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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Research Project #422176

Research Project: IMMUNOLOGICAL APPROACHES TO CONTROLLING SWINE INTESTINAL PARASITES AND MUCOSAL PATHOGENS

Location: Animal Parasitic Diseases Laboratory

2015 Annual Report


1a. Objectives (from AD-416):
Objective 1: Determine the immune relationship between parasites and the mucosal immune response concentrating on epigenetic targets and the innate immune system. The goal of the proposed research project is to evaluate the influence of parasitic infection during gestation and in the pre-weaning period on mucosal macrophages and to explore dietary effects that regulate mucosal immune responses in pigs. Objective 2: Evaluate the ability of nutritional supplements and pathogen-associated molecules in modulating the immune response. Macrophages and related dendritic cells at mucosal surfaces provide the first line of defense as they respond to pathogen-associated molecular pattern (PAMP) molecules that bind toll-like receptors (TLRs) and trigger innate immune responses that link them to components of acquired immunity. They also respond to danger-associated molecular pattern (DAMP) molecules that trigger responses to cell injury and inflammation. The inherent potential of molecules from the parasite to modulate immune function to secure the parasitic relationship with the host may be met by nutritional conditions that influence host immunity. This objective will begin to evaluate these features of macrophage biology as they contribute to resistance to parasitic infection and the influence of nutrients on this process.


1b. Approach (from AD-416):
The approach for Objective 1 is to determine the immune relationship between parasites and the mucosal immune response concentrating on epigenetic targets and the innate immune system. Stimulation of primary pig alveolar macrophages (AM) by all-trans retinoic acid (ATRA), parasites, or parasite-derived products in vitro will provide information on transcriptomic markers and epigenetic sites to evaluate in later in vivo-treatment studies of pigs given ATRA and infected with Ascaris suum. Exposure of sows during gestation and neonates during the first 21 days of life to ATRA or infection with A. suum will polarize pig AM and imprint epigenetic traits that influence functional activity at mucosal surfaces. The approach used for Objective 2 is to evaluate the ability of nutritional supplements and pathogen-associated molecules in modulating the immune response. The aim is to identify parasite-derived nucleotide metabolizing enzymes, and in particular apyrases, that may control local inflammatory responses by modulating ATP levels in surrounding tissues. The AM will be used as a functional readout cell for parasite products and metabolites derived from parasite enzymatic activity. ATRA acting as a supplemental nutrient in the presence of adenosine will modulate adenosine receptor signaling of primary pig AM leading to synergistic effects on macrophage function, cytokine production, and gene expression. The study is designed to determine if ATRA co-stimulation with adenosine alters pig AM function in vitro.


3. Progress Report:
Immunization of mice with cloned Ascaris suum apyrase, as a model to detect immunological activity for use in pigs, failed to show immune protection against a challenge infection with A. suum infective eggs. To test an alternative strategy, we used a computational based approach to predict candidate proteins with immunogenic potential by screening orthologous protein families of a pan-nematode secretome database. Among the top 17 candidates derived from our prioritization scheme, three were chosen for cloning, expression and purification of recombinant proteins for a vaccine trial in mice challenged with three parasitic worms that inhabit different regions of the intestine; A. suum, Heligmosomoides polygyrus bakeri and Trichuris muris. Parasite recovery data showed a significant reduction in A. suum larvae in the lungs and H. p. bakeri adults in the intestines of mice vaccinated with the cloned parasite proteins; there was no significant reduction in the recovery of T. muris larvae. This very promising result is now being tested in a repeat study to confirm the initial results. The work is partially supported through a reimbursable cooperative agreement with Washington University, St. Louis, MO with funding from the National Institute of Food and Agriculture (8042-32000-094-03R).


4. Accomplishments
1. Parasite modulation of host immunity. Ascaris suum, the large roundworm, is a zoonotic parasite of swine and closely related to a roundworm species that infects nearly 25% of the world’s population. It and related parasites have evolved methods for controlling the host immune response. Agricultural Research Service (ARS) scientists in Beltsville, Maryland, studied a class of parasite enzymes (called ATP diphosphohydrolases) and determined that they modify the innate immune responses by removing key nucleotide substrates that are released from damaged cells and that would otherwise provoke an inflammatory response. Thus, proteins secreted by the parasites degrade factors that mediate the host’s cell-to-cell signaling and stress responses. Finding parasite proteins that potentiate colonization and survival may hasten progress towards vaccines or drugs that prevent or minimize the damage caused by these infections. Vaccines are being developed for other livestock parasites using this technology because this feature of parasite inhibition of signals that activate the host are common to other parasites and hosts.

2. Resource limitation has consequences on co-infection for both hosts and parasites. Most animals are concurrently infected with multiple parasite species and live in environments with fluctuating resource availability. Resource limitation can influence host immune responses and the degree of competition between co-infecting parasites. To test how resource limitation affects immune trade-offs and co-infection outcomes, a factorial experiment using laboratory mice was conducted where mice were given a standard or low protein diet, dosed with two species of parasitic worms (alone and in combination), and then challenged with a micro-parasite. Co-infection was found to influence parasite survival and reproduction via host immunity, but the magnitude and direction of responses depended on both resources and the combination of co-infecting parasites. These findings highlight that resources and their consequence for host defenses are a key context that shapes the magnitude and direction of parasite interactions. The work, a collaborative effort with scientists from the University of Georgia, will be used to determine how changes in diet can improve the health of animals co-infected with different parasites.

