RESUMO
In livestock species, the monolayer of epithelial cells covering the digestive mucosa plays an essential role for nutrition and gut barrier function. However, research on farm animal intestinal epithelium has been hampered by the lack of appropriate in vitro models. Over the past decade, methods to culture livestock intestinal organoids have been developed in pig, bovine, rabbit, horse, sheep and chicken. Gut organoids from farm animals are obtained by seeding tissue-derived intestinal epithelial stem cells in a 3-dimensional culture environment reproducing in vitro the stem cell niche. These organoids can be generated rapidly within days and are formed by a monolayer of polarized epithelial cells containing the diverse differentiated epithelial progeny, recapitulating the original structure and function of the native epithelium. The phenotype of intestinal organoids is stable in long-term culture and reflects characteristics of the digestive segment of origin. Farm animal intestinal organoids can be amplified in vitro, cryopreserved and used for multiple experiments, allowing an efficient reduction of the use of live animals for experimentation. Most of the studies using livestock intestinal organoids were used to investigate host-microbe interactions at the epithelial surface, mainly focused on enteric infections with viruses, bacteria or parasites. Numerous other applications of farm animal intestinal organoids include studies on nutrient absorption, genome editing and bioactive compounds screening relevant for agricultural, veterinary and biomedical sciences. Further improvements of the methods used to culture intestinal organoids from farm animals are required to replicate more closely the intestinal tissue complexity, including the presence of non-epithelial cell types and of the gut microbiota. Harmonization of the methods used to culture livestock intestinal organoids will also be required to increase the reproducibility of the results obtained in these models. In this review, we summarize the methods used to generate and cryopreserve intestinal organoids in farm animals, present their phenotypes and discuss current and future applications of this innovative culture system of the digestive epithelium.
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Animais Domésticos/anatomia & histologia , Técnicas de Cultura de Células/veterinária , Criopreservação/veterinária , Intestino Grosso/citologia , Intestino Delgado/citologia , Organoides/citologia , Animais , Técnicas de Cultura de Células/métodos , Criopreservação/métodos , Células Epiteliais/citologia , Mucosa Intestinal/citologiaRESUMO
BACKGROUND: Comparative genomics studies are central in identifying the coding and non-coding elements associated with complex traits, and the functional annotation of genomes is a critical step to decipher the genotype-to-phenotype relationships in livestock animals. As part of the Functional Annotation of Animal Genomes (FAANG) action, the FR-AgENCODE project aimed to create reference functional maps of domesticated animals by profiling the landscape of transcription (RNA-seq), chromatin accessibility (ATAC-seq) and conformation (Hi-C) in species representing ruminants (cattle, goat), monogastrics (pig) and birds (chicken), using three target samples related to metabolism (liver) and immunity (CD4+ and CD8+ T cells). RESULTS: RNA-seq assays considerably extended the available catalog of annotated transcripts and identified differentially expressed genes with unknown function, including new syntenic lncRNAs. ATAC-seq highlighted an enrichment for transcription factor binding sites in differentially accessible regions of the chromatin. Comparative analyses revealed a core set of conserved regulatory regions across species. Topologically associating domains (TADs) and epigenetic A/B compartments annotated from Hi-C data were consistent with RNA-seq and ATAC-seq data. Multi-species comparisons showed that conserved TAD boundaries had stronger insulation properties than species-specific ones and that the genomic distribution of orthologous genes in A/B compartments was significantly conserved across species. CONCLUSIONS: We report the first multi-species and multi-assay genome annotation results obtained by a FAANG project. Beyond the generation of reference annotations and the confirmation of previous findings on model animals, the integrative analysis of data from multiple assays and species sheds a new light on the multi-scale selective pressure shaping genome organization from birds to mammals. Overall, these results emphasize the value of FAANG for research on domesticated animals and reinforces the importance of future meta-analyses of the reference datasets being generated by this community on different species.
