Effect of sainfoin (Onobrychis viciifolia) on cyathostomin eggs excretion, larval development, larval community structure and efficacy of ivermectin treatment in horses.

Alternative strategies to chemical anthelmintics are needed for the sustainable control of equine strongylids. Bioactive forages like sainfoin (Onobrychis viciifolia) could contribute to reducing drug use, with the first hints of in vitro activity against cyathostomin free-living stages observed in the past. We analysed the effect of a sainfoin-rich diet on cyathostomin population and the efficacy of oral ivermectin treatment. Two groups of 10 naturally infected horses were enrolled in a 78-day experimental trial. Following a 1-week adaptation period, they were either fed with dehydrated sainfoin pellets (70% of their diet dry matter) or with alfalfa pellets (control group) for 21-days. No difference was found between the average fecal egg counts (FECs) of the two groups, but a significantly lower increase in larval development rate was observed for the sainfoin group, at the end of the trial. Quantification of cyathostomin species abundances with an ITS-2-based metabarcoding approach revealed that the sainfoin diet did not affect the nemabiome structure compared to the control diet. Following oral ivermectin treatment of all horses on day 21, the drug concentration was lower in horses fed with sainfoin, and cyathostomin eggs reappeared earlier in that group. Our results demonstrated that short-term consumption of a sainfoin-rich diet does not decrease cyathostomin FEC but seems to slightly reduce larval development. Consumption of dehydrated sainfoin pellets also negatively affected ivermectin pharmacokinetics, underscoring the need to monitor horse feeding regimes when assessing ivermectin efficacy in the field.

Inter-breed diversity and temporal dynamics of the faecal microbiota in healthy horses.

Understanding gut microbiota similarities and differences across breeds in horses has the potential to advance approaches aimed at personalized microbial modifications, particularly those involved in improving sport athletic performance. Here, we explore whether faecal microbiota composition based on faecal 16S ribosomal RNA gene sequencing varies across six different sport breeds at two time points 8 months apart within a cohort of 189 healthy horses cared for under similar conditions. Lusitano horses presented the smallest and Hanoverians the greatest bacterial diversity. We found subtle but significant differences in ß-diversity between Lusitano, Anglo Arabian and the central European breeds, and we reproduced these results across the two time points. Repeat sampling of subjects showed community to be temporally more stable in Lusitano and Anglo Arabian breeds. Additionally, we found that 27 genera significantly varied in abundance across breeds. Overall, 33% of these taxa overlapped with previously identified taxa that were associated with genetic variation in humans or other species. However, a non-significant correlation was observed between microbial composition and the host pedigree-based kinship. Despite a notable variation in the diversity and composition of the faecal microbiota, breed exerted limited effects on the equine faecal microbiota.

Identification of rumen microbial biomarkers linked to methane emission in Holstein dairy cows.

Mitigation of greenhouse gas emissions is relevant for reducing the environmental impact of ruminant production. In this study, the rumen microbiome from Holstein cows was characterized through a combination of 16S rRNA gene and shotgun metagenomic sequencing. Methane production (CH4 ) and dry matter intake (DMI) were individually measured over 4-6 weeks to calculate the CH4 yield (CH4 y = CH4 /DMI) per cow. We implemented a combination of clustering, multivariate and mixed model analyses to identify a set of operational taxonomic unit (OTU) jointly associated with CH4 y and the structure of ruminal microbial communities. Three ruminotype clusters (R1, R2 and R3) were identified, and R2 was associated with higher CH4 y. The taxonomic composition on R2 had lower abundance of Succinivibrionaceae and Methanosphaera, and higher abundance of Ruminococcaceae, Christensenellaceae and Lachnospiraceae. Metagenomic data confirmed the lower abundance of Succinivibrionaceae and Methanosphaera in R2 and identified genera (Fibrobacter and unclassified Bacteroidales) not highlighted by metataxonomic analysis. In addition, the functional metagenomic analysis revealed that samples classified in cluster R2 were overrepresented by genes coding for KEGG modules associated with methanogenesis, including a significant relative abundance of the methyl-coenzyme M reductase enzyme. Based on the cluster assignment, we applied a sparse partial least-squares discriminant analysis at the taxonomic and functional levels. In addition, we implemented a sPLS regression model using the phenotypic variation of CH4 y. By combining these two approaches, we identified 86 discriminant bacterial OTUs, notably including families linked to CH4 emission such as Succinivibrionaceae, Ruminococcaceae, Christensenellaceae, Lachnospiraceae and Rikenellaceae. These selected OTUs explained 24% of the CH4 y phenotypic variance, whereas the host genome contribution was ~14%. In summary, we identified rumen microbial biomarkers associated with the methane production of dairy cows; these biomarkers could be used for targeted methane-reduction selection programmes in the dairy cattle industry provided they are heritable.

