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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

2019 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
In the last National Animal Health Monitoring System (NAHMS) survey, nematode parasites (worms) ranked among the top concerns of U.S. cattle producers. This issue has been exacerbated because drug resistance, once relegated to a small subset of worms, is now pervasive and has been observed in all major groups of parasitic nematodes infecting cattle, sheep and goats. Thus, drug resistance is now the single biggest deterrent to controlling nematodes of large and small ruminants on North American pastures (Objective 3). Managing the problem is not only relegated to on farm control, but involves controlling transmission to and from local wildlife that can act as reservoirs to reintroduce pathogens back onto production facilities. A large scale, epidemiological study was completed to garner information on the prevalence of worms within U.S. wild ruminants that are also common in domestic livestock. Nationwide, Wildlife Departments from all 50 states were contacted with a request for wildlife fecal samples from which we could identify infecting species that are commonly found in the U.S. cattle industry. Twenty-five departments participated in the survey that spanned one hunting season. Over 600 fecal samples were received and analyzed for parasites. Samples from white-tailed deer, Caribou and Mule deer harbored the greatest percentage of infections. Among these, 180 positive samples were especially using molecular methods developed in our lab and compared to sequencing methods of collaborators at the Department of Veterinary Medicine, University of Calgary. Among the 14 different parasite species found in wild animals, 9 are commonly found in U.S. livestock, 7 of which have been documented as drug resistant. Ostertagia ostertagi, the most devastating nematode to the U.S. cattle industry was also the most prevalent in U.S. wildlife (52% of all positive samples contained this parasite). Likewise, the most harmful small ruminant parasites i.e., Haemonchus contortus and Trichostrongylus colubriformis, were found on average, in 25% of all samples tested. Trichostrongylus colubriformis is a parasitic nematode most frequently found in domestic sheep and goats. It is also commonly found in cattle along with the cattle species Trichostrongylus axei and among U.S. wild ruminants. It has been reported as resistant to not one, but all three major classes of anthelmintic drugs. In attempts to collate data from the drug resistant forms of Haemonchus, Cooperia and Teladorsagia, we embarked on a project to sequence the genome and transcriptome of Trichostrongylus colubriformis for comparative studies (Objective 2). To date, the genome has been sequenced and assembled. Transcriptomic and proteomic approaches for identifying genes and proteins that are regulated in drug–resistant parasites using RNA from the various stages of parasite development have been sequenced as well and are being compared (mapped) to the parasite genome. Within this population of genes and proteins, we hypothesize using this information to validate genetic markers 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 U.S. large and small ruminant production systems. It is well documented that cattle exposed to and infected with the parasitic worm, Ostertagia ostertagi, develop immunity slowly, possibly due to successful evasion of the host immune surveillance by the parasite. One possible mechanism is host immunosuppression by the parasite and its products. Studies have been performed to phenotype and enumerate immune cells present at the site of infection i.e., the host abomasum. Preliminary results showed that specialized immune cells, i.e., T cells (lymphocytes), are present in infected tissues, however, CD4 T cells that play an important role in adaptive immunity, appear more abundant than CD8 T cells which have a cytotoxic function. Ongoing research is to determine the relative abundance of T cells, B cells (immunity via antibodies) and antigen-presenting cells by surface-marker phenotyping. These cells will be further defined by the cytokines (immune-related macromolecules) they produce. We know the type of immunity (Th2) that is required for protections for GI nematode parasites. With the knowledge of the functional phenotypes of immune cells present at the site of infection with O. ostertagi, we will be able to design immune interventions capable of boosting and biasing host immunity towards the correct protective Th2 response against ostertagiasis. Ostertagia ostertagi is an important abomasal parasite of cattle. Adult worms produce excretory-secretory (ES) antigens and cause significant tissue damage. Understanding how these worms regulate early immune responses is a critical step in developing novel methods to stop parasite invasion. We know that O. ostertagi modulate local (abomasum) and systemic immunity to avoid a host protective response. However, we do not know how this happens. To this end, we first investigated the effects of O. ostertagi ES components on cultured bovine macrophage cells (important first responders to infection) by examining the expression of pro-inflammatory (TNFa, IL-1 and IL-6, co-stimulatory molecules CD40, 80 and 86) and anti-inflammatory (TGFß and IL-10) genes, and by morphological analysis. Second, we used these same macrophage cells to examine the potential for synergy among gut specific microbial proteins, Pathogen Associated molecular patterns (PAMPs or TLR ligands 2, 4 and 5) and ES products. PAMPs are molecules or ligands expressed by gut microbes, that bind to host cells to initiate intracellular signaling pathways and thus activate immune responses. Third, we studied the overall effect of O. ostertagi infection on the cells of the peripheral blood circulation by looking at immune receptors (Toll like receptors; TLRs 3,4, 5, 7, 8, 9, 10) at key time points after infection. Fourth, we characterized immune-related, genetic changes in peripheral blood mononuclear cells (PBMCs) collected from calves at key time points following infection hypothesizing that the parasite modulates the host systemic immunity during infection. Finally, high throughput, next generation sequencing will be utilized to ascertain a more holistic view of the host response to Ostertagia infection. Our results from the first study suggest that changes in cell morphology coupled with transcriptional expression in pro and anti-inflammatory genes coincide with macrophage activation by parasite secreted products. Second, the bacterial PAMPs enhanced (TLR-4 and TLR-2 ligands) and suppressed (TLR5 ligand) the pro-inflammatory activity of macrophages when used with O. ostertagi ES products implicating a link between TLR ligands and pro-inflammation during O. ostertagi infection. Third, quantitative PCR demonstrated that the peripheral immune cells display a mixed response as the infection progresses indicating active involvement of both pro and anti-cellular immune responses and may give cause to the absence of host protective immunity. Finally, future RNA sequencing analyses will dissect the overall functional and pathway genes analysis related to cell activation, that may underpin the drivers of impaired protective response against this parasite. Having a better understanding of these early stages of infection will improve our understanding of disease pathogenesis, vaccine development strategies and will present set of markers for diagnostic potential. Haemonchus contortus, is a blood sucking parasitic nematode of small ruminants that causes significant economic losses. Incorrect and excessive chemical deworming have resulted in drug resistance. Vaccine targets continue to be sought as cheap and alternative measures for control. Parasite-derived proteases and protease inhibitors have been used for controlling host responses to infection and therefore make good vaccine candidates. We therefore identified a class of proteases called “Cathepsin B-like” (CBP) that belong to family of proteins known as cysteine proteases. CBPs are proteins produced by parasites (intracellularly or extracellularly) that promote digesting host defense proteins that advance survival, host evasion and regulating blood coagulation. Controlling the parasite’s ability to digest proteins and evade the host will therefore protect the host against infection. Our analyses showed that identified CBP proteins possessed canonical active sites and classic secretory signals that may be secreted during infection. To this end, we have cloned and expressed three novel CBP genes from H. contortus, designated as Hc-CBP-1, Hc-CBP-2 and Hc-CBP-3 for further investigation. Preliminary analyses showed that one of the three CBP-1 is active and may be involved in nutrition, development and pathogenicity of H. contortus. The studies involving the testing of the functional activity and role in host evasion are currently ongoing. Following functional characterization, we will utilize a cocktail vaccine strategy to target these proteins against H. contortus infection in sheep.

