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Many livestock and human vaccines are leaky because they block symptoms but do not prevent infection or onward transmission. This leakiness is concerning because it increases vaccination coverage required to prevent disease spread and can promote evolution of increased pathogen virulence. Despite leakiness, vaccination may reduce pathogen load, affecting disease transmission dynamics. However, the impacts on post-transmission disease development and infectiousness in contact individuals are unknown. Here, we use transmission experiments involving Marek disease virus (MDV) in chickens to show that vaccination with a leaky vaccine substantially reduces viral load in both vaccinated individuals and unvaccinated contact individuals they infect. Consequently, contact birds are less likely to develop disease symptoms or die, show less severe symptoms, and shed less infectious virus themselves, when infected by vaccinated birds. These results highlight that even partial vaccination with a leaky vaccine can have unforeseen positive consequences in controlling the spread and symptoms of disease.

Gene regulatory elements are central drivers of phenotypic variation and thus of critical importance towards understanding the genetics of complex traits. The Functional Annotation of Animal Genomes consortium was formed to collaboratively annotate the functional elements in animal genomes, starting with domesticated animals. Here we present an expansive collection of datasets from eight diverse tissues in three important agricultural species: chicken ( Gallus gallus ), pig ( Sus scrofa ), and cattle ( Bos taurus ). Comparative analysis of these datasets and those from the human and mouse Encyclopedia of DNA Elements projects reveal that a core set of regulatory elements are functionally conserved independent of divergence between species, and that tissue-specific transcription factor occupancy at regulatory elements and their predicted target genes are also conserved. These datasets represent a unique opportunity for the emerging field of comparative epigenomics, as well as the agricultural research community, including species that are globally important food resources. In order to interpret non-coding variants, information about regulatory elements in the genome is essential. Here, the authors annotate regulatory elements in chicken, pig and cattle, and characterize conservation of these elements between species.

Marek’s disease (MD), a T cell lymphoma disease in chickens, is caused by the Marek’s disease virus (MDV) found ubiquitously in the poultry industry. Genetically resistant Line 6 3 (L6) and susceptible Line 7 2 (L7) chickens have been instrumental to research on avian immune system response to MDV infection. In this study we characterized molecular signatures unique to splenic immune cell types across different genetic backgrounds 6 days after infection. Using three populations, L6, L7, and an F1 cross between L6xL7, we evaluated the immune cell transcriptome of responding cell types using single cell RNA sequencing. Several MDV genes were found expressed mainly in cytotoxic T cells while ICP4 and MEQ MDV genes were expressed across infected cell types. Using the F1 we quantified allele specific expression (ASE) of biallelic SNPs and found biased expression of parental alleles specific to immune cell subtypes. We identified 22 SNPs with ASE in response to MDV infection mapped to gene rich regions surrounding 59 genes of critical importance for chromatin remodeling and transcriptional regulation. Histone deacetylase genes (HDAC1 and HDAC8) had increased expression of L6 alleles, while small nuclear RNA genes (SNORA68 and SNORA72) expressed higher levels of L7 alleles with infection in T cell subsets. SNPs with ASE also mapped genes important for an adequate immune response including GNLY (cytotoxic activity) and PDIA3 (component of MHC class I peptide loading complex), and genes known to promote viral replication (MCM5 and EIF3M). These results show that functional variants associated with susceptibility to MD may have a bigger impact in subsets of immune cell types, and by characterizing the transcriptomes of these subtypes we can unravel molecular signatures specific to MD genomic resistance.

