Location: Animal Parasitic Diseases Laboratory
2024 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
In refining the drug-truncated infection (DTI) procedure in O. ostertagi infected cattle, researchers in Beltsville, Maryland, found that anthelmintic treatment on day 15 post infection produces the maximum protection with long lasting immune responses. Due to the lack of a critical piece of equipment, the irradiator, researchers in Beltsville, Maryland, were unable to establish T cell lines for antigens screening. Instead, researchers in Beltsville, Maryland, performed a vaccine trial using the 15-day old O. ostertagi worm extract, based on a hypothesis that the protective antigens must be rich at this stage. Immunized animals had very strong immune responses. Unfortunately, researchers in Beltsville, Maryland, failed to detect protection with reduced fecal egg counts and increased weight gain in immunized animals. The possible reasons that attribute the failure in protection include 1) presence of inhibitory molecules in the total worm extract and 2) the type of adjuvant which was unable to promote a Th2 response. The progress is related to Objective 1, Sub-objective 1A.
Immunosuppression slows development of anti-worm immunity, enabling parasites like O. ostertagi to repeatedly infect cattle in successive grazing seasons. Attempts to understand and address such problems rely on untested assumptions that the immune system of cattle mirrors that of people and mice, which scientists best understand. Working with scientists at University of Maryland College Park, researchers in Beltsville, Maryland, documented an important difference in the signaling molecules that govern T-cell responses to infectious stimuli. The team learned that CD4 T cells central to host defense do not express interleukin 4, a hallmark of such cells in human and murine models in anti-worm immunity. The findings implicate the complexity of the anti-worm immune responses in ruminants, and unveiling alternate immune pathways is likely the key to resistance against parasite infections. The progress is related to Objective 1, Sub-objective 1A.
Using RNA sequencing technology, researchers in Beltsville, Maryland, analyzed the global immune responses of abomasal mucosa, the internal surface lining of the 4th chamber of the bovine stomach, of O. ostertagi infected cattle. The results show that most of the differentially expressed genes are associated with immune responses, cellular reorganization, and cell migration and proliferation. In particular, numerous genes associated with T cell exhaustion, Th1, Th2 and Th17 responses were upregulated. Overall, the naïve cattle exposed O. ostertagi for the first time do not develop a typical Th2-dominant response which is required for protection. The results may in part explain why animals infected by the parasite or immunized by total worm extract are incapable of initiating immune responses fostering a protective immunity. The progress is related to Objective 1, Sub-objective 1A.
To understand the genetic and epigenetic bases of host resistance, researchers in Beltsville, Maryland, collaborated with scientists in The Commonwealth Scientific and Industrial Research Organization (CSIRO) in Armidale, Australia, challenging merino sheep with Trichostrongylus colubriformis. They did this knowing that host genetics modestly control parasite resistance (heritability estimates ranging between 0.01 to 0.65 for traits influencing fecal egg counts). Decades of selective breeding produced lambs resistant to either of the two most important parasites in ruminants: Haemonchus contortus and Trichostrongylus colubriformis. The fecal egg counts in the grazing season differed markedly between resistant and susceptible lambs (P = 1.45 x 10-9). Resistant lambs produced 12.8-fold fewer eggs per gram of feces. The sheep lines also significantly differed in their susceptibility to parasite establishment, when challenged with infection. On average, only 87.9 worms were recovered from resistant lambs, compared to 7421.3 from susceptible lambs (N = 20 per groups, P = 1.85 x 1015). ARS researchers characterized gut microbiome samples collected in the rumen and proximal colon tissue using full-length 16S rRNA gene and employed advanced bioinformatics tools including machine learning. Doing so identified several microbial predictors with high accuracy for the resistant. They also characterized the host intestinal transcriptome of these samples using RNAseq and unraveled the microRNA fraction of the transcriptome using deep sequencing. Using sophisticated algorithms, they identified novel biological pathways underlying resistance. These findings may help select selecting for resistance in other ruminants, contributing to sustainable parasite control. The progress is related to Objective 2.
