2008 Annual Report 1a.Objectives (from AD-416) The goals of this research are to evaluate the host pathogen relationship using genomics and immunologically-based approaches, and use this information to develop methods that complement and/or reduce drug intervention as a means to control gastrointestinal (GI) nematodes of cattle. In this regard, we will identify structural variations in the bovine genome that influence host resistance to GI nematodes, pinpoint functional and/or structural variations in parasite genes associated with the drug resistant phenotype, identify and enhance immune factors that regulate the host-pathogen interface as an alternative to anthelmintic treatment, and finally use this collective knowledge to develop integrated parasite control programs that incorporate marker-assisted selection schemes that reduce economic loss caused by the parasites. These goals will be attained through a systems-based approach that will employ molecular, immunological, and genomic techniques to studies at the gene, cell and whole animal levels. 1b.Approach (from AD-416) Studies will focus on using genomic approaches to develop novel means to control parasitic diseases in cattle, and on-farm application of these techniques. These studies will investigate both the parasite genome and the host genome. Studies focusing on the parasite will evaluate genetic factors associated with drug-resistant and drug-susceptible phenotypes in parasitic nematodes. Investigations of the host genome will focus on the identification of host genetic loci that affect resistance to parasite infection and will develop and implement genome-wide selection or marker-assisted selection programs based on high density SNP information. Additionally the project will use SNP haplotypes to refine and characterize QTL for parasite resistance that were identified in earlier studies. At the same time studies will continue to characterize the host immune response to parasitic infection using microarrays and real time PCR to discern gene expression patterns in cattle demonstrating different levels of resistance and/or immunity to the parasites. Finally, the information gleaned from these studies will be applied on-farm through co-operative research efforts with producers to establish practical management programs to control and diminish the effects of parasites on production effeiciency. 3.Progress Report Working with USDA, APHIS, National Animal Health Monitoring System (NAHMS), a survey was developed as part of the NAHMS Beef 2007-08 Study to identify the incidence of gastrointestinal nematode infections in US cattle and to assess the effectiveness of currently used anthelmintics. Preliminary results indicate that current anthelmintic practices have changed both the historic distribution of parasites and are resulting in treatment failures at numerous locations. This study is the first comprehensive look at nematode parasitism in cattle in any developed country in the world. The results will provide important guidelines concerning proper management and treatment programs for the American cattle industry. This research supports National Program 103 Component 7: Countermeasures to Prevent and Control Parasitic Diseases. Completed genotyping of over 700 phenotypically characterized Angus cattle using the Bovine SNP50 chip. Initial analyses indicated QTL for the level of parasite eggs passed in the feces on 7 bovine chromosomes with 2 chromosomes carrying multiple loci. These data will provide loci for further analyses aimed at identification of structural variations that after resistance and tolerance to the parasites and also will yield molecular markers that can be used in cattle selection programs. This research supports National Program 103 Component 2: Genetic and Biological Determinants of Disease Susceptibility The function of P-glycoprotein (Pgp), which confers multi-drug resistance by active efflux of drug, is dependent upon phosphorylation. Nematode apyrase genes were identified in Ostertagia and Trichostrongylus, cloned and expressed as a functionally active proteins capable of dephosphorylating di- and trinucleotide phosphates. We have begun to sequence Pgp from drug susceptible and resistant forms of Cooperia punctata to determine if there is a link among variants of this gene to the drug resistant phenotype. This research supports National Program 103 Component 7, countermeasures to prevent and control parasitic diseases, problem statement a) drug resistant gastrointestinal (GI) parasitic disease. Experimentally controlled infection with Cooperia oncophora was conducted to identify recognition and inflammatory pathways in the host-parasite relationship. A total of 310 sequences were differentially expressed during the course of infection, and 22 canonical pathways and 9 regulatory networks were significantly affected by the infection. Goblet cells, columnar epithelial cells, smooth muscle cells, and other cell types have been individually isolated using laser capture microarray (LCM) from the bovine small intestine of both the infected and control calves. Regulation of mucin biosynthesis pathway in goblet cells during the parasite infection is being investigated using real-time RT-PCR, western blotting, immunohistochemistry, and comparative genomics. These studies represent the first attempt to understand the host-parasite relationship using this cutting-edge genomics tool in cattle. This research supports National Program 103 Component 7: Countermeasures to Prevent and Control Parasitic Diseases 4.Accomplishments 1. Library construction from susceptible and resistant nematode lines. The rise and persistence of nematode resistance and selection against the most widely used anthelmintics, in particular, the avermectins and milbemycins (AM), highlight the importance of understanding genetic variation in the parasite populations. Identifying markers that differentiate the resistant and susceptible phenotypes will provide a mechanism by which parasites circumvent drug intervention. To this end, pure populations of drug resistant and drug susceptible Cooperia punctata were generated and sufficient genetic material has been isolated. One BAC library comprising 10,00 primary clones from drug-resistant Cooperia punctata and 2 normalized cDNA libraries comprising 25,000 clones each from drug susceptible Cooperia oncophora and drug resistant Cooperia punctata were constructed to identify genes differentially expressed as a result of drug treatment, and to identify molecular markers for the drug resistant phenotype. The back library and genomic DNA will be used in next generation sequencing to produce a 1-3X coverage of the Cooperia punctata genome. These are critical first steps to using population genetics for identifying genetic markers associated with drug resistant nematodes. This research supports National Program 103 Component VII, countermeasures to prevent and control parasitic diseases, problem statement a) drug resistant gastrointestinal (GI) parasitic disease. 2. Development of a national survey of cattle nematode parasitism and the effectiveness of currently used anthelmintic treatment practices in controlling parasitism. Working with our sister agency, USDA, APHIS and 2 University collaborators a comprehensive National Survey was designed and initiated. Selected cattle operations throughout the U.S. were given the opportunity to participate. This is the first comprehensive survey of this kind in any cattle producing region of the world. This research supports National Program 103 Component VII, countermeasures to prevent and control parasitic diseases, problem statement a) drug resistant gastrointestinal (GI) parasitic disease. 6.Technology Transfer Number of Active CRADAs 1 Review Publications Sonstegard, T.S., Marcos, D., Gasbarre, L.C., Van Tassell, C.P. 2006. Use of box-cox transformation in analysis of fecal egg count data. World Congress of Genetics Applied in Livestock Production. 8(15-35):1-4. Silva, M.V., Van Tassell, C.P., Sonstegard, T.S., Cobuci, J.A., Gasbarre, L.C. 2006. Estimates of genetic parameters using random regression models for gastrointestinal helminthoid data in angus cattle. World Congress of Genetics Applied in Livestock Production. 8:15-34. Silva, M.V., Van Tassell, C.P., Sonstegard, T.S., Cobuci, J.A., Gasbarre, L.C. 2008. Estimates of (co) variance components using random regression models for gastrointestinal helminthoid data in Angus cattle. Brazilian Animal Science Society. CD-ROM MEL036. p. 105.