1. Comparative genomics of the major parasitic worms. Parasitic roundworms and flatworms cause debilitating chronic infections of humans and animals, decimate crop production and are a major impediment to socioeconomic development. USDA scientists in Beltsville, Maryland, and University collaborators undertook a major survey of genes that modulate host immune responses, enable parasite migration though host tissues, or allow parasites to feed. They identified extensive, lineage-specific differences in core metabolism and protein families that have historically been targeted for drug development. From wide-ranging analyses involving computer modeling and simulations, they identified and prioritized new drug targets and compounds for testing. This is the broadest and most comprehensive such comparative study to date utilizing the genomes of parasitic and non-parasitic worms, providing a transformative new resource for the research community to understand and combat the diseases that parasitic worms cause.

2. Discovery of a key to parasitism in bovine nematodes. Traits shared by otherwise unrelated parasitic nematodes could be key to their ability to infect and harm livestock, so discovering them may provide new avenues for their control. By searching the genomes of parasitic and non-parasitic nematodes, ARS scientists in Beltsville, Maryland, discovered an enzyme (cyanase) exclusive to parasitic nematodes. Surprisingly, some nematodes appear to have acquired this ancient enzyme from plants, whereas others acquired it from bacteria, implying that completely unrelated events produced the same outcome. Interrupting the function of this evidently important component may assist in preventing or treating parasitic infection, which would be a great benefit to veterinary medicine and livestock production.