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Animais Domésticos/genética , Cromatina/genética , Anotação de Sequência Molecular , Transcriptoma , Animais , Bovinos , Galinhas , Cabras , Filogenia , Sus scrofaRESUMO
BACKGROUND: Improving functional annotation of the chicken genome is a key challenge in bridging the gap between genotype and phenotype. Among all transcribed regions, long noncoding RNAs (lncRNAs) are a major component of the transcriptome and its regulation, and whole-transcriptome sequencing (RNA-Seq) has greatly improved their identification and characterization. We performed an extensive profiling of the lncRNA transcriptome in the chicken liver and adipose tissue by RNA-Seq. We focused on these two tissues because of their importance in various economical traits for which energy storage and mobilization play key roles and also because of their high cell homogeneity. To predict lncRNAs, we used a recently developed tool called FEELnc, which also classifies them with respect to their distance and strand orientation to the closest protein-coding genes. Moreover, to confidently identify the genes/transcripts expressed in each tissue (a complex task for weakly expressed molecules such as lncRNAs), we probed a particularly large number of biological replicates (16 per tissue) compared to common multi-tissue studies with a larger set of tissues but less sampling. RESULTS: We predicted 2193 lncRNA genes, among which 1670 were robustly expressed across replicates in the liver and/or adipose tissue and which were classified into 1493 intergenic and 177 intragenic lncRNAs located between and within protein-coding genes, respectively. We observed similar structural features between chickens and mammals, with strong synteny conservation but without sequence conservation. As previously reported, we confirm that lncRNAs have a lower and more tissue-specific expression than mRNAs. Finally, we showed that adjacent lncRNA-mRNA genes in divergent orientation have a higher co-expression level when separated by less than 1 kb compared to more distant divergent pairs. Among these, we highlighted for the first time a novel lncRNA candidate involved in lipid metabolism, lnc_DHCR24, which is highly correlated with the DHCR24 gene that encodes a key enzyme of cholesterol biosynthesis. CONCLUSIONS: We provide a comprehensive lncRNA repertoire in the chicken liver and adipose tissue, which shows interesting patterns of co-expression between mRNAs and lncRNAs. It contributes to improving the structural and functional annotation of the chicken genome and provides a basis for further studies on energy storage and mobilization traits in the chicken.
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Tecido Adiposo/metabolismo , Galinhas/genética , Fígado/metabolismo , RNA Longo não Codificante/genética , Transcriptoma , Animais , Galinhas/metabolismo , Sequência Conservada , Evolução Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma , Genótipo , Humanos , Metabolismo dos Lipídeos/genética , Fases de Leitura Aberta , Especificidade de Órgãos , Fenótipo , Locos de Características Quantitativas , RNA Antissenso , RNA Longo não Codificante/química , RNA Mensageiro/genéticaRESUMO
UNLABELLED: The alphaherpesvirus pseudorabies virus (PrV) establishes latency primarily in neurons of trigeminal ganglia when only the transcription of the latency-associated transcript (LAT) locus is detected. Eleven microRNAs (miRNAs) cluster within the LAT, suggesting a role in establishment and/or maintenance of latency. We generated a mutant (M) PrV deleted of nine miRNA genes which displayed properties that were almost identical to those of the parental PrV wild type (WT) during propagation in vitro. Fifteen pigs were experimentally infected with either WT or M virus or were mock infected. Similar levels of virus excretion and host antibody response were observed in all infected animals. At 62 days postinfection, trigeminal ganglia were excised and profiled by deep sequencing and quantitative RT-PCR. Latency was established in all infected animals without evidence of viral reactivation, demonstrating that miRNAs are not essential for this process. Lower levels of the large latency transcript (LLT) were found in ganglia infected by M PrV than in those infected by WT PrV. All PrV miRNAs were expressed, with highest expression observed for prv-miR-LLT1, prv-miR-LLT2 (in WT ganglia), and prv-miR-LLT10 (in both WT and M ganglia). No evidence of differentially expressed porcine miRNAs was found. Fifty-four porcine genes were differentially expressed between WT, M, and control ganglia. Both viruses triggered a strong host immune response, but in M ganglia gene upregulation was prevalent. Pathway analyses indicated that several biofunctions, including those related to cell-mediated immune response and the migration of dendritic cells, were impaired in M ganglia. These findings are consistent with a function of the LAT locus in the modulation of host response for maintaining a latent state. IMPORTANCE: This study provides a thorough reference on the establishment of latency by PrV in its natural host, the pig. Our results corroborate the evidence obtained from the study of several LAT mutants of other alphaherpesviruses encoding miRNAs from their LAT regions. Neither PrV miRNA expression nor high LLT expression levels are essential to achieve latency in trigeminal ganglia. Once latency is established by PrV, the only remarkable differences are found in the pattern of host response. This indicates that, as in herpes simplex virus, LAT functions as an immune evasion locus.
