2009 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 efficiency.
Nematode apyrase (AP) genes in Ostertagia and Trichostrongylus, were functionally characterized cloned and expressed as recombinant proteins capable of dephosphorylating di- and trinucleotide phosphates. Results demonstrated that the parasite secretes and utilizes these proteins to enrich energy production following its emergence from dormant growth stages. Results further suggest that the presence of this protein within the esophageal bulb make this a prime candidate for vaccine targeting. Work has begun to evaluate TGF-beta homologues and other proteins involved in the growth and development of parasitic nematodes as targets for parasite control. Sequencing of the Ostertagia genome has begun with accompanying data being generated on all 3 key life-cycle stages of the parasite. Collaborations have been advanced looking at transcription of Glutamate chloride ion channels as targets for parasite resistance to drug intervention.
Completed the Ostertagia challenge experiment in order to understand molecular basis of protective immunology and initiated transcriptome characterization. Using metagenomics, characterization of gut microbiota was initiated to understand rumen microbial dynamics in response to Ostertagia infection. Analysis of regulatory/genetic network and pathways of the bovine small intestine during Cooperia infection were completed. Using laser capture microdissection we completed cell-specific analysis of mucin-related genes in individual cells during a Cooperia infection.
Genome-wide association analysis using the SNP data to identify regions of the genome affecting parasite indicator traits was completed; 8 chromosomal candidate regions were identified. Ongoing targeted re-sequencing is being performed to identify the genes or genetic variation affecting different host response to parasite infection; however, this relies on completion of haplotype maps of the BARC Angus herd.
A survey was conducted as part of the NAHMS Beef 2007-08 Study to identify the incidence of gastrointestinal nematode infections in U.S. cattle operations and to assess the effectiveness of currently used antihelmintics to control these parasites using both parasitological and genetic analyses to determine the species of parasites resistant to drug treatment. Results clearly demonstrated that overuse of antihelmintic treatments have changed the historic distribution of parasite species infecting cattle in the US and are resulting in increased treatment failures. This study was the first comprehensive look at nematode parasitism and drug resistance 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.
National survey of effectiveness of current anthelmintic treatment practices in controlling parasitism commpleted. A comprehensive national survey of cattle nematode parasitism and the effectiveness of currently used antihelmintic treatment practices in controlling parasitism has been completed in collaboration with APHIS and 2 University collaborators. Selected cattle operations throughout the U.S. were given the opportunity to participate. Results are currently being evaluated for the presence and distribution of drug resistance in cattle nematodes. Genetic studies are being performed to evaluate the species of parasites infecting these animals.
Identification of canonical pathways and regulatory networks that were significantly affected in the bovine small intestine during Cooperia infection. In continuing with understanding the relationship between host immune responses and parasite infections, we identified a total of 22 canonical pathways and 9 regulatory networks that were significantly affected in the bovine small intestine during Cooperia infection. This work led to the characterization of a novel class of cell adhesion molecules involved in recognition of carbohydrates on parasitic nematodes.
Fecal egg count (FEC) is used to identify and quantify gastrointestinal parasite infestations. FEC values are not “normally” distributed and a small percentage of the herd is responsible for the majority of parasite transmission. In an attempt to bring FEC distribution close to normality, logarithmic transformations have been used for these data. Unfortunately, normalization of the distribution is not achieved in the majority of the cases, resulting in the necessity of using less sensitive non-parametric statistics analyses, and the inability to fit the data into more complex genetic analyses. In this study we compared an extension of the Box-Cox transformation to determine the efficiency of this transformation compared to log transformation. Between 1992 and 2006, 12,450 observations of FEC were collected from 498 animals (males and females) from the BARC Angus herd. Contemporary group was defined as animals entering in experiment together. There were 19 contemporary groups in the study. The statistical model used in ANOVA included the contemporary groups, sex of the animal and age at test, as fixed effect, and error, as random effect. Results show that Box-Cox transformation reduced coefficients of asymmetry in all the variables studied. Plotting the residuals (stem-leaf and Normal plot graphs for normal distribution and box-plot for homogeneity of variance) for each trait and checking the parameters of the normal distribution and homogeneity of variance, Box-Cox was always superior to log. As the log transformation is a special case of the Box-Cox, this transformation will be, at least, equal to log, never worse.
Library construction from susceptible and resistant nematode lines. The rise and persistence of nematode resistance and selection against the most widely used antihelmintics, 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 10,000 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.
5.Significant Activities that Support Special Target Populations
In collaboraton with scientists from West Virginia University and Schering Plough-Intervet, variation was studied in the effectiveness of different classes of anthelmintics on fecal egg counts in cattle entering the West Virginia Southern Bull and Replacement Heifer Test Program. Each year, the West Virginia University Extension Service, the West Virginia Department of Agriculture, and the West Virginia Cattleman’s Association conduct the Southern Bull and Replacement Heifer Test Program. In 2006, researchers observed, that in spite of a strict vaccination program on arrival, test cattle developed more health problems than expected. Fecal samples were found to have high egg counts in many cases, even though the animals had been treated multiple times with cattle wormers. In 2007, fecal samples were collected from the rectum of 86 heifers from 9 different producers, and 103 bulls from 13 producers on delivery to the test station. At this time, the bulls were treated with an oral benzimidazole (fenbendazole) and the heifers with a pour-on endectocide (ivermectin). Fecal samples were taken from the animals for a second time, 14 days after treatment. Initial egg counts did not differ between the bulls and heifers (49.4 and 42.4 respectively). In contrast, endectocide treatment reduced fecal eggs counts by only 45 %, whereas treatment with an oral benzimidazole reduced the mean fecal egg count by 95% ( 26.9 and 2.2 respectively). In the heifers the effectiveness of the treatment varied according to the source of the animals (range of 83 to 26% reduction). These results indicate that the resistance to endectocides can be demonstrated in cattle operations in West Virginia, and that the effectiveness of endectocides can vary markedly among cattle operations. Producers need to carefully monitor the effectiveness of their anthelmintic treatments.