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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Research Project #441382

Research Project: Molecular, Immune and Microbiome Approaches for Mitigating GI Nematode Infections of Livestock

Location: Animal Parasitic Diseases Laboratory

2022 Annual Report

Objective 1: Characterize host immune responses and identify vaccine candidates and therapeutics that mitigate the impact of parasite infections. Sub-objective 1A: Characterize truncated infection-induced host immune responses and identify T cell-stimulating antigens as vaccine candidates. Sub-Objective 1B. Test and optimize the efficacy of an enterically coated Cry5B formulation. Objective 2: Characterize and modulate the host microbiome to enhance resilience to parasitic infections. Objective 3. Identify genetic markers that discriminate between drug susceptible and resistant strains of nematodes. Objective 4. Use molecular epidemiology to investigate the role of wild ruminants as sources of nematode infections in domestic livestock. Sub-Objective 4A. Establish sampling procedures for population genetic analyses of GI nematodes of domestic and wild ungulates. Sub-Objective 4B: Survey the biodiversity of parasites in sympatric wild and domestic ruminants of the United States. Sub-Objective 4C. Population genetic structure of multiple GI nematode species across the United States.

Identify the most effective infection and drug treatment strategies that allow for maximized in-tissue immune stimulation by killed O. ostertagi, thereby increasing protection against challenge infection. Characterize the synergy between rumen protected methionine and probiotics on growth in parasite naïve goats. Test the synergistic effect of sequential exposure of parasites to two therapeutic treatments, the conventional anthelmintic drug Cydectin and Cry5B paraprobiotics, in reducing worm burden in sheep under natural infection on pasture. At necropsy, worm burden in the entire GI tract will be determined from actual counts of worms collected from the abomasum and small intestine. Parasite species, sex, and developmental stages will be recorded. Longitudinal repeat measure fecal egg count data will be transformed using the square root, log, or other means, and then be analyzed using a mixed model (linear or non-linear) in the R nlme package. Focus on Cooperia punctata, given that resistance is well documented and we possess two strains resistant to Ivermectin (IVM) or Doramectin (DM). fecal samples will be collected from 10 cattle from four locations that are separated by at least 50 miles and 10 white-tailed deer collected within 10 miles of the sampled farms. Fecal cultures will be prepared using a modified coproculture technique and DNA extracted. We will assess the abundance of various coinfecting species using the Nemabiome technique. use non-invasive techniques to assay wild and domestic parasite species in six regions of the United States based on the concentration of cattle production, natural geographic divisions, and wild ungulate species: the six regions refer to the Southeast, Northeast, Upper, Central, and Southern Midwest, and Southwest. Sequence individuals from these collections using RADseq technology and bioinformatic tools to examine population genetic structure for as many species as we have sufficient sampling (at least 30 individuals from multiple regions), taking advantage of the samples collected for the above discussed biodiversity assay.

