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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Research Project #431901

Research Project: Evaluation of Swine Immunity and Development of Novel Immune and Genomic Intervention Strategies to Prevent and/or Treat Respiratory Diseases of Swine

Location: Animal Parasitic Diseases Laboratory

2017 Annual Report

1a. Objectives (from AD-416):
Highly infectious diseases of pigs present exceptional challenges to producers. Novel approaches are required to maintain animal health and welfare, particularly as scientists work to develop alternatives to the use of antibiotics for pathogen control. This project will explore immune and genomic-based approaches for understanding host-pathogen interactions. Probing the genetic variations associated with infection, immune evasion, innate and adaptive immune responses, and disease susceptibility and resistance will lead to improved animal health and alternatives for disease control and vaccine design. The goal of this research project is to develop effective countermeasures for preventing and controlling important respiratory diseases of pigs, such as Porcine Reproductive and Respiratory Syndrome (PRRS). New genetic and immune markers will help producers and animal health professionals to prevent and control swine viral diseases; they will provide basic data to use for design of alternate control and vaccine strategies, thus decreasing production costs and improving trade potential. As a result of this work, animal health companies will have alternatives for discovering biotherapeutics and vaccines for swine respiratory diseases; pig breeding companies will have new tools to identify disease-resistant stock. Overall this project will stimulate advances in pig health that may be of broad economic importance. Objective 1. Develop immunologic tools to evaluate swine immunity, including using immunological tools to enhance our understanding of swine immune system development [C4, PS4B], and using immunological tools to inform the design of novel innate immune intervention strategies to treat respiratory diseases of swine. [C2, PS2B] Objective 2. Elucidate host response associated with swine respiratory disease and protective immunity, including discovering genetic and biological determinants associated with swine respiratory disease susceptibility, tolerance, or resistance, and discovering genetic and biologic determinants associated with good responders to swine respiratory disease vaccines. [C4, PS4B]

1b. Approach (from AD-416):
Characterize swine immune proteins (cytokines, chemokines) and monoclonal antibodies (mAbs) to these proteins and their receptors, and to antigens that define swine cell subsets and activation markers (CD antigens). To speed progress on reagent development, collaborate with commercial partners for protein expression and mAb production. Coordinate ARS efforts with NIFA supported US UK Swine Toolkit progress. Once panels of mAbs reactive with swine targets are available, test them for specificity and identify epitope reactivities to help develop sandwich ELISAs and Bead Based Multiplex Assays (BBMA). Work with USDA ARS and NIFA leadership to establish a veterinary immune reagent repository for relevant hybridomas and cell lines from various livestock species as well as to provide an updated website highlighting the availability of these reagents. Emerging and re-emerging infectious diseases heighten the need to use the expanded swine toolkit to facilitate veterinary and biomedical research. Complex immune interactions determine the efficacy of a pig's response to infection, vaccination and therapeutics. New tools developed through this project, and the US UK Swine Toolkit grant, will expand options for probing mechanisms involved in disease and vaccine responses. Continue to assess samples archived through the PRRS Host Genetics Consortium (PHGC) for protein and metabalome alterations that may be predictive of PRRS viral levels or weight gain changes at different time-points post infection. Expand efforts to use pig as an important biomedical model including tuberculosis (TB) research. For TB test whether vaccination in neonatal minipigs leads to the development of immune responses similar to those described in human infants. Results from these trials will allow study of infant TB and TB vaccine efficacy. address biomedical Following up on PHGC studies, as part of a USDA NIFA translational genomics grant, a more complex model, testing vaccination for PRRS followed by PRRSV and porcine circovirus (PCV) challenge [a PCV associated disease (PCVAD) model] was pursued. This approaches typical farm conditions and enables us to ask about the effectiveness of vaccination prior to PRRSV and PCV2 challenge. Additionally, data was collected on genetically defined pigs in true field trial conditions, providing data that is essential for transfer (and affirmation) of our disease genetic results to pig breeders. We expect that the combined models and genomic approaches will lead to identification of chromosomal regions, putative candidate genes and mechanisms involved in regulating pig responses to viral infections, vaccinations, and associated growth effects.For this ARS project we will evaluate the effect of anti-viral response pathways and biomarkers on vaccine and infection responses. We will use RNAseq analyses to provide a more complete picture and reveal details of regulatory mechanisms impacting pig responses to vaccination, viral infection, and differential growth effects. Our proposed studies will expand analyses of samples collected on the grant funded 4 vaccination/PCVAD trials and 6 field trials (Appendix). As we identify

