfaces, water via an open surface and long-cut roughage in racks. Regarding specific recommendations for veal systems, calves should be kept in small groups (2–7 animals) within the first week of life, provided with ~ 20 m2/calf and fed on average 1 kg neutral detergent fibre (NDF) per day, preferably using long-cut hay. Recommendations on cow–calf contact include keeping the calf with the dam for a minimum of 1 day post-partum. Longer contact should progressively be implemented, but research is needed to guide this implementation in practice. The ABMs body condition, carcass condemnations, abomasal lesions, lung lesions, carcass colour and bursa swelling may be collected in slaughterhouses to monitor on-farm welfare but should be complemented with behavioural ABMs collected on farm. info:eu-repo/semantics/publishedVersion

Antibiotic resistance has exponentially increased during the last years. It is necessary to develop new antimicrobial drugs to prevent and treat infectious diseases caused by multidrug- or extensively-drug resistant (MDR/XDR)-bacteria. Host Defense Peptides (HDPs) have a versatile role, acting as antimicrobial peptides and regulators of several innate immunity functions. The results shown by previous studies using synthetic HDPs are only the tip of the iceberg, since the synergistic potential of HDPs and their production as recombinant proteins are fields practically unexplored. The present study aims to move a step forward through the development of a new generation of tailored antimicrobials, using a rational design of recombinant multidomain proteins based on HDPs. This strategy is based on a two-phase process, starting with the construction of the first generation molecules using single HDPs and further selecting those HDPs with higher bactericidal efficiencies to be combined in the second generation of broad-spectrum antimicrobials. As a proof of concept, we have designed three new antimicrobials, named D5L37βD3, D5L37D5L37 and D5LAL37βD3. After an in-depth exploration, we found D5L37D5L37 to be the most promising one, since it was equally effective against four relevant pathogens in healthcare-associated infections, such as methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis (MRSE) and MDR Pseudomonas aeruginosa, being MRSA, MRSE and P. aeruginosa MDR strains. The low MIC values and versatile activity against planktonic and biofilm forms reinforce the use of this platform to isolate and produce unlimited HDP combinations as new antimicrobial drugs by effective means. info:eu-repo/semantics/publishedVersion

20 pages, 8 figures, supplementary material https://doi.org/10.1093/molbev/msad071.-- Data Availability: Data underlying this article are available in the article and in its online Supplementary material, Aquaporin-mediated oocyte hydration is considered important for the evolution of pelagic eggs and the radiative success of marine teleosts. However, the molecular regulatory mechanisms controlling this vital process are not fully understood. Here, we analyzed >400 piscine genomes to uncover a previously unknown teleost-specific aquaporin-1 cluster (TSA1C) comprised of tandemly arranged aqp1aa-aqp1ab2-aqp1ab1 genes. Functional evolutionary analysis of the TSA1C reveals a ∼300-million-year history of downstream aqp1ab-type gene loss, neofunctionalization, and subfunctionalization, but with marine species that spawn highly hydrated pelagic eggs almost exclusively retaining at least one of the downstream paralogs. Unexpectedly, one-third of the modern marine euacanthomorph teleosts selectively retain both aqp1ab-type channels and co-evolved protein kinase-mediated phosphorylation sites in the intracellular subdomains together with teleost-specific Ywhaz-like (14-3-3ζ-like) binding proteins for co-operative membrane trafficking regulation. To understand the selective evolutionary advantages of these mechanisms, we show that a two-step regulated channel shunt avoids competitive occupancy of the same plasma membrane space in the oocyte and accelerates hydration. These data suggest that the evolution of the adaptive molecular regulatory features of the TSA1C facilitated the rise of pelagic eggs and their subsequent geodispersal in the oceanic currents, This work was supported by the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033), the European Regional Development Fund (ERDF) “A way of making Europe” (European Union), Grant no. AGL2016-76802-R (to J.C.), and the Norwegian Research Council (RCN) Grant no. 294768/E40 (to R.N.F). F.C. and A.F. were supported, respectively, by the “Ramon y Cajal” programe (RYC-2015-17103) and a predoctoral (BES-2014-068745) contract from Spanish MCIN. R.N.F. was also supported by the University of Bergen (Norway), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Longevity is an economically important trait, since extending the functional life of a doe would allow us to keep the most productive females in the herd as long as possible, and this could result in the increased profitability of dairy farms. Thus, the objectives of this study were to determine the most important factors that influence the length of productive life (LPL) of female Florida goats and to estimate its genetic additive variance using a Cox proportional hazards model. The data consisted of 70,695 productive life records from 25,722 Florida females kidding between 2006 and 2020. A total of 19,495 does had completed their productive life while 6227 (24.2%) does had censored information. The pedigree contained information on 56,901 animals. The average censoring age and average failure age after first kidding for LPL were 36 and 47 months respectively. The model included, as time-independent effects, the age at first kidding and the interaction between herd, year and season of birth of the doe, and as time-dependent effects, the age at kidding, the interaction between herd, year and season of kidding, the within-herd class of milk production deviation, and the interaction between the lactation number and the stage of lactation. All fixed effects had a significant effect on LPL (p