Progress Report
Drug-truncated infection (DTI) in cattle. Ruminants only gradually acquire immunity to gastrointestinal (GI) nematodes and typically get re-infected each grazing season. ARS scientists in Beltsville, Maryland, have shown that repeatedly truncating cattle infections elicits protective immunity to Ostertagia ostertagi. This immunity is stronger than when worms undergo uninterrupted development. Drug treatment evidently induces dying worms to release protective antigens. Peripheral T cells respond to stimulation by Ostertagia antigens and produce cytokines and chemokines promoting Th2 immune response. Ongoing studies will optimize the timing and dosage of drugs to maximize protective immunity against re-infections. Identification and characterization of parasite cathepsin B-like proteins. Haemonchus contortus (Hc) is a blood-feeding gastrointestinal worm that causes significant economic losses to the small ruminant industry worldwide. ARS scientists in Beltsville, Maryland, are exploring cathepsin B-like proteins (CBPs) as vaccine candidates. ARS scientists identified and characterized two novel CBPs, Hc-CBP-1 and Hc-CBP-2, in the excretory secretory products (ESP) and in tissues of adult worms. ARS scientists screened peptide arrays of recombinant proteins with antibodies to identify key immunogenic peptides. ARS scientists also analyzed transcription of the genes encoding Hc-BBP-1 and Hc-CBP-2 and assessed the function of mature recombinant proteins in regulating cell function. Each protein localized to the brush borders of the intestine. Peptide displays, screened with antibodies, revealed dominant and overlapping epitopes. Adult worms secrete Hc-CBP-1. Worms in immunosuppressed animals increased their expression of Hc-cbp-2 three-fold; immune suppression did not influence expression of Hc-cbp-1. Recombinant Hc-CBP-1 suppressed mRNA expression of bovine peripheral blood mononuclear cell cytokines/activation markers. These results suggest that Hc-CBP-1 and Hc-CBP-2 influence cellular and immunological activities and may provide viable targets for attenuating H. contortus infections. Bovine peripheral blood immune responses analyzed by single-cell RNA sequencing. The general responses of cows to immune stimuli reflect the distinct activities of numerous cell types, whose independent actions remain largely uncharacterized. Single cell transcriptional analysis opens new doors for understanding these constituent responses. Therefore, ARS scientists collaborated to define immune cell compositions and immune responses by functional subsets of peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharides (LPS), using single-cell RNA sequencing (scRNA-seq) technology. ARS scientists discerned seven major cell types, including CD4 T cells, CD8 T cells, B cells, monocytes, natural killer cells, innate lymphoid cells, and dendritic cells using the cell subset-specific gene markers. ARS scientists tracked differential gene expression, cell cycle progression, cellular differentiation, and chromatin accessibility and trait-relevant cell types. These results advance the understanding of bovine immune response and demonstrate the usefulness of scRNAseq technology to define functional subsets of immune cells. Exploring parasite transcriptome for mechanisms of anthelmintic resistance. The emergence of anthelmintic resistance in GI nematodes of cattle drives a need to understand the molecular basis of resistance. GI nematodes respond to anthelmintic drugs, in part, by changing their expression of genes. ARS scientists in Beltsville, Maryland, therefore investigated differential gene expression of ivermectin-resistant worms in the presence of absence of the drug. ARS scientists isolated adult Cooperia punctata from ivermectin-treated calves. ARS scientists left some of these worms untreated and exposed others to increasing concentrations of ivermectin (three replicates of four drug concentrations). ARS scientists then extracted RNA from the worms, converted RNA to cDNA, and sequenced these using short read next generation sequencing technology. ARS scientists assembled the sequences into a novel transcriptome and evaluated global gene expression, finding significant treatment effects, and identified 2457 transcripts that were differentially expressed between the treated and untreated worms. Ongoing analyses seek to identify the biological and metabolic pathways most affected by drug treatment. Sequencing worm genomes to understand parasite species distribution in livestock. Identifying geographic trends in the species composition of livestock parasite populations will improve integrated parasite management plans. Previous efforts to identify genetic differences among parasite populations have not succeeded, but recent advances in DNA sequencing technologies may overcome previous limitations. In preparation for investigations of genetic diversity in different GI nematode populations, ARS scientists in Beltsville, Maryland, developed genomic resources and techniques for three species that infect cattle. ARS scientists produced draft genomes for Ostertagia ostertagi and Trichostrongylus colubriformis and the first transcriptome assembly for Cooperia punctata. ARS scientists established long-read sequencing methods to pursue more complete genomes for these species. ARS scientists also established protocols for restriction site associated DNA sequencing (RADseq) for Haemonchus contortus, benefitting from an available genome reference. The protocols provide genomic information even in the presence of contaminating fungal and bacterial sequences commonly occurring in fecal collections. ARS scientists are also evaluating a newer technique, called low-pass sequencing, which may prove superior to RADseq in future population genetic analyses. ARS scientists also developed metagenomic techniques to examine species diversity within a single host. To do so, ARS scientists configured custom local BLAST databases to identify nematodes and protists using DNA sequences stored in GenBank. ARS scientists contacted University of Maryland faculty and organizers of the Maryland State Fair to acquire samples from across the state. Sample collection in August and September of 2022 will include animals raised on both sides of Chesapeake Bay, which may subdivide parasite populations.

1. Improved computational tools for assessing how microbiome composition changes through time. The composition of the microbiome changes through time, confounding attempts to understand animal responses to changes in diet, veterinary treatment, or husbandry conditions. However, irregular sampling intervals and missing data sometimes plague efforts to understand these responses. ARS scientists in Beltsville, Maryland, developed user-friendly workflows for longitudinal genomic and microbiome analyses. The compared and improved batch correction and group difference detection algorithms to better discern changes through time. These improvements enable microbiologists, veterinarians, and livestock producers to harvest better data, from fewer animals, when seeking to understand how the microbiome changes through time.

Review Publications
Bilska-Zajak, E., Thompson, P.C., Rosenthal, B.M., Rozycki, M., Cencek, T. 2021. Infection, genetics, and evolution of Trichinella: Historical insights and applications. Infection, Genetics and Evolution. 95:105080.
Zarlenga, D.S., Barone, C., Hebert, D.A., Santin, M., Newcomb, H. 2021. A simple method for identifying and quantifying gastrointestinal nematodes of cattle. Parasitology Research. 120(12):3979-3986.
Bilska-Zajac, E., Tonanzi, D., Pozio, E., Rozycki, M., Cencek, T., Thompson, P.C., Rosenthal, B.M., La Rosa, G. 2021. Genetic structure uniformity substantiates transmission of Trichinella spiralis from one swine farm to another. Parasites & Vectors. 14:359.
Oh, S., Li, R.W. 2022. Large-scale meta-longitudinal microbiome data with a known batch factor. Nature Methods.