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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Research Project #431978

Research Project: Immune, Molecular, and Ecological Approaches for Attenuating GI Nematode Infections of Ruminants

Location: Animal Parasitic Diseases Laboratory

2021 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.

Progress Report
Host cells and genes play important roles during infections with gastrointestinal nematodes. To date, attempts to evaluate cellular transcriptional responses in parasite-infected cows lack holistically. Therefore, changes in host peripheral blood mononuclear cells to infection with the pathogenic cattle parasite, Ostertagia ostertagi, have been monitored at 5 key timepoints by whole transcriptome sequencing. Data analysis of gene expression and biological pathways is currently underway with an endpoint of producing a detailed, comprehensive view of cattle immune response to infection. Initial analyses indicate that immune responses differed most at days 7 and 27 post-infection with pathways involved in the interferon-alpha expression, sialic-acid synthesis, and T-cell differentiation affected by nematode infections. More detailed analyses will be conducted after the collection of additional data. Such a dataset and subsequent analysis will aid understanding of how the disease progresses and suggest avenues for new vaccines. A recent publication compared the genetic makeup of over 80 different nematode species to identify a subset of common genes and proteins that may be used as vaccine targets or targets for new antiparasitic drugs. However, because evolution has shown us that nematode parasitism arose independently at least 13 different times in the past 500 million years, parasitic nematodes share fewer than half of their genes, limiting commonalities in this group. Thus, we endeavored to identify new vaccine and drug targets 1) by focusing rather than expanding our target species to only those parasites that show commonalities in livestock hosts, 2) by selecting only worms with economic importance to the U.S. cattle and sheep industries, and 3) selecting worms that closely share evolutionary trajectories. By analyzing annotated genomic and transcriptomic sequences of the cattle and sheep parasites Trichostrongylus, Ostertagia, Cooperia, Teladorsagia, and Haemonchus we identified 224 sequences conserved only among these 5 worm groups. Further assimilation of these proteins to the national drug databases has identified at least 10 chemicals that should interact with these proteins and may therefore possess anthelmintic activity for this subpopulation of livestock parasites. Work has now begun to lab test the efficacy of these drugs to kill the parasite worms of interest as a terminal step to identifying new anthelmintics before animal trials can begin. Ostertagia is an abomasal parasite that causes significant economic harm to cattle producers. Early immune responses to this parasite are poorly understood. ARS scientists at Beltsville, Maryland, showed that early immune responses to products secreted by parasites activate conflicting innate immune responses. Some of these responses favor inflammation and others disfavor, yet both are induced by the same subset of secreted parasite proteins, which is beneficial to the infection process. This benefit to the parasite may persist even after long-term and repeated parasite exposure. This may explain why cows can sustain infections with Ostertagia years after their initial exposure. Thus, improving vaccination may require focusing on production traits rather than the presence or absence of the worm. A manuscript was submitted and accepted on this subject matter Haemonchus contortus, a blood-sucking parasitic nematode of small ruminants, causes significant economic loss. Widespread drug resistance renders traditional deworming approaches ineffective. Vaccines hold promise as alternative measures for control. Parasite-derived proteases and protease inhibitors merit consideration as vaccine candidates because they influence host responses to infection. To this end, we have identified two H. contortus cathepsin B-like proteases (CBP). These help the parasite digest host defense proteins, evade host immunity, and prevent blood coagulation. Blocking these activities may enhance livestock immunity. Therefore, we cloned and expressed two novel CBP genes from H. contortus (Hc-CBP-1 and Hc-CBP-2) and showed one may be linked to parasite growth and pathogenicity. Using protein arrays, we showed the regions these proteins that induce host immune responses and may be important in protection. The work has culminated in one submitted manuscript. Progress was also made on PIM research by sequencing the Ostertagia L4 transcriptome using PacBio methodology on high-quality RNA. The obstacle to discovering additional PIMs (especially short peptides) has been the lack of a draft genome sequence and incomplete transcriptome data on key stages of Ostertagia ostertagi. Initial analysis by mass spectrometry of reverse-phase HPLC fractionated Ostertagia small peptides did not produce candidates. Therefore, we opted to develop complete Ostertagia transcriptomes. These will significantly facilitate future vaccine discovery research. The diagnosis of GI nematodes in production facilities is critical for ascertaining the need for drug intervention and if drug resistance is present on the farm and in the surrounding environment. A test was developed to differentiate and quantify mixed infections of GI nematodes using fluorescently-labeled polymerase-chain reaction products and a capillary-based sequencer. Tests on monospecific infections, experimentally mixed infections, and environmentally-derived samples have shown that the test meets all criteria as an antemortem assay for diagnosing nematode infections. The assay can identify Haemonchus, Ostertagia, Cooperia, Trichostrongylus, Oesophagostomum, and Nematodirus parasites at the genus level. All these parasite groups infect cattle and small ruminants, and drug resistance has been identified in most of them. Such a test will greatly enhance producers' ability to manage better their herds and the parasites that populate their pastures. Due to the emergence of anthelmintic resistance, there is an urgent need to develop alternative means to control gastrointestinal (GI) nematodes in cattle. The GI nematode, Ostertagia ostertagi poses a particularly challenging problem in that protective immunity induced by repeated natural infections is slow to develop. Studies were conducted to investigate the effects of repeated, experimental, drug-truncated infections (rDTI) on the development of protective immunity to Ostertagia. In this project, drug-truncated infections were achieved by terminating artificial infections prior to the parasite reaching full adult stage, i.e., 14-16 days post-infection. This process (DTI) kills the parasite in the tissues and allows the dead worms to decompose and serve as a source of antigens to the host. Repeating this process no less than 3 times (rDTI) boosts the Ostertagia-specific immunity induced by initial DTI. The advantage of such a procedure is that the immune responses are stimulated at the gastric mucosal site of infection, where vaccine delivery is highly challenging. Our studies demonstrated that eggs per gram of feces (EPG), a readout of infection, in the rDTI-treated group were significantly reduced relative to control animals. Both humoral and cell-mediated immune responses were involved in the response. The results indicate that contrary to natural infections, rDTI can induce protection against challenge infection. Current studies are focusing on identifying the parasite antigens responsible for eliciting the host protective responses. Understanding the diversity of GI nematode parasites in domestic livestock is important for developing comprehensive, integrated parasite management plans for farmers. A partnership has been initiated between the ASDA-APHIS National Animal Health Monitoring Survey, South Dakota State University, and USDA-ARS to examine GI nematodes in bison herds across the nation. The study will include a survey to understand anthelmintic treatment in these herds, followed by the collection of fecal samples. Molecular methods will enumerate resident GI nematode species and examine trends in species abundance following anthelmintic treatment. Where anthelmintic resistance is observed, population genetic techniques will be utilized to look for markers associated with resistance. Furthermore, these samples can be used to examine the genetic diversity within each parasite species to understand gene flow among parasite populations across the United States. The data produced can also be linked to future planned parasite collections from wild ungulate hosts to understand the links and potential transmission between wild ruminants and domestic livestock. This study is in the initial phases of execution and will provide an overview of parasites in bison herds and seek to identify subdivisions among parasite populations that could be treated as distinct management units.