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Herpesvirus Suídeo 1/fisiologia , Interações Hospedeiro-Patógeno , Pseudorraiva/imunologia , Pseudorraiva/virologia , Gânglio Trigeminal/imunologia , Gânglio Trigeminal/virologia , Latência Viral , Animais , Perfilação da Expressão Gênica , Herpesvirus Suídeo 1/imunologia , Imunidade Celular , MicroRNAs , Deleção de Sequência , Suínos , Replicação ViralRESUMO
The Functional Annotation of Animal Genomes (FAANG) Consortium recently held a Gathering On FAANG (GO-FAANG) Workshop in Washington, DC on October 7-8, 2015. This consortium is a grass-roots organization formed to advance the annotation of newly assembled genomes of domesticated and non-model organisms (www.faang.org). The workshop gathered together from around the world a group of 100+ genome scientists, administrators, representatives of funding agencies and commodity groups to discuss the latest advancements of the consortium, new perspectives, next steps and implementation plans. The workshop was streamed live and recorded, and all talks, along with speaker slide presentations, are available at www.faang.org. In this report, we describe the major activities and outcomes of this meeting. We also provide updates on ongoing efforts to implement discussions and decisions taken at GO-FAANG to guide future FAANG activities. In summary, reference datasets are being established under pilot projects; plans for tissue sets, morphological classification and methods of sample collection for different tissues were organized; and core assays and data and meta-data analysis standards were established.
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Animais Domésticos/genética , Genoma , Genômica , Animais , Congressos como Assunto , District of Columbia , Cooperação Internacional , Padrões de ReferênciaRESUMO
Gene atlases for livestock are steadily improving thanks to new genome assemblies and new expression data improving the gene annotation. However, gene content varies across databases due to differences in RNA sequencing data and bioinformatics pipelines, especially for long non-coding RNAs (lncRNAs) which have higher tissue and developmental specificity and are harder to consistently identify compared to protein coding genes (PCGs). As done previously in 2020 for chicken assemblies galgal5 and GRCg6a, we provide a new gene atlas, lncRNA-enriched, for the latest GRCg7b chicken assembly, integrating "NCBI RefSeq", "EMBL-EBI Ensembl/GENCODE" reference annotations and other resources such as FAANG and NONCODE. As a result, the number of PCGs increases from 18,022 (RefSeq) and 17,007 (Ensembl) to 24,102, and that of lncRNAs from 5789 (RefSeq) and 11,944 (Ensembl) to 44,428. Using 1400 public RNA-seq transcriptome representing 47 tissues, we provided expression evidence for 35,257 (79%) lncRNAs and 22,468 (93%) PCGs, supporting the relevance of this atlas. Further characterization including tissue-specificity, sex-differential expression and gene configurations are provided. We also identified conserved miRNA-hosting genes with human counterparts, suggesting common function. The annotated atlas is available at gega.sigenae.org.
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RNA Longo não Codificante , Animais , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Galinhas/genética , Galinhas/metabolismo , Transcriptoma , Anotação de Sequência Molecular , Análise de Sequência de RNARESUMO
The Farm Animal Genotype-Tissue Expression (FarmGTEx) project has been established to develop a public resource of genetic regulatory variants in livestock, which is essential for linking genetic polymorphisms to variation in phenotypes, helping fundamental biological discovery and exploitation in animal breeding and human biomedicine. Here we show results from the pilot phase of PigGTEx by processing 5,457 RNA-sequencing and 1,602 whole-genome sequencing samples passing quality control from pigs. We build a pig genotype imputation panel and associate millions of genetic variants with five types of transcriptomic phenotypes in 34 tissues. We evaluate tissue specificity of regulatory effects and elucidate molecular mechanisms of their action using multi-omics data. Leveraging this resource, we decipher regulatory mechanisms underlying 207 pig complex phenotypes and demonstrate the similarity of pigs to humans in gene expression and the genetic regulation behind complex phenotypes, supporting the importance of pigs as a human biomedical model.
