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United States Department of Agriculture

Agricultural Research Service

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Location: Animal Genomics and Improvement Laboratory

2012 Annual Report

1a. Objectives (from AD-416):
Objective 1. Develop genomic tools for small ruminants to study structural and functional genetic variation. A de novo goat genome assembly will be constructed using next-generation sequencing technologies. Single-nucleotide polymorphisms in small ruminant genomes will be analyzed and their utility in genome-wide association studies will be established. Objective 2. Characterize genetic variation in small ruminants to develop selection tools for host resistance to parasites and their pathogenic effects. Genes and QTL in small ruminant genomes that influence host resistance to gastrointestinal nematodes will be identified. Objective 3. Investigate the ruminant host transcriptome and immune responses to identify similarities and differences in response to parasitic infection across ruminants. Inter- and intra-species and breed differences in parasite-induced changes in the host transcriptome will be analyzed, and biological pathways underlying host resistance and their regulatory processes will be characterized.

1b. Approach (from AD-416):
The project will focus on using integrated approaches to develop genomic tools in ruminants and information to better understand how to implement selection for parasite tolerance while also increasing meat and milk production. The studies will attempt to better understand livestock biology of parasite resistance through a combination of quantitative genetics, marker-assisted selection, genome annotation, and gene expression analyses. First, studies will focus on the development of genomic tools for small ruminants to study structural and functional genetic variation, including the construction of a de novo goat reference genome assembly and identification of approximately 45 million single-nucleotide polymorphisms (SNP) to facilitate genetic analysis. Additionally, the project will use combined linkage and linkage disequilibrium (LD) mapping in small ruminant resource populations to identify previously unidentified genes and QTL for parasite tolerance. Finally, efforts will be made to investigate the ruminant host transcriptome and immune responses to identify similarities and differences in host immunity, growth characteristics and nutrient utilization in response to parasitic infection across ruminants.

3. Progress Report:
Sequence production for a de novo genome assembly of the goat genome was nearly completed. In order to ensure the best assembly, a group of 100 goats from the several U.S. breeds (Kiko, San Clemente, Boer, Myotonic, Spanish) were genotyped using the Illumina Caprine50K single nucleotide polymorphism (SNP) beadchip assay. This assay became commercially available in 2011, and interrogates about 50,000 different SNP locations across the genome. The results from this genotyping experiment revealed the most inbred goat, and this animal was selected for genome assembly. Multiple next generation sequence (NGS) libraries were constructed. From one of these libraries, 100 bp paired end reads were sequenced on the Illumina HiSeq instrument to generate 120 billion base pairs (40 X genome coverage) and completed the milestone for Objective 1A. However, other library types containing long inserts (3-20 Kbp) will be sequenced during 2012 to provide the sequence information needed to assembly sequence contigs into longer pieces (scaffolds) and build the best assembly using data from multiple sequencing platforms. For SNP discovery in goats, more than 100 samples have been collected in the U.S. and more than 300 samples have been collected from locally adapted populations in Africa. We are in the process of genotyping these animals. Genome analysis of this dataset will allow us to refine selection of truly unique animals for future sequence-based SNP discovery efforts. Genotyping of 384 animals from a double-backcross population of Red Maasai and Dorper sheep was completed and analysis for parasite-related regions of the genome is underway. Samples from native sheep of Africa were also obtained and genotyped for comparative analysis of signatures of selection for parasite resistance. Genome-wide association analysis of fecal egg count phenotypes for an Angus resource population was completed using BovineSNP50 data. Significant associations affecting parasite infection in Angus cattle were found on Chromosomes 3 and 15. In collaboration with university partners, two breeds of sheep endemic to the Canary Islands, the Canaria Hair Breed and the Canaria sheep, were experimentally challenged with 10,000 larvae of Haemonchus contortus for 7 and 21 days, respectively. These breeds represent two distinct resistance phenotypes in terms of mean fecal egg counts, adult worm counts, number of eggs in utero, and female worm stunting. The Canaria Hair Breed has been shown to display a greater resistance to H. contortus infection than Canaria sheep. Total RNA samples from these animals were extracted and transcriptomics and pathway analyses are currently underway.

4. Accomplishments
1. Development of resources and bioinformatic tools that enable studies to understand host-parasite relationships. We identified local complement activation as a key pathway responsible for the development of long-term protective immunity and host resistance against Ostertagia infection in cattle. We characterized complicated interactions among the host, its microbiota, and parasites mediated by nutrients in the cattle-Ostertagia system. We showed that serpins produced by the abomasal microbiota prevent commensal bacteria from attack by host proteases. Our results clearly demonstrated that Ostertagia infection in immune cattle induces a minimal disruption in the host microbiome, which contributes to the development of protective immunity. In addition, we applied a bioinformatic pipeline and tools to a porcine model and characterized the porcine gut microbiota in response to helminth infection. The impact of helminth infection on gut microbial diversity has thus been quantified. Our findings demonstrate for the first time that changes in abundances of Succinivibrio and Mucispirillum in the proximal colon relate to alterations in carbohydrate metabolism and niche disruptions in mucosal interfaces induced by parasites. Our research provided strong evidence that helminth infection alters host susceptibility to secondary bacterial infection because the abundance of Campylobacter species is significantly different among naïve animals, infected animals with adult worms, and infected animals without worms.