Inferring the evolution of the major histocompatibility complex of wild pigs and peccaries using hybridisation DNA capture-based sequencing.

The major histocompatibility complex (MHC) is a key genomic model region for understanding the evolution of gene families and the co-evolution between host and pathogen. To date, MHC studies have mostly focused on species from major vertebrate lineages. The evolution of MHC classical (Ia) and non-classical (Ib) genes in pigs has attracted interest because of their antigen presentation roles as part of the adaptive immune system. The pig family Suidae comprises over 18 extant species (mostly wild), but only the domestic pig has been extensively sequenced and annotated. To address this, we used a DNA-capture approach, with probes designed from the domestic pig genome, to generate MHC data for 11 wild species of pigs and their closest living family, Tayassuidae. The approach showed good efficiency for wild pigs (~80% reads mapped, ~87× coverage), compared to tayassuids (~12% reads mapped, ~4× coverage). We retrieved 145 MHC loci across both families. Phylogenetic analyses show that the class Ia and Ib genes underwent multiple duplications and diversifications before suids and tayassuids diverged from their common ancestor. The histocompatibility genes mostly form orthologous groups and there is genetic differentiation for most of these genes between Eurasian and sub-Saharan African wild pigs. Tests of selection showed that the peptide-binding region of class Ib genes was under positive selection. These findings contribute to better understanding of the evolutionary history of the MHC, specifically, the class I genes, and provide useful data for investigating the immune response of wild populations against pathogens.

Understanding the response to endurance exercise using a systems biology approach: combining blood metabolomics, transcriptomics and miRNomics in horses.

BACKGROUND: Endurance exercise in horses requires adaptive processes involving physiological, biochemical, and cognitive-behavioral responses in an attempt to regain homeostasis. We hypothesized that the identification of the relationships between blood metabolome, transcriptome, and miRNome during endurance exercise in horses could provide significant insights into the molecular response to endurance exercise. For this reason, the serum metabolome and whole-blood transcriptome and miRNome data were obtained from ten horses before and after a 160 km endurance competition. RESULTS: We obtained a global regulatory network based on 11 unique metabolites, 263 metabolic genes and 5 miRNAs whose expression was significantly altered at T1 (post- endurance competition) relative to T0 (baseline, pre-endurance competition). This network provided new insights into the cross talk between the distinct molecular pathways (e.g. energy and oxygen sensing, oxidative stress, and inflammation) that were not detectable when analyzing single metabolites or transcripts alone. Single metabolites and transcripts were carrying out multiple roles and thus sharing several biochemical pathways. Using a regulatory impact factor metric analysis, this regulatory network was further confirmed at the transcription factor and miRNA levels. In an extended cohort of 31 independent animals, multiple factor analysis confirmed the strong associations between lactate, methylene derivatives, miR-21-5p, miR-16-5p, let-7 family and genes that coded proteins involved in metabolic reactions primarily related to energy, ubiquitin proteasome and lipopolysaccharide immune responses after the endurance competition. Multiple factor analysis also identified potential biomarkers at T0 for an increased likelihood for failure to finish an endurance competition. CONCLUSIONS: To the best of our knowledge, the present study is the first to provide a comprehensive and integrated overview of the metabolome, transcriptome, and miRNome co-regulatory networks that may have a key role in regulating the metabolic and immune response to endurance exercise in horses.

Developing a 670k genotyping array to tag ~2M SNPs across 24 horse breeds.

BACKGROUND: To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. RESULTS: Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of ~5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix, now Thermo Fisher Scientific) a set of ~2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of ~670 thousand SNPs (MNEc670k), was designed for genotype imputation. CONCLUSIONS: Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.

Novel equine tissue miRNAs and breed-related miRNA expressed in serum.

