2013 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.
Ascaris suum is a widespread and prevalent parasite of pigs, and is closely related to Ascaris lumbricoides, which infects nearly 1/3 of the global human population. These parasites are highly capable of adapting to, and modifying, their host environments. Their ability to persist in pigs and people depends, in part, on being able to degrade excreted products that would otherwise provoke strong innate and acquired immune responses. Nucleotide metabolizing enzymes comprise one class of molecules that enables such parasites to blunt host immunity. These enzymes are generally secreted into the surrounding space and, by degrading interstitial nucleotides, prevent initiation of the innate immune response. ARS researchers at the Beltsville Agricultural Research Center and the Beltsville Human Nutrition Center cloned and expressed the gene encoding this enzyme and showed it to be biologically functional. The protein specifically degrades those nucleotides that would otherwise initiate stress responses. The team identified the active site within the enzyme by demonstrating that changes to it abrogated enzymatic function. Establishing the biological function and mechanistic basis for this enzyme’s action lays the groundwork for vaccines that might substantially limit the damage incurred by these prevalent and damaging parasites to veterinary and human health.
Description of a new class of anti-parasite drugs against pig roundworms. Ascaris suum is an intestinal parasitic roundworm of pigs that is very closely related to Ascaris lumbricoides, a major intestinal parasitic roundworm of humans that infects more than one billion people worldwide. Because of reduced efficacy and the threat of resistance to the current small set of approved drugs to treat Ascaris infections, new treatments are needed. ARS scientists at the Beltsville Agricultural Research Center and the Beltsville Human Nutrition Center in collaboration with scientists at the Division of Biological Sciences, University of California, San Diego tested the effectiveness of Cry5B, a roundworm-killing protein made by the natural soil bacterium Bacillus thuringiensis which represents a promising new class of anti-worm proteins (anthelmintics). They demonstrated, for the first time, that this drug binds receptors on A. suum and kills cultured larval and adult worms. Moreover, oral administration of the drug to infected pigs resulted in near elimination of the infection from the intestine. This novel approach holds excellent potential to protect swine and human health.
Culture-based testing of anti-parasite drugs against economically important worm species. Parasitic worms infect humans and livestock worldwide with negative effects on health and productivity. Little research has been conducted to develop new anti-worm drugs (athelmintics) owing to low perceived returns on such investment; nevertheless, diminishing efficacy of available drugs is cause for concern among livestock producers and human health professionals. Testing new drugs in livestock or humans can be prohibitively expensive. Reliable, low-cost systems for preliminarily screening new drugs would therefore be of tremendous value. ARS Researchers working at the Beltsville Agricultural Research Center and the Beltsville Human Nutrition Center in collaboration with scientists at the Division of Biological Sciences, University of California, San Diego demonstrated the feasibility of this approach by testing a number of established anti-parasitic drugs in culture, and then in rodents. This approach holds promise for evaluating many new compounds.
Comparing the human and pig genome to model disease. The domestic pig is known as an excellent model for human immunology and the two species are infected by many similar pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the gene complex that regulates the immune system, known as the pig immunome, is not well-characterized. The complete sequencing of the pig genome has provided the opportunity to annotate and categorize the pig immunome, and compare and contrast pig and human immune systems. This extensive annotation dramatically extends the genome-based knowledge of a major portion of the porcine immunome. The functional approach of using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Analysis of this core immunome cluster confirmed rapid evolutionary change in this set of genes, and the importance of the pig’s adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response. This information will be useful for modeling human disease in pigs because of the close evolutionary and functional features of the two species.