2. Completed a study to unravel the mechanisms of protective immunity to Cooperia infection in cattle. Infections by Cooperia species and associated parasite drug resistance are arguably the most serious health concern in ruminants. We characterized transcriptome dynamics in response to Cooperia infection in cattle and identified a total of 34 biological pathways significantly impacted by infection. Among them, smooth muscle hyper-contractility was recognized as a novel mechanism of the host response. Our data clearly demonstrate that while infection invokes host responses throughout the infection cycle, underlying mechanisms in the host are different during key stages of the infection, often coinciding with important developmental events in the parasite life cycle.

3. Characterized the response of the abomasal transcriptome to gastrointestinal parasites in parasite-susceptible and parasite-resistant Angus cattle using RNA-seq technology. These cattle displayed distinctly separate resistance phenotypes as assessed by fecal egg counts. Of 15,432 bovine genes collectively expressed in the bovine fundic abomasum, 13,758 genes were expressed in all samples tested and likely represent core components of the bovine abomasal transcriptome. PIGR, Complement C3, and Immunoglobulin J chain were among the most abundant transcripts in the transcriptome. Using stringent criteria, 64 genes were identified as significantly over-expressed in resistant animals. Among 94,224 gene splice junctions identified, 133 were uniquely present: 90 were observed only in resistant animals, and 43 were present only in susceptible animals. Gene Ontology enrichment of the genes under study uncovered an association with fat metabolism, which was confirmed by an independent pathway analysis. Several specific biological pathways were found to be impacted in resistant animals, which are potentially involved in the development of parasite resistance in cattle.

Review Publications
Hoorens, P., Rinaldi, M., Mihi, B., Dreseen, L., Grit, G., Meeusen, E., Li, R.W., Geldhof, P. 2011. Galectin-11 induction in the gastrointestinal tract of cattle following nematode and protozoan infections. Parasite Immunology. 33:669–678.

Li, R.W., Rinaldi, M., Capuco, A.V. 2011. Characterization of the abomasal transcriptome for mechanisms of resistance to gastrointestinal nematodes in cattle. Veterinary Research. 42(1):114.

Yang, Z., Wu, R., Li, L., Xiong, Z., Zhao, H., Li, R.W., Guo, D., Pan, Z. 2012. Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral particles as a non-transmissible bivalent marker vaccine candidate against CSF and JE infections. Virus Research. 165(1):61-70.

Li, R.W., Wu, S., Li, W., Hill, D.E., Urban, Jr., J.F., Navarro, K., Couch, R.D. 2012. Alterations in the Porcine Colon Microbiota Induced by the Gastrointestinal Nematode Trichuris suis. Infection and Immunity. 80(6):2150-2157.

Li, R.W., Schroeder, S.G. 2011. Cytoskeleton remodling and alterations in smooth muscle contractility in the bovine jejunum during the early stage of Cooperia oncophora infection. Functional and Integrative Genomics. 12(1):35-44.

Wu, S., Li, R.W., Li, W., Urban Jr, J.F., Dawson, H.D., Beshah, E. 2012. Worm burden-dependent disruption of the porcine colon microbiota by Trichuris suis infection. PLoS One. 7(4):e35470.

Silva, M.V., Van Tassell, C.P., Sonstegard, T.S., Cobuci, J.A., Gasbarre, L.C. 2012. Box-Cox Transformation and Random Regression Models for Fecal Egg Count Data. Frontiers in Genetics. 2:112.

Li, R.W., Tuo, W. 2010. Comparative gene expression profiling of Neospora caninum strains. Experimental Parasitology. 129:346-354.

Silva, M.V., Sonstegard, T.S., Hanotte, O., Mugambi, J., Garcia, J.F., Nagda, S., Gibson, J., Iraqi, F., Mcclintock, A.E., Kemp, S., Boettcher, P., Malek, M., Van Tassell, C.P., Baker, R.L. 2012. Identification of quantitative trait loci affecting resistance to gastro-intestinal parasites in a double backcross population of Red Maasai and Dorper sheep. Animal Genetics. 43(1):63-71.

Li, R.W., Huang, Y., Gasbarre, L. 2011. Metagenome plasticity of the bovine abomasal microbiota in immune animals in response to Ostertagia ostertagi infection. PLoS One. 6:e24417.

Hou, Y., Liu, G., Bickhart, D.M., Matukumalli, L.K., Li, C., Song, J., Gasbarre, L.C., Van Tassell, C.P., Sonstegard, T.S. 2011. Genomic regions showing copy number variations associate with resistance or susceptibility to gastrointestinal nematodes in Angus Cattle. Functional and Integrative Genomics. 12(1):81-92.

Last Modified: 06/25/2017
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