BACKGROUND: MiRNAs regulate multiple genes at the post-transcriptional level and therefore play an important role in many biological processes. It has been suggested that miRNA exported outside the cells contribute to inter-cellular communication. Consequently, circulating miRNAs are of particular interest and are promising biomarkers for many diseases. The number of miRNAs annotated in the horse genome is much lower compared to model organisms like human and mouse. We therefore aimed to identify novel equine miRNAs for tissue types and breed in serum. RESULTS: We analysed 71 small RNA-seq libraries derived from nine tissues (gluteus medius, platysma, masseter muscle, heart, liver, cartilage, bone, total blood and serum) using miRDeep2 and miRdentify tools. Known miRNAs represented between 2.3 and 62.9 % of the reads in 71 libraries. A total of 683 novel miRNAs were identified. Breed and tissue type affected the number of miRNAs detected and interestingly, affected its average intensity. A total of 50 miRNAs in serum proved to be potential biomarkers to differentiate specific breed types, of which miR-122, miR-200, miR-483 were over-expressed and miR-328 was under-expressed in ponies compared to Warmbloods. The different miRNAs profiles, as well as the differences in their expression levels provide a foundation for more hypotheses based on the novel miRNAs discovered. CONCLUSIONS: We identified 683 novel equine miRNAs expressed in seven solid tissues, blood and serum. Additionally, our approach evidenced that such data supported identification of specific miRNAs as markers of functions related to breeds or disease tissues.

RNA sequencing-based analysis of the spleen transcriptome following infectious bronchitis virus infection of chickens selected for different mannose-binding lectin serum concentrations.

BACKGROUND: Avian infectious bronchitis is a highly contagious disease of the upper-respiratory tract caused by infectious bronchitis virus (IBV). Understanding the molecular mechanisms involved in the interaction between innate and adaptive immune responses to IBV infection is a crucial element for further improvements in strategies to control IB. To this end, two chicken lines, selected for high (L10H line) and low (L10L line) serum concentration of mannose-binding lectin (MBL) were studied. In total, 32 birds from each line were used. Sixteen birds from each line were infected with IBV and sixteen were left uninfected. Eight uninfected and infected birds from each line were euthanized at 1 and 3 weeks post infection. RNA sequencing was performed on spleen samples from all 64 birds and differential gene expression analysis was performed for four comparisons: L10L line versus L10H line for uninfected birds at weeks 1 and 3, respectively, and in the same way for infected birds. Functional analysis was performed using Gene Ontology (GO) Immune System Process terms specific for Gallus gallus. RESULTS: Comparing uninfected L10H and L10L birds, we identified 1698 and 1424 differentially expressed (DE) genes at weeks 1 and 3, respectively. For the IBV-infected birds, 1934 and 866 DE genes were identified between the two lines at weeks 1 and 3, respectively. The two most enriched GO terms emerging from the comparison of uninfected birds between the two lines were "Lymphocyte activation involved in immune response" and "Somatic recombination of immunoglobulin genes involved in immune response" at weeks 1 and 3, respectively. When comparing IBV-infected birds between the two lines, the most enriched GO terms were "Alpha-beta T cell activation" and "Positive regulation of leukocyte activation" at weeks 1 and 3, respectively. CONCLUSIONS: Healthy birds from the two lines showed significant differences in expression profiles for subsets of adaptive and innate immunity-related genes, whereas comparison of the IBV-infected birds from the two lines showed differences in expression of immunity-related genes involved in T cell activation and proliferation. The observed transcriptome differences between the two lines indicate that selection for MBL had influenced innate as well as adaptive immunity.

The forecasting power of the mucin-microbiome interplay in livestock respiratory diseases.

Complex respiratory diseases are a significant challenge for the livestock industry worldwide. These diseases considerably impact animal health and welfare and cause severe economic losses. One of the first lines of pathogen defense combines the respiratory tract mucus, a highly viscous material primarily composed of mucins, and a thriving multi-kingdom microbial ecosystem. The microbiome-mucin interplay protects from unwanted substances and organisms, but its dysfunction may enable pathogenic infections and the onset of respiratory disease. Emerging evidence also shows that noncoding regulatory RNAs might modulate the structure and function of the microbiome-mucin relationship. This opinion paper unearths the current understanding of the triangular relationship between mucins, the microbiome, and noncoding RNAs in the context of respiratory infections in animals of veterinary interest. There is a need to look at these molecular underpinnings that dictate distinct health and disease outcomes to implement effective prevention, surveillance, and timely intervention strategies tailored to the different epidemiological contexts.

