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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

2020 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
Cattle suffer infections with the parasite Ostertagia ostertagi. Each local parasite population harbors tremendous genetic diversity. This constrains efforts to understand how extensively one population differs from another. Moreover, high background variation conceals variants that contribute to important traits, such as drug resistance. Understanding gene flow among populations could assist herd management. We therefore sought to develop genetic markers capable of defining parasite movement among locations. First, we generated a robust genome sequence with accurate gene annotation. We also sequenced approximately 37,000 genes and nearly 20,000 isoforms from Ostertagia messenger RNA (mRNA) coding for proteins from fourth stage larvae (L4). We will soon sequence mRNA from adult and third stage larvae. Together, these data will provide a comprehensive view of developmental changes in gene expression. We will use this information to define how much these parasites vary local, regional, and national scales to understand to properly bound parasite subdivisions. We will also compare parasites sampled from cattle to parasites from wildlife. Doing so will help establish whether wild ungulates are reservoirs for cattle infections. Recent progress sheds light on how cattle immunity responds to infection with Ostertagia. Particular cells and genes play important roles. But no one has yet attempted global assessment of cellular transcriptional responses. Therefore, monitored responses to large and small parasite doses, collecting peripheral blood mononuclear cells (PBMCs) at five intervals. We characterized gene expression at each timepoint. To understand how these responses change over time, we need to perform additional sequencing. We will analyze gene expression and biological pathways, producing a detailed, comprehensive view of cattle immune responses. This will aid understanding of how the disease progresses, and may suggest avenues for new vaccines. No vaccine against Ostertagia now exists. Subunit vaccines confer only partial protection. Host immunity develops slowly (in animals infected once, and in pasture-raised, naturally-infected animals subjected to recurrent exposure for years). We therefore sought to identify parasite-derived molecules that normally suppress host immune responses. Our working hypothesis holds that worms elevate host regulatory T cells (Tregs) which suppress anti-worm immunity. We found the parasite induces an important regulatory cytokine, interleukin-10 (IL-10) in neutrophils (white blood cells). This cytokine may regulate other immune cells (T-cells), suppressing immune responses to infection. We will therefore try to identify parasite molecules that induce IL-10 or Tregs as candidates for vaccination. We are also identifying vaccine targets by searching protein databases. Expression may differ among developmental stages of a parasite. Comparing expression among parasite species may identify shared proteins that regulate host immune responses. We made progress analyzing small molecular weight proteins, excretory/secretory proteins and whole worm proteins of Ostertagia using High-performance liquid chromatography and mass spectrometry. We also sequenced the transcriptome of Ostertagia and assembled and annotated a reference database capable of supporting future search efforts. Although gastrointestinal nematodes infect cattle continuously, these hosts do develop a modest level of immune protection. The worm’s cuticle, containing collagen-like proteins, change with each stage of development. This may help them evade immune responses. We experimentally infected animals with repeated, abbreviated infections of O. ostertagi (truncating each infection with anti-parasite drugs after 14 days). Doing so meant eliminating infections before parasites matured beyond the L4 stage. We observed reduced parasite egg shedding and increased/sustained body weight gain. This illustrates that cattle can be partially protected when the infection course is manipulated and/or host immune responses are enhanced. Future experiments will focus on improving immunity with reduced immunization time and frequency. We are comparing recently completed and annotated genomic and transcriptomic sequences of the cattle and sheep parasite Trichostrongylus colubriformis to those of other livestock parasitic nematodes (Ostertagia, Cooperia, Teledorsagia, and Haemonchus) in search of common drug targets. A recent paper analyzed over 56 nematode species to find common targets. But because parasitism arose at least 13 times in nematodes, parasitic nematodes share fewer than half of their genes. To identify new vaccine targets, we are targeting subsets of related parasites. We thereby identified 224 sequences conserved in 5 worms but absent from closely-related outgroups. Among these we are seeking candidates that share strong similarity, perform similar biological functions, and retain common antigenic sites susceptible to host antibodies. With international collaborators, we edited a special issue of “Frontiers in Immunology” entitled “Immunoparasitology: a unique interplay between host and pathogen.” The more than 30 high-quality papers explored cutting-edge research in parasite immunology, covering various aspects of parasite biology, host-parasite interactions, and control measures. This special issue highlighted how vaccines must function in hosts that are continuously exposed to multiple, interacting infections.

1. Parasitic infections downregulate cattle immune responses. Ostertagia parasites evade bovine immune responses. ARS scientists in Beltsville, Maryland, showed that the parasite facilitates its survival by decreasing production of an important immune-regulating protein (the cytokine interleukin-10) production in bovine white-blood cells. This protein regulates other immune cells in cattle. Thus, the parasite uses white blood cells, and the immune proteins they secrete, to suppress host immune responses. These data suggest new strategies for strengthening host responses to vaccination.

2. Allergic asthma does not require a suspect protein. Nematode parasites of the gut induce local (mucosal) immune responses. These parasites also induce systemic immune responses. Intestinal infections can therefore affect the lungs. Allergic asthma commonly induces scarring and damage in the lungs (pulmonary fibrosis). ARS scientists at Beltsville, Maryland, worked with University collaborators to investigate a protein suspected as promoting pulmonary fibrosis. They found this protein, TRPV4, is not required for developing allergic asthma, and should not be pursued as a target to diagnose or treat this type of lung disease.

3. Parasitic infections in cattle confuse early host immune responses. 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 from parasites activate conflicting innate immune responses. Some of these responses favor, and others disfavor, inflammation. The parasite may benefit by confusing early responses. The benefit may last even after long-term and repeated parasite exposure. This may explain why cows sustain infection years after their initial exposure. Improving vaccination may require focusing the immune response on productive responses.

Review Publications
Li, L., Si, H., Wu, S., Mendez, J.O., Zarlenga, D.S., Tuo, W., Xiao, Z. 2019. Characterization of IL-10 producing neutrophils in cattle infected with Ostertagia ostertagi. Scientific Reports. 9:20292.
Barone, C.D., Wit, J., Gilleard, J.S., Hoberg, E.P., Zarlenga, D.S. 2020. Wild ruminants as reservoirs of domestic livestock gastrointestinal nematodes. Veterinary Parasitology.
Suo, X., Wu, Z., Lillehoj, H.S., Tuo, W. 2020. Editorial: Immunoparasitology: A unique interplay between host and pathogen. Frontiers in Immunology. 11:880.
Palaniyandi, S., Rajendrakumar, A.M., Periasamy, S., Goswami, R., Tuo, W., Zhu, X., Rahaman, S.O. 2019. TRPV4 is dispensable for the development of airway allergic asthma. Laboratory Investigation.
Liu, J., Tuo, W., Wu, X., Xiong, J., Yu, E., Yin, C., Ma, Z., Liu, L. 2020. Immunoproteomic and mass spectrometric analysis of Eimeria acervulina antigens recognized by antisera from chickens infected with E. acervulina, E. tenella or E. necatrix. Veterinary Parasitology. 13:93.