Anthropogenic global change is driving an increase in the frequency and intensity of drought and flood events, along with associated imbalances and limitation of several soil nutrients. In the context of an increasing human population, these impacts represent a global-scale challenge for biodiversity conservation and sustainable crop production to ensure food security. Plants have evolved strategies to enhance uptake of soil nutrients under environmental stress conditions; for example, symbioses with fungi (mycorrhization) in the rhizosphere and the release of exudates from roots. Although crop cultivation is managed for the effects of limited availability of nitrogen (N) and phosphorus (P), there is increasing evidence for limitation of plant growth and fitness because of the low availability of other soil nutrients such as the metals potassium (K), calcium (Ca), magnesium (Mg), and iron (Fe), which may become increasingly limiting for plant productivity under global change. The roles of mycorrhizas and plant exudates on N and P uptake have been studied intensively; however, our understanding of the effects on metal nutrients is less clear and still inconsistent. Here, we review the literature on the role of mycorrhizas and root exudates in plant uptake of key nutrients (N, P, K, Ca, Mg, and Fe) in the context of potential nutrient deficiencies in crop and non-crop terrestrial ecosystems, and identify knowledge gaps for future research to improve nutrient-uptake capacity in food crop plants. info:eu-repo/semantics/acceptedVersion

IntroductionThe impact of parasites on gut microbiota of the host is well documented, but the role of the relationship between the parasite and the host in the formation of the microbiota is poorly understood. This study has focused on the influence that trophic behavior and resulting parasitism has on the structure of the microbiome.MethodsUsing 16S amplicon sequencing and newly developed methodological approaches, we characterize the gut microbiota of the sympatric pair of whitefish Coregonus lavaretus complex and the associated microbiota of cestodes parasitizing their intestine. The essence of the proposed approaches is, firstly, to use the method of successive washes of the microbiota from the cestode’s surfaces to analyze the degree of bacterial association to the tegument of the parasite. Secondly, to use a method combining the sampling of intestinal content and mucosa with the washout procedure from the mucosa to understand the real structure of the fish gut microbiota.Results and discussionOur results demonstrate that additional microbial community in the intestine are formed by the parasitic helminths that caused the restructuring of the microbiota in infected fish compared to those uninfected. Using the desorption method in Ringer’s solution, we have demonstrated that Proteocephalus sp. cestodes possess their own microbial community which is put together from “surface” bacteria, and bacteria which are weakly and strongly associated with the tegument, bacteria obtained after treatment of the tegument with detergent, and bacteria obtained after removal of the tegument from the cestodes.