Fecal Microbial Communities of Nellore and Crossbred Beef Calves Raised at Pasture.

This study aimed to investigate the effect of age and genetics on the fecal microbiota of beef calves. Ten purebred Nellore (Bos taurus indicus) and ten crossbreed 50% Nellore-50% European breed (Bos taurus taurus) calves co-habiting on the same pasture paddock had fecal samples collected on days five (5 d), 14 d, 28 d, 60 d, 90 d, 180 d, 245 d (weaning) and 260 d after birth. All calves were kept with their mothers, and six Nellore dams were also sampled at weaning. Microbiota analysis was carried out by amplification of the V4 region of the 16S rRNA gene following high-throughput sequencing with a MiSeq Illumina platform. Results revealed that bacterial richness increased with age and became more similar to adults near weaning. Differences in microbiota membership between breeds were found at 60 d and 90 d and for structure at 60 d, 90 d, 245 d, and 260 d (p < 0.05). In addition, crossbreed calves presented less variability in their microbiota. In conclusion, the genetic composition significantly impacted the distal gut microbiota of calves co-habiting in the same environment, and further studies investigating food intake can reveal possible associations between microbiota composition and performance.

Chicory (Cichorium intybus) reduces cyathostomin egg excretion and larval development in grazing horses.

Cyathostomins are the most prevalent parasitic nematodes of grazing horses. They are responsible for colic and diarrhea in their hosts. After several decades of exposure to synthetic anthelmintics, they have evolved to become resistant to most compounds. In addition, the drug-associated environmental side-effects question their use in the field. Alternative control strategies, like bioactive forages, are needed to face these challenges. Among these, chicory (Cichorium intybus, Puna II cultivar (cv.)) is known to convey anthelmintic compounds and may control cyathostomins in grazing horses. To challenge this hypothesis, we measured fecal egg counts and the rate of larval development in 20 naturally infected young saddle horses (2-year-old) grazing either (i) a pasture sown with chicory (n = 10) or (ii) a mesophile grassland (n = 10) at the same stocking rate (2.4 livestock unit (LU)/ha). The grazing period lasted 45 days to prevent horse reinfection. Horses in the chicory group mostly grazed chicory (89% of the bites), while those of the control group grazed mainly grasses (73%). Cyathostomins egg excretion decreased in both groups throughout the experiment. Accounting for this trajectory, the fecal egg count reduction (FECR) measured in individuals grazing chicory relative to control individuals increased from 72.9% at day 16 to 85.5% at the end of the study. In addition, larval development in feces from horses grazed on chicory was reduced by more than 60% from d31 compared to control individuals. Using a metabarcoding approach, we also evidenced a significant decrease in cyathostomin species abundance in horses grazing chicory. Chicory extract enriched in sesquiterpenes lactones was tested on two cyathostomins isolates. The estimated IC50 was high (1 and 3.4 mg/ml) and varied according to the pyrantel sensitivity status of the worm isolate. We conclude that the grazing of chicory (cv. Puna II) by horses is a promising strategy for reducing cyathostomin egg excretion and larval development that may contribute to lower the reliance on synthetic anthelmintics. The underpinning modes of action remain to be explored further.

The peripheral blood transcriptome reflects variations in immunity traits in swine: towards the identification of biomarkers.

BACKGROUND: Immune traits (ITs) are potentially relevant criteria to characterize an individual's immune response. Our aim was to investigate whether the peripheral blood transcriptome can provide a significant and comprehensive view of IT variations in pig. RESULTS: Sixty-day-old Large White pigs classified as extreme for in vitro production of IL2, IL10, IFNγ and TNFα, phagocytosis activity, in vivo CD4⁻/CD8⁺ or TCRγδ + cell counts, and anti-Mycoplasma antibody levels were chosen to perform a blood transcriptome analysis with a porcine generic array enriched with immunity-related genes. Differentially expressed (DE) genes for in vitro production of IL2 and IL10, phagocytosis activity and CD4⁻/CD8⁺ cell counts were identified. Gene set enrichment analysis revealed a significant over-representation of immune response functions. To validate the microarray-based results, a subset of DE genes was confirmed by RT-qPCR. An independent set of 74 animals was used to validate the covariation between gene expression levels and ITs. Five potential gene biomarkers were found for prediction of IL2 (RALGDS), phagocytosis (ALOX12) or CD4⁻/CD8⁺ cell count (GNLY, KLRG1 and CX3CR1). On average, these biomarkers performed with a sensitivity of 79% and a specificity of 86%. CONCLUSIONS: Our results confirmed that gene expression profiling in blood represents a relevant molecular phenotype to refine ITs in pig and to identify potential biomarkers that can provide new insights into immune response analysis.