Background: In livestock species like the chicken, high throughput single nucleotide polymorphism (SNP) genotyping assays are increasingly being used for whole genome association studies and as a tool in breeding (referred to as genomic selection). To be of value in a wide variety of breeds and populations, the success rate of the SNP genotyping assay, the distribution of the SNP across the genome and the minor allele frequencies (MAF) of the SNPs used are extremely important. Results: We describe the design of a moderate density (60k) Illumina SNP BeadChip in chicken consisting of SNPs known to be segregating at high to medium minor allele frequencies (MAF) in the two major types of commercial chicken (broilers and layers). This was achieved by the identification of 352,303 SNPs with moderate to high MAF in 2 broilers and 2 layer lines using Illumina sequencing on reduced representation libraries. To further increase the utility of the chip, we also identified SNPs on sequences currently not covered by the chicken genome assembly (Gallus_gallus-2.1). This was achieved by 454 sequencing of the chicken genome at a depth of 12x and the identification of SNPs on 454-derived contigs not covered by the current chicken genome assembly. In total we added 790 SNPs that mapped to 454-derived contigs as well as 421 SNPs with a position on Chr_random of the current assembly. The SNP chip contains 57,636 SNPs of which 54,293 could be genotyped and were shown to be segregating in chicken populations. Our SNP identification procedure appeared to be highly reliable and the overall validation rate of the SNPs on the chip was 94%. We were able to map 328 SNPs derived from the 454 sequence contigs on the chicken genome. The majority of these SNPs map to chromosomes that are already represented in genome build Gallus_gallus-2.1.0. Twenty-eight SNPs were used to construct two new linkage groups most likely representing two micro-chromosomes not covered by the current genome assembly. Conclusions: The high success rate of the SNPs on the Illumina chicken 60K Beadchip emphasizes the power of Next generation sequence (NGS) technology for the SNP identification and selection step. The identification of SNPs from sequence contigs derived from NGS sequencing resulted in improved coverage of the chicken genome and the construction of two new linkage groups most likely representing two chicken micro-chromosomes.

Marek’s disease (MD) is a lymphoproliferative disease of chickens induced by Marek’s disease virus (MDV), an oncogenic α-herpesvirus. MDV has increased in virulence, prompting continued efforts in both improved vaccines and enhanced genetic resistance. Model pairs of genetically MD-resistant and MD-susceptible chickens that were either MHC-matched or MHC-congenic allowed characterization of T cell receptor (TCR) repertoires associated with MDV infection. MD-resistant chickens showed higher usage of Vβ-1 TCRs than susceptible chickens in both the CD8 and CD4 subsets in the MHC-matched model, and in the CD8 subset only in the MHC-congenic model, with a shift towards Vβ-1+ CD8 cells during MDV infection. Long and short read sequencing identified divergent TCRβ loci between MHC-matched MD-resistant and MD-susceptible chickens, with MD-resistant chickens having more TCR Vβ1 genes. TCR Vβ1 CDR1 haplotype usage in MD-resistant x MD-susceptible F1 birds by RNAseq indicated that the most commonly used CDR1 variant was unique to the MD-susceptible line, suggesting that selection for MD resistance in the MHC-matched model optimized the TCR repertoire away from dominant recognition of one or more B2 haplotype MHC molecules. Finally, TCR downregulation during MDV infection in the MHC-matched model was strongest in the MD-susceptible line, and MDV reactivation downregulated TCR expression in a tumor cell line.

Necrotic enteritis (NE) is an important intestinal infectious disease of commercial poultry flocks caused by Clostridium perfringens. Using an experimental model of NE involving co-infection with C. perfringens and Eimeria maxima, transcriptome profiling and functional genomics approaches were applied to identify the genetic mechanisms that might regulate the host response to this disease. Microarray hybridization identified 1,049 transcripts whose levels were altered (601 increased, 448 decreased) in intestinal lymphocytes from C. perfringens/E. maxima co-infected Ross chickens compared with uninfected controls. Five biological functions, all related to host immunity and inflammation, and 11 pathways were identified from this dataset. To further elucidate the role of host genetics in NE susceptibility, two inbred chicken lines, ADOL line 6 and line 7 which share an identical B2 major histocompatibility complex haplotype but differ in their susceptibility to virus infection, were compared for clinical symptoms and the expression levels of a panel of immune-related genes during experimental NE. Line 6 chickens were more susceptible to development of experimental NE compared with line 7, as revealed by decreased body weight gain and increased E. maxima oocyst shedding. Of 21 immune-related genes examined, 15 were increased in C. perfringens/E. maxima co-infected line 6 vs. line 7 chickens. These results suggest that immune pathways are activated in response to experimental NE infection and that genetic determinants outside of the chicken B complex influence resistance to this disease.

Genetically resistant or susceptible chickens to Marek’s disease (MD) have been widely used models to identify the molecular determinants of these phenotypes. However, these prior studies lacked the basic identification and understanding of immune cell types that could be translated toward improved MD control. To gain insights into specific immune cell types and their responses to Marek’s disease virus (MDV) infection, we used single-cell RNA sequencing (scRNAseq) on splenic cells from MD resistant and susceptible birds. In total, 14,378 cells formed clusters that identified various immune cell types. Lymphocytes, specifically T cell subtypes, were the most abundant with significant proportional changes in some subtypes upon infection. The largest number of differentially expressed genes (DEG) response was seen in granulocytes, while macrophage DEGs differed in directionality by subtype and line. Among the most DEG in almost all immune cell types were granzyme and granulysin, both associated with cell-perforating processes. Protein interactive network analyses revealed multiple overlapping canonical pathways within both lymphoid and myeloid cell lineages. This initial estimation of the chicken immune cell type landscape and its accompanying response will greatly aid efforts in identifying specific cell types and improving our knowledge of host response to viral infection.