Influenza A viruses (IAVs) can infect a wide variety of bird and mammal species. Their genome is characterized by 8 RNA single stranded segments. The low proofreading activity of their polymerases and the genomic reassortment between different IAVs subtypes allow them to continuously evolve, constituting a constant threat to human and animal health. In 2009, a pandemic of an IAV highlighted the importance of the swine host in IAVs adaptation between humans and birds. The swine population and the incidence of swine IAV is constantly growing. In previous studies, despite vaccination, swine IAV growth and evolution were proven in vaccinated and challenged animals. However, how vaccination can drive the evolutionary dynamics of swine IAV after coinfection with two subtypes is poorly studied. In the present study, vaccinated and nonvaccinated pigs were challenged by direct contact with H1N1 and H3N2 independent swine IAVs seeder pigs. Nasal swab samples were daily recovered and broncho-alveolar lavage fluid (BALF) was also collected at necropsy day from each pig for swine IAV detection and whole genome sequencing. In total, 39 swine IAV whole genome sequences were obtained by next generation sequencing from samples collected from both experimental groups. Subsequently, genomic, and evolutionary analyses were carried out to detect both, genomic reassortments and single nucleotide variants (SNV). Regarding the segments found per sample, the simultaneous presence of segments from both subtypes was much lower in vaccinated animals, indicating that the vaccine reduced the likelihood of genomic reassortment events. In relation to swine IAV intra-host diversity, a total of 239 and 74 SNV were detected within H1N1 and H3N2 subtypes, respectively. Different proportions of synonymous and nonsynonymous substitutions were found, indicating that vaccine may be influencing the main mechanism that shape swine IAV evolution, detecting natural, neutral, and purifying selection in the different analyzed scenarios. SNV were detected along the whole swine IAV genome with important nonsynonymous substitutions on polymerases, surface glycoproteins and nonstructural proteins, which may have an impact on virus replication, immune system escaping and virulence of virus, respectively. The present study further emphasized the vast evolutionary capacity of swine IAV, under natural infection and vaccination pressure scenarios. This research was funded by grants AGL2016–75280-R from Ministerio de Ciencia, Innovación y Universidades from the Spanish government. ÁL-V has a pre-doctoral fellowship FPI 2017, Ministerio de Ciencia, Innovación y Universidades from the Spanish government. The authors would like to thank Laura Baioni for the sequencing libraries preparation. The authors also want to thank the IRTA-CReSA animal facilities staff for their work during the in vivo experiment. Lastly, we would like to thank Marta Pérez and all the people from CReSA who were involved in the experiment during the COVID-19 lockdowns. info:eu-repo/semantics/publishedVersion

Weaning is a stressful period for pigs that causes gastrointestinal disruption and low growth rates. For a long time, zinc oxide at pharmacological doses, along with different antibiotics, has been used prophylactically during this phase to reduce the incidence of these gastrointestinal problems. Nowadays, the increasing concern about the environment, along with the global and constant growth of bacterial resistance to antibiotics, has led to the prohibition of the prophylactic use of zinc oxide in diets for piglets in the EU, along with the implementation of new regulations on the use of antibiotics. Consequently, the pig sector faces the important challenge that supposes developing alternatives to the classical system based on the use of these antimicrobial compounds. This study is the first step to achieving this goal by minimizing the use of various antibiotics and zinc oxide in weanling pigs by supplementing citrus flavonoids and only one antibiotic (amoxicillin). Accordingly, the influence of zinc oxide plus antibiotics and citrus flavonoids plus amoxicillin in weaned pigs has been investigated and its impact on growth performance, gut microbiology profile, gut signaling, intestinal architecture, and serum biomarkers indicative of stress and inflammatory responses have been evaluated. Citrus flavonoids plus amoxicillin improved growth performance and gut health, evidencing a positive microbial modulation, stress status reduction, and a positive effect on the gastrointestinal barrier, and other digestive functions. Additionally, the expression of some bitter taste receptors in the intestine has been increased when supplementing both dietary strategies, the one based on zinc oxide or the one based on citrus flavonoids supplementation. Consequently, the present study shows that in weanling piglets, the supplement of citrus flavonoids with amoxicillin might be a promising alternative to the dietary use of pharmacological doses of zinc oxide with more than two antibiotics, therefore minimizing the use of antimicrobial compounds without detrimental effects on performance. Since citrus flavonoids have antioxidant and anti-inflammatory properties, it was hypothesized that these compounds would become a suitable alternative to the use of therapeutic doses of zinc oxide at weaning. A total of 252 weaned pigs ([LargeWhite × Landrace] × Pietrain) were distributed according to BW (5.7 kg ± 0.76) into 18 pens (6 pens per diet, 14 pigs/pen). Three experimental diets for the prestarter (0-14 d postweaning) and starter (15-35 d postweaning) period were prepared: (i) a nonmedicated (CON) diet, (ii) a CON diet supplemented with zinc oxide at 2500 mg/kg, amoxicillin at 0.3 mg/kg and apramycin at 0.1 mg/kg (ZnO), and (iii) CON diet with the addition of a commercial citrus flavonoid extract at 0.3 mg/kg and amoxicillin at 0.3 mg/kg (FLAV). Pig BW, ADG, ADFI, and FCR were assessed on d7, d14, and d35. Samples of intestinal tissue, cecal content, and serum were collected on day seven (18 piglets). FLAV treatment achieved greater BW and ADG during the starter and for the entire experimental period compared with the CON diet (p < 0.05), whereas ZnO pigs evidenced intermediate results. Jejunum tissue analysis showed that pigs fed the FLAV diet overexpressed genes related to barrier function, digestive enzymes, and nutrient transport compared to those pigs fed the CON diet (p < 0.05). An increase in the abundance of bacterial genera such as Succinivibrio, Turicibacter, and Mitsuokella (p < 0.05) was observed in the FLAV compared with the CON and ZnO piglets. ZnO and FLAV increased the expression of TAS2R39, while ZnO pigs also expressed greater TAS2R16 than CON (p < 0.05) in the intestine. FLAV treatment improved the gut function, possibly explaining a higher performance at the end of the nursery period. Consequently, citrus flavonoids supplementation, together with amoxicillin, is a promising alternative to the use of zinc oxide plus amoxicillin and apramycin in weanling pigs, minimizing the use of antibiotics.

