Location: Animal Parasitic Diseases Laboratory2017 Annual Report
Objective 1. Identify and characterize parasitic immune modulators and local immune cell responses associated with GI nematodes of livestock. There is a pressing need for alternative control measures, such as vaccines, to complement/reduce antiparasite drug usage. Parasites evade host immunity by down-regulating or manipulating immune responses in favor of their own survival. Regulatory immune (T and B) cells that are up-regulated during infection may actually control an otherwise hostile environment and, in so doing, limit the host protective response. We propose to characterize parasite-provoked regulatory T/B cells. Further, parasitic immune modulators (PIMs) will be identified and characterized for their ability to induce host regulatory cells. Those PIMs responsible for cross-regulation will be selected as vaccine candidates. Objective 2. Identify proteomic and molecular markers for defining anthelmintic resistance among GI nematodes of livestock. Identifying genetic markers that differentiate the resistant and susceptible parasites will assist in herd management and long-term control. Our approach will be to utilize proteomic analyses and high-throughput transcript sequencing to discern genotypic differences that occur when resistant parasites are placed under drug selection. This will involve comparing drug treated vs. non-drug treated parasites that are resistant to macrocylic lactone class of drugs. This approach will minimize our risk of pursuing coincidentally-associated markers, and provide targets for new therapies. Putative new markers will be confirmed from environmentally-derived samples and will assist in reducing treatments with ineffective drugs. Objective 3. Explore the effects of accelerating climate change and ecological perturbation on managed and wild systems with emphasis on complex host-parasite relationships. Drug resistance cannot be viewed only as a problem of domestic livestock, but must include an evaluation of wild ungulates and the effects that environmental change can have on parasite transmission. With environmental change will come the movement of hosts and therefore exotic parasites into more temperate climates. Comprehensive definitions of parasite faunal diversity serve as the basis for exploring the impact of environmental change on complex systems, and are dependent on accurate baselines for host and parasite distributions. To discern the effects of accelerating climate change, information on parasite diversity will be obtained and summarized based on available georeferenced data from the literature, and on suitable biological collections. Basic studies in taxonomy, systematics and phylogeny of specific nematode groups will enable us to define species diversity in complex faunas among wild and domesticated North American ruminants. Parasite distributions will be mapped using geographic information systems (GIS) and Species Distribution Models (SDM) to assess the effects of global change on how invasion, colonization, and climate change influence the dissemination and persistence of drug resistance genes in the GI nematodes of ruminants.
Objective 1. Hypothesis: Parasite infection-elicited host regulatory cells are directly or indirectly induced by the parasitic immune modulators (PIMs). Rationale: Our recent report indicates that B cells and T cells with regulatory phenotypes are expanded in abomasa and the draining lymph nodes (dLN) in cattle raised on pastures. The proposed studies will investigate if single-infection by O. ostertagi, C. oncophora, or H. contortus, and mixed infections thereof induce a similar change in immune cell phenotypes. The ability of PIMs from ES products to enrich host regulatory cells will be investigated. Objective 2: Hypothesis: Drug treatment uniquely alters the genetic and proteomic profiles in resistant worms, enabling the identification of parasite targets associated with the resistance phenotype. Rationale: It is anticipated that the mechanism of resistance to macrocyclic lactones (ML) will be conserved among this broad group of parasites. We intend to focus on C. punctata, given that resistance is well documented and we possess two strains resistant to Ivermectin or Dectomax. Also, once putative markers have been identified, these can be tested against drug resistant forms of Trichostrongylus and Haemonchus, which are also available at our facility. Given the high level of genetic variation in and between nematode populations, resistant and sensitive isolates will also differ genetically in ways unrelated to resistance. To overcome this problem, we will compare proteomic and genomic data from a given, resistant isolate in the presence or absence of ML, hypothesizing that drug treatment will alter proteomic and gene expression profiles. This approach will focus on those genes and gene products regulated by drug treatment and which may differ from those isolates that are not drug resistant. Preliminary data will be generated using Ivermectin and the data will be validated using Dectomax. Objective 3: Hypothesis: Goal: Characterize diversity among species of Haemonchus in North American ruminants. Rationale: Surveys continue to broaden our understanding of parasite diversity, and establish baselines to assess changing patterns of distribution. Several key taxa remain poorly known in North America, and important biogeographic zones have been poorly documented. Borderland areas between managed and natural systems remain a concern given the poor understanding of species diversity for nematodes and their exchange between free-ranging and domestic hosts. Haemonchus nematodes are present in the Nearctic by recent anthropogenic introduction. Thus, radiation in tropical environments suggests this fauna is currently constrained in distribution by patterns of temperature and humidity. Global distributions are attributable to human-related translocation with domestic stock and would be anticipated to respond to accelerating climate warming and environmental change. To better assess change and distributions, we will collect and characterize isolates of Haemonchus and other parasites both morphologically and genetically, then use GIS and SDM’s to model parasite distributions based applications and mapping for ruminant helminth faunas.