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Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Suínos/genética , Animais , Humanos , Genótipo , Fenótipo , Análise de Sequência de RNARESUMO
BACKGROUND: The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems. RESULTS: The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome. CONCLUSIONS: This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig's adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.
Assuntos
Genômica , Imunidade/genética , Anotação de Sequência Molecular , Suínos/genética , Suínos/imunologia , Animais , Bovinos , Evolução Molecular , Duplicação Gênica , Humanos , Imunoglobulinas/genética , Camundongos , Modelos Moleculares , Conformação Proteica , Receptores de Antígenos de Linfócitos T/genética , Receptores KIR/genética , Seleção Genética , Especificidade da EspécieRESUMO
BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine diseases worldwide. Despite its relevance, serum biomarkers associated with early-onset viral infection, when clinical signs are not detectable and the disease is characterized by a weak anti-viral response and persistent infection, have not yet been identified. Surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) is a reproducible, accurate, and simple method for the identification of biomarker proteins related to disease in serum. This work describes the SELDI-TOF MS analyses of sera of 60 PRRSV-positive and 60 PRRSV-negative, as measured by PCR, asymptomatic Large White piglets at weaning. Sera with comparable and low content of hemoglobin (< 4.52 µg/mL) were fractionated in 6 different fractions by anion-exchange chromatography and protein profiles in the mass range 1-200 kDa were obtained with the CM10, IMAC30, and H50 surfaces. RESULTS: A total of 200 significant peaks (p < 0.05) were identified in the initial discovery phase of the study and 47 of them were confirmed in the validation phase. The majority of peaks (42) were up-regulated in PRRSV-positive piglets, while 5 were down-regulated. A panel of 14 discriminatory peaks identified in fraction 1 (pH = 9), on the surface CM10, and acquired at low focus mass provided a serum protein profile diagnostic pattern that enabled to discriminate between PRRSV-positive and -negative piglets with a sensitivity and specificity of 77% and 73%, respectively. CONCLUSIONS: SELDI-TOF MS profiling of sera from PRRSV-positive and PRRSV-negative asymptomatic piglets provided a proteomic signature with large scale diagnostic potential for early identification of PRRSV infection in weaning piglets. Furthermore, SELDI-TOF protein markers represent a refined phenotype of PRRSV infection that might be useful for whole genome association studies.
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BACKGROUND: Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific. RESULTS: Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1.The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response. CONCLUSIONS: This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.
Assuntos
Infecções por Escherichia coli/veterinária , Perfilação da Expressão Gênica , Doenças das Cabras/genética , Mastite Bovina/genética , Mastite/veterinária , Doenças dos Ovinos/genética , Infecções Estafilocócicas/veterinária , Infecções Estreptocócicas/veterinária , Animais , Bovinos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Infecções por Escherichia coli/genética , Feminino , Doenças das Cabras/microbiologia , Cabras , Mastite/genética , Mastite/microbiologia , Mastite Bovina/microbiologia , Redes e Vias Metabólicas , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Transcrição de Fator Regulador X , Ovinos , Doenças dos Ovinos/microbiologia , Infecções Estafilocócicas/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Infecções Estreptocócicas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In order to generate an atlas of the functional elements driving genome expression in domestic animals, the Functional Annotation of Animal Genome (FAANG) strategy was to sample many tissues from a few animals of different species, sexes, ages, and production stages. This article presents the collection of tissue samples for four species produced by two pilot projects, at INRAE (National Research Institute for Agriculture, Food and Environment) and the University of California, Davis. There were three mammals (cattle, goat, and pig) and one bird (chicken). It describes the metadata characterizing these reference sets (1) for animals with origin and selection history, physiological status, and environmental conditions; (2) for samples with collection site and tissue/cell processing; (3) for quality control; and (4) for storage and further distribution. Three sets are identified: set 1 comprises tissues for which collection can be standardized and for which representative aliquots can be easily distributed (liver, spleen, lung, heart, fat depot, skin, muscle, and peripheral blood mononuclear cells); set 2 comprises tissues requiring special protocols because of their cellular heterogeneity (brain, digestive tract, secretory organs, gonads and gametes, reproductive tract, immune tissues, cartilage); set 3 comprises specific cell preparations (immune cells, tracheal epithelial cells). Dedicated sampling protocols were established and uploaded in https://data.faang.org/protocol/samples. Specificities between mammals and chicken are described when relevant. A total of 73 different tissues or tissue sections were collected, and 21 are common to the four species. Having a common set of tissues will facilitate the transfer of knowledge within and between species and will contribute to decrease animal experimentation. Combining data on the same samples will facilitate data integration. Quality control was performed on some tissues with RNA extraction and RNA quality control. More than 5,000 samples have been stored with unique identifiers, and more than 4,000 were uploaded onto the Biosamples database, provided that standard ontologies were available to describe the sample. Many tissues have already been used to implement FAANG assays, with published results. All samples are available without restriction for further assays. The requesting procedure is described. Members of FAANG are encouraged to apply a range of molecular assays to characterize the functional status of collected samples and share their results, in line with the FAIR (Findable, Accessible, Interoperable, and Reusable) data principles.