The gut mucin-microbiota interactions: a missing key to optimizing endurance performance.

Endurance athletes offer unique physiology and metabolism compared to sedentary individuals. Athletes training at high intensities for prolonged periods are at risk for gastrointestinal disturbances. An important factor in endurance performance is the integrity and function of the gut barrier, which primarily depends on heavily O-glycosylated mucins. Emerging evidence shows a complex bidirectional dialogue between glycans on mucins and gut microorganisms. This review emphasizes the importance of the crosstalk between the gut microbiome and host mucus mucins and some of the mechanisms underlying this symbiosis. The contribution of mucin glycans to the composition and functionality of the gut microbiome is discussed, as well as the persuasive impact of the gut microbiome on mucin composition, thickness, and immune and metabolic functions. Lastly, we propose natural and synthetic glycans supplements to improve intestinal mucus production and barrier function, offering new opportunities to enhance endurance athletes' performance and gut health.

Patterns of variation in equine strongyle community structure across age groups and gut compartments.

BACKGROUND: Equine strongyles encompass more than 64 species of nematode worms that are responsible for growth retardation and the death of animals. The factors underpinning variation in the structure of the equine strongyle community remain unknown. METHODS: Using horse-based strongyle community data collected after horse deworming (48 horses in Poland, 197 horses in Ukraine), we regressed species richness and the Gini-Simpson index upon the horse's age, faecal egg count, sex and operation of origin. Using the Ukrainian observations, we applied a hierarchical diversity partitioning framework to estimate how communities were remodelled across operations, age groups and horses. Lastly, strongyle species counts collected after necropsy (46 horses in France, 150 in Australia) were considered for analysis of their co-occurrences across intestinal compartments using a joint species distribution modelling approach. RESULTS: First, inter-operation variation accounted for > 45% of the variance in species richness or the Gini-Simpson index (which relates to species dominance in communities). Species richness decreased with horse's age (P = 0.01) and showed a mild increase with parasite egg excretion (P < 0.1), but the Gini-Simpson index was neither associated with parasite egg excretion (P = 0.8) nor with horse age (P = 0.37). Second, within-host diversity represented half of the overall diversity across Ukrainian operations. While this is expected to erase species diversity across communities, community dissimilarity between horse age classes was the second most important contributor to overall diversity (25.8%). Third, analysis of species abundance data quantified at necropsy defined a network of positive co-occurrences between the four most prevalent strongyle genera. This pattern was common to necropsies performed in France and Australia. CONCLUSIONS: Taken together, these results show a pattern of ß-diversity maintenance across age classes combined with positive co-occurrences that might be grounded by priority effects between the major species.

Transcriptome-wide mapping of milk somatic cells upon subclinical mastitis infection in dairy cattle.

BACKGROUND: Subclinical intramammary infection (IMI) represents a significant problem in maintaining dairy cows' health. Disease severity and extent depend on the interaction between the causative agent, environment, and host. To investigate the molecular mechanisms behind the host immune response, we used RNA-Seq for the milk somatic cells (SC) transcriptome profiling in healthy cows (n = 9), and cows naturally affected by subclinical IMI from Prototheca spp. (n = 11) and Streptococcus agalactiae (S. agalactiae; n = 11). Data Integration Analysis for Biomarker discovery using Latent Components (DIABLO) was used to integrate transcriptomic data and host phenotypic traits related to milk composition, SC composition, and udder health to identify hub variables for subclinical IMI detection. RESULTS: A total of 1,682 and 2,427 differentially expressed genes (DEGs) were identified when comparing Prototheca spp. and S. agalactiae to healthy animals, respectively. Pathogen-specific pathway analyses evidenced that Prototheca's infection upregulated antigen processing and lymphocyte proliferation pathways while S. agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle, and carbohydrate and lipid metabolism. The integrative analysis of commonly shared DEGs between the two pathogens (n = 681) referred to the core-mastitis response genes, and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells (r2 = 0.72), followed by the udder health (r2 = 0.64) and milk quality parameters (r2 = 0.64). Variables with r ≥ 0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cytohubba plug-in. The genes in common between DIABLO and cytohubba (n = 10) were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals (sensitivity > 0.89, specificity > 0.81, accuracy > 0.87, and precision > 0.69). Among these genes, CIITA could play a key role in regulating the animals' response to subclinical IMI. CONCLUSIONS: Despite some differences in the enriched pathways, the two mastitis-causing pathogens seemed to induce a shared host immune-transcriptomic response. The hub variables identified with the integrative approach might be included in screening and diagnostic tools for subclinical IMI detection.