Background Numerous long non-coding RNAs (lncRNAs) have been identified and their roles in gene regulation in humans, mice, and other model organisms studied; however, far less research has been focused on lncRNAs in farm animal species. While previous studies in chickens, cattle, and pigs identified lncRNAs in specific developmental stages or differentially expressed under specific conditions in a limited number of tissues, more comprehensive identification of lncRNAs in these species is needed. The goal of the FAANG Consortium (Functional Annotation of Animal Genomes) is to functionally annotate animal genomes, including the annotation of lncRNAs. As one of the FAANG pilot projects, lncRNAs were identified across eight tissues in two adult male biological replicates from chickens, cattle, and pigs. Results Comprehensive lncRNA annotations for the chicken, cattle, and pig genomes were generated by utilizing RNA-seq from eight tissue types from two biological replicates per species at the adult developmental stage. A total of 9393 lncRNAs in chickens, 7235 lncRNAs in cattle, and 14,429 lncRNAs in pigs were identified. Including novel isoforms and lncRNAs from novel loci, 5288 novel lncRNAs were identified in chickens, 3732 in cattle, and 4870 in pigs. These transcripts match previously known patterns of lncRNAs, such as generally lower expression levels than mRNAs and higher tissue specificity. An analysis of lncRNA conservation across species identified a set of conserved lncRNAs with potential functions associated with chromatin structure and gene regulation. Tissue-specific lncRNAs were identified. Genes proximal to tissue-specific lncRNAs were enriched for GO terms associated with the tissue of origin, such as leukocyte activation in spleen. Conclusions LncRNAs were identified in three important farm animal species using eight tissues from adult individuals. About half of the identified lncRNAs were not previously reported in the NCBI annotations for these species. While lncRNAs are less conserved than protein-coding genes, a set of positionally conserved lncRNAs were identified among chickens, cattle, and pigs with potential functions related to chromatin structure and gene regulation. Tissue-specific lncRNAs have potential regulatory functions on genes enriched for tissue-specific GO terms. Future work will include epigenetic data from ChIP-seq experiments to further refine these annotations.

Marek’s disease (MD) is a lymphoproliferative disease of chickens caused by Marek’s disease virus (MDV), an oncogenic α-herpesvirus. Since 1970, MD has been controlled by widespread vaccination; however, more effective MD vaccines are needed to counter more virulent MDV strains. The bivalent vaccine combination of SB-1 and herpesvirus of turkey (HVT) strain FC126 has been widely used. Nonetheless, the mechanism(s) underlying this synergistic effect has not been investigated. Three experiments were conducted where SB-1 or HVT were administered as monovalent or bivalent vaccines to newly hatched chickens, then challenged five days later with MDV. In Experiment 1, levels of MDV replication in PBMCs were measured over time, and tumor incidence and vaccinal protection determined. In Experiment 2, MDV and vaccine strains replication levels in lymphoid organs were measured at 1, 5, 10, and 14 days post-challenge (DPC). In Experiment 3, to verify that the bursa was necessary for HVT protection, a subset of chicks were bursectomized and these birds plus controls were similarly vaccinated and challenged, and the levels of vaccinal protection determined. The efficacy of bivalent SB-1 + HVT surpasses that of either SB-1 or HVT monovalent vaccines in controlling the level of pathogenic MDV in PBMCs until the end of the study, and this correlated with the ability to inhibit tumor formation. SB-1 replication in the spleen increased from 1 to 14 DPC, while HVT replicated only in the bursa at 1 DPC. The bursa was necessary for immune protection induced by HVT vaccine. Synergy of SB-1 and HVT vaccines is due to additive influences of the individual vaccines acting at different times and target organs. And the bursa is vital for HVT to replicate and induce immune protection.

We describe the organization of a nascent international effort, the Functional Annotation of Animal Genomes (FAANG) project, whose aim is to produce comprehensive maps of functional elements in the genomes of domesticated animal species.