Researchers in Beltsville, Maryland, characterized transcriptional changes induced in cattle peripheral mononuclear cells responding to Ostertagia ostertagi infection and presented their findings at the Gordon Research Conferences series on the biology of host-parasite interactions. The feedback received from experts in the field of parasitology will enhance future work to distill lessons and applications from these immunological responses. The progress is related to Objective 3.
Understanding the molecular bases of drug resistance may offer methods to better manage such parasites. One means to do so entails exposing resistant parasites to drugs to identify which genes thereby undergo increased or decreased expression. Researchers in Beltsville, Maryland, therefore investigated transcriptional responses to the presence of drugs in worms resistant to doramectin, a variant of ivermectin. To do so, they isolated adult Cooperia punctata from calves treated with doramectin. They employed four concentrations of doramectin to worm subsets, leaving a fifth group untreated, before snap-freezing them in liquid nitrogen. Once characterized, gene expression will be compared among groups and to worms previously treated with ivermectin. The progress is related to Objective 3.
Accurate genome assemblies underpin a wide range of research aimed at better parasite control. Therefore, researchers in Beltsville, Maryland, endeavored to improve genomic resources for the most pathogenic cattle worm, Ostertagia ostertagi, by generating copious long-read sequencing data. They improved the genome assembly 6-fold (reducing it from ~19,000 to ~3,000 fragments). Long stretches of shared gene organization in a related nematode parasite, Haemonchus contortus, enabled construction of a chromosomal hypothesis for the new O. ostertagi genome assembly. This scaffold will serve as a reference for future population genetic analyses. The team is undertaking similar efforts for the Cooperia punctata genome, which is proving more difficult to resolve using this approach. The progress is related to Objective 4.
Boson herd health can be influenced by the diversity of parasites they harbor. To understand local diversity, researchers in Beltsville, Maryland, collected nematodes from 953 bison living on 56 farms across the United States. They extracted DNA from pools of worms and generated DNA sequences, which identify each species infecting each host. A signature collection of parasite species typified each farm. Bison on each farm differed in the relative proportion of each such parasite. Parasite with no regional difference in parasite species composition. These data suggest that each farm may require a parasite management plan customized to its parasite community, all the more so where worms have acquired resistance to anthelmintic drugs. The researcher team in Beltsville, Maryland, is therefore evaluating these samples for the occurrence of anthelmintic resistance genes, to provide producers with actionable information to better manage parasitic disease. The progress is related to Objective 4.
In order to understand the evolution of populations, DNA sequences from individual animals are required. Unfortunately, individual worms from the main species of nematodes infecting cattle, sheep, and goats provide very little DNA as they are so small. Consequently, most DNA sequencing efforts have focused on extracting DNA from pools of hundreds or even thousands of individuals making it difficult to understand informative signals in the DNA sequence as the history of mutation is confounded by histories in multiple individuals that may have been separated for long periods of time. Researchers in Beltsville, Maryland, pioneered new techniques to acquire DNA from individuals and sequence their genomes to uncover what was hidden in previous sequencing efforts. After sequencing multiple individuals from multiple hosts on the same farm, it is apparent that certain nuclear locations are more suitable for analysis than others for understanding population subdivision and adaptation. This has provided a path forward for subsequent investigations into the movement of parasites among locations in the United States. The progress is related to Objective 4.
Accomplishments
1. Development of potent yet versatile mucosal vaccines. Many parasites and viruses use mucosal surfaces as their portal of entry. Blocking their transmission therefore requires tools that elicit protective mucosal immunity, which differs from the humoral immunity elicited from typical intramuscular vaccines. ARS researchers in Beltsville, Maryland, collaborated with researchers at University of Maryland to demonstrate efficacy of a novel Covid-19 vaccine delivered through the mucosal surface immunoglobulin receptor. Importantly, it conferred protection and reduced transmission. This vaccine delivery platform also counters influenza transmission. These results herald a new means to combat viruses and parasites acquired through mucosal surfaces in the airway and the gut, such as stomach worms and avian influenza, in cattle.