3. Stomach worms induce, but escape, extracellular traps secreted by cow neutrophils. Gastrointestinal parasite cause significant losses to the cattle industry. Despite suffusing the infected area with immune cells, cows generally fail to achieve a protective response. Neutrophils are some of the immune system’s “first responders,” and recent light has been shone on extracellular “traps” which can hinder the movement of pathogens. USDA researchers in Beltsville, Maryland, found that extracts of Ostertagia worms induced the formation of such traps, as did live parasites and also the free-living nematode Caenorhabditis elegans. This suggests neutrophil traps may be a conserved, early response mechanism against nematodes which cattle stomach worms, however, circumvent. Discovering this component of the immune response provides new avenues for refining vaccines targeting these and other parasitic worms, which are urgently needed as the major deworming drugs are waning in efficacy.

4. A new way parasites manipulate immunity. Understanding immune responses associated with host parasite relationships is key to developing alternatives to drug treatment i.e. vaccination. Comparing highly pathogenic and non-pathogenic parasite isolates, USDA scientists in Beltsville, Maryland, identified a protein (cytoplasmic dynein LC8 light chain) that was elevated in pathogenic forms of the parasite and downregulated in the non-pathogenic clones. They determined that this class of proteins is highly conserved in other parasites species, responds to calcium, a salt found in abundance in host intestinal tracts, is upregulated by agents that augment protein secretions, and is internalized by the host immune cells, stimulating messengers important to both the innate and the adaptive immune systems. Taken together, these results define this factor as a secreted immune modulator that cross-regulates host immunity. Ongoing work will determine the occurrence and function of similar proteins in other parasite types.

Review Publications
Greiman, S., Cook, J., Tkach, V., Hoberg, E.P., Menning, D., Hope, A., Sonsthagen, S., Talbot, S. 2018. Museum metabarcoding: A novel method revealing gut helminth communities of small mammals across space and time. International Journal for Parasitology. 48(13):1061-1070.
Zarlenga, D.S., Mitreva, M., Thompson, P., Tyagi, R., Tuo, W., Hoberg, E.P. 2018. A tale of three kingdoms: members of the Phylum Nematoda independently acquired the detoxifying enzyme cyanase through horizontal gene transfer from plants and bacteria. Parasitology. 146(4):445-452.
Beasely, H., Bennett, H.M., Coghlan, A., Cotton, J., Doyle, S.R., Gordon, D., Harsha, B., Huckvale, T., Lomax, J., Holroyd, N., Reid, A.J., Ribeiro, D., Rinaldi, G., Shafie, M., Stanley, E., Tracey, A., Berriman, M., Hallsworth-Pepin, K., Martin, J., Ozersky, P., Rosa, B.A., Tyagi, R., Zhang, X., Mitreva, M., Laetsch, D.R., Koutsovoulos, G., Kumar, S., Kaur, G., Blaxter, M., Howe, K.L., Leach, A.R., Mutowo, P., Rawlings, N., Kuo, T., Lee, T.J., Ke, H., Tsai, I.J., Wheeler, N.J., Day, T.A., Zamanian, M., Beech, R.N., Parkinson, J., Seshadri, S.L., Kikuchi, T., Maizels, R.M., Partono, F., Babayan, S., Allen, J.E., O'Boyle, N., Wang, L., Osuna, A., Cruz-Bustos, T., Samblas, M.G., Cuellar, C., Cooper, P.J., Devaney, E., Harcus, Y., Hodgkinson, J., Bah, G., Tanya, V.N., Eberhard, M.L., Asano, K., Rodriguez, P.F., Sato, H., Gilleard, J.S., Matthews, J.B., Cook, J., Toldeo, R., Scholz, T., Schnyder, M., Allan, F., Emery, A., Olson, P.D., Rollinson, D., Castillo, E., Kalbe, M., Eom, K.S., Horak, P., Mitreva, M., Hawdon, J.M., Urban Jr, J.F., Hill, D.E., Zarlenga, D.S., Bisset, S.A., Pfarr, K., Makepeace, B., Taylor, D.W. 2018. Comparative genomics of the major parasitic worms. Nature Genetics. 5:163-174.
Cao, L., Qu, G., Fetterer, R.H., Zhang, X., Tuo, W. 2019. Neospora caninum dynein LC8 light chain 2 is identical to that of Toxoplasma gondii and differentially produced by pathogenically distinct isolates. Parasitology.
Mendez, J., Sun, D., Tuo, W., Zhengguo, X. 2018. Bovine neutrophils form extracellular traps in response to the gastrointestinal parasite Ostertagia ostertagi. Scientific Reports. 8:17598.