Location: Animal Parasitic Diseases Laboratory
2021 Annual Report
Objectives
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]
Approach
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 www.vetimm.org 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
Progress Report
A major focus of this project has been the development of new reagents for swine immunity. Scientists at Beltsville, Maryland, cloned and expressed swine immune proteins (cytokines, chemokines) working with a commercial partner. A second commercial partner developed monoclonal antibodies (mAbs) to selected proteins. We characterized reactivities of 10 panels of mAbs to different swine interleukins, interferons, and chemokines, testing for intracellular staining and cross-species reactivity in collaboration with university partners. These mAbs will enable each protein to be quantified in body fluids and culture supernatants and their intracellular expression to be measured. Characterized sets of mAbs are being offered for transfer to commercial partners; the tools and reagents generated by this project support swine immune, disease, and biomedical research efforts worldwide.
Documenting the availability of essential swine immune reagents will advance international pig health and vaccination research efforts. In collaboration with .K.U.K. partners, scientists at Beltsville, Maryland, added swine data previously housed at www.vetimm.org to the new Pirbright/Roslin .K.U.K. Immunological Toolbox website and database (http://www.immunologicaltoolbox.co.uk). In addition, we updated genomic maps of swine immune markers using the newly refined swine genome build (Sus scrofa 11.1) and information collected in an exhaustive search of the internet and commercial resources. Our efforts resulted in the identification and verified availability of monoclonal and polyclonal antibodies that react with porcine cytokines, chemokines, growth factors, or cell subset markers, as well as cloned recombinant proteins and assays for their quantitation.
Our international team organized a compendium of swine leukocyte antigens (SLA). Genes within the major swine histocompatibility, or SLA, complex regulates T cell immunity, determining the intensity of disease and vaccine responses. The SLA complex maps to swine chromosome 7 and encodes approximately 150 loci, at least 120 presumed functional. ARS scientists at Beltsville, Maryland, worked with scientists from the U.S., Austria, Japan, and France to annotate all recognized SLA genes. The team updated SLA nomenclature, reviewed SLA genetic typing methods, defined SLA protein expression, and described the roles of each SLA genetic locus in antigen presentation and immune, disease, and vaccine responses as well as in xenotransplantation (cross-species transplantation). Overall, these data provide swine researchers essential information on SLA genes and proteins and underscore their importance in swine health, disease, and vaccine responses and their future importance for xenotransplantation.
We continued efforts to improve analyses of porcine reproductive and respiratory syndrome (PRRS) virus infection responses by profiling gene expression in blood and tissues of nursery pigs as part of the PRRS Host Genomics Consortium. These efforts identified biomarkers and pathways involved in controlling PRRS resistance and susceptibility. We refined methods to confirm the importance of specific biomarkers by targeting gene expression using swine immune-focused NanoString arrays, improving our ability to interrogate details of anti-viral responses. These approaches further helped to validate immune gene expression in flow-sorted blood cell subsets by ARS scientists at Beltsville, Maryland, and Ames, Iowa, working with Iowa State University researchers. Blood cell subset gene expression was confirmed and expanded using flow cytometry for the separation of single cells, which was followed by single-cell RNAseq gene expression analyses. Finally, we used advanced biostatistical analyses to describe mechanisms regulating cell cell-specific immune responses more completely.
Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes major losses to U.S. pork producers, with an estimated $300 million in annual losses due to reproductive PRRS. Fetal responses to congenital PRRSV infection are variable within litters, from uninfected fetuses to dead neighbors or those with high viral levels. ARS scientists in Beltsville, Maryland, worked with scientists at the University of Saskatchewan to probe these disparities, concentrating on maternal and fetal factors that could predict disease severity and fetal resilience. Twelve days after the mother was exposed in her third trimester, fetuses were harvested and grouped by preservation status and viral level (VLV.L.) in the fetal placenta, serum, and thymus. We used NanoString codesets to assess targeted immune gene expression patterns in the fetal placenta and thymus. We found that the anti-viral immune response was initiated only after PRRSV reached detectable levels in the fetal thymus, when a core set of interferon-inducible genes was strongly upregulated in both tissues. High .L.V.L. occurred in fetuses expressing notably low levels of several protective innate and adaptive immune pathways. Gene expression in the placenta differentiated fetal demise. Overall, this work indicates that fetal responses are governed by activities in the fetus and at the maternal-fetal interface. The newly discovered biomarkers may help breeders improve animal health. These markers also suggest new anti-viral therapeutic approaches.
Accomplishments
1. Breeding pigs to resist viral infections. Pigs with complete resistance to porcine reproductive and respiratory syndrome (PRRS) virus can only be produced by gene editing the CD163 gene. As an alternate, ARS scientists in Beltsville, Maryland, worked with Iowa State University researchers to search for naturally occurring CD163 genes and discovered three natural genes that can improve a pig's resistance to PRRS virus infection. Unfortunately, none of the three genes lead to complete resistance. These genes identified can now be used to select for pigs increased natural resistance to viral infections without gene editing.
2. Piglets are an important model for developing tuberculosis vaccines for human babies. The only vaccine available against human tuberculosis (T.B.) is Bacillus Calmette-Guerin (BCG). Better vaccines are needed, especially for newborns. But human neonatal responses to BCG vaccination are only poorly understood. ARS scientists at Beltsville, Maryland, worked with researchers at Colorado State University to determine that piglets and human infants respond similarly to BCG vaccination. By affirming that the pig serves as an effective neonatal animal model for .B.T.B. vaccine development, this advanced work progress towards developing better human vaccines to a major global disease.
Review Publications
Ramos, L., Lunney, J.K., Gonzalez-Juarrero, M. 2020. Neonatal and infant immunity for vaccine development against tuberculosis: importance of age-matched animal models. Disease Models and Mechanisms. 13: dmm045740. https://doi.org/10.1242/dmm.045740.
Van Goor, A.G., Pasternak, A., Walker, K.E., Hong, L., Malgarin, C., Macphee, D.J., Harding, J.C., Lunney, J.K. 2020. Differential responses in placenta and fetal thymus at 12 days post infection elucidate mechanisms of viral level and fetal compromise following PRRSV2 infection. BMC Genomics. 21:763. https://doi.org/10.1186/s12864-020-07154-0.
Jeon, R., Cheng, J., Putz, A., Dong, Q., Harding, J., Dyck, M., Plastow, G., Fortin, F., Lunney, J.K., Rowland, R., Dekkers, J. 2021. Effect of genotype at a genetic marker for the GBP5 gene on resilience to a polymicrobial natural disease challenge in pigs. Livestock Science. 244:104399. https://doi.org/10.1016/j.livsci.2021.104399.
Dong, Q., Lunney, J.K., Lim, K., Nguyen, Y., Hess, A.S., Beiki, H., Rowland, R., Walker, K.E., Reecy, J., Tuggle, C., Dekkers, J. 2021. Gene expression in tonsils in swine following infection with porcine reproductive and respiratory syndrome virus. BioMed Central (BMC) Veterinary Research. 17:88. https://doi.org/10.1186/s12917-021-02785-1.