RESUMO
In addition to their common usages to study gene expression, RNA-seq data accumulated over the last 10 years are a yet-unexploited resource of SNPs in numerous individuals from different populations. SNP detection by RNA-seq is particularly interesting for livestock species since whole genome sequencing is expensive and exome sequencing tools are unavailable. These SNPs detected in expressed regions can be used to characterize variants affecting protein functions, and to study cis-regulated genes by analyzing allele-specific expression (ASE) in the tissue of interest. However, gene expression can be highly variable, and filters for SNP detection using the popular GATK toolkit are not yet standardized, making SNP detection and genotype calling by RNA-seq a challenging endeavor. We compared SNP calling results using GATK suggested filters, on two chicken populations for which both RNA-seq and DNA-seq data were available for the same samples of the same tissue. We showed, in expressed regions, a RNA-seq precision of 91% (SNPs detected by RNA-seq and shared by DNA-seq) and we characterized the remaining 9% of SNPs. We then studied the genotype (GT) obtained by RNA-seq and the impact of two factors (GT call-rate and read number per GT) on the concordance of GT with DNA-seq; we proposed thresholds for them leading to a 95% concordance. Applying these thresholds to 767 multi-tissue RNA-seq of 382 birds of 11 chicken populations, we found 9.5 M SNPs in total, of which â¼550,000 SNPs per tissue and population with a reliable GT (call rate ≥ 50%) and among them, â¼340,000 with a MAF ≥ 10%. We showed that such RNA-seq data from one tissue can be used to (i) detect SNPs with a strong predicted impact on proteins, despite their scarcity in each population (16,307 SIFT deleterious missenses and 590 stop-gained), (ii) study, on a large scale, cis-regulations of gene expression, with â¼81% of protein-coding and 68% of long non-coding genes (TPM ≥ 1) that can be analyzed for ASE, and with â¼29% of them that were cis-regulated, and (iii) analyze population genetic using such SNPs located in expressed regions. This work shows that RNA-seq data can be used with good confidence to detect SNPs and associated GT within various populations and used them for different analyses as GTEx studies.
RESUMO
BACKGROUND: DNA barcodes are a global standard for species identification and have countless applications in the medical, forensic and alimentary fields, but few barcoding methods work efficiently in samples in which DNA is degraded, e.g. foods and archival specimens. This limits the choice of target regions harbouring a sufficient number of diagnostic polymorphisms. The method described here uses existing PCR and sequencing methodologies to detect mitochondrial DNA polymorphisms in complex matrices such as foods. The reported application allowed the discrimination among 17 fish species of the Scombridae family with high commercial interest such as mackerels, bonitos and tunas which are often present in processed seafood. The approach can be easily upgraded with the release of new genetic diversity information to increase the range of detected species. RESULTS: Cocktail of primers are designed for PCR using publicly available sequences of the target sequence. They are composed of a fixed 5' region and of variable 3' cocktail portions that allow amplification of any member of a group of species of interest. The population of short amplicons is directly sequenced and indexed using primers containing a longer 5' region and the non polymorphic portion of the cocktail portion. A 226 bp region of CytB was selected as target after collection and screening of 148 online sequences; 85 SNPs were found, of which 75 were present in at least two sequences. Primers were also designed for two shorter sub-fragments that could be amplified from highly degraded samples. The test was used on 103 samples of seafood (canned tuna and scomber, tuna salad, tuna sauce) and could successfully detect the presence of different or additional species that were not identified on the labelling of canned tuna, tuna salad and sauce samples. CONCLUSIONS: The described method is largely independent of the degree of degradation of DNA source and can thus be applied to processed seafood. Moreover, the method is highly flexible: publicly available sequence information on mitochondrial genomes are rapidly increasing for most species, facilitating the choice of target sequences and the improvement of resolution of the test. This is particularly important for discrimination of marine and aquaculture species for which genome information is still limited.