Host-parasite interactions determine the outcome of host protection from the infection. Such interactions are mediated by parasite-regulated and -biased host immune cells as well as immune-related proteins i.e., cytokines (Objective 1). Our recent work demonstrated that cattle can respond to Ostertagia ostertagi infection as early as 3 days post infection, with a massive immune cell mobilization in the abomasum and draining lymph nodes. The response is very rapid, considering that the parasite only resides within the gastric glands for a few days during the parasitic stage of its life cycle; however, it never invades the host tissue. Cattle raised on pasture can get exposed to gastrointestinal (GI) nematode parasites, including O. ostertagi which is considered the most pathogenic cattle GI nematode of the abomasum; though the blood feeding and drug-resistant Haemonchus is on the rise. Both T and B cells are up-regulated in the abomasum and lymph nodes in cattle when raised on pastures or when experimentally infected. B cell expansion and/or migration may account for most of the changes occurring during the infection. Cytokine genes expressed by the infected abomasum and the draining lymph nodes are indicative of mixed T helper cell profiles, including both anti- and pro-inflammatory responses. Ongoing work will continue to determine the phenotypes of immune cells and the cytokines regulated by GI nematodes and define bovine protective immune responses against the GI nematodes. These data will be used as the basis for future vaccine design and formulation. Annexins are a group of proteins that are ubiquitous in nature. As a family of proteins they are important in various cellular and physiological processes such as providing a membrane scaffold for cell shape. Also, annexins are involved in trafficking and organization of vesicles, exocytosis, endocytosis and calcium ion channel formation. Ion channels are well known drug targets in nematodes. Annexins are not only intracellular but also exist in the extracellular space where they have been associated with inflammation and apoptosis among other things. We identified and characterized a family of annexin-like proteins in Ostertagia as potential vaccine candidates (Objective 1). More than 20 of the Ostertagia annexin-like gene transcripts are present throughout the parasitic life cycle stages; however, they are most abundant during the adult stage. They are now being investigated as vaccine candidates targeting the adult stage of the worm. Parasites harbor and secrete proteins that allow them to modulate host immune responses (Objective 1). In so doing this provides an environment within the host that is conducive to parasite longevity. We identified a class of secreted enzymes in nematodes i.e., apyrases, that degrade ATP and AFP and in so doing control inflammatory responses and blood coagulation. Parasites of the genus Haemonchus require blood meals beginning as early as the fourth stage and feeding continues into and throughout the adult stage. Infections with Haemonchus cause severe anemia in moderately to heavily infected animals. The apyrase specific to Haemonchus was cloned, expressed, purified and is currently being use in a pilot study to vaccinate cattle against the parasite. The hypothesis is that controlling the parasite’s ability to degrade ATP and ADP will result in blood coagulation and interfere with the parasite’s feeding process thereby protecting the host against the parasite and reduce blood loss. If successful, the approach will be tested for use on other common worm parasites of cattle using the parasite specific recombinant protein. We have identified from Beltsville pastures, a drug-resistant strain of the small and large ruminant parasite, Trichostrongylus colubriformis. In attempts to collate data from the drug resistant forms of Haemonchus and Cooperia we currently have in storage, we have begun to sequence the genome and transcriptome of Trichostrongylus for comparative genomics. Also, the parasite samples have been generated to advance both transcriptomic and proteomic approaches for identifying genes and proteins that are regulated in drug–resistant parasites as a result of drug treatment (Objective 2). Within this population of genes and proteins, we hypothesize that genetic markers will exist for differentiating drug-resistant from drug-sensitive worm populations. This will provide the means to identify and manage the early stages of drug-resistant parasites within US beef herd production systems. Assessment of Haemonchus specimens (Objective 3) and insuring we have pure stocks of nematodes (Objective 2) demand accurate, sensitive and specific methods of identification; in particular stages which are morphologically indistinguishable i.e. eggs. Diagnosing GI nematodes is also critical to determining if drug intervention is necessary and if drug resistance is present on the farm and the surrounding environment. Work has been advanced in developing a test to differentiate and quantify mixed infections of GI nematodes, using fluorescently-labeled polymerase-chain reaction (PCR) products and a capillary-based sequencer. Tests on monospecific infections, experimentally-mixed infections and environmentally-derived samples have now shown the test to meet all criteria as an antemortem assay for nematode infections. The test can identify at the genus level Haemonchus, Ostertagia, Cooperia, Trichostrongylus and Oesophagostomum parasites. All these parasite groups