3. Immune antibodies and helminth (parasitic worm) products drive chemokine and inflammatory cell crosstalk to promote intestinal repair. Worm parasites can cause damage when migrating through host tissues then rapid tissue repair is needed to prevent bleeding and bacterial dissemination, particularly from the intestine. Mice lacking antibodies or activating antibody receptors had impaired intestinal tissue repair following worm infection, but supplying antibody-rich immune serum partially rescued wound healing even in infected mice lacking antibodies. Impaired healing was associated with inflammatory cells expressing messenger proteins and chemokines at reduced levels, and by smooth muscle cells within intestinal lesions. Antibodies and worms together triggered chemokine production by inflammatory cells in test tube studies via engagement of Fc receptors, and chemokine secretion by intestinal muscle cells was directly activated by worms. Blocking chemokine production during worm infection reproduced the delayed wound repair observed mice lacking endogenous antibodies and receptors. Conditioned media used to grow human inflammatory cells stimulated with worms that infect humans together with immune serum, promoted chemokine-dependent wound closure human muscle cells in the test tube. These findings suggest that worms and antibodies instruct a chemokine driven crosstalk between inflammatory cells and muscle cells in the intestine to promote intestinal repair. This work, in collaboration with colleagues in Switzerland, will be expanded to look for parasite products that can be harnessed in clinical settings of impaired wound healing.

4. Immune and inflammatory responses in pigs concurrently infected with two parasitic worms. The immune response mounted by pig to a parasitic infection can vary considerably depending on the presence or absence of other parasites. For example, whipworm elicits a strong immune response in the large intestine leading to its rapid expulsion from the pig, but also has a strong antagonistic effect on hookworms when pigs are co-infected. ARS scientists in Beltsville, Maryland, in collaboration with scientists in Denmark examined the basis of such interactions by comparing components of the immune responses mounted by pigs harboring one or a pair of infections. Whipworm was found to markedly enhance antibody responses against hookworm. Pigs infected with only whipworm exhibited a strong immune response whereas those infected only with hookworm displayed a weaker, delayed response. Interactions were found between the two worm parasites with regard to the production of several immune activating proteins or cytokines. An understanding of the interactions between these two common worm infections in pigs can help devise more efficacious vaccines to protect animal and human health.

5. Expanded scope of the Porcine Translational Research Database. The Porcine Translational Research Database fosters comprehensive and integrated analysis of the pig genome and provides important tools for global analysis and data-mining of the pig immune response. ARS scientists in Beltsville, MD increased its content approximately 1.5 fold by adding more than 1,600 gene entries since last year and by manually assembling and annotating 5,820 other transcripts of immune and metabolism related genes as well as all known epigenetic regulators from 5,428 genes (28% of the pig genome and including 931 genes not found in the current publically available pig genome assembly); 1,395 of these full-length gene sequences were submitted to Genbank (Bioproject: PRJNA80971). These sequences were also used to perform several transcriptomic and epigenetic–based studies (miRNA and mRNASeq expression profiling) including responses of pig inflammatory cells from the lung to vitamin A or to protein messenger cytokines like interleukin-4 (IL-4) or interferon-gamma (IFN-g) and the bacterial product LPS, as well as in the pig response to whipworm. We observed selective high level induction of the epigenetic regulator histone deacetylase 9 (HDAC9) and the histone demethylase KDM6B by IL-4 and numerous histone demethylases and methyltransferases by LPS/IFN-g. This information will be useful for modeling human disease in pigs because of the close evolutionary and functional features of the two species. The database is part of a collaborative initiative with scientists in the Beltsville Human Nutrition Research Center and has been accessed over 45,000 times by investigators from more than 30 laboratories worldwide.


Review Publications
Andreasen, A., Petersen, H., Kringel, H., Iburg, T., Skovgaard, K., Dawson, H.D., Urban Jr, J.F., Thamsborg, S. 2015. Immune and inflammatory responses in pigs infected with Trichuris suis and Oesophagostomum dentatum. Veterinary Parasitology. 207(3-4):249-258.

Budischak, S.A., Sakamoto, K., Megow, L.C., Cummings, K.R., Urban Jr, J.F., Ezenwa, V. 2015. Resource limitation alters the consequences of co-infection for both hosts and parasites. International Journal for Parasitology. 45(7):455-463.

Dong, B., Zarlenga, D.S., Ren, X., 2014. An overview of live attenuated recombinant pseudorabies viruses for use as novel vaccines. Journal of Immunology, 2014. 1-10. Article ID 824630, DOI:10.1155/2014/824630.

Von Bieren, J.E., Volpe, B., Sutherland, D.B., Burgi, J., Verbeek, S., Marsland, B.J., Urban Jr, J.F., Harris, N.L. 2015. Immune antibodies and helminth products promote CXCR2-dependent repair of parasite-induced injury. PLoS Pathogens. 11(3):e1004778.

Zarlenga, D.S., Hoberg, E.P., Rosenthal, B.M., Mattiucci, S., Nascetti, G. 2014. Anthropogenics: Human influence on global and genetic homogenization of parasite populations. Journal of Parasitology. 100(6):756-772. doi: 10.1645/14-622.1.