Research Project: Molecular Approaches to Control Intestinal Parasites that Affect the Microbiome in Swine and Small Ruminants

Location: Animal Parasitic Diseases Laboratory

2019 Annual Report

Objectives
Objective 1. Determine the change in the intestinal metabolome and microbiome during parasitic nematode infection and after anti-parasitic clearance. Sub-objective #1. Characterize parasite-induced molecular mechanisms that modulate intestinal inflammation. Sub-objective #2. Evaluate the potential impact of Cry5B on the native and parasitized gut microbiome. Objective 2. Identify pan-nematode secretome products with immune modulating activity that along with nutritional supplements eliminate parasites and enhance enteric health. Sub-objective #1. Use antibodies from parasite infected pigs and goats to select for immunogenic cloned parasite products that have been computationally identified as vaccine targets. Sub-objective #2: Test for cloned parasite products that induce innate immune responses at the mucosal surface of explanted intestinal tissues from pigs and goats.

Approach
Objective 1. Determine the change in the intestinal metabolome and microbiome during parasitic nematode infection and after anti-parasitic clearance. Sub-objective #1. Characterize parasite-induced molecular mechanisms that modulate intestinal inflammation. Hypothesis #1: Parasitic infections alter the relative abundance of butyrate-producing bacteria in the gut and change the composition and concentration of total short-chain fatty acids (SCFA) as well as anti-inflammatory butyrate, which in turn modulates intestinal inflammation and host immunity. Sub-objective #2. Evaluate the potential impact of Cry5B on the native and parasitized gut microbiome. Hypothesis #2: The administration of the Cry5B anthelmintic will have minimal effects on the native microbial community in the gut due to its transient nature and invertebrate gut targets. Hypothesis #3: Parasite-induced changes in the microbiome will be restored to the native structure and function after treatment with Cry5B that reduces worm burden. Experimental design: Quantifying changes in the intestinal metabolome and gut microbiome induced by parasitic infection, and characterizing the abilities of anti-parasitic treatments to restore altered gut microbiota, are important in dissecting mechanisms of host pathophysiology and immunity. We will conduct an in-depth comparison of the gut metabolome and microbiome between animals randomly assigned to two conditions (naive and infected) and exposed to Cry5B in an optimally determined delivery system. Objective 2. Identify pan-nematode secretome products with immune modulating activity that along with nutritional supplements eliminate parasites and enhance enteric health. Sub-objective #1. Use antibodies from parasite infected pigs and goats to select for immunogenic cloned parasite products that have been computationally identified as vaccine targets. Hypothesis #1: Immunization of target host species with computationally selected immunogenic cloned parasite products will disrupt parasitism and prevent infections. Sub-objective #2: Test for cloned parasite products that induce innate immune responses at the mucosal surface of explanted intestinal tissues from pigs and goats. Hypothesis #2: Selected cloned immunogens that also have innate immune features defined by responses in intestinal explants will enhance vaccine efficacy and disrupt parasitism. Experimental design: Powerful new technologies to characterize the transcriptomes from multiple life stages of parasitic nematodes (Heizer et al., 2013) can be used to predict secreted peptides common to the pan-secretome. Combining this bioinformatics approach with antibody detection systems of immune peptides and innate responses of intestinal explanted tissues from pigs and goats will be used to identify vaccine candidates for immunization in the target host species of interest.

Progress Report
Anthelmintic (anti-worm) drug treatment partially restored the structure and function of the intestinal microbiome altered by the parasitic barber's pole worm (Haemonchus contortus) in goats. ARS scientists in Beltsville, Maryland, examined the effect of drug treatment on the microbial composition along the gastrointestinal tract of parasite-infected goats and found similarities between proximal colon (PC) and fecal (FC) microbiomes of the same animals are significantly higher than those from the same microbial habitats of different animals. However, the structure of the stomach (abomasal) microbiome is unique and distinct from that of the midgut and hindgut. A single-dose moxidectin treatment of goats experimentally inoculated with a drug-resistant Haemonchus strain resulted in a significant reduction in both fecal egg counts and worm counts. Compared to the untreated group, the drug treatment significantly affected the abundance of 42 bacterial operational taxonomic units in the abomasum. The effect of the drug appeared to be amplified in the hindgut where more favorable microbial habitats exist and in which the treatment significantly improved various microbial diversity indices, such as species richness and evenness. Furthermore, the drug treatment had a significant impact on several basic pathways such as the bacterial secretion system and replication and recombination. These findings support the use of fecal measurements to reflect microbial changes in the hindgut and will be useful to develop treatment protocols that eliminate parasites and enhance performance of the animal.

