Location: Animal Parasitic Diseases Laboratory2012 Annual Report
1a. Objectives (from AD-416):
Objective 1: Identify genetic markers associated with parasite phenotypes that are resistant to anthelmintic drugs. Objective 2: Develop state-of-the-art assays to diagnose and quantify GI nematodes in ruminants resulting in better pasture management and reductions in anthelmintic dependency. Sub-Objective 2a: Determine the relative contribution of parasite genera in eggs from cattle harboring mixed nematode infections. Sub-Objective 2b: Develop a simple and rapid test for identifying nematode eggs. Objective 3: Identify parasite products that function as modulators of the host immune response.
1b. Approach (from AD-416):
Objectives will be attained using molecular/genomic, immunological, and epidemiological techniques. The drug resistant phenotype in bovine nematodes results from mutation(s) and/or change(s) in transcriptional patterns in a subset of genes. Transcriptome sequencing will be used to develop markers for the resistance phenotype. State-of-the-art assays are required to diagnose and quantify GI nematodes to help reduce drug use. Two diagnostic tests will be developed: one using size fragment analysis, an offshoot of Sanger Sequencing technology to quantify infection levels of each parasite species, and a second using loop-mediated isothermal amplification (LAMP) technology for rapid testing in veterinary or regional clinical labs. Finally, parasite products that function as modulators of the host immune response will be identified by their ability to stimulate CD4+ T cell responses, and elicit cytokines known to be important in protection. Changes in host regulatory cells (Tregs) induced by the presence of parasites will also be studied to understand how the parasite down regulates or manipulates the host immune response to its benefit.
3. Progress Report:
Fundamental information on the biology, evolution, and genetics of nematodes is essential to identifying new drug targets. The transcriptomes of two parasitic nematodes that are important pathogens of cattle, Cooperia and Ostertagia, were sequenced focusing on all available larval stages (L1+L2, L3, L4) and male and female adult worms. The stages were arbitrarily partitioned into "free-living" and "parasitic" life stages to better evaluate the differences between the external parasite environment and the infection process. Sequences were compared to each other and to available databases for transcriptional differences and similarities. Because of the rise in parasite resistance to drug intervention, it is important to understand the level of dissemination of resistance in the U.S. A beef cattle survey was conducted in 2008-2009 to identify the incidence of gastrointestinal (GI) nematode infections in U.S. cattle operations, and to assess the effectiveness of commercial drugs to control these parasites. In the current year, "scrubbing" the data was completed and manuscripts were prepared for publication. Understanding protein evolution is important for deciphering processes that drive species diversity and adaptation, and that will eventually assist in parasite control. The dynamics of change in protein families and protein domains (conserved functional regions within proteins) over the course of metazoan evolution were explored by large scale sequencing. Change, as defined by birth/death (appearance and disappearance) and duplication/deletion events within protein families and domains, was analyzed using representatives of the three major metazoan groups i.e. vertebrates, arthropods, and nematodes. A manuscript was prepared and submitted. Accurately and rapidly diagnosing GI nematodes is critical to determining if drug intervention is necessary and if drug resistance is present on the farm. Work was advanced to develop a test for differentiating and quantifying mixed infections of GI nematodes, using fluorescently-labeled polymerase-chain reaction (PCR) products and a capillary-based sequencer. Preliminary tests were performed on monospecific infections to validate the ability to discern Haemonchus, Ostertagia, Cooperia, Trichostrongylus and Oesophagostomum. All these parasite groups infect cattle and drug resistance has been identified in most of them. Understanding the relationship between nematode pathogens and the host immune response is critical to developing methods to attenuate infections. To this end, the process of obtaining sufficient parasite sample was initiated. Sufficient adult worms from the cattle nematodes, Ostertagia and Cooperia, were obtained and processed for whole worm extract and excretory secretory antigens for use in developing immune assays and studying the parasite's ability to regulate host immune cells. The macrophage migration inhibition factor (OoMIF) from Ostertagia was cloned, expressed, purified, and characterized. Recombinant OoMIF was demonstrated to have tautomerase and oxidoreductase activities. Its potential to regulate bovine T cell responses is now underway.
1. Sequencing of the Cooperia and Ostertagia transcriptomes. Gastrointestinal nematodes of cattle cause an estimated $2 billion in lost annual revenue to producers. Efforts have been advanced to sequence the transcriptomes (all genes that encode proteins) in the Cooperia and Ostertagia genomes because Ostertagia is among the most pathogenic cattle-infecting species and because drug resistant forms of Cooperia are now surfacing among U.S. cattle producers. Transcriptome data are important resources for studying the genetics and the biology of parasitic worms. Comparisons were generated between expressed sequences of these two parasites as a way to better identify their similarities and differences; these comparisons also include other parasitic and non-parasitic nematodes whose genomes and transcriptomes have been sequenced to near completion. ARS scientists at Beltsville, MD, in collaboration with scientists in Melbourne, Australia, Ghent, Belgium, and St. Louis, MO characterized the transcriptomes from multiple life stages of both Cooperia oncophora and Ostertagia ostertagi. The results of this study showed distinct differences in the enzymes involved in the free-living and parasitic stages of their life cycles. As an example, domains previously implicated in parasite growth and development were more prevalent in the free-living stages; whereas domains potentially involved in feeding dominated in the parasitic stages. These data will allow future comparative analyses on the biology, evolution, and adaptation to parasitism in nematodes. Further examination of these differences may reveal conservation in genes important to infection, immune response, and host recognition, and thus provide targets for the development of new and broad-based anthelmintics.
2. A national survey of cattle gastrointestinal (GI) nematodes and the effectiveness of anthelmintic treatment was completed. Prior to this study, drug resistance had been observed predominantly in nematode parasites of sheep and goats, but only anecdotal information pointed to resistance in cattle species. Scientists at Beltsville, MD in collaboration with those at the University of Minnesota, Colorado State University, Merck Animal Health, APHIS, and cattle operations randomly selected throughout the U.S., participated in an effort to assess the mode, regiment, and effectiveness of drug treatment, and its relationship to clearing nematode infections. Results unequivocally demonstrated that overuse of drugs has: 1) increased treatment failures; 2) changed the distribution of parasite species; and, 3) caused resistance to develop in parasite species previously responsive to treatment. Results also showed that certain nematode species, in particular Cooperia punctata, originally believed to be innocuous can cause harm to animal health. Genetic studies revealed in nearly all instances that resistance was due to Cooperia punctata and/or Haemonchus. As a result, overuse of drugs has not only selected for drug-resistant nematodes, but also has selected for species with elevated pathology in the host. Furthermore, this has changed the population dynamics of parasites on pastures. This study was the first comprehensive look at nematode parasitism and drug resistance in cattle in any developed country in the world. The results have been submitted for publication and will advance guidelines to help manage GI nematodes in the American cattle industry. Our results clearly demonstrate that there has been a rapid rise in the prevalence of cattle GI nematodes that are resistant to the most commonly used anthelmintics. This is important and needed information to assist in altering management practices to address the absence of available drug intervention strategies.