Location: Animal Parasitic Diseases Laboratory
2018 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
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. All milestones were “Met” or “Substantially met” in FY18.
Under Objective 1, swine immune proteins (cytokines, chemokines) were expressed and monoclonal antibodies (mAbs) to these proteins were developed and characterized 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. Refined molecular technologies, including NanoString arrays and 3'RNAseq, were used 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 viral resistance allele. 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. A model of reproductive PRRS has been updated to determine how fetuses resist congenital infection.
Genomic prediction of response to PRRSV infection improved when all genomic markers are tested. Because Porcine Reproductive and Respiratory Syndrome (PRRS) is a devastating disease for the swine industry, a tool to provide genomic predictions for resistance to PRRS virus (PRRSV) infection would be very important. ARS scientists in Beltsville, Maryland, partnered with Iowa State and Kansas State University researchers to investigate whether they could improve accuracy of genomic prediction for both serum viral load and weight gain by testing datasets from populations with different genetic backgrounds that were challenged with 2 different PRRS viruses (data collected by the PRRS Host Genetics Consortium trials). To improve the accuracy of these predictions, subsets of genetic markers [termed single nucleotide polymorphisms (SNPs)] were tested. Inclusion of SNPs that are close to a known viral protective gene on chromosome 4 was vital for accurate prediction of viral load, but accuracy was best when SNPs across the whole genome were used. Results show that genomic prediction of response to PRRSV infection is moderately accurate and, when using all SNPs in the genome, is not very sensitive to the different PRRS viruses tested.
Predicting genetic variation in tolerance to PRRSV infection requires extensive data collection. Breeding pigs that better survive Porcine Reproductive and Respiratory Syndrome (PRRS) would greatly limit the costs incurred by PRRS, one of the most devastating swine diseases worldwide. ARS researchers, working with a broad consortium of industry and academic partners, aimed to determine whether genetic selection could improve the ability of pigs to thrive (be “tolerant), despite having been infected with PRRS virus. They discovered that breeding programs can, indeed, increase such “tolerance,” but only through intensive recording schemes involving repeated measurements. Absent such detailed records, one promising alternative entails selecting for a marker previously discovered by the PRRS Host Genetics Consortium research team (WUR, which confers PRRS “resistance” by limiting viral load early in the course of infection). Different models identified significant genetic variation in tolerance. Influence of the WUR marker was found in each model, but its best effect was evident when the model focused on genetically more resistant, WUR+ pigs who were also more tolerant to PRRS. The WUR marker was shown to not only increase resistance, but also improve tolerance to infection: infected pigs ultimately grew better if they had the selected variant. These results will be of great interest to the swine industry, swine breeders, veterinarians and epidemiologists.
Definitive characterization of 359 swine immune cell subset cluster of differentiation (CD) markers. Improved technologies and genomics have contributed to dramatic increases in our knowledge of the immune molecules regulating interactions involved in successful infectious disease and vaccination responses. The international community has established a unique terminology for identifying individual molecules expressed on immune cell subsets; these are called cluster of differentiation (CD) markers. Our latest review identified 359 swine CD proteins corresponding to known human CD markers, with gene names, sequences, and nucleotide identity to human proteins. It presented documented reactivity of more than 800 reagents that identify swine CD markers and affirmed their reactivity with immune cell subsets. Overall, the data presented on reagents to quantitate CD marker expression will be used by researchers to enhance their swine immune studies, leading to improved understanding of swine health and disease and of the pig as a biomedical model.
Accomplishments
1. Identification of alternate genetic markers for anti-PRRS responses. It would be very valuable to more effectively identify those pigs best capable of responding to Porcine Reproductive and Respiratory Syndrome (PRRS) virus infection and vaccination since. PRRS is the most economically important disease of pigs in the U.S. and worldwide. ARS scientists in Beltsville, Maryland, partnered with Iowa State and Kansas State University researchers to assess whether the level of serum anti-PRRS antibody response 42 days after being infected is passed from parent to offspring and could predict a decreased level of virus in the blood and improved weight gain. Mapping studies of the pigs’ genomes identified 3 markers on swine chromosome 7 associated with antibody response level. These findings suggest antibody response to PRRS can help select pigs with increased resistance to PRRS virus infection, providing another tool for improving the health of swine herds.
