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:
A goat buck of San Clemente breed with a genomic inbreeding coefficient of 0.42 was identified by genotyping for genome sequencing. Genomic DNA from this buck was used to produce fosmid, Illumina paired-end, and PacBio libraries. Sequencing produced about 60X coverage of Illumina paired-end reads and 5X coverage of 1-15kb long reads. De novo genome assembly has not been initiated due the need for more sequence production using a new sequencing technology to be released shortly. Animals for whole genome resequencing for single nucleotide polymorphism(SNP) discovery have not been identified to date due to delays in receiving samples from African collaborators. A double backcross population of Red Maasai and Dorper sheep was genotyped using an OvineSNP50K BeadChip. Sophisticated statistical methods were developed and employed to ensure valid inference of the results from this study. Specifically, permutation testing was used to guard against deviations from normally distributed trait data. Data for three parasitological parameters (average fecal egg counts, packed cell volume, and animal live weight), were adjusted for fixed effects prior to setting the threshold for the tails of the distributions. Genome-wide significance threshold were defined after 100,000 permutations. Association analyses were calculated and statistically significant SNP markers were observed. Chromosomal regions were detected for association with parasite parameters. A set of 7, 10, and 9 SNP explained approximately 29, 19, and 22% of the phenotypic variation of those traits, respectively. As a practical result, a panel with such genetic markers could be used in Red Maasai x Dorper breeding programs to select for sheep with greater parasite resistance. The transcriptome characterization of two breeds of sheep endemic to the Canary Islands, the Canaria Hair Breed and the Canaria sheep, was partially completed. The Canaria Hair Breed displays a greater resistance to barber's pole worm (Haemonchus contortus) infection than Canaria sheep, as manifested by reduced counts of fecal eggs and adult worm counts, as well as increased female worm stunting. Bioinformatics analysis of obtained sequence reads is in progress. However, a complete ovine transcriptome characterization, including transcript abundance estimation and detection of RNA editing and alternative splicing events, is hampered by the lack of a well-annotated ovine genome. A NIFA grant proposal was submitted to seek funding to improve genome annotation and to develop and integrate bioinformatics tools and methods needed for RNA sequencing analysis. Parasite-naïve dairy goats of the Alpine breed were obtained at birth and thirty were orally infected with barber's pole worm larvae. The remaining six goat kids were not infected and will serve as controls. The experimental infection is expected to be completed by mid-July and will be used to study correlations between parasitological variables and transcriptomics parameters during the next year.
1. Mechanisms underlying host resistance to parasitic infection in cattle were unraveled. Parasitic infections in ruminants represent a serious threat to livestock production worldwide. Knowledge on mechanisms underlying host resistance remains largely unknown. Towards this end, we characterized the transcriptome of the parasite-resistant cattle using high-throughput sequencing technology. We identified accurate cellular networks and biological pathways related to parasite resistance. We identified three ancient antimicrobials that are likely involved in regulating host-parasite interactions. Our results provide insights into the immune regulation of host-parasite interactions and molecular mechanisms of host resistance in cattle. This information facilitates applied breeding for parasite-resistant animals and development of vaccines that confer protection against all major parasite species.
2. The factors enhancing vaccine-induced host protection to parasitic infections were identified. Vaccination is one of the most rational and cost-effective strategies for parasite control in livestock species. An experimental vaccine against Ostertagia ostertagi (brown stomach worm), arguably the most important parasite in cattle, was developed by our European collaborators. Intramuscular injection of this vaccine resulted in a significant reduction (59%) of parasite eggs in cattle. To understand mechanisms triggered by the vaccine, we compared transcriptome responses of cattle treated with the vaccine and the controls. Sixty-nine genes were significantly impacted in the cattle protected by the vaccine. A correlation study demonstrated that two genes, granulysin and granzyme B, were negatively correlated with fecal egg counts and total worm counts, which are two of the most important indicators of parasitic infections. Our findings on granulysin production as host defense mechanism will help the development of more effective vaccines. This will benefit both producers and customers who desire meat and dairy products with reduced drug residues.
Van, M., Van, C., Borloo, J., Rinaldi, M., Li, R.W., Chiers, K., Van Den Broeck, W., Vercruysse, J., Claerebout, E., Geldhof, P. 2013. Granule-exocytosis of granulysin and granzyme B as a potential key mechanism in vaccine-induced immunity in cattle against the nematode ostertagia ostertagi. Infection and Immunity. 81(15):1798-1809.