Species interactions, stability, and resilience of the gut microbiota - Helminth assemblage in horses.

The nature and strength of interactions entertained among helminths and their host gut microbiota remain largely unexplored. Using 40 naturally infected Welsh ponies, we tracked the gut microbiota-cyathostomin temporal dynamics and stability before and following anthelmintic treatment and the associated host blood transcriptomic response. High shedders harbored 14 species of cyathostomins, dominated by Cylicocyclus nassatus. They exhibited a highly diverse and temporal dynamic gut microbiota, with butyrate-producing Clostridia likely driving the ecosystem steadiness and host tolerance toward cyathostomins infection. However, anthelmintic administration sharply bent the microbial community. It disrupted the ecosystem stability and the time-dependent network of interactions, affecting longer term microbial resilience. These observations highlight how anthelmintic treatments alter the triangular relationship of parasite, host, and gut microbiota and open new perspectives for adding nutritional intervention to current parasite management strategies.

Comparison of two molecular barcodes for the study of equine strongylid communities with amplicon sequencing.

Basic knowledge on the biology and epidemiology of equine strongylid species still needs to be improved to contribute to the design of better parasite control strategies. Nemabiome metabarcoding is a convenient tool to quantify and identify species in bulk samples that could overcome the hurdle that cyathostomin morphological identification represents. To date, this approach has relied on the internal transcribed spacer 2 (ITS-2) of the ribosomal RNA gene, with a limited investigation of its predictive performance for cyathostomin communities. Using DNA pools of single cyathostomin worms, this study aimed to provide the first elements to compare performances of the ITS-2 and a cytochrome c oxidase subunit I (COI) barcode newly developed in this study. Barcode predictive abilities were compared across various mock community compositions of two, five and 11 individuals from distinct species. The amplification bias of each barcode was estimated. Results were also compared between various types of biological samples, i.e., eggs, infective larvae or adults. Bioinformatic parameters were chosen to yield the closest representation of the cyathostomin community for each barcode, underscoring the need for communities of known composition for metabarcoding purposes. Overall, the proposed COI barcode was suboptimal relative to the ITS-2 rDNA region, because of PCR amplification biases, reduced sensitivity and higher divergence from the expected community composition. Metabarcoding yielded consistent community composition across the three sample types. However, imperfect correlations were found between relative abundances from infective larvae and other life-stages for Cylicostephanus species using the ITS-2 barcode. While the results remain limited by the considered biological material, they suggest that additional improvements are needed for both the ITS-2 and COI barcodes.

Improving Species Level-taxonomic Assignment from 16S rRNA Sequencing Technologies.

Analysis of the bacterial community from a 16S rRNA gene sequencing technologies requires comparing the reads to a reference database. The challenging task involved in annotation relies on the currently available tools and 16S rRNA databases: SILVA, Greengenes and RDP. A successful annotation depends on the quality of the database. For instance, Greengenes and RDP have not been updated since 2013 and 2016, respectively. In addition, the nature of 16S sequencing technologies (short reads) focuses mainly on the V3-V4 hypervariable region sequencing and hinders the species assignment, in contrast to whole shotgun metagenome sequencing. Here, we combine the results of three standard protocols for 16S rRNA amplicon annotation that utilize homology-based methods, and we propose a new re-annotation strategy to enlarge the percentage of amplicon sequence variants (ASV) classified up to the species level. Following the pattern (reference) method: DADA2 pipeline and SILVA v.138.1 reference database classification (Basic Protocol 1), our method maps the ASV sequences to custom nucleotide BLAST with the SILVA v.138.1 (Basic Protocol 2), and to the 16S database of Bacteria and Archaea of NCBI RefSeq Targeted Loci Project databases (Basic Protocol 3). This new re-annotation workflow was tested in 16S rRNA amplicon data from 156 human fecal samples. The proposed new strategy achieved an increase of nearly eight times the proportion of ASV classified at the species level in contrast to the reference method for the database used in the present research. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample inference and taxonomic profiling through DADA2 algorithm. Basic Protocol 2: Custom BLASTN database creation and ASV taxonomical assignment. Basic Protocol 3: ASV taxonomical assignment using NCBI RefSeq Targeted Loci Project database. Basic Protocol 4: Definitive selection of lineages among the three methods.