3. Progress Report:
Progress was made on both project objectives and their subobjectives, all of which fall under National Program 103, Animal Health. They address NP103 Component 4: Respiratory Diseases, Problem Statement 4B: Porcine Respiratory Diseases and NP103 Component 2: Antimicrobial Resistance, Problem Statement 2B: Alternatives to Antibiotics. This new project builds on progress made in the recently completed 8042-32000-098-00D project. All milestones were “Met” or “Substantially met” in FY17. Under Objective 1, swine immune proteins (cytokines, chemokines) were expressed and monoclonal antibodies (mAbs) to these proteins were developed in collaboration with university and commercial partners. These reagents are essential for advancing international pig health and vaccination research efforts which require a broad range of immune reagents, and unfortunately are not widely available for pigs. New molecular technologies, including NanoString arrays and 3'RNAseq, were developed to more effectively assess expression of important genes controlling immune responses to porcine reproductive and respiratory syndrome virus (PRRSV) infection and vaccination. Under Objective 2, genetic and biological determinants associated with porcine reproductive and respiratory syndrome virus (PRRSV) infection were explored, expanding on the previously discovered swine chromosome 4 (SSC4) genetic allele, and responsible gene [guanylate binding protein 5 (GBP5)], that was associated with 15% decrease in viral load and 11% weight gain following infection. An updated coinfection [PRRSV and porcine circovirus (PCV2)] model of swine respiratory disease was used to assess susceptibility and resistance as well as determinants associated with good vaccine responses.

4. Accomplishments
1. Discovery of genetic variants that characterize pigs with superior responses to viral infection or vaccination. ARS scientists in Beltsville, Maryland, partnered with Iowa State and Kansas State University researchers to work on the most economically important disease of pigs, porcine reproductive and respiratory syndrome (PRRS). Our goal was to identify genetic markers of pigs that respond especially well to vaccination against PRRS and that thrive in the face of co-infection with PRRS virus and a second virus of major concern, the porcine circovirus. Specific regions in the pig genome were identified that resulted in improved weight gain while controlling viral load (level of virus in the bloodstream) of commercial crossbred nursery pigs. Based on these results pig breeders will be able to select for more disease-resistant pigs, improving the health of swine and reducing economic losses attributable to viral infection.

2. Genomics helps predict a pig’s resistance to respiratory viral infections. Porcine Reproductive and Respiratory Syndrome (PRRS) is a devastating disease for the swine industry, causing $600 million in losses/year for U.S. pork producers. ARS scientists in Beltsville, Maryland partnered with researchers at Iowa State and Kansas State Universities to investigate whether they could accurately predict genes that control both PRRS virus (PRRSV) load, i.e., level of virus in the bloodstream, and weight gain. To do this PRRS resistance prediction they set up statistical groups (training and validation datasets) from pig populations with different genetic backgrounds that had been infected with two different PRRSV isolates; further, to improve the accuracy of their predictions, sets of genetic markers from across the whole genome were used as genetic tags. The response of individual pigs to PRRSV infection was predicted by these data with moderate accuracy with limited influence due to PRRSV isolate. This PRRS resistance prediction tool will aid pig breeders in improving the health of the swine herd, and reduce losses associated with these infections.