1. Lessons from a previous period of climate change. The last ice age performed a “natural experiment” that drove geographic and genetic changes in mammals and their parasites. Many crossed the land bridge that once spanned the Bering Straits. To understand the lasting consequences of those changing climates and rising seas, USDA scientists worked for fifteen years with a team of University researchers (funded by the National Science Foundation) to map mammals and their parasites across Alaska and far Eastern Russia. They discovered how ancient climate changes shaped host and pathogen diversification, colonization, coevolution, and community assembly. The team collected over 53,000 mammal specimens and 14,000 parasite lots at nearly 9,000 locales. They produced over 200 publications and 20 graduate theses or dissertations. They illustrated how ancient events continue to shape our world. The team identified many cases where climate changes encouraged pathogens to ‘jump’ from one species of host to another. The work has great resonance as we reckon with a pandemic derived from a non-human host. The team’s collections were carefully archived and have continued to serve as a key resource for predicting and interpreting biotic responses to rapidly shifting environments. The work has been widely cited by many scientists. The data and concepts produced have value for wildlife biologists, veterinarians, epidemiologists, and ranchers contending with livestock parasites.

Review Publications
Cook, J.A., Galbreath, K.E., Bell, K.C., Campbell, M.L., Carriere, S., Colella, J.P., Dawson, N.G., Dunnum, J.L., Eckerlin, R.P., Geriman, S.E., Fedorov, V., Haas, G.M., Haukisalmi, V., Henttonen, H., Hope, A.G., Jackson, D., Jung, T., Koehler, A., Kinsella, M., Krejsa, D., Kutz, S.J., Liphardt, S., Macdonald, S.O., Malaney, J.L., Makarikov, A., Martin, J., Mclean, B., Mulders, R., Batsaikhan, N., Talbot, S.L., Tkach, V., Tsvetkova, A., Toman, H.M., Waltari, E., Whitman, J., Hoberg, E.P. 2016. The Beringian Coevolution Project: Holistic collections of mammals and associated parasites reveal novel perspectives on changing environments in the north. Arctic Science.
Braga, M.P., Araujo, S.B., Agosta, S., Brooks, D., Hoberg, E.P., Nylin, S., Janz, N., Boeger, W.A. 2018. Host use dynamics in a heterogeneous fitness landscape generates oscillations and diversification. Evolution.
Wiese, J.D., Caven, A.J., Zarlenga, D.S., Topliff, C.L. 2021. Gastrointestinal parasites of a reintroduced semi-wild Plains bison (Bison bison bison) herd: Examining effects of demographic variation, deworming treatments, and management strategy. International Journal of Parasitology. 14:216-227.
Bakshi, M., Hebert, D.A., Gulbronson, C., Bauchan, G.R., Tuo, W., Zarlenga, D.S. 2021. Ostertagia ostertagi mediates early host immune responses via macrophage and Toll like receptor pathways. Infection and Immunity. 1-42.