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Análise de Alimentos/métodos , Alimentos Marinhos/análise , Análise de Sequência de DNA/métodos , Atum/genética , Animais , Primers do DNA/genética , DNA Mitocondrial/análise , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , Especificidade da EspécieRESUMO
Anisakids are nematodes whose larval stages are often present in fish, molluscs, and crustaceans. Members of the family Anisakidae belonging to the genera Anisakis and Pseudoterranova are implicated in human infections caused by the consumption of raw or undercooked fish. Adequate cooking will kill anisakid larvae, however, killed or inactivated larvae can still cause sensitization and immunoglobulin E-dependent hypersensitivity in human. This work describes the development of DNA-based tests to detect and quantify the presence of Anisakis spp. and Pseudoterranova spp. larvae in fish and fish-derived products, including fish fillets, surimi, fish sticks, canned fish, and baby food. Primers and TaqMan MGB probes recognizing only Anisakis spp. and Pseudoterranova spp. were designed on the first internal transcribed spacer 1 regions of rDNA for a real-time polymerase chain reaction assay. A commercial probe for 18S rDNA was used to detect and quantify the total eukaryotic DNA of the samples. The specificity and sensitivity of the assays were tested using reference samples prepared from mixtures made of Anisakis larvae in different quantity of codfish, and subsequent dilutions. Studies were performed to assess the ability of the test to detect and quantify anisakids in various products. Results showed that this test is able to detect anisakid DNA contained in a proportion of 1:10(5) in 1 ng of total DNA. The high prevalence of anisakids reported in main fishery species was confirmed by frequently detecting anisakids DNA in fish muscle and fish-derived products. A partial correlation was found between the number of larvae present in the viscera and the level of contamination of fish fillets. In conclusion, this molecular test is useful to detect the presence of Anisakis spp. and Pseudoterranova spp. in fish and fish-derived products and to quantify the level of contamination along the food chain, with potential applications for fish farms, fish markets, and food producers.
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Anisakis/isolamento & purificação , Produtos Pesqueiros/parasitologia , Parasitologia de Alimentos , Animais , Anisakis/classificação , Anisakis/genética , DNA de Helmintos/análise , DNA Espaçador Ribossômico , Peixes/parasitologia , Conservação de Alimentos , Doenças Transmitidas por Alimentos/prevenção & controle , Humanos , Lactente , Alimentos Infantis/parasitologia , Larva , Limite de Detecção , Reação em Cadeia da Polimerase , Controle de Qualidade , Alimentos Marinhos/parasitologia , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
Long non-coding RNAs (LNC) regulate numerous biological processes. In contrast to human, the identification of LNC in farm species, like chicken, is still lacunar. We propose a catalogue of 52,075 chicken genes enriched in LNC ( http://www.fragencode.org/ ), built from the Ensembl reference extended using novel LNC modelled here from 364 RNA-seq and LNC from four public databases. The Ensembl reference grew from 4,643 to 30,084 LNC, of which 59% and 41% with expression ≥ 0.5 and ≥ 1 TPM respectively. Characterization of these LNC relatively to the closest protein coding genes (PCG) revealed that 79% of LNC are in intergenic regions, as in other species. Expression analysis across 25 tissues revealed an enrichment of co-expressed LNC:PCG pairs, suggesting co-regulation and/or co-function. As expected LNC were more tissue-specific than PCG (25% vs. 10%). Similarly to human, 16% of chicken LNC hosted one or more miRNA. We highlighted a new chicken LNC, hosting miR155, conserved in human, highly expressed in immune tissues like miR155, and correlated with immunity-related PCG in both species. Among LNC:PCG pairs tissue-specific in the same tissue, we revealed an enrichment of divergent pairs with the PCG coding transcription factors, as for example LHX5, HXD3 and TBX4, in both human and chicken.