Crassostrea gigas oysters represent a significant global food source, with 4.7 million tons harvested per year. In 2001, the bacterium V. aesturianus francensis emerged as a pathogen that causes adult oyster mortality in France and Ireland. Its impact on oyster aquaculture has increased in Europe since its reemergence in 2012. To better understand the evolutionary mechanisms leading to the emergence and persistence over time of this pathogen, we conducted a survey of mollusk diseases through national reference laboratories (NRLs) across Europe. We analyzed 54 new genomes of V. aestuarianus (Va) isolated from multiple environmental compartments since 2001, in areas with and without bivalve mortalities. We used a combination of comparative genomics and population genetics approaches to show that Va francensis lineages have undergone clonal expansion in Europe, likely after a recent selective bottleneck. Low mutation and recombination rates may have selected particular virulent genotypes. Furthermore, we identified a specific cus-cop-containing island conferring copper resistance to Va francensis whose acquisition may have favored the emergence of pathogenic lineages adapted to oysters.

Fungi in the order Chaetothyriales are renowned for their ability to cause human infections. Nevertheless, they are not regarded as primary pathogens, but rather as opportunists with a natural habitat in the environment. Extremotolerance is a major trend in the order, but quite different from black yeasts in Capnodiales which focus on endurance, an important additional parameter is advancing toxin management. In the ancestral ecology of rock colonization, the association with metabolite-producing lichens is significant. Ant-association, dealing with pheromones and repellents, is another mainstay in the order. The phylogenetically derived family, Herpotrichiellaceae, shows dual ecology in monoaromatic hydrocarbon assimilation and the ability to cause disease in humans and cold-blooded vertebrates. In this study, data on ecology, phylogeny, and genomics were collected and analyzed in order to support this hypothesis on the evolutionary route of the species of Chaetothyriales. Comparing the ribosomal tree with that of enzymes involved in toluene degradation, a significant expansion of cytochromes is observed and the toluene catabolism is found to be complete in some of the Herpotrichiellaceae. This might enhance human systemic infection. However, since most species have to be traumatically inoculated in order to cause disease, their invasive potential is categorized as opportunism. Only in chromoblastomycosis, true pathogenicity might be surmised. The criterion would be the possible escape of agents of vertebrate disease from the host, enabling dispersal of adapted genotypes to subsequent generations.

The epidemiological situation of SARS-CoV-2 in humans and animals is continually evolving. To date, animal species known to transmit SARS-CoV-2 are American mink, raccoon dog, cat, ferret, hamster, house mouse, Egyptian fruit bat, deer mouse and white-tailed deer. Among farmed animals, American mink have the highest likelihood to become infected from humans or animals and further transmit SARS-CoV-2. In the EU, 44 outbreaks were reported in 2021 in mink farms in seven MSs, while only six in 2022 in two MSs, thus representing a decreasing trend. The introduction of SARS-CoV-2 into mink farms is usually via infected humans; this can be controlled by systematically testing people entering farms and adequate biosecurity. The current most appropriate monitoring approach for mink is the outbreak confirmation based on suspicion, testing dead or clinically sick animals in case of increased mortality or positive farm personnel and the genomic surveillance of virus variants. The genomic analysis of SARS-CoV-2 showed mink-specific clusters with a potential to spill back into the human population. Among companion animals, cats, ferrets and hamsters are those at highest risk of SARS-CoV-2 infection, which most likely originates from an infected human, and which has no or very low impact on virus circulation in the human population. Among wild animals (including zoo animals), mostly carnivores, great apes and white-tailed deer have been reported to be naturally infected by SARS-CoV-2. In the EU, no cases of infected wildlife have been reported so far. Proper disposal of human waste is advised to reduce the risks of spill-over of SARS-CoV-2 to wildlife. Furthermore, contact with wildlife, especially if sick or dead, should be minimised. No specific monitoring for wildlife is recommended apart from testing hunter-harvested animals with clinical signs or found-dead. Bats should be monitored as a natural host of many coronaviruses. info:eu-repo/semantics/publishedVersion

