Location: Animal Parasitic Diseases Laboratory
2023 Annual Report
Objectives
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.
Approach
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
Downregulation of the polymeric immunoglobulin receptor during Ostertagia ostertagi infection as a way to evade the host immunity and antigen-specific cytokine production by cattle immunized by drug-truncated infections. The polymeric immunoglobulin receptor (pIgR) mediates transport of immunoglobulin A (IgA) across the mucosal membrane. IgA is an essential antibody against any pathogens that enter the host and initiate infections from a mucosal site. Our research revealed the bovine stomach worm, O. ostertagi which parasitizes the gastric glands during the parasitic phase, prevents pIgR expression on the gastric epithelium immediately surrounding the worms without affecting the overall pIgR levels in the stomach. These findings implicate that the worm manipulates the host response or immunity by eliminating key molecules responsible for delivering important anti-worm antibodies, such as IgA. Further research to demonstrate the mechanisms of pIgR downregulation would facilitate vaccine development against the parasite.
Drug-truncated infections (DTI) elicit protective immunity in cattle. The cytokines induced by DTI represent an overall T helper 2 (Th2) immune response with Th1 and Th17 components. Overall, the ex vivo O. ostertagi antigen stimulates a dominating Th2 phenotype, characterized by elevated interleukin-4 (IL-4), IL-13, normal T cell expressed and presumably secreted (RANTES), monokine induced by interferon gamma (MIG), and Interferon gamma-induced protein 10 (IP-10). The upregulated IL-17A and IFNg are also consistent with their key role in anti-worm immunity. Some of these cytokines can be excellent biomarkers for the study of protective antigens. Further research is warranted to understand the host protective role of these cytokines regulated O. ostertagi.
Assessing the efficacy of natural products and repurposed drugs as novel alternatives to dewormers for animal and human parasites. Parasitic worms are a major threat to food animal production and have been a public health issue in developing countries. Anthelmintic resistance has abolished the effectiveness of classical drugs in parasite control. To tackle these issues, ARS scientists in Beltsville, Maryland, are evaluating the dozens of natural products for their antiparasitic activities using in vitro and in vivo models. In collaboration with researchers from several international institutions, ARS scientists found that several azole class compounds, particularly the modified compounds from the antifungal fluconazole, are potent against both fungal pathogens and parasitic worms such as Brugia and Trichuris. The broad-spectrum activity of these compounds represents new avenues for the treatment of various infections in humans and animals. Together, these chemically modified compounds and several new classes of natural products have the promise to be low-cost alternatives to traditional dewormers.
Understanding the molecular basis of resistance to identify potential paths to managing resistant parasites. One aspect of the response of gastrointestinal nematodes to anthelmintic drugs is differential expression of genes when the drug is present or absent. Studies were conducted to investigate the differential gene expression of worms that are resistant to ivermectin when in the presence or absence of the drug. Adult Cooperia punctata were isolated from ivermectin treated calves. Subsamples of these worms were left untreated or exposed to one of four different concentrations of ivermectin. Ribonucleic acid (RNA) was extracted from the worms following treatment, converted to cDNA, and then sequenced. The sequences were assembled into a novel transcriptome for Cooperia punctata and then expression levels for each treatment were determined. Subsequent analysis showed significant differences among treatments and identified 2,457 transcripts that were differentially expressed between the treated and untreated worms. Expression differences among different treatment concentrations have been evaluated and it was found that there were substantial overlaps between the two highest drug treatment concentrations and the two lower concentrations of drug, with little commonality between the low and high concentration treatments. It is suspected that worm death in the high concentration treatments is responsible for this difference. Confirmation and validation of this finding is ongoing as worm passages have been limited by availability of large animal experiments. The microRNAs have been collected but they have not yet been sequenced and analyzed.
Identifying geographic trends in the species composition of livestock parasite populations will allow for informed integrated parasite management plans. Previous efforts to identify genetic differences among parasite populations have not succeeded but recent advances in deoxyribonucleic acid (DNA) sequencing technologies may overcome previous limitations. In preparation for investigations of genetic diversity in different GI nematode populations, genomic resources and techniques were developed for three species of interest that infect cattle. Draft genomes were produced for Ostertagia ostertagi and Trichostrongylus colubriformis and the first transcriptome for Cooperia punctata was assembled. Currently, efforts are being made to produce more complete genomes for these species using long-read sequencing techniques that are now established in the lab. Genome assemblies have improved but remain in many fragments. New sequencing techniques are being pursued to improve these assemblies.
Establishing restriction site associated DNA sequencing (RADseq) protocols for Haemonchus contortus samples. The protocols have been shown to consistently provide information across the genome of target species even in the presence of contaminating fungal and bacterial sequences associated with fecal collection of worms. Low-pass genomic sequencing was shown to be an effective substitute for RADseq for population genetic analyses based on tracing epidemiological samples of a nematode species. Concurrently, metagenomic techniques were developed for examining species diversity within a single host. In association with this effort, custom local BLAST databases were configured to identify known nematodes based on DNA sequences stored in GenBank. For larger collection goals, substantial numbers of samples from wild and domestic ungulate hosts have been collected from multiple regions of the country through the National Animal Health Monitoring Survey. The parasites from these hosts were cultured to larval stages and counted. DNA was extracted from a portion of the pooled worms and subjected to metagenomic sequencing. General parasite species composition and abundance were determined for each of the samples using metagenomic techniques. Individual worms have been identified for low-pass whole genome sequencing including those that may represent novel or undescribed species.
Accomplishments
Review Publications
Liu, F., Smith, A.D., Wang, T.T., Pham, Q., Yang, H., Li, R.W. 2023. Ellagitannin punicalagin disrupts the pathways related to bacterial growth and affects multiple pattern recognition receptor signaling by acting as a selective histone deacetylase inhibitor. Journal of Agricultural and Food Chemistry. 71(12):5016-5026. https://doi.org/10.1021/acs.jafc.2c08738.
Liu, F., Smith, A.D., Wang, T.T., Pham, Q., Yang, H., Li, R.W. 2023. Multi-omics analysis detected multiple pathways by which pomegranate punicalagin exerts its biological effects in modulating host–microbiota interactions in murine colitis models. Food & Function. 14:3824. https://doi.org/10.1039/D3FO00286A.
Zhang, M., Li, R.W., Yang, H., Tan, Z., Liu, F. 2022. Recent advances in developing butyrogenic functional foods to promote gut health. Critical Reviews in Food Science and Nutrition. 1-22. https://doi.org/10.1080/10408398.2022.2142194.
Liu, J., Bai, Y., Liu, F., Kohn, R.A., Tadesse, D.A., Sarria, S., Li, R.W., Song, J. 2022. Rumen microbial predictors for short-chain fatty acid levels and the grass-fed regimen in angus cattle. Animals. 12(21). Article 2995. https://doi.org/10.3390/ani12212995.
Liu, F., Smith, A.D., Wang, T.T., Pham, Q., Cheung, L., Yang, H., Li, R.W. 2022. Biological pathways via which the anthocyanin malvidin alleviated the murine colitis induced by Citrobacter rodentium. Food & Function. 14(2):1048-1061. https://doi.org/10.1039/D2FO02873E.
Liu, J., Li, R.W. 2022. Tools and resources enabling marker gene-based microbiome studies. In: Wu, V.C.H., editor. The Gut Microbiome: Bench to Table. 1st edition. Boca Raton, FL: CRC Press. p. 3-44. https://doi.org/10.1201/b22970.