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Galinhas/genética , Biologia Computacional/métodos , Anotação de Sequência Molecular/métodos , RNA Longo não Codificante/genética , Animais , Atlas como Assunto , Proteínas Aviárias/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , MicroRNAs/genética , Especificidade de Órgãos , Análise de Sequência de RNA , Distribuição TecidualRESUMO
Porcine reproductive and respiratory syndrome (PRRS) has an extensive impact on pig production. The causative virus (PRRSV) is divided into two species, PRRSV-1 (European origin) and PRRSV-2 (North American origin). Within PRRSV-1, PRRSV-1.3 strains, such as Lena, are more pathogenic than PRRSV-1.1 strains, such as Flanders 13 (FL13). To date, the molecular interactions of PRRSV with primary lung mononuclear phagocyte (MNP) subtypes, including conventional dendritic cells types 1 (cDC1) and 2 (cDC2), monocyte-derived DCs (moDC), and pulmonary intravascular macrophages (PIM), have not been thoroughly investigated. Here, we analyze the transcriptome profiles of in vivo FL13-infected parenchymal MNP subpopulations and of in vitro FL13- and Lena-infected parenchymal MNP. The cell-specific expression profiles of in vivo sorted cells correlated with their murine counterparts (AM, cDC1, cDC2, moDC) with the exception of PIM. Both in vivo and in vitro, FL13 infection altered the expression of a low number of host genes, and in vitro infection with Lena confirmed the higher ability of this strain to modulate host response. Machine learning (ML) and gene set enrichment analysis (GSEA) unraveled additional relevant genes and pathways modulated by FL13 infection that were not identified by conventional analyses. GSEA increased the cellular pathways enriched in the FL13 data set, but ML allowed a more complete comprehension of functional profiles during FL13 in vitro infection. Data indicates that cellular reprogramming differs upon Lena and FL13 infection and that the latter might keep antiviral and inflammatory macrophage/DC functions silent. Although the slow replication kinetics of FL13 likely contribute to differences in cellular gene expression, the data suggest distinct mechanisms of interaction of the two viruses with the innate immune system during early infection.
Assuntos
Monócitos/imunologia , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína , Animais , Feminino , Pulmão/citologia , Monócitos/virologia , Suínos , TranscriptomaRESUMO
Functional annotation of genomes is a prerequisite for contemporary basic and applied genomic research, yet farmed animal genomics is deficient in such annotation. To address this, the FAANG (Functional Annotation of Animal Genomes) Consortium is producing genome-wide data sets on RNA expression, DNA methylation, and chromatin modification, as well as chromatin accessibility and interactions. In addition to informing our understanding of genome function, including comparative approaches to elucidate constrained sequence or epigenetic elements, these annotation maps will improve the precision and sensitivity of genomic selection for animal improvement. A scientific community-driven effort has already created a coordinated data collection and analysis enterprise crucial for the success of this global effort. Although it is early in this continuing process, functional data have already been produced and application to genetic improvement reported. The functional annotation delivered by the FAANG initiative will add value and utility to the greatly improved genome sequences being established for domesticated animal species.