2. Safe antimicrobial alternatives for gut health. Excessive use of antimicrobials in food animal production may have contributed to the emergence and rapid spread of antimicrobial resistant genes. Dietary modulation of gut microbiota can lower the need for antibiotics, but many plant-derived antimicrobial alternatives lack efficacy and specificity. Therefore, ARS scientists in Beltsville, Maryland, demonstrated that a predominant compound in pomegranate, punicalagin, possesses potent antimicrobial activities against a broad range of pathogens, including viruses, bacteria, fungi, and parasites, at physiologically relevant concentrations. The team also unraveled how this natural product dampens intestinal inflammation. The findings will facilitate the development of pomegranate related natural products to promote gut health.
3. Harnessing parasite worms for human wellness. Graft-versus-host disease poses a potentially lethal complication of bone marrow transplantation, requiring methods to regulate host immunity. Because certain parasites downregulate inflammation and modulate gut microbiota, ARS researchers in Beltsville, Maryland, collaborated with university partners to investigate their potential to help transplant patients by promoting transplant tolerance. These findings provide further evidence that parasites and their proteins can offer clear benefits to patients as a complementary therapy.
4. Blueberry anthocyanins benefit children with diarrheal diseases. Diarrheal diseases caused by enteric pathogenic Escherichia coli infection represent a severe threat to public health, particularly in children younger than five. The disease is traditionally controlled by antibiotics, but antibiotic resistant strains and severe side effects of antibiotic therapy require safe and sustainable alternatives. Therefore, ARS scientists in Beltsville, Maryland, evaluated the efficacy of a blueberry-derived flavonoid for its potential as a complementary therapy. They discovered that this flavonoid acts as a key regulator of intestinal inflammation and tissue repair in animal models. The consumption of fruits rich in the flavonoid promotes the growth of probiotic Bifidobacterium species and regulates microbiota interactions. These results will provide a roadmap for developing blueberry-related health-promoting products.
Review Publications
Fleming, D.S., Liu, F., Li, R.W. 2023. Differential correlation of transcriptome data reveals gene pairs and pathways involved in treatment of Citrobacter rodentium infection with bioactive punicalagin. Molecules. 28(21). Article e28217369. https://doi.org/10.3390/molecules28217369.
Kandel, A., Li, L., Wang, Y., Tuo, W., Xiao, Z. 2024. Differentiation and regulation of bovine Th2 cells in vitro. Cells. 13(9). Article e13090738. https://doi.org/10.3390/cells13090738.
Liu, F., Smith, A.D., Wang, T.T., Pham, Q., Hou, P., Cheung, L., Yang, H., Li, R.W. 2024. Phospholipid-rich krill oil promotes intestinal health by strengthening beneficial gut microbial interactions in an infectious colitis model. Food & Function. 15(5):2604-2615. https://doi.org/10.1039/D3FO04980A.
Lin, Y., Jung, H., Bulman, C.A., Ng, J., Vinck, R., O'Beirne, C., Moser, M.S., Tricoche, N., Peguero, R., Li, R.W., Urban, J., Le Pape, P., Pagniez, F., Moretto, M., Weil, T., Lustigman, S., Mitreva, M., Sakanari, J.A., Gassera, G. 2023. Discovery of new broad-spectrum anti-infectives for eukaryotic pathogens using bioorganometallic chemistry. Journal of Medicinal Chemistry. 66(23):15867-15882. https://doi.org/10.1021/acs.jmedchem.3c01333.
Li, R.W., Moon, C.D., Morgavi, D.P. 2023. Rumen microbiome dynamics and their implications in health and environment. Frontiers in Microbiology. 14. Article e1223885. https://doi.org/10.3389/fmicb.2023.1223885.
Li, W., Wang, T., Rajendrakumar, A.M., Acharya, G., Miao, Z., Varghese, B.P., Yu, H., Dhakal, B., Leroith, T., Karunakaran, A., Tuo, W., Zhu, X. 2023. An FcRn-targeted mucosal vaccine against SARS-CoV-2 infection and transmission. Nature Communications. 14(1). Article e7114. https://doi.org/10.1038/s41467-023-42796-0.