Modeling the spread of viral and bacterial pathogens. Modeling is an important way to anticipate how diseases spread and to intervene early to curtail or eliminate dissemination. Insufficient epidemiological data on animal pathogens makes testing new algorithms on this broad group of organisms problematic. As such, developing systems for modeling animal pathogens can often be tested on human diseases then adapted to livestock when sufficient data comes available. Herein, we developed an algorithm (D-R model) and tested it using data related to the spread of ceftazidime-resistant Escherichia coli. The availability of extensive human data provided support that such a model can be adapted to swine viral diseases. Results showed that the D-R model, which was originally created to define trends in the transmission of swine viral diseases, could be successfully modified to evaluate trends in the appearance of ceftazidime-resistant E. coli. Predictions based on limited data successfully mirrored actual increases in drug resistance through 2005, a decrease between 2005 and 2008, and a dramatic inflection point and abrupt increase beginning in 2008. This is consistent with a resistance profile where changes in drug intervention temporarily delayed the upward trend in the appearance of the resistant phenotype; however, resistance quickly resumed its upward momentum in 2008. The same model was successfully tested in evaluating the incidence and death rates caused by tuberculosis in the U.S. and in Germany. The results exceeded the current algorithms used by the World Health Organization to monitor common human diseases. The success of this algorithm suggests it can be applied to a wide range of viral and bacterial infections and can be adapted to animal infections. Its strength lies in the predictive capabilities using minimal amounts of high quality data and its ability to “self-correct” amidst data anomalies. Current algorithms falter under both these nature conditions.
Ding, F., Zarlenga, D.S., Ren, Y., Li, G., Luan, J., Ren, X. 2011. Use of the D-R model to define trends in the emergence of Ceftazidime-resistant Escherichia coli in China. PLoS One. 6(12):e27295.
Ren, Y., Ding, F., Zarlenga, D.S., Ren, X. 2012. Incidence rates and deaths of tuberculosis in HIV-negative patients in the United States and Germany as analyzed by new predictive model for infection. PLoS One. 7(10):e42055.
Yang, Y., Qina, W., Zarlenga, D.S., Cao, L., Tian, G. 2013. TsDAF-21/Hsp90 is expressed in all examined stages of Trichinella spiralis. Veterinary Parasitology. 194(2-4):171-174.
Urban, Jr., J.F., Hu, Y., Miller, M.M., Scheib, U., Yiu, Y.Y., Aroian, R.V. 2013. Bacillus thuringiensis-derived Cry5B has potent anthelmintic activity against Ascaris suum. PLOS Neglected Tropical Diseases. 7(6):e2263.
Blum, A.M., Hang, L., Setiawan, T., Urban Jr, J.F., Stoyanoff, K., Leung, J., Weinstock, J.V. 2012. Heligmosomoides induces tolerogenic dendritic cells that block colitis and prevent antigen-specific gut Tcell responses. Journal of Immunology. Sept 1:189(5):2512-2520.
Smith, A.D., Cheung, L., Shea-Donohue, T., Urban Jr, J.F. 2013. Selenium status alters the immune response and expulsion of adult Heligmosomodies bakeri in mice. Infection and Immunity. 81(7):2546-2553.
Yang, Z., Grinchuk, V., Smith, A.D., Qin, B., Bohl, J.A., Sun, R., Notari, L., Zhang, Z., Sesaki, H., Urban Jr, J.F., Shea-Donahue, T., Zhao, A. 2013. Parasitic nematode-induced modulation of body weight and associated metabolic dysfunction in mouse models of obesity. Infection and Immunity. 81(6):1905-1914.
Zhao, A., Yang, Z., Sun, R., Grinchuk, V., Netzel-Arnett, S., Anglin, I.E., Driesbaugh, K.H., Notari, L., Bohl, J.A., Madden, K.B., Urban Jr, J.F., Antalis, T.M., Shea-Donahue 2013. SerpinB2 is critical to Th2 immunity against enteric nematode infection. Journal of Immunology. 190(11):5779-87.
Yang, Z., Grinchuk, V., Urban Jr, J.F., Bohl, J., Sun, R., Notari, L., Yan, S., Ramalingam, T., Keegan, A.D., Wynn, T.A., Shea-Donohue, T., Zhao, A. 2013. Macrophages as IL-25/IL-33-responsive cells play an important role in the induction of type 2 immunity. PLoS One. 8(3):e59441,1-11.