Dinoflagellates belonging to the Perkinsus genus are OIE (World Organization for animal Health)-listed pathogens extremely virulent for clams and oysters in many marine ecosystems throughout the world. During the monitoring activities of the Mediterranean mussel (Mytilus galloprovincialis) in Campania region (Italy), the presence of typical trophozoites of Perkinsus sp. was observed in mussels from farms and natural banks. Simultaneously, following mussel mortality in the Spanish waters of Catalonia, histopathological studies revealed the presence of the same parasite. Although perkinsosis is an endemic disease in clams in Italy (with prevalence from 40 to 80%), there are no reports to date of its presence in Mediterranean mussels and of the effect on this species. For this study, histopathology, Ray’s Fluid Thioglycollate Medium (RFTM), and molecular diagnostics with conventional Polimerase Chain Reaction (PCR) and qPCR were performed. In samples from Italy, histopathology in the mussel from one farm revealed a prevalence of 26% in February 2019, 40% in February 2020, 16% in November 2020, and 23% in April 2021. In a natural bank, Perkinsus was also detected in May 2020 but in lower prevalence. In Spain, in July 2020, the presence of the parasite was 20% in one site and 10% in a second site and related to animal mortality. In both areas, Perkinsus sp. elicited multiple inflammatory capsules of different size or infiltrates at the level of the digestive gland and gonad. Molecular diagnostics of the Internal Transcriber Spacer (ITS) region of the rDNA (ITS1, 5.8S, and ITS2) showed a 97% similarity of P. olseni from Italy with samples from New Zealand, Australia, and Uruguay and in bivalves such as Pitar rostrata, Astrovenus sp., and Haliotis sp., whereas in Spain the identity was 99% samples from South Korean venerids such as Anadara granosa. Phylogenetic analysis group together P. olseni from Italian and Spanish mussels but place them distant from other P. olseni described in the clams from Europe (Italy, France, and Spain). Direct impact of transboundary animal diseases in aquaculture constitutes a serious consequence for export living animals and their products, as well for international trade. This compromises food security, also causing a high socioeconomic impact on aquaculture exporting nations. P. olseni is a generalist pathogen able to infect different bivalve species, possibly passing from clams to oysters and mussels. Recognized international organizations should take this into account in the view of possible cross-infection. Other studies are needed to define pathogen virulence in this species.

The single and comparative intradermal tuberculin tests (SITT and CITT) are official in vivo tests for bovine tuberculosis (TB) diagnosis using bovine and avian purified protein derivatives (PPD-B and PPD-A). Infection with bacteria other than Mycobacterium tuberculosis complex (MTC) can result in nonspecific reactions to these tests. We evaluated the performance of the skin test with PPDs and new defined antigens in the guinea pig model. A standard dose (SD) of Rhodococcus equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis, M. avium subsp. avium, M. avium subsp. hominissuis, M. scrofulaceum, M. persicum, M. microti, M. caprae and M. bovis, and a higher dose (HD) of M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis were tested using PPD-B, PPD-A, P22, ESAT-6-CFP-10-Rv3615c peptide cocktail long (PCL) and fusion protein (FP). The SD of R. equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare and M. avium subsp. paratuberculosis did not cause any reactions. The HD of M. nonchromogenicum, M. monacense, M. intracellulare, and M. avium subsp. paratuberculosis and the SD of M. avium subsp. hominissuis, M. scrofulaceum and M. persicum, caused nonspecific reactions (SIT). A CITT interpretation would have considered M. avium complex and M. scrofulaceum groups negative, but not all individuals from M. nonchromogenicum HD, M. monacense HD and M. persicum SD groups. Only animals exposed to M. bovis and M. caprae reacted to PCL and FP. These results support the advantage of complementing or replacing PPD-B to improve specificity without losing sensitivity.