3. Certain pigs better tolerate viral burdens. Pig responses to viral infection were probed to answer the question: Does an animal survive an infection due to resistance (reduced level of virus in the bloodstream) and/or due to tolerance (lower impact of virus infection on performance, e.g. weight gain, meat quality)? This is quite important for Porcine Reproductive and Respiratory Syndrome virus (PRRSV) infections since they are economically a devastating disease for the swine industry; therefore, ARS scientists in Beltsville, Maryland, partnered with others at Iowa State University, Kansas State University and the Roslin Institute in Scotland to develop a mathematical model describing serum PRRSV responses. Our past work has provided evidence in support of a genetic basis for PRRS resistance but it was not known whether pigs also differed genetically in PRRS tolerance. Evidence for genetic variation in tolerance of pigs to PRRS was weak when based on data from only PRRSV infected piglets. However, simulations indicated that differences in genetic background may impact tolerance and this could be detected if comparable data on uninfected relatives were available. We concluded that unlike the proven genetics of resistance to PRRS virus infection, genetics of tolerance can be predicted but will be more difficult to verify.

4. Susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) infection has a heritable component, yet little is known about the underlying causes. The PRRS Host Genetics Consortium of researchers from ARS in Beltsville, Maryland, Iowa State University, the University of Alberta and PigGen Canada explored how different pigs responded to viral infection. We performed complex analyses, integrating pig genotypes, blood cell gene expression and post infection phenotypes (viral load and weight gain) over 42 days to reveal differences, termed quantitative trait loci, for 560 PRRSV-responsive genes. These studies revealed that several immune-related pathways including cytokine signaling, interferon signaling and antigen presentation were important and contribute to poor responses (higher viral load or lower weight gain) after PRRSV infection. As a result of this effort, we expect to provide breeders and producers information on selecting pigs with superior anti-PRRS responses and increased resistance to swine respiratory infections.

5. PRRS virus induces pathology in fetuses as well as at the uterine maternal-fetal attachment site. Porcine reproductive and respiratory syndrome virus (PRRSV) infections during pregnancy cause U.S. producers losses of over $300 million annually. ARS scientists at Beltsville, Maryland, the University of Saskatchewan and the University of Alberta, probed maternal and fetal factors that could be predictive of PRRS severity and resilience in newborn piglets. Fetal death and high viral load clustered in litters suggesting viral transmission between fetuses starting from a few index fetuses in each litter, with large fetuses at greater risk; also, gene expression profiles indicated an active, adaptive immune response to the viral infection with both antibody and cell-mediated immune components in the sow; in contrast, the fetus exhibited an immature, innate and inflammatory immune response with up-regulation of genes implicated in infectious disease pathology. Sows with higher viral load and lower T cell immune signaling were more likely to have virus infected fetuses. Thus, fetal pathology is influenced by events occurring at both the maternal-fetal attachment site as well as within the PRRSV-infected fetus. Efforts are underway to improve reproductive PRRS control methods.

6. In the fight against human tuberculosis, piglets prove an excellent research model. Numerous groups have attempted to develop vaccines to control human tuberculosis (TB) andto date the only vaccine available against TB is Bacillus Calmette-Guerin (BCG), however, there have been major failures in the development of new pediatric vaccines against TB due to incomplete knowledge of the immune response elicited in the neonate after vaccination. Since the pig is known to have an immune system similar to humans, ARS scientists at Beltsville, Maryland, worked with researchers at Colorado State University to determine if piglets become infected with TB and have similar immunological responses to those found in infants vaccinated with BCG. Both vaccinated and non-vaccinated pigs were challenged via the aerosol route with Mycobacterium tuberculosis (Mtb). As compared to human infants, neonatal pigs demonstrated a similar course of TB infection as well as similar immune response to BCG and to TB challenge. Overall, our results affirmed that the pig should be a good model for the human neonate for development of diagnostics, drugs and vaccines against TB.

