Submitted to: BARC Poster Day
Publication Type: Abstract only
Publication Acceptance Date: 3/28/2008
Publication Date: 4/23/2008
Citation: Wysocki, M., Ernst, C.W., Steibel, J.P., Ramos, A.M., Mccaw, M., Petry, D., Johnson, R.K., Rothschild, M.F., Fahrenkrug, S.C., Elsik, C.G., Kuhar, D.J., Lunney, J.K. 2008. Investigation of immune pathways involved in regulating responses to porcine reproductive and respiratory syndrome using swine protein-annotated oligonucleotide microarrays. 19th Annual BARC Poster Day. Interpretive Summary:
Technical Abstract: Porcine reproductive and respiratory syndrome (PRRS) costs U.S. swine producers around $560 million annually. PRRS virus (PRRSV)-infected pigs are susceptible to pneumonia and reproductive losses; infected sows have increased rates of abortions, stillbirths, mummifications, and give birth to weak piglets with chronic respiratory problems. Our goal is to uncover genetic components involved in early regulatory immune response during PRRSV infection. Therefore, we decided to use microarrays, which provide a great source of information about differentially expressed genes, to identify immune regulatory and protective pathways. In our initial experiment animals were divided into three groups: (1) pigs infected with a virulent PRRSV isolate MNW2B; (2) pigs vaccinated using a contemporary PRRS ATP vaccine (Ingelvac®); and (3) control pigs. Tissues were collected between days 3-6 post infection/vaccination. Because PRRS is a respiratory disease we decided to collect the following tissues: Cranial lung, Distal lung, Tracheobronchial lymph nodes (TBLN) and Tonsil. Total RNA was isolated and labeled using Alexa Fluor® 555 and Alexa Fluor® 647 dye (Invitrogen). A microarray loop design was applied to compare gene expression between individuals from all three groups and additionally to investigate differences due to day post infection within the first group. For this study we used the Swine Protein-Annotated Oligonucleotide Microarray (SPAM; www.pigoligoarray.org). Our first task was to validate these arrays; this was performed by analyzing transcriptional profiles for TBLN, Lung and longissimus dorsi muscle from normal or PRRSV infected pigs. Additionally, our laboratory conducted Real-Time RT-PCR for 12 selected candidate genes to confirm the microarray results. Final statistical analyzes of both the validation and PRRSV study are underway, however our diagnostic analyses, based on R software and on the unique SPAM mismatch oligonucleotide design, revealed no evidence of non-specific hybridization. This confirmed the value of the SPAM design and usefulness of the mismatch hybridization controls. Our studies serve as essential, foundational steps for our investigation of immune pathways and identification of candidate genes involved in PRRSV resistance. Our approach combines different methods, molecular and protein expression, that are necessary for the full study of a complex, destructive disease like PRRS.