Location: Animal Parasitic Diseases Laboratory2013 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:
The availability of tests to accurately and rapidly diagnose GI nematodes from animal feces is critical to determining if drug intervention is necessary and if drug resistance is present in the herd. A DNA-based test has been developed using fluorescently-labeled products and a capillary-based sequencer. Preliminary data indicates that the test is able to differentiate and quantify mixed infections of parasite using fecal eggs. To date, tests to validate “proof-of-principle” were performed on monospecific infections of nematodes that commonly infect U.S. cattle i.e. Haemonchus, Ostertagia, Cooperia, Trichostrongylus and Oesophagostomum, among which drug resistance has been identified. The test has been shown to be both specific and semi-quantitative in both lab-prepared and field samples. Fundamental information on the biology, evolution, and genetics of nematodes is essential to identifying new therapies. Information is lacking on the roles that specific groups of proteins i.e. protein families, and specific inheritable regions within these protein families i.e. “domains”, play in the evolution of parasites and their abilities to adapt to new environments. To this end, the transcriptomes of two cattle nematodes, 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 after it enters the host. Sequences were compared to each other and to available databases for transcriptional differences and similarities. In addition, protein families and domains among 11 nematodes were investigated to identify pan-specific targets that delineate parasitic nematodes from free-living nematodes and that can act as new targets for drug development. Two manuscripts were written and submitted on these areas of study. Internal parasites modulate the host immune responses, enhancing their local environments and promoting infection. As such, understanding the relationship between nematode pathogens and the host immune response is critical to developing methods to attenuate infections. One parasite, Ostertagia ostertagi is capable of eliciting a massive immune response in cattle, yet finding a way to prevent a protective response from developing. A number of Ostertagia-derived immune modulators were characterized. Macrophage migration inhibition factor (OoMIF) is one such modulator that is a parasite secreted protein with an apparent mass of 12kDa. The gene was cloned from Ostertagia and the recombinant protein was expressed. OoMIF has been localized to the hypodermis/muscle cells and to reproductive systems. Studies demonstrated that OoMIF possesses tautomerase and immunoregulatory activities, and the ability to form dimmers and trimers in vitro. Given its function, OoMIF is now being investigated for its role in cross-regulating host immunity during the host infection process in the hope of identifying a new target for drug intervention.
1. Widespread drug resistance in cattle nematodes. In collaboration with Academic, Industry, and Government partners, ARS scientists conducted a broad, scientific survey to assess the effectiveness of drug treatment on nematode infections. Prior to this study, drug resistance had been observed in nematode parasites of sheep and goats, but only anecdotal information pointed to resistance in cattle. In approximately one-third of the surveyed operations, early resistance to treatment was documented. Although some of these treatment failures might have been the result of improper drug application, follow-up studies established drug resistance as having contributed most of the time. Results from this study unequivocally demonstrated that overuse of drugs: 1) increased treatment failures; 2) changed the distribution of parasite species in U.S. production facilities; and 3) caused resistance to develop in parasite species previously responsive to treatment. This study was the first comprehensive look at nematode parasitism and drug resistance in cattle in any developed country in the world. This study provided needed information to help assess management practices and address the loss of available drug intervention strategies and clearly demonstrated a rapid rise in the prevalence of cattle GI nematodes that are resistant to the most commonly used anthelmintics.
Yin, J., Qu, G., Cao, L., Li, Q., Fetterer, R.H., Feng, X., Liu, Q., Wang, G., Qi, D., Zhang, X., Miramontes, E.N., Jenkins, M.C., Zhang, N., Tuo, W. 2012. Characterization of Neospora caninum microneme protein 10 (NcMIC10) and its potential use as a diagnostic marker for neosporosis. Veterinary Parasitology. 187(1-2):28-35.