2. Differential immunity of pig fetuses to congenital PRRSV infection. Porcine reproductive and respiratory syndrome virus (PRRSV) infections cause major reproductive losses, with an estimate of over $300 million annual losses in the U.S. alone. Joint studies of ARS scientists at Beltsville, Maryland, with scientists at the University of Saskatchewan, probed responses to PRRSV infection in first time pregnant pigs, in their third-trimester, and assessed maternal and fetal factors that could be predictive of PRRS severity and resilience in fetal pigs. Viral load in maternal and associated- fetal tissues was a strong predictor of viral load in fetal thymus. The expression of immune-related genes in fetuses with no, low or high viral load at 5 to 12 days post maternal PRRSV infection was investigated. Disease progression in fetuses was accompanied by changes in inflammatory and early protective immune responses. These studies have affirmed the diversity of fetal pig anti-PRRSV response within each litter. These results set the stage for more detailed analyses now underway to probe for key markers of fetal pig PRRS resilience.
3. Pig model for exploring human tuberculosis vaccines. Development of effective vaccines for human tuberculosis (TB) is a worldwide goal. To date, the only vaccine available against TB is Bacillus Calmette-Guerin (BCG). There have been major failures in the development of new pediatric vaccines against TB due to incomplete knowledge of the immune response elicited after neonatal vaccination and to testing vaccine efficacy in adults rather than in neonatal animal models. 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 and determined that piglets become infected with TB, and have similar immunological responses to those found in infants vaccinated with BCG. We challenged both vaccinated and non-vaccinated animals via the aerosol route with Mycobacterium tuberculosis (Mtb) and characterized changes in immune responses between the two groups. Our results demonstrated a similar course of TB infection and similar immune response to BCG and to Mtb challenge in pigs as compared to humans. Thus, our results affirm that the pig model can be used for development of diagnostics, drugs and vaccines against TB.
4. New tools for measuring swine immunity. 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 Tennessee State Universities and the University of Bristol, U.K., to develop new reagents [monoclonal antibodies (mAbs )]. Reactivity of panels of mAbs reactive with 6 different swine immune proteins have now been characterized. The mAbs for each protein were then compared and tested for intracellular binding reactivity. Sets of these mAbs are being transferred to commercial partners so new tests can be produced. Tools and reagents generated by this project are being made available for swine immune, disease and biomedical research efforts worldwide.
Review Publications
Waide, E., Tuggle, C.K., Serao, N., Schroyen, M., Hess, A., Rowland, R., Lunney, J.K., Plastow, G., Dekkers, J. 2018. Genomic prediction of piglet response to infection with one of two porcine reproductive and respiratory syndrome virus isolates. Genetics Selection Evolution. 50:3. https://doi.org/10.1186/s12711-018-0371-4.
Dunkelberger, J., Serao, N., Weng, Z., Waide, E.H., Niederwerder, M., Kerrigan, M., Lunney, J.K., Rowland, R., Dekkers, J. 2017. Genomic regions associated with host response to porcine reproductive and respiratory syndrome vaccination and co-infection in nursery pigs. BMC Genomics. 18:865. https://doi.org/10.1186/s12864-017-4182-8..
Lough, G., Hess, A.S., Hess, M.K., Rashidi, H., Dekkers, J.C., Mulder, H., Kyriazakis, I., Matika, O., Lunney, J.K., Rowland, R.R., Doeschl-Wilson, A.B. 2017. Harnessing longitudinal information to identify genetic variation in tolerance of pigs to Porcine Reproductive and Respiratory Syndrome virus infection. Genetic Selection Evolution. 49:37
Rowland, R.R., Lunney, J.K. 2017. Alternative strategies for the control and elimination of PRRS. Veterinary Microbiology. 209:1-4. https://doi.org/10.1016/j.vetmic.2017.09.006.
Ramos, L., Obregon-Henao, A., Henao-Tamayo, M., Bowen, R., Lunney, J.K., Gonzalez-Juarrero, M. 2017. The minipig as an animal model to study Mycobacterium tuberculosis infection and natural transmission. Tuberculosis. 106:91-98.
Dawson, H.D., Lunney, J.K. 2018. Porcine cluster of differentiation (CD) Markers 2017 Update. Research in Veterinary Science. 118:199-246.
Hong, L., Han, K., Wu, K., Lunney, J.K., Ruize, L., Huang, J., Zhao, S., Yu, M. 2017. E-cadherin and its regulator ZEB2 control the development of the placental folds by modulating trophoblast cell differentiation in pigs. Reproduction. 154:765-775. https://doi.org/10.1530/REP-17-0254