Accomplishments
1. The pig intestinal roundworm disrupts gut microbial interactions and metabolism. Parasitic infections drain resources from food animals and can cause tissue damage, but can they also alter the composition of myriad gut microbes? Agricultural Research Service scientists in Beltsville, Maryland, in collaboration with colleagues at the University of Ghent, Belgium, found that that infection with parasitic worms (Ascaris suum) significantly decreases the diversity of bacterial microbes in the swine gut and disrupts modular global networks, decreasing short-chain fatty acids such as butyrate, a potent inhibitor of intestinal inflammation. Worm eggs also promoted certain bacteria in ways that suggest a novel and unexpected dependency. These findings better explain the impact of parasitic infection and suggest new strategies to improve pig performance. The results may also advance efforts to ameliorate widespread harms induced by globally-prevalent human parasitic infections.

2. User-friendly genomic tools enable biologists to support livestock research. Collaborating with scientists from Korea, Agricultural Research Service scientists in Beltsville, Maryland, developed new tools for measuring changes in livestock gene expression. Compared with conventional methods, this tool allows for more rapid identification of more genes undergoing more subtle changes in relative abundance. These procedures provide easy analysis of complex molecular data to improve animal health and performance, and should have widespread impact on animal science, leading to improvements in animal health, welfare, and production.

3. Diverse mechanisms contribute to parasite resistance in sheep. In collaboration with Commonwealth Scientific Industrial Research Organization (CSIRO) scientists in Australia, ARS scientists in Beltsville, Maryland, compared resistant and susceptible sheep lines in their responses to parasite infection. These source populations are the result of efforts from over 40 years’ selective breeding. The expression of nearly one thousand genes differed among resistant and susceptible animals in response to a primary infection. Different mechanisms, including coagulation, complement cascades, and tissue repair, contribute most to resistance in distinct lines of sheep. These findings elucidate fundamental aspects of livestock immunology and will support the development of genetically resistant sheep via selective breeding.

4. Comparative genomics of the major parasitic worms. Parasitic nematodes (roundworms) and platyhelminths (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 potential drug targets and compounds for testing. This study is the broadest and most comprehensive 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.

5. 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 the prevention or treatment of parasitic infection, which would be a great benefit to veterinary medicine and livestock production.

6. International agreement on diagnosing Trichinella. Trichinosis was once a major concern for pork safety, and remains a concern for consumers of wild game, which might be infected with one or more of several parasite species. Discerning which of these species is present aids epidemiological studies and outbreak control, but they all look alike, creating a need for genetic diagnostic methods. A simple and effective diagnostic assay developed by ARS researchers in Beltsville, Maryland, was endorsed by the International Commission on Trichinellosis (ICT) for use in all outbreaks and whenever Trichinella has been found in consumable foods. This will assist food inspectors and veterinarians better understand and manage an important zoonotic disease.