Assuntos
Animais Domésticos/genética , Genoma/genética , Genômica , Anotação de Sequência Molecular , Animais , Epigenômica , Edição de Genes , Genótipo , FenótipoRESUMO
Host miRNAs are known to modulate the cell response to virus infections. We characterized the miRNA-targeted transcriptome of porcine alveolar macrophages (PAMs) at early times after infection with a subtype 1.1 strain of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus). We performed the immunoprecipitation of RISC (RNA-induced Silencing Complex) followed by microarray analysis of the RISC-bound miRNA targets (RIP-Chip) to evaluate the relative enrichment or depletion of expressed genes in RISC. The miRNA-mediated regulation occurred early after PRRSV infection and decreased fast (1,241 and 141 RISC-bound genes at 7 h and 10 h post-infection, respectively); it affected several cell functions with evidence of miRNA buffering of upregulated interferon-related genes. Eight miRNAs were highly enriched in RISC of both control and infected cells with no evidence of differential expression. Although miR-335-5p was the miRNA with most predicted targets among enriched RISC-bound genes, no effects on surface markers, cytokine expression and PRRSV replication were detected upon miR-335-5p mimics of primary PAMs. Our results do not point to specific miRNA-driven mechanisms regulating the early response to infection with this PRRSV 1.1 strain and indicate that the miRNome expressed by steady-state PAMs reacts promptly to counterbalance PRRSV infection by a pervasive modulation of host functions.
Assuntos
MicroRNAs/genética , Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Transcriptoma/genética , Animais , Regulação da Expressão Gênica/genética , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , MicroRNAs/isolamento & purificação , Análise de Sequência com Séries de Oligonucleotídeos , Síndrome Respiratória e Reprodutiva Suína/patologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Transdução de Sinais/genética , SuínosRESUMO
Swine lymph nodes (LN) present an inverted structure compared to mouse and human, with the afferent lymph diffusing from the center to the periphery. This structure, also observed in close and distant species such as dolphins, hippopotamus, rhinoceros, and elephants, is poorly described, nor are the LN macrophage populations and their relationship with B cell follicles. B cell maturation occurs mainly in LN B cell follicles with the help of LN macrophage populations endowed with different antigen delivery capacities. We identified three macrophage populations that we localized in the inverted LN spatial organization. This allowed us to ascribe porcine LN MΦ to their murine counterparts: subcapsular sinus MΦ, medullary cord MΦ and medullary sinus MΦ. We identified the different intra and extrafollicular stages of LN B cells maturation and explored the interaction of MΦ, drained antigen and follicular B cells. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen that infects tissue macrophages (MΦ). PRRSV is persistent in the secondary lymphoid tissues and induces a delay in neutralizing antibodies appearance. We observed PRRSV interaction with two LN MΦ populations, of which one interacts closely with centroblasts. We observed BCL6 up-regulation in centroblast upon PRRSV infection, leading to new hypothesis on PRRSV inhibition of B cell maturation. This seminal study of porcine LN will permit fruitful comparison with murine and human LN for a better understanding of normal and inverted LN development and functioning.
Assuntos
Linfócitos B/imunologia , Linfonodos/imunologia , Macrófagos/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Animais , Vírus da Síndrome Respiratória e Reprodutiva Suína , SuínosRESUMO
The cestode Molicola horridus is a muscle parasite of teleost fish. The ability of molecules present in this parasite to induce allergic response is not known yet. Since fish-borne parasitic allergens can induce allergic manifestations even when the parasitized fish is well cooked, the knowledge of potential allergens present in food is important in order to provide a save products for consumers. The aim of the study was to determine the allergenic potential of the components present in the crude larval extract (CLE) of M. horridus. Two mouse models were exposed to the CLE: adult BALB/c mice that were intraperitoneally (i.p.) immunized and newborn BALB/c mice that were orally exposed. Specific antibody levels in serum and faeces were measured by ELISA. The cellular immune response was determined by proliferation assay of splenocytes from sensitized mice. The protein profile of CLE was analysed by SDS-PAGE and western blot. In adult mice, specific IgG and IgA were detected in sera and faeces, whereas specific IgE were detected in sera only. In newborn mice, specific IgG were detected in sera and a low level of IgA was detected in faeces. SDS-PAGE revealed the CLE protein profile, with most of the proteins running from 15 to 50kDa. Specific IgG recognized mainly the 26 and 75kDa proteins and a molecular complex below 100kDa by immunoblot. Specific IgE recognized the same 26kDa protein as IgG did, and, with less intensity, another protein at 30kDa. Splenocytes from CLE-immunized mice proliferated when stimulated with CLE in a dose-dependent manner. The crude larval extract from M. horridus has potential allergenic molecules which can represent a risk for fish consumers.