7. New tools for studying swine immunology. Analyses of disease and vaccine responses require sophisticated immune tools yet those for pigs are limited. ARS scientists at Beltsville, Maryland, worked with commercial partners and researchers at Ohio State and Kansas State Universities and the University of Bristol, U.K., to address this issue, supported in part by a USDA U.S.-U.K. collaboration grant. Numerous swine immune proteins (cytokines, chemokines) were expressed and used for immunizations to develop specific reagents [monoclonal antibodies (mAbs)] reactive with these proteins. Panels of mAbs reactive with swine immune proteins, interleukin-6 (IL-6), IL-13, IL-17A, interferon-beta (IFNb) and IFNg, have now been produced. Tools and reagents generated by this project will be made available for swine immune, disease and biomedical research efforts worldwide.

Review Publications
Tuggle, C.K., Giuffra, E., White, S.N., Clarke, L., Zhou, H., Ross, P.J., Acloque, H., Reecy, J.M., Archibald, A., Boichard, M., Chamberlain, A., Cheng, H.H., Crooijmans, R., Delany, M., Groenen, M.A., Hayes, B., Lunney, J.K., Plastow, G.S., Silverstein, J., Song, J., Watson, M. 2016. GO-FAANG meeting: A gathering on functional annotation of animal genomes. Animal Genetics. 47(5):528-533.

Waide, E.H., Tuggle, C.K., Serao, N., Schroyen, M., Hess, A., Rowland, R., Lunney, J.K., Plastow, G., Dekkers, J. 2017. Genome-wide association analyses of piglet responses to infection with two porcine reproductive and respiratory syndrome virus isolates. Journal of Animal Sciences. 95:16-38.

Hay, E.A., Choi, I., Xu, L., Zhou, Y., Rowland, R., Lunney, J.K., Liu, G. 2017. CNV analysis of host responses to porcine reproductive and respiratory syndrome virus infection. Journal of Genomics. 5:58-63.

Dunkelberger, J., Serao, N., Neiderwerder, M., Kerrigan, M., Lunney, J.K., Rowland, R., Dekkers, J. 2017. Effect of a major QTL for PRRS-resistance on response to co-infection with PRRS and PCV2b in commercial pigs, with or without prior vaccination for PRRS. Journal of Animal Science. 95:584-598.

Lough, G., Rashidi, H., Kyriazakis, I., Dekkers, J.C., Hess, A.S., Hess, M.K., Deeb, N., Kause, A., Lunney, J.K., Rowland, R.R., Mulder, H., Doeschl-Wilson, A. 2017. Using multi-trait and random regression models to identify genetic variation in tolerance of pigs to Porcine Reproductive and Respiratory Syndrome virus. Genetics Selection Evolution. 49:37.

Kommadath, A., Bao, H., Choi, I., Reecy, J.M., Koltes, J.E., Fritz-Waters, E., Eisley, C.J., Grant, J.R., Rowland, R.R., Tuggle, C.K., Dekkers, J.C., Lunney, J.K., Guan, L., Stothard, P., Plastow, G.S. 2017. Genetic architecture of gene expression underlying variation in susceptibility to porcine reproductive and respiratory syndrome virus infection. Scientific Reports. 7:46203. doi:10.1038/srep46203.

Harding, J., Ladininig, A., Novakovic, P., Detmer, S., Wilkinson, J., Yang, T., Lunney, J.K., Plastow, G. 2017. Novel insights into host responses and the reproductive pathophysiology of type 2 porcine reproductive and respiratory syndrome (PRRS). Veterinary Microbiology. 209: 114-123. 10.1016/j.vetmic.2017.02.019.

Dekkers, J., Rowland, R., Lunney, J.K., Plastow, G. 2017. The use of host genetics to control porcine reproductive and respiratory syndrome. Veterinary Microbiology. S0378-1135(16)30678-2. doi: 10.1016/j.vetmic.2017.03.026.