Review Publications
Liao, Y., Liu, F., Sun, X., Li, R.W., Wu, V.C. 2018. Complete genome sequence of Escherichia coli Phage vB_EcoS Sa179lw isolated from surface water in a produce-growing area in northern California. Genome Announcements. 6(27):e00337-18. https://doi.org/10.1128/genomeA.00337-18.
Oh, S., Li, C., Baldwin, R.L., Song, S., Liu, F., Li, R.W. 2019. Temporal dynamics in meta longitudinal RNA-Seq data. Scientific Reports. 9(1):763. https://doi.org/10.1038/s41598-018-37397-7.
Liu, F., Horton-Sparks, K., Hull, V., Li, R.W., Martinez-Cerdeno, V. 2018. The valproic acid rat model of autism present with gut bacterial dysbiosis similar to that in human autism. Molecular Autism. 9:61. https://doi.org/10.1186/s13229-018-0251-3.
Wang, Y., Liu, F., Urban Jr, J.F., Paerewijck, O., Geldhof, P., Li, R.W. 2019. Ascaris suum infection was associated with a worm-independent reduction in microbial diversity and altered metabolic potential in the porcine gut microbiome. International Journal for Parasitology. 49(3-4):247-256. https://doi.org/10.1016/j.ijpara.2018.10.007.
Liu, F., Li, Z., Wang, Z., Xue, C., Tang, Q., Li, R.W. 2019. Network analysis detected microbial co-occurrence patterns and identified keystone species in the gut microbial community of mice in response to stress and chondroitin sulfate disaccharide dietary supplement. International Journal of Molecular Sciences. 20(9):2130. https://doi.org/10.3390/ijms20092130
Zhang, R., Liu, F., Hunt, P., Li, C., Zhang, L., Ingham, A., Li, R.W. 2019. Transcriptome analysis unraveled potential mechanisms of resistance to Haemonchus contortus infection in Merino sheep populations bred for parasite resistance. Veterinary Research. 50:7. https://doi.org/10.1186/s13567-019-0622-6.
Coelho, C.H., Gazzinelli-Guimaraes, P.H., Howard, J., Barnafo, E., Alani, N.A., Muratova, O., Mccormack, A., Kelnhofer, E., Urban Jr, J.F., Narum, D., Anderson, C., Langhorne, J., Nutman, T.B., Duffy, P.E. 2019. Chronic helminth infection does not impair immune response to malaria transmission blocking vaccine Pfs230D1M-EPA/Alhydrogel® in mice. Scientific Reports. 37(8):1038-1045. https://doi.org/10.1016/j.vaccine.2019.01.027.
Solano Aguilar, G., Jang, S., Lakshman, S., Gupta, R., Beshah, E., Sikaroodi, M., Vinyard, B.T., Molokin, A., Gillevet, P., Urban Jr, J.F. 2018. The effect of dietary Agaricus bisporus mushroom on intestinal microbiota composition and host immunological function. Nutrients. 10(11):1721. https://doi.org/10.3390/nu10111721.
Leroux, L., Nasr, M., Valanparamabil, R., Tam, M., Rosa, B.A., Siciliani, E., Hill, D.E., Zarlenga, D.S., Jaramillo, M., Weinstock, J.V., Geary, T.G., Stevenson, M.M., Urban Jr, J.F., Makedonka, M., Jardim, A. 2018. Analysis of the Trichuris suis excretory/secretory proteins as a function of life cycle stage and their immunomodulatory properties. Scientific Reports. 8:15921. https://doi.org/10.1038/s41598-018-34174-4.
Jarjour, N.N., Schwarzkopf, E.A., Bradstreet, T.R., Shchukina, I., Lin, C., Huang, S., Lai, C., Cook, M.E., Taneja, R., Stappenbeck, T., Randolph, G.J., Artyomov, M.N., Urban Jr, J.F., Edelson, B.T. 2019. Bhlhe40 mediates tissue-specific control of macrophage proliferation in homeostasis and type 2 immunity. Nature Immunology. 20:687-700. https://doi.org/10.1038/s41590-019-0382-5.
Metwali, A., Thorne, P., Winckler, S., Metwali, N., Urban Jr, J.F., Elliott, D.E., Ince, M., Guan, X., Beyatli, S., Truscott, J. 2018. Mechanisms of exacerbating lung inflammation in inflammatory bowel disease. Digestive Disease and Science. https://doi.org/10.1007/s10620-018-5196-z.
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. https://doi.org/10.1017/S0031182018001701.
Pozio, E., Zarlenga, D.S. 2019. Recommendations for genotyping Trichinella muscle stage larvae. Food and Waterborne Parasitology. 15:e00033. https://doi.org/10.1016/j.fawpar.2018.e00033.
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. https://doi.org/10.1038/s41588-018-0262-1.
Li, S., Bostick, J.W., Ye, J., Qiu, J., Zhang, B., Urban Jr, J.F., Avram, D., Zhou, L. 2018. Aryl hydrocarbon receptor signaling cell intrinsically inhibits intestinal group 2 innate lymphoid cell function. Immunity. 49(5):915-928,e5. https://doi.org/10.1016/j.immuni.2018.09.015.