Vibrio parahaemolyticus is a bacterium present in estuarine environments. Since the first outbreak in Japan in 1950, it has been a dominant cause of foodborne infections throughout the world. Vibrio parahaemolyticus strains can accumulate in shellfish and cause gastroenteritis. The thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes, respectively, are considered major virulence factors in V. parahaemolyticus. Conventional methods used for V. parahaemolyticus monitoring in seafood, based on microbiological counts, are time consuming and laborious, and they cannot identify virulence genes. Therefore, a duplex electrochemical biosensor for the detection of the tdh and trh genes of V. parahaemolyticus in oysters was developed. Oyster homogenates were prepared, enriched for bacterial content, and DNA was extracted. Then, a duplex PCR with tailed primers was performed to amplify both genes. Afterwards, sandwich hybridisation assays were carried out on magnetised working electrodes of an array. The electrochemical biosensor was able to detect the tdh and trh genes from V. parahaemolyticus strains, and no cross-contaminations were observed between electrodes. The limit of detection for both genes was 10 pg/μL and no cross-reactivity was observed when using other Vibrio species and non-Vibrio pathogens. The biosensor was able to detect as low as 1 CFU of V. parahaemolyticus in oyster homogenate. Screening of oysters from an infectivity experiment and comparison with other techniques proved the proper performance of the biosensor and its applicability to the analysis of natural samples, with added advantages of specificity, duplexing capability, portability and provided virulence information. info:eu-repo/semantics/acceptedVersion

The prevalence and inter-observer repeatability of the four most valid and feasible indicators of consciousness after waterbath stunning in turkeys were evaluated before bleeding (i.e., tonic convulsion, breathing, spontaneous blinking and vocalization) and during bleeding (i.e., fluttering, breathing, spontaneous swallowing and head shaking). Furthermore, correlations between them were calculated to further understand the association between such indicators. This study compared the assessments of four observers on 7877 turkeys from 28 flocks slaughtered in eight different abattoirs. Repeatability between observers was assessed by combining the crude percentage of agreement and the Fleiss’ kappa. Before bleeding, tonic seizure was observed to be not reliable under commercial conditions and spontaneous blinking and vocalization had poor repeatability. During bleeding, spontaneous swallowing was not observed and head shaking had poor repeatability. Hence, the most relevant indicators of consciousness before bleeding is breathing while during bleeding is breathing and wing flapping. Moreover, the stunning efficiency of the key electrical parameters used in the different slaughterhouses was assessed. Therefore, a refined list of indicators of the state of consciousness after waterbath stunning is proposed to be used in commercial turkey slaughterhouses to ensure consistency of controls. On the other hand, the range of the prevalence of turkeys with indicators of consciousness within a batch found before bleeding [0–16%] and after bleeding [0–18%] highlights the importance of controls. info:eu-repo/semantics/publishedVersion

The contribution of cross-presenting XCR1+ dendritic cells (DCs) and SIRPα+ DCs in maintaining T cell function during exhaustion and immunotherapeutic interventions of chronic infections remains poorly characterized. Using the mouse model of chronic LCMV infection, we found that XCR1+ DCs are more resistant to infection and highly activated compared with SIRPα+ DCs. Exploiting XCR1+ DCs via Flt3L-mediated expansion or XCR1-targeted vaccination notably reinvigorates CD8+ T cells and improves virus control. Upon PD-L1 blockade, XCR1+ DCs are not required for the proliferative burst of progenitor exhausted CD8+ T (TPEX) cells but are indispensable to sustain the functionality of exhausted CD8+ T (TEX) cells. Combining anti-PD-L1 therapy with increased frequency of XCR1+ DCs improves functionality of TPEX and TEX subsets, while increase of SIRPα+ DCs dampened their proliferation. Together, this demonstrates that XCR1+ DCs are crucial for the success of checkpoint inhibitor-based therapies through differential activation of exhausted CD8+ T cell subsets. This work was supported by grants from the Spanish Ministry of Science and Innovation (grant No. PID2019-106323RB-I00 AEI//10.13039/501100011033), the “Unidad de Excelencia María de Maeztu” funded by the MCIN and the AEI (DOI: 10.13039/501100011033; Ref: CEX2018-000792-M), “la Caixa” Foundation (HR17-00199), the Russian Science Foundation (grant No. 18-11-00171), and the Research Council of Norway (grant No. 250884).