Liver transcriptome profile in pigs with extreme phenotypes of intramuscular fatty acid composition.

BACKGROUND: New advances in high-throughput technologies have allowed for the massive analysis of genomic data, providing new opportunities for the characterization of the transcriptome architectures. Recent studies in pigs have employed RNA-Seq to explore the transcriptome of different tissues in a reduced number of animals. The main goal of this study was the identification of differentially-expressed genes in the liver of Iberian x Landrace crossbred pigs showing extreme phenotypes for intramuscular fatty acid composition using RNA-Seq. RESULTS: The liver transcriptomes of two female groups (H and L) with phenotypically extreme intramuscular fatty acid composition were sequenced using RNA-Seq. A total of 146 and 180 unannotated protein-coding genes were identified in intergenic regions for the L and H groups, respectively. In addition, a range of 5.8 to 7.3% of repetitive elements was found, with SINEs being the most abundant elements. The expression in liver of 186 (L) and 270 (H) lncRNAs was also detected. The higher reproducibility of the RNA-Seq data was validated by RT-qPCR and porcine expression microarrays, therefore showing a strong correlation between RT-qPCR and RNA-Seq data (ranking from 0.79 to 0.96), as well as between microarrays and RNA-Seq (r=0.72). A differential expression analysis between H and L animals identified 55 genes differentially-expressed between groups. Pathways analysis revealed that these genes belong to biological functions, canonical pathways and three gene networks related to lipid and fatty acid metabolism. In concordance with the phenotypic classification, the pathways analysis inferred that linolenic and arachidonic acids metabolism was altered between extreme individuals. In addition, a connection was observed among the top three networks, hence suggesting that these genes are interconnected and play an important role in lipid and fatty acid metabolism. CONCLUSIONS: In the present study RNA-Seq was used as a tool to explore the liver transcriptome of pigs with extreme phenotypes for intramuscular fatty acid composition. The differential gene expression analysis showed potential gene networks which affect lipid and fatty acid metabolism. These results may help in the design of selection strategies to improve the sensorial and nutritional quality of pork meat.

[Relationship between nutritional status and the risk of having pressure ulcers in patients included in a home care program]. / Relación entre el estado nutricional y el riesgo de presentar úlceras por presión en pacientes incluidos en el programa de atención domiciliaria.

OBJECTIVE: The objective of the present study was to evaluate the relationship between the nutritional status and the risk of pressure ulcers (PU) in patients within home care programs (ATDOM). We also evaluated the relationship between the level of cognitive impairment, physical dependence, underlying diseases and the nutritional status. SCOPE: Patients in home care program in Primary Health Care. PATIENTS: 100 home care patients. MAIN VARIABLES: age, sex, caregiver, illness, BMI, haemoglobin, haematocrit, lymphocyte count, albumin, cholesterol, Barthel index, Pfeiffer, nutritional assessment (MNA) and Braden scale. RESULTS: Fourteen percent of the ATDOM patients had malnutrition and 46% a high risk of malnutrition. The degree of dependency, and the level of cognitive impairment increased (P<001) the risk of pressure ulcers. Furthermore, the nutritional status affected the risk of pressure ulcers (P<001) with OR 3.73 higher in malnourished patients. Values of 3.76±0.05g/dL albumin and cholesterol of 176.43±6.38 were associated with an increased risk of ulceration. There was a significant relationship between nutritional status (P<01) and the degree of dependence. In malnourished patients albumin levels decreased to 3.46±0.098, with averages of 11.41±154.95mg/dL cholesterol. Finally, a lower BMI was significantly related to malnutrition. CONCLUSIONS: The present study demonstrates that 14% of the ATDOM patients showed malnutrition, and 46% a high risk of malnutrition. Malnutrition, the degree of physical dependence and severity of cognitive impairment is associated with an increased risk of ulceration, which justify the need for carrying out some personalised measurements on ATDOM patients.