infect cattle and small ruminants and drug resistance has been identified in most of them. Explorations of parasite diversity in ungulates involve first developing a database that collates a dispersion of literature documenting the distribution of nematode parasites in domestic and free-ranging ungulates in North America (Objective 3). We have focused on the period of time following 1964 and the report by Willard Becklund documenting the diversity of nematodes and there distributions. Our new database also incorporates data populated in the former US National parasite Collection. These will form the basis for ecological niche modeling needed to explore the spatial and host distributions for these parasites. We originally proposed to evaluate additional isolates of Haemonchus that show morphological and genetic variation inconsistent with current documentation on this pathogen (Objective 3). New isolates of Haemonchus spp., especially from the region of west Texas and New Mexico have not become available as yet. Our existing lots of specimens that have been the basis for characterizing the current problems in diagnostics and potentially taxonomy, remain incomplete. Thus, we have shifted to studies needed to characterize the recently recognized invasive species, Lamnema chavezi, in exotic South American camelids and that have established in North America. In collaborations with veterinary diagnostic services at several universities, we acquired larval nematodes from the livers of camelids from California at necropsy, and also a large number of adult specimens from Argentina. These studies involve comparative morphology and DNA sequencing based on multi-locus nuclear and mitochondrial genes. NexGen methods are being used to characterize the genome of this species as a basis for identification, diagnosis and completion of a phylogenetic placement of this stomach parasite among related nematodes.
1. Cattle raised on pasture have higher levels of gastro-intestinal inflammation than those raised on grain. When compared to cattle raised indoors and on grain, cattle raised on pasture have higher levels of abomasal (stomach) and lymph node inflammation. Pasture-raised cattle were also diagnosed with Ostertagia ostertagi, a cattle abomasum-specific nematode parasite. Among other cell types, white blood cells (B cells and CD4 T cells) with a potential regulatory phenotype were elevated at the site of infection and in peripheral blood in pasture-raised cattle. ARS scientists in Beltsville, Maryland, determined that infection and pathology caused by nematode parasites such as Ostertagia ostertagi contribute to reduced weight gain in cattle raised on pastures as a result of the inflammatory response and reduced feed intake. The results of this study emphasize the need for GI nematode parasite control in pasture-raised animals, and the development and application of vaccines which are compatible with organic cattle production systems.
2. Identification of a South American nematode of camelids in North American llamas and alpacas. The exotic trichostrongyline nematode parasite of llamas and alpacas, Lamema chavezi, was recently discovered for the first time among North American populations of these Southern Hemisphere camelids and the parasites were not characterized when originally reported. Molecular data necessary for accurate diagnosis of migrating larvae (in the liver) and adult nematodes in the upper small intestine had not been developed. Working with academic collaborators, ARS scientists in Beltsville, Maryland, developed the first authoritative DNA markers to definitively identify adults and larval parasites of this species, providing a rapid pathway for identifying larvae during migration in the liver, and a basis for confirmation of identity for eggs in feces. These techniques substantially add to the toolkit used by veterinarians and disease ecologists in federal, state, and private facilities for understanding the distribution of these parasites and the potential for disease. This assay will assist in assessing the dissemination of this once exotic pathogen in North America, and determine the source of introduction into Western and South Western U.S.
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Bell, K.C., Kendall, C., Hoberg, E.P., Demboski, J.R., Cook, J.A. 2016. Temporal and spatial mosaics: deep host association and shallow geographic drivers shape genetic structure in a widespread pinworm, Rauschtineria eutamii. Biological Journal of the Linnean Society, London. 119:397-413.
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Cook, J.A., Greiman, S., Agosta, S., Anderson, R.P., Arbogast, B.S., Baker, R.J., Boeger, W., Bradley, R.D., Demboski, J.R., Dobson, A.P., Dunnum, J.L., Eckerlin, R.P., Esselstyn, J., Galbreath, K., Hawdon, J., Hoesktra, H., Kutz, S., Light, J., Olson, L., Patterson, B.D., Patton, J.L., Philips, A.J., Rickart, E., Rogers, D.S., Siddall, M., Tkach, V., Hoberg, E.P. 2016. Transformational principles for NEON sampling of mammalian parasites and pathogens: a response to Springer et al. (2016). Bioscience. 54:787-799.
Hoberg, E.P., Makarikov, A., Tkach, V., Meagher, S., Nims, T., Eckerlin R.P., Galbreath, K.E. 2016. Insights on the host associations and geographic distribution of Hymenolepis folkertsi (Cestoda: Hymenolepididae) among rodents across temperate latitudes of North America. Parasitology Research. 115:4627-4638. doi:10.1